The COVID-19 pandemic has posed significant challenges to healthcare systems,
highlighting the need for a better understanding of the factors influencing disease
susceptibility and severity. While factors such as advanced age, male sex, and high
body mass index are recognized as aggravating conditions, there remains variability
in disease manifestation that is not yet fully explained. Previous genetic studies have
identified polymorphisms associated with severe COVID-19 outcomes, but the
representation of the Brazilian population in these studies remains limited. This study
aimed to analyze genetic variants previously associated with COVID-19 in 64 capillary
blood samples stored on filter paper. The comparison groups consisted of 41
symptomatic individuals and 23 oligosymptomatic/asymptomatic individuals. DNA was
extracted and analyzed by Mendelics using the BeadChip® Infinium Global Screening
Array-24 version 3.0 (Illumina®) genotyping technology, which can identify 654,000
SNPs per sample. In the data analysis, a total of nine samples did not meet quality
criteria, resulting in a final genomic dataset of 55 samples from 34 symptomatic and
21 oligosymptomatic/asymptomatic individuals. The initial screening included 108
SNPs previously described in the literature as associated with infection risk,
hospitalization, and COVID-19 severity. From this dataset, 17 SNPs were identified in
the studied population. Data analysis was conducted using the GenomeStudio
software, leading to the exclusion of nine samples due to unsatisfactory quality criteria,
leaving 34 symptomatic and 21 oligosymptomatic/asymptomatic participants.
Regarding demographic data, participants ranged in age from 14 to 79 years (mean
age of 38 years; median of 34 years), with a predominance of females (64.7%). Clinical
analysis indicated that symptomatic individuals had a higher frequency of cough
(79.4%), fever (70.6%), headache (67.6%), and sore throat (61.8%), while fever and
headache occurred in 22.2% of oligosymptomatic individuals. Regarding genetic
analyses, the screened SNPs were located in the following loci: 1q21.3 (rs35154152),
2p16.1 (rs1123573), 3p21.31 (rs2531743), 4p15.2 (rs16877005), 4q24 (rs13107325),
6p21.1 (rs1886814), 6p21.32 (rs35122968), 9p21.3 (rs28368148), 9q34.2 (rs8176719
and rs687289), 9q34.13 (rs505922), 11p15.5 (rs35705950), 19p13.2 (rs73510898 and
rs34536443), 19p13.3 (rs2109069 and rs12610495), and 19q13.33 (rs1405655). The
frequency distribution of the SNPs between the analyzed groups was not statistically
significant, and thus, an Odds Ratio estimation for the risk alleles reported in the
literature and the clinical outcomes of this study was not performed. These findings
reinforce the need for broader genomic analyses to better understand the influence of
genetic factors on the response to SARS-CoV-2 infection and potential genetic
biomarkers for COVID-19 susceptibility.

Culicidae are the most important arthropod vectors in public health, as they are involved in the transmission of pathogens that cause viral and parasitic diseases that affect humans and animals. The blood obtained by mosquitoes during hematophagy has been used to detect pathogens that may or may not be transmitted by vectors (molecular xenomonitoring) and to identify host feeding preferences. Another tool used in pathogen surveillance is the FTA card with sugar bait, to detect them in saliva samples from vectors. This thesis aimed to (i) compare the abundance and richness of mosquitoes in riparian vegetation areas and in dwelling outdoors; (ii) evaluate whether the size of the riparian vegetation fragment affects the abundance and richness of mosquitoes; (iii) evaluate the possible circulation of pathogens (dengue virus, Zika, chikungunya, Mayaro, Oropouche, Plasmodium spp, and trypanosomatids) from female mosquitoes (iv) and in FTA cards; (v) verify the females' food source; (vi) produce and distribute educational material on insect vectors and disease prevention. Collections were made quarterly for two consecutive years, covering the rainy and dry seasons in ten locations with riparian vegetation in the urban area of Porto Velho, Rondonia, and in 30 associated outdoors. The sampling of the culicidae was performed using the electric aspiration technique for 15 minutes. In the laboratory, all mosquitoes were taxonomically identified, and the female mosquitoes were given an FTA card with a sugar solution. After being sacrificed, the mosquitoes were stored at minus -80°C for subsequent RNA/DNA extraction and analysis by real-time PCR (qPCR) or conventional PCR to detect the pathogens and verify the food source. Abundance and richness data were analyzed using generalized linear mixed models (GLMM), and the infection rate was estimated using the minimum infection rate (MIR). A total of 3,914 mosquitoes were collected, distributed among 11 genera (Aedes, Aedeomyia, Anopheles, Coquillettidia, Culex, Limatus, Mansonia, Psorophora, Trichoprosopon, Uranotaenia, and Wyeomyia) and 21 species. The most abundant species were Culex quinquefasciatus (73.93%), Aedes albopictus (8.91%), and Aedes aegypti (7.05%). The outdoor habitat had a higher abundance of mosquitoes (2,285 - 58.38%) compared to riparian vegetation (1,629 - 41.62%). However, riparian vegetation exhibited more mosquito species than peridomicile vegetation. Mosquitoes were significantly more abundant in the dry season compared to the rainy season; however, the dry season showed a significant difference with lower species richness. A non-significant trend was observed between the size of the forest fragment and the abundance of mosquitoes in both environments. We observed a significant correlation of more species in locations with larger forest fragments of riparian vegetation. The molecular analysis of 221 pools resulted in one positive sample for ZIKV (Ct: 37.20 - MIR: 1.08), containing two females of Ae. aegypti collected in riparian vegetation. Of the 95 FTA cards, ZIKV was detected in four cards offered to Ae. albopictus, Cx. quinquefasciatus, Psorophora albipes, and Psorophora ferox, collected in three locations and in both environments. The booklet “Insects of Medical Interest” was produced and distributed in dwellings (n = 266 booklets) and in six public schools (n = 576 booklets) near the riparian vegetation areas. The integration of vector surveillance with molecular surveillance can be useful for monitoring the occurrence of emerging and re-emerging pathogens, as well as for directing control and prevention methods. Furthermore, scientific dissemination and popularization activities are an important strategy for preventing disease-carrying insects, having a social impact that can increase the involvement of academia with society, as well as the community's contact with science.

Snakebite envenomation, caused by the accidental injection of venom from
venomous snakes into humans, is a serious public health issue, especially in tropical
regions of developing countries. Each year, 5.4 million people suffer snakebites,
resulting in 1.8 to 2.7 million envenomations. In Brazil, between 2010 and 2023,
343,403 cases were reported, with the Northern region leading in notifications
(124,055 cases). Among the four genera of venomous snakes in Brazil, the Bothrops
genus is of significant medical interest, accounting for approximately 86% of
snakebites. Within this genus, Bothrops jararacussu ranks among the species that
cause the most incidents and inject the largest amounts of venom, leading to severe
damage. Although antivenom is effective against systemic effects, it has limited
efficacy against local damage. Given this context, the aim of this study was to
evaluate the dynamics of the inflammatory and hemorrhagic processes following
experimental envenomation with the P-I metalloproteinase isolated from Bothrops
jararacussu venom, named BjussuMP-II. To this end, the experimental
envenomation was conducted in the gastrocnemius muscles of male Swiss mice,
weighing between 18 and 22 grams (divided into groups of 5 animals), through
intramuscular injection of 50 μL of PBS or 50 μg of BjussuMP-II. After 30 minutes,
treatments with antivenom, LED, or a combination of antivenom and LED were
administered. Three hours later, blood and muscle samples were collected for
myotoxicity assays, histological sections, RT-qPCR to evaluate microRNA-223 gene
expression, and immunoenzymatic assays for IL-1β detection. Other groups of
animals were used to evaluate rolling and adhesion in the vascular endothelium via
intravital microscopy and to analyze the hemorrhagic process by inducing a halo on
the dorsal skin. The results reaffirmed that antivenom therapy alone is not effective
in treating the local damage caused by snakebites. In contrast, photobiomodulation
with LED, whether alone or in combination with antivenom, was shown to reduce
myotoxicity, tissue damage, leukocyte influx, the number of rolling or adherent
leukocytes in the vessels of inoculated muscles, as well as hemorrhage on the dorsal
skin. When applied alone, LED was able to reduce microRNA-223 gene expression;
however, none of the treatments were effective in reducing IL-1β levels in vivo. It
was concluded, therefore, that LED treatment, when applied alone, reduces
myotoxicity, hemorrhage, tissue damage, and leukocyte influx into the tissues, while
also demonstrating potential for reducing microRNA-223 in vivo. Furthermore, the
combination of antivenom and LED proved to be equally effective.

The Trypanothione Reductase (TR) is a validated molecular target for the development of new inhibitors to treat diseases caused by Trypanosomatids. On another front, snake venom toxins, due to their structural and functional diversity, are important sources of bioactive molecules for the development of new drugs, including the treatment of diseases such as Leishmaniasis. In this context, the present study aims to analyze the interaction of toxins (Giroxin and Convulxin from Crotalus durissus terrificus) with the Trypanothione Reductase of L. braziliensis (TRLb) through in silico models and evaluate their inhibition through in vitro models. To achieve this, molecular docking between TRLb and toxins was performed using the ClusPro2.0 tool, and molecular dynamics were conducted using the GROMACS tool. Subsequently, the gene for TRLb was inserted into the pET 28(a+) vector and expressed in E. coli. The relative molecular mass and isoelectric point were determined by 12.5% one-dimensional and two-dimensional SDS-PAGE, and enzymatic activity was evaluated using colorimetric methods. The venom from Crotalus durissus terrificus was obtained from the LABIOPROT-FIOCRUZ-RO venom bank. It was fractionated by affinity chromatography on a Tricorn 10/20 column filled with Benzamidine-Sepharose resin (GE Healthcare). In the inhibition assays, the IC50 was calculated using the non-linear regression model in the GraphPad Prism program. As results, in silico assays indicated that Convulxin and Giroxin are capable of interacting with the enzyme's catalytic site, opening possibilities for further research in the field. TRLb was obtained with a high degree of purity, and its catalytic activity was preserved, with a molecular mass and isoelectric point consistent with those described in the literature. The purification of Giroxin from C.d.t resulted in three fractions, named 1, 2, and Giroxin, with electrophoretic profiling showing a single protein band with a molecular mass of approximately 30 kDa. Giroxin was used at different concentrations for the assessment of its inhibitory capacity, namely 5 μM, 1 μM, and 0.2 μM, resulting in a decrease in the catalytic activity of TRLb by 88%, 41.2%, and 18%, respectively.

The hepatitis delta virus (HDV) is highly pathogenic and is linked to the progression of serious liver diseases such as cirrhosis and cancer. HDV requires the presence of hepatitis B virus (HBV) for its replication and spread. An estimated 12 to 72 million people are infected worldwide with HDV. Despite high endemic indicators, infections caused by HDV represent a condition neglected by health services. The diagnosis of HDV has limitations due to the availability of inputs applicable to the detection and determination of viral infection. Aiming to produce relevant biotechnological resources for the purpose of detecting HDV infection, recombinant antigenic targets were developed from sequences of the delta antigen (HDVAg) of HDV genotypes I and III. The characterization of recombinant HDVAg (rHDVAg) was carried out and different variables were analyzed with a view to evaluating the application of this target for the indirect detection of HDV infections. To this end, serological assays were conducted to detect the presence of anti-HDVAg antibodies, using ELISA and a panel containing 170 serological samples. Of these, 66 sera were previously characterized by qRT-PCR, of which 46 were identified as seropositive for HDV and 20 as seronegative. Of these, 66 sera were previously characterized by qRT-PCR, of which 46 were identified as seropositive for HDV and 20 as seronegative. Likewise, to investigate possible cross-reactions, 104 samples previously characterized in relation to different viral infectious agents were used. Preliminary results from serological assays using rHDVAg-I and rHDVAg-III revealed a sensitivity of 53.66% and specificity of 90.0%, and a sensitivity of 95.7% and specificity of 90.0%, respectively. It was possible to demonstrate the effectiveness of rHDVAg-III in terms of its ability to specifically recognize infections caused by HDV. The partial data obtained in the enzymatic assays demonstrate that the product developed in this study has potential for diagnosing HDV. The phase I proof of concept demonstrated the ability to apply rHDVAg, for the purpose of developing diagnostic prototypes, which can comprise immunoenzymatic and chemiluminescent assays and constitute a rapid test device for detecting viral infection. Perspectively, it will be necessary to increase the number of samples tested from both endemic and non-endemic areas to complete the analysis comprehensively and advance to phase II of the study. It is important to highlight that the development of different diagnostic strategies with inputs of high sensitivity and specificity for the detection and/or screening of the rHDVAg antigenic target can assist in the prognosis and, with attention to preventing the evolution of the infection and reducing general costs related to complications. due to chronicity and will also allow attention to the patient in order to limit liver worsening caused by the HDV virus.

Ticks are obligatory ectoparasites of vertebrate animals, which could accidentally parasitize humans. In Brazil, only rickettsiae are known to cause illness to humans, however, there are lack of studies about other pathogens in this country, such as the bacteria Borrelia and Ehrlichia, especially in the Amazon region, where there are a great number of anthropophilic tick species. The Borrelia genus are spirochaete bacteria that infect mammals and birds worldwide, and some species can cause the human Lyme disease. There are recent records of Borrelia-infected ticks in Brazil, but the occurrence of any human borreliosis still lack of evidence, although cases of a Lyme-like have been reported. The relapsing fever is a zoonosis also provoked by Borrelia spirochaetes that are associated to argasid ticks from the Ornithodoros genus. Ehrlichia spp. is a genus of obligatory intracellular Gram-negative bacteria that infect phagocytes of humans and animals. Ehrlichia chaffeensis and Ehrlichia ewingii are etiological agents of, respectively, monocytic and granulocytic ehrlichiosis in North America. This study aimed to survey the incidence of Borrelia spp. and Ehrlichia spp. infecting freeliving and parasitic ticks in the region of the Porto Velho Natural Park (PVNP), Rondônia state. The collection of free-living and wild animals parasitic ticks were performed within trails in the forest area of the PVNP, whereas the ticks collections from domestic animals (dogs and horses) were performed in private properties in the surrounding areas of the Park. After collected, the ticks were identified and submitted to DNA extraction and nested-PCR to search for borrelial Flab gene DNA, and semi nested-PCR targeting ehrlichial dsb gene DNA. The PCR products were visualized in a 1.5% agarose gel. A total of 153 free-living ticks were collected and the most abundant species were Amblyomma scalpturatum, followed by Amblyomma sp., A. naponense and Amblyomma calcaratum. Regarding the method of collection, 70% of the ticks were collected by dragging, 18% by dragg-flagging and 12% by visual inspection. An infection rate of 4.6% of Ehrlichia canis was found in Rhipicephalus linnaei. The infection rates of E. canis were 2% in Amblyomma scalpturatum and 1% in Dermacentor nitens. The positive samples were confirmed to the ehrlichial species by DNA sequencing of dsb gene partial sequence. There were no Flab gene fragment detection in the tested ticks, suggesting that there were no borrelial infection in the ticks. Although Ehrlichia infection have been reported in horses, the present study reports E. canis in D. nitens parasitizing horses for the first time. This study is the first to report E. canis in A. scalpturatum. The results of this study indicate that non-native pathogens may be introduced to the wild tick fauna in Brazil, and could help in a better understanding of the tick-pathogen-host relationships in the Western Amazon Region.

The enzyme Cathepsin D (CatD) and Phospholipase A2 (PLA2) play an important role in
various pathophysiological conditions, being involved in processes ranging from
neurodegenerative and inflammatory conditions to the life cycle of various parasites.
Moraes et al. (2022) demonstrated the interaction of human CatD with BthTX-II, a PLA2
from Bothrops jararacussu, forming a stable complex that modulates the activity of CatD,
rendering it active at higher pH values. This phenomenon may be capable of assisting in
the elucidation of some biochemical events related to CatD in the tumor
microenvironment and Alzheimer's disease. Considering the high cost of obtaining
human CatD for the continuity of studies, the present work proposes the adaptation of a
new protocol for obtaining CatD from bovine liver (Bos indicus) and the partial evaluation
of its interaction with a BthTX-II, aiming to enable future biophysical and structural
studies. The methodology used to obtain the enzyme consisted of grinding 500g of
bovine liver, followed by two solubilization/precipitation steps and subsequent
fractionation on a Sepharose affinity column + Pepstatin A previously equilibrated with
0.1M sodium acetate pH 5.0 and eluted in a step gradient of 0 to 100% sodium acetate
pH 5.0 + 0.5M NaCl (buffer B) and 0.1M Tris-HCl pH 8.5 (buffer C) at a flow rate of
1mL/min for 40 minutes. Subsequently, the sample was subjected to reverse-phase C18
column chromatography (25 cm x 0.45 cm, Discovery) previously equilibrated with 0.1%
trifluoroacetic acid and eluted under a gradient of 0 to 70% ACN 99.9% (v/v) and 0.1%
TFA (v/v) at a flow rate of 1mL/min. The evaluation of relative mass, purity degree, and
isoelectric point was observed on 12.5% SDS-PAGE and two-dimensional
electrophoresis. The enzymatic complex was evaluated for its enzymatic activity on the
substrate casein at different pH values, subjected to reading at 660 nm in a
spectrophotometer. The predicted interaction between the proteins was observed in silico
through molecular docking using the ClusPro tool and molecular dynamics simulations
(MD) performed using the GROMACS program with the CHARMM36m force field. The
bovine liver CatD exhibited a relative molecular mass of ~32 kDa, an approximate pI of
5.5, and optimal activity on casein at pH 3 and temperature 50°C. The CatD/BthTX-II
complex showed modulation in its catalytic activity, extending its activity on the casein
substrate up to pH 6, with the optimal activity value at pH 5. In silico data highlight that
the most predominant amino acid residues related to protein binding consist of
hydrophobic residues. The complex showed little variation in its stability, suggesting
significant structural resistance. Thus, the bovine liver CatD forms the same complex
observed with its human analog and BthTX-II, enabling its use for subsequent structural
studies.

Biolarvicides are agents that stand out worldwide in the biological control of mosquitoes of medical importance, reinforcing the initiative of harmless control and thus avoiding the accelerated use of chemical means, which minimizes problems related to resistance in various mosquito species. Given this versatility, we evaluated the effectiveness of two products, Vectobac-WG® and Vectomax-FG®, against the larvae of two species of Mansoniini in the laboratory. First-stage larvae (L1) of Ma. humeralis and Ma. titillans were obtained from field females and L1 larvae of Aedes aegypti from the laboratory were used as a comparison parameter for bioassays, which followed the World Health Organization (WHO) protocol (with adaptations), in an environment with controlled temperature (T) and relative humidity (RH) (T 25 - 27ºC / RH -70 - 80%). Different concentrations of Vectobac-WG 3. 000 UTI/mg (0.00025ppm, 0.0005ppm, 0.001ppm, 0.002ppm, 0.004ppm and 0.008ppm), and Vectomax-FG 50 UTI/mg (0.00031ppm, 0.00062ppm, 0.00125ppm, 0.0025ppm, 0.005ppm and 0.01ppm) were tested as larvicides using 50 L1 larvae in triplicates, with four repetitions at different times. Larval mortality was monitored for up to 48 hours. The persistence of the same products was tested in two lighting conditions (light and dark) for 35 days. The dose-effect and persistence of the products on the larvae were analyzed using the Kruskal-Wallis test (non-parametric ANOVA), and Dunn's test was used for multiple comparisons at a 5% significance level. To obtain the lethal concentrations (CL50 and CL90) after 24 and 48 hours, Probit analysis was carried out (concentration x mortality). Vectobac-WG caused an average mortality of 61.1% for Ma. humeralis, 70.8% for Ma. titillans and 37.7% for Ae. aegypti. Treatment with Vectomax-FG resulted in mortality of 50.1% for Ma. humeralis, 53.7% for Ma. titillans and 51.1% for Ae. aegypti. The estimated lethal concentrations (LC) of Vectobac-WG for Ma. humeralis and Ma. titillans were LC50 from 0.0004ppm to 0.0008ppm and LC90 from 0.0019ppm to 0.0029ppm, respectively. The CLs estimated for Vectomax-FG for Ma. humeralis and Ma. titillans were higher, i.e., CL50 0.0016ppm to 0.0018 ppm and CL90 0.0051ppm to 0.0061ppm, respectively. After an evaluation of 5 weeks from the start of application in the laboratory, the product Vectomax-FG caused mortality of > 66% up to the 3rd week for the two lighting conditions evaluated and Vectobac-Wg caused larval mortality of more than 50% under the use of direct light up to the 3rd week, this product showed mortality of 43% up to the 2nd week in the dark. In view of the results, we concluded that the larvae of Ma. humeralis and Ma. titillans were susceptible to the products Vectobac-WG and Vectomax-FG. The mortality of the larvae depended on the concentration of the product used, the type of bacilli and the species of mosquito used. The products persisted for 21 days, with mortality ranging from 100% to 58%. From the second week onwards, there was a noticeable difference between the lighting conditions.

The mosquito Aedes aegypti is an arthropod vector of viruses such as Dengue,
Zika, Chikungunya, and urban Yellow Fever. Only Dengue and Yellow Fever
have vaccines as preventive treatments, with vector control still being a method
for suppressing arboviral diseases and preventing new outbreaks. Therefore,
discovering substances and their applicability for vector control is an ongoing
pursuit. Given this context, the objective of this study was to evaluate the
insecticidal effects of essential oil and vegetable oil on different life stages of Ae.
aegypti. Essential oil from Morinda citrifolia fruit (OE noni) and vegetable oil from
Carapa guianensis seed (OV andiroba) were used to assess ovicidal (20
eggs/treatment), larvicidal (10 L3 larvae/treatment), pupicidal (10
pupae/treatment), and adulticidal activity through the ingestion of sugar baits (20
females/treatment). The protocol adapted from the World Health Organization
was used in tests with concentrations ranging from 1000, 750, 500, 250, 125, 50,
25, 10, and 5 ppm, with distilled water and 1% ethanol controls, and three
replicates and three repetitions with different generations. The tests were
observed for 24 to 72 hours. No ovicidal effect was observed for either oil at the
tested concentrations, except for the OE noni higher concentration. The larvicidal
activity of OE noni resulted in LC50 (24 h) 90.1 ppm and LC90 (24 h) 146.1 ppm,
causing 100% mortality at concentrations from 250 to 150 ppm in the first 24
hours of the bioassay. OV andiroba had LC50 (24 h) 646.7 ppm and LC90 (24 h)
3048.3 ppm, causing 66.67% mortality at 250 ppm and 58.33% at 400 ppm after
48 hours of observation. The pupicidal activity of OE noni resulted in LC50 387.6
ppm and LC90 588.3 ppm, causing 82.22% mortality at 500 ppm and 64.44% at
450 ppm in the first 24 hours of the bioassay. OV andiroba had LC50 (24 h)
1460.02 ppm and LC90 (24 h) 2701.7 ppm, causing 98.89% mortality at 3000
ppm and 2500 ppm in the first 24 hours. No adulticidal activity was observed from
the ingestion of either oil (OE noni and OV andiroba) at the tested concentrations,
despite high ingestion. It is concluded that both oils exhibited insecticidal activity
against the larval and pupal stages of Ae. aegypti. Additionally, the consumption
of sugar baits containing both oils did not result in adult mortality at the tested
concentrations, regardless of the high level of engorgement.

The Oropouche virus - OROV, is characterized as a re-emerging arbovirus of great concern to public health due to its capacity for genetic rearrangement, and since its discovery has been responsible for several outbreaks of acute fever identified in Central and South American countries, with at least 500,000 reported cases. The western Amazon region is already notorious for outbreaks of acute febrile illnesses, for which the lack of specific diagnoses for different pathogens makes patient management difficult. The OROV was already recorded in the region in the 1990s, and this is the first study since that record to detect individuals with acute febrile illness using a screening test to exclude Zika, dengue and chikungunya, confirming by sequencing the circulation of the Oropouche virus in the state of Rondonia and the border. Reports of the incidence of this virus are intrinsically favored by environmental and climatic conditions, which can favor the increase and distribution of the vector population to areas of human traffic. In addition, there is a problem related to the lack of distribution of the specific diagnosis established in routine screening for arboviruses in Brazil. Thus, through molecular techniques of RT-qPCR (Reverse Transcription quantitative Polymerase Chain Reaction) used as a primary exclusion methodology for Zika, dengue and Chikungunya, and evaluating profiles of OROV co-infections with other arboviruses. Sanger sequencing confirmed the circulation of the Oropouche virus in the state of Rondonia and the border. 20.99% (421/2006) positive OROV cases were identified, with detections of 1.43% (6/421) for co-infections with dengue (serotypes 1 and 2) circulating in border locations in western Amazonia, among serum samples, with 95.81% (366/382) of the cohort characterized as infected individuals with reported symptoms, mainly fever, headache, myalgia, and back pain. Among the positive samples, it was possible to obtain a total of 3.40% (13/382) of samples for analysis of the S and M segments of Oropouche, which showed similarities with Brazilian sequences. It was thus possible to verify the circulation of OROV in Rondonia and border areas, strengthening genomic surveillance of emerging and re-emerging viruses.

Candida spp. they colonize the human organism in a commensal way, only, depending
on the states of the host, these microorganisms can to comport as opportunistic agents
and cause from superficial infections to systemic infections. These infections result in
prolonged hospitalizations, increased hospital costs, and high rates of morbidity and
mortality. Despite this, when it comes to the northern region of Brazil, studies on
candidiasis are scarce, and in Rondônia, they are non-existent. Therefore, the present
study aimed to carry out the molecular characterization of clinical isolates of Candida
spp. obtained from patients in the Porto Velho/RO region, as well as to phenotypically
determine virulence factors and antifungal susceptibility. For this, the isolates were
obtained from clinical specimens of patients attended at the Laboratory of Medical
Mycology of the Centro de Pesquisa em Medicina Tropical de Rondônia (CEPEM/RO),
which were first identified through the CHROMagar Candida medium. Molecular
characterization of the species was performed through PCR of the ITS region of fungal
rDNA coupled to genetic sequencing. The disk diffusion method was used following
the recommendations of the M44-P protocol to determine antifungal susceptibility to
nystatin, amphotericin B, fluconazole, itraconazole and miconazole. To determine the
hemolysin activity, the sheep blood agar assay was used and, to determine the
phospholipase activity, the egg yolk agar method was used. The present study
evidenced the presence of five species of Candida as causing infections in patients in
the Porto Velho/RO region, of which C. albicans 22/31 (71%) was the most frequent,
followed by C. tropicalis with 6/31 (19,4%), C. parapsilosis with 1/31 (3,2%),
Cyberlindnera jadinii with 1/31 (3,2%) and C. duobushaemulonii with 1/31 (3,2%).
Resistance to fluconazole was observed in 26/30 (86,7%) isolates and to itraconazole
in 24/30 (80%) isolates, mainly in species of C. albicans, C. tropicalis and
Cyberlindnera jadinii, evidencing a reduction in the sensitivity of Candida spp. to first
option triazole antifungals. Besides, the present study demonstrated that 30/30 (100%)
of Candida isolates were hemolysin producers and that only C. albicans 9/22 (40,9%)
and Cyberlindnera jadinii 1/1 (100%) were phospholipase producers. These results
inedited provide important information for the management of infections in relation to
strategies of the treatment, control and prevention of the cases of candidiasis
diagnosed in the region.

COVID-19 is the disease caused by the severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) that had the first confirmed case in November 2019 and in March 2020 it was officially considered a pandemic. With this rapid spread, rapid diagnosis is a key part of actions to combat the disease. SARS-CoV-2 is expected to become a regular seasonal epidemic agent in the calendar of respiratory viruses. Thus, even outside the pandemic period, referral hospitals and health centers will receive patients suspected of COVID-19, requiring sensitive, accurate and adequate tests for low demand. Point of care (POC) tests can bring advantages such as savings in materials and supplies and a reduction in diagnosis time. The objective of this work is to standardize a POC molecular test to aid in the diagnosis of COVID-19 using a portable qPCR device called Q3-Plus, verifying the performance of this equipment in comparison to bench equipment used as the gold standard for diagnosis. And finally, compare the results obtained using a commercial RT-qPCR protocol and an adapted one. The study population consisted of 50 patients from the state of Rondônia with samples of nasopharyngeal secretion collected between March and April 2020 for SARS-CoV-2 research, processed using the RT-qPCR technique using the ABI 7500 fast equipment, com its results released by Lacen Rondônia through the Gal System. The first stage of the tests consisted in the standardization of the qPCR reaction in the Q3 plus equipment using a multiplex that uses the N1 region (CDC) as a viral target and the RNP as an endogenous target. In this step, a plasmid was used. After that, the 50 RNAs were used in the sensitivity and specificity, repeatability and reproducibility tests. Using a 6-point plasmid dilution curve as a sample, the Q3 plus was able to detect up to 1.25 copies of the viral target in the reaction, exhibiting a slope of -2.98 and an efficiency of 116%, in addition to 100% of agreement between the results of reproducibility and repeatability tests. The Point of care molecular test has been shown to be sensitive and specific for the SARS-CoV-2 virus, as it has shown agreement with the results obtained with other equipment or kits and is still reproducible for more than one year operator with 100% agreement on the results.

The COVID-19 pandemic showed the importance of genomic surveillance for rapid responses to
decision-making in the control of emerging and re-emerging diseases. SARS-CoV-2 had an
increase in mutations and infection cases during the second wave of infections that started in
December 2020 in Manaus, observing an increase of subvariants in circulation in subsequent
periods. Among the most critical features of the virus is the ability to evade the adaptive immune
response mounted against SARS-CoV-2. Thus, even with the increase in vaccination rates, there is
a great and justifiable need to continue surveillance for SARS-CoV-2 and subsequent lineages that
may emerge. Thus, the objective of this study was to analyze the epidemiological data and the
genetic diversity of SARS-CoV-2 in the state of Rondônia. Samples collected from different units
in the state were screened by qualitative diagnostic amplification ct<25 for SARS-CoV-2 at
LACEN/RO. The epidemiological and clinical data of the individuals were collected through
electronic medical records, subsequently the samples were extracted using a commercial kit, then
evaluated for viral load and sequenced using the COVIDSEQ Kit (Illumina). Analysis of sequence
quality and mutations were performed by Pangolin and Nextclade, and at the end, phylogeny was
performed using the maximum likelihood method. The results showed the circulation of 5 lineages
in Rondônia between 2021 and 2022, where Gama, Delta and Omicron were the most prevalent
variants, and to a lesser extent, the Zeta and Mu clades. Regarding sex, it was found that there was
a prevalence of female sex and the individuals had a median age of 40 years. The most commonly
reported symptoms were sore throat, cough, and fever, with no statistical differences in relation to
the variants present in the study. The viral load was measured, presenting a median of 7.35 log10
copies/ml. Among the variants, the VOC Omicron had the highest number of subvariants and
mutations in the S gene. Moreover, the gamma variant had the highest prevalence of mutations
when the ORFab, S, E, and N genes were evaluated. There was a higher number of unvaccinated
individuals in this cohort, where statistical analyses showed that vaccination was a protective factor
for hospitalizations. ML phylogeny confirmed the clades circulating in the state of Rondônia. In
conclusion, the results showed the clinical epidemiological profile compiled with the data
generated by genomic surveillance of SARS-CoV-2 infections, tracing a profile of how the
infections were in the state of Rondônia in the years of the study. This work was an important
initiative for genomic surveillance in the state of Rondônia, which helped the state government
make decisions in the face of SARS-CoV-2 epidemic waves.

Studies carried out in vivo and in vitro demonstrated that the P53 protein and the cytokine IFN-γ may be involved in protective mechanisms against Plasmodium infection. The objective of this study was to analyze the expression level of the TP53 and IFNG genes in infection caused by P. vivax in different parasitemias in patients treated at the Tropical Medicine Research Center – CEPEM. A total of 52 samples were collected, which were classified into 4 groups with different parasitemias: +/- (half a cross), + (one cross), ++ (two crosses) and +++ (three crosses). Extraction and purification of total RNA from whole blood and cDNA synthesis were performed. The optimal primer concentration and cDNA efficiency were determined to standardize the real-time PCR technique. To evaluate the variation in expression, the comparative Ct methodology was used, given by the search ΔCT: Ct gene of interest – Ct of the control gene. Ct values of the TP53 and IFNG genes were obtained, using the GAPDH gene as an endogenous control.

Note a reduction in TP53 gene expression in patients with high parasitemia +++ (three crosses) compared to those with lower parasitemia. However, no statistically significant difference was found when comparing the other groups to each other. Expression of the IFNG gene did not vary significantly with different levels of parasitemia. The results support studies that indicate that the parasite can suppress P53 levels in the host to continue its life cycle.

Antibiotics, whether natural or synthetic, are the main tools in combating bacterial infections. However, the inappropriate and extensive use of antibiotics has favored the emergence of strains of bacteria that are multi-resistant to these medications, which has become a serious public health problem. One of the most prevalent species of pathogens in the hospital environment, causing infections, is the Gram-negative bacterium Acinetobacter baumannii. The World Health Organization identifies carbapenem-resistant A. baumannii as a priority in the development of new antibiotics, as these bacteria have resistance mechanisms that allow them to escape the most adverse classes of antibiotics. Therefore, in the present work, using bioinformatics and medicinal chemistry tools, we identified molecules with antimicrobial potential to be tested against A. baumannii. As strategy I, the computational construction of a hybrid molecule of antibiotics from the β-lactam and tetracycline classes, potentially effective against the penicillin-binding protein and the 30S ribosomal subunit of A. baumannii, was carried out. This hybrid molecule, called IM-TP, was used as a model for a similarity search in the PubChem database, in order to find molecules that retain the desirable characteristics of the hybrid model. The search found 295 molecular structures that retained characteristics of tetracyclines and β-lactams. As strategy II, through a partnership with Dr. Geisa Caprini Evaristo and the Center for Studies on Biomolecules Applied to Health (CEBio/FIOCRUZ-RO), they carried out in silico bioprospecting of the molecules present in the skin secretion of Phyllomedusa camba, a species of anuro, of which there are records of antimicrobial activity for its skin secretion. Using Strategy II, a total of 149 molecular structures were obtained from the PubChem database. The molecular structures found in strategies I and II were used in molecular docking against the penicillin-binding protein (PDB 3UDX) and the 30S ribosomal unit (PDB 7RYG) of A. baumannii. The molecular docking results highlighted promising molecules, both from strategy I and strategy II, with the potential to be tested in vitro against A. baumannii. For strategy I molecules, despite good of in silico results against both A. baumannii targets, no suppliers were found for these molecules, that made it impossible to perform in vitro antimicrobial activity assays against A. baumannii. Based on the in silico results of the molecules presented in the skin secretion of P. camba against the penicillin-binding protein and the 30S ribosomal subunit of A. baumannii, antimicrobial activity assays were carried out for the skin secretion of P. camba, which were discovered in MIC and IC50 data. For the skin secretion of P. camba against A. baumannii (ATCC 19606), the result was an MIC of 50 μg/mL and IC50 of 28.92 μg/mL, which seem promising when compared with information from the literature.

Recent WHO data report the occurrence of up to 5 million envenoming caused by venomous snakes in the world, with up to 0.15 million fatalities, leaving 0.4 million victims with injuries irreversible. The Bothrops genus is responsible for more than 90% of snakebites in Brazil. To minimize its effects, the only specific treatment is serum therapy, which does not adequately reverse the local effects, making it necessary to search for adjuvant therapies associated with serum therapy to improve the treatment of these effects. In this context, photobiomodulation with LED is considered an important therapeutic resource with anti-inflammatory, analgesic, and healing effects. Despite its indication as an associative treatment, its mechanism of action is still the subject of studies. Thus, the general objective of this study was to analyze the oxidative redox potential of the application of photobiomodulation with LED and its action on the inflammatory profile of murine macrophages, stimulated with Bothrops jararacussu venom and bothropstoxins I and II. To this end, several methodological approaches were carried out, such as: cell marking and cell proliferation assays, analyzes of cell death by necrosis and apoptosis, dosage of cytokines and antioxidant enzymes, gene expression for markers of M1 and M2 phenotypes and PGE₂ receptors as well as COX, PGE₂ dosage and study membrane potential study. The results showed that photobiomodulation with LED modulated macrophages to the M2 phenotype, induced an increase in antioxidant enzymes and cytokine release, decreased cell death by necrosis and apoptosis, increased mitochondrial membrane potential, and modulated gene expression of PGE2 and COX 1 and 2 receptors. Together, the data obtained contribute to the understanding of the mechanism of action of Photobiomodulation with LED and reaffirm its important oxidative and modulatory redox characteristics, sufficiently capable of acting as necessary associative therapy with serum therapy to reduce the local effects of snakebite envenoming and may be used as a therapeutic resource teo the treatment of this important condition.

Plasmodium falciparum is known to cause severe malaria. Current treatment consists in artemisinin-based combination therapy, but resistance can lead to treatment failure. The knowledge of essential proteins of P. falciparum can be used on the search for new antimalarials, among these is shikimate dehydrogenase (SDH), part of a pathway responsible for the production of endogenous aromatic amino acids. SDH from P. falciparum (PfSDH) is unknown to the scientific community, therefore, this study aims to establish the first protocol for active PfSDH expression. Putative PfSDH nucleotide sequence was used to construct the optimized expression vector pET28a+PfSDH inserted in E. coli BL21(DE3). Optimal expression conditions were acquired by varying IPTG and time while temperature was fixed at 37 °C. Western Blot analysis was applied to verify appropriate PfSDH expression. Solubilization and purification started with lysis followed by double IMAC purification. SDS-PAGE was applied over various steps. Enzyme activity was measured spectrophotometrically by NADPH oxidation. Tanimoto coefficient and pharmacophore pattern similarity were applied to the in silico search for ligands employing the substrate DHS as template. Optimal PfSDH expression occur at 0.1 mM IPTG for 48 hours of growth at 37 °C and shaking at 200 rpm. Recombinant PfSDH obtained after purification was soluble, pure and its physiological catalysis was confirmed. Among all possible ligands obtained, 15 molecules had greater relevance when combining both methods applied. Thus, this study describes the first protocol for heterologous expression of PfSDH in soluble and active form, likewise instigating for a higher comprehension related to target-ligant interactions.

Phospholipases A2 (PLA2s) are proteins found in abundance in the venom of snakes of the genus Bothrops and play an important role in the inflammatory reaction and leukocyte activation. Monocytes and lymphocytes are cells of the immune system characterized by mediating and presenting effective responses during an inflammatory response. The NLRP3 inflammasome is a multiprotein complex that is present in cells of the immune system and is activated by stimuli such as pathogen-associated molecular patterns (PAMPs) and damageassociated molecular patterns (DAMPs). The action of PLA2s against human peripheral blood mononuclear cells (PBMCs) is still not well understood. In this sense, the present study aimed to evaluate the role of PLA2s (BthTX-I Lys49) or (BthTX-II Asp49) from Bothorps jararacussu on NLRP3 inflammasome activation. Thus, viability tests were carried out from human PBMCs in a period of 4 hours by DNA labeling with 7AAD. The results showed that at concentrations of 5 and 10 μg/mL there was no cell death. The relative gene expression of NLRP3, Caspase1, ASC, IL-1β and GAPDH (internal control) gene were performed by RT-qPCR. Furthermore, the protein expression of these components (NLRP3, Caspase-1, ASC), gasdermin D (GSDMD) and β-actin (internal control) was verified by Western Blot. The results obtained showed significant amplification when compared to the GAPDH control of NLRP3, ASC, Caspase-1 and IL-6 in 1 hour and, of IL-1β, in 2 hours. Protein expression was performed after 4 hours of PBMC stimulation, and the results obtained showed significant expression of NLRP3 and GSDMD inflammasome complex proteins. Another method of proving the activation of the NLRP3 inflammasome was the counting of punctas formed by the NLRP3 sensor component by immunofluorescence. Then, to verify the cell death dependent on the activation of caspase-1, pyroptosis, LDH was measured in the supernatant of the PBMCs incubated with the different stimuli after 4 and 6 hours. The release of LDH occurred in 6 hours, as well as the expression of GSDMD, suggesting that the release of this molecule occurs due to the formation of pores in the membrane, a consequence of pyroptosis. Taken together, the data obtained so far show that the PLA2s, BthTX-I and BthTX-II, isolated from Bothrops jararacussu venom, activate the NLRP3 inflammasome complex culminating in the formation of membrane pores through GSDMD in human PBMCS that contribute to the inflammatory response observed in envenomation by this snake.

Streptococcus agalactiae (GBS) is the main etiological agent of neonatal infections today, and colonization of the rectovaginal site of pregnant women is the main risk factor for the development of the pathology. This bacterial species is classified into ten serotypes (Ia, Ib and II-IX), which vary in terms of prevalence, geographic and temporal distribution and degree of virulence. GBS exhibits a repertoire of surface proteins essential for adhering to and invading host tissues, as well as hindering their recognition by the immune system and ability to form bacterial biofilms. In addition, an increase in the rate of resistance to prophylactic antimicrobials has been reported. Brazil fits in the list of countries where GBS is not recognized as a relevant etiological agent of neonatal diseases. Therefore, the present study aimed to carry out the identification and phenotypic and molecular characterization of serotypes, virulence factors and antimicrobial resistance of Streptococcus agalactiae isolates in pregnant women in the city of Porto Velho-RO. GBS samples were isolated from rectovaginal cultures of pregnant women aged 35 to 37 weeks attended in the basic health network. GBS was identified in the samples by PCR (Polymerase Chain Reaction) with specific primers for the cfb gene, and later by sequencing the 16S ribosomal region. Serotyping was performed by conventional multiplex PCR assay. The genes associated with virulence (hylB, cylE, lmb, bac, bca, scpB and hvgA) and resistance (erm(A), erm(B) and mef(A/E)) were evaluated by PCR. The antimicrobial susceptibility profile was determined by the disk-diffusion method. The ability to form bacterial biofilms in-vitro was also measured. Of the total number of pregnant women included in the study, 23% (114/496) were positive for GBS colonization, and of these it was possible to recover 85 isolates. We evidenced the presence of six serotypes in the region, with 36.5% (31/85) belonging to Ia, 14.1% (12/85) to Ib and II, 11.8% (10/85) to III, 15 .3% (13/85) at V and 8.2% (7/85) at VI. Regarding virulence factors, the following frequencies were observed: scpB in 100% (85/85), lmb in 97.6% (83/58), bca in 84.7% (72/85), hylB in 64.7% (55/85), clyE in 60% (51/85) and bac in 57.6% (49/58). A high rate of resistance to tetracycline, erythromycin and clindamycin was observed with 74.1% (63/85), 14.1% (12/85) and 3.5% (3/85), respectively. Already 100% (85/85) of the isolates were sensitive to penicillin, ampicillin, cefazillin, ceftriaxone, chloramphenicol and vancomycin. Regarding resistance genes, it was observed that of the isolates not sensitive to macrolides and lincosamines, 64.7% (11/17) had the erm(A) gene, 41.2% (7/17) had the erm(B) gene, and 88.2% (14/17) carried the mef(A/E) gene. It was shown that 91.8% (78/85) of the isolates had a strong/moderate ability to produce biofilms. It was evidenced that 100% of the serotype III isolates are carriers of the hvgA gene and, therefore, classified as hypervirulent. The second description of serotype VI in Brazil stands out here, with a higher prevalence rate than that found previously. The present study points to a high rate of genes associated with virulence and resistance in the isolates from the region, associated with a homogeneous spread among the serotypes found, and underlines the importance of establishing active surveillance systems to understand the infections caused by GBS.

Snake venoms contain a mixture of molecules capable of mediating directly or indirectly inflammatory, hemorrhagic, neurotoxic, myotoxic and other cellular changes. In this context the metalloproteases, which are enzymes highly toxic profile, zinc dependent and of variable molecular mass, playing a relevant role in the complex and multifactorial response characteristic of the snake poisoning. In this pointing of view, several studies with snake venoms seek to find substances with possible potential therapeutic application for the development and drug improvement, demonstrating a beneficial characteristic for these biological molecules. Therefore, this work aims to evaluate in vitro the antimicrobial action from the Bothrops jararacussu snake venom, as well as from an isolated metalloprotease. To obtain the metalloprotease, it was necessary to fractionate the venom by liquid chromatography, using a CM-Sepharose-type ion-exchange column and the choice of fraction corresponding to the protein of interest was based on its electrophoretic characteristics (SDS-PAGE), molecular mass (mass spectrometry) and enzyme activity (fibrinogenolytic and caseinolytic). The results showed that the metalloprotease was purified with a satisfactory yield and adequate purity, representing about 5.4% of the total venom and presenting a single polypeptide chain with a molecular mass between 23 and 24 kDa, classifying it as a P1 class metalloprotease. This enzyme showed metal ion-dependent proteolytic activity, confirming that it belongs to the class of metalloproteases from snake venoms. It was also observed that this metalloprotease was capable of inducing hemorrhage in vivo in the cremaster muscle of mice analyzed by intravital microscopy. In antimicrobial activities, Bothrops jararacussu venom, showed inhibition at some concentrations tested in two ATCCs strains (100, 50, 25, 12.5 μL/mL for Klebsiella pneumoniae and 100 and 50 μL/mL for Stapylococcus aureus), however, the different concentrations of metalloprotease tested were not able to inhibit the bacterial growth. Finally, the data presented in this study support the biochemical and functional characterization of the protein under study, but they differ from other published studies regarding antimicrobial activity. Other work must be carried out to verify the pharmacological potential of metalloproteases, as well as other components of snake venom, which have a significant contribution to scientific knowledge with important biotechnological applications for human health.

Human T-lymphotropic virus type 1 (HTLV-1) is one of the etiologic agents of T-cell leukemia/lymphoma in adults and of HTLV-1-associated myelopathy, called tropical spastic paraparesis (HAM/TSP). About 5-10 million individuals are infected with the virus worldwide. In Brazil, the tests applied for the diagnosis of the disease are restricted to the screening steps in blood donor samples. Thus, the availability of supplies to assist in the detection of infection, as well as its implementation at the public health level aimed at the Unified Health System (SUS) would be an alternative to facilitate the diagnosis of HTLV-1 infection. The use of molecules with high stability and ability to recognize molecular targets in a specific and sensitive way is a criterion for the effectiveness of diagnostic tests. From this perspective, camelid VHHs stand out for having such characteristics. Therefore, this work proposes the use of anti-p24 VHHs for the construction of biosensors applicable to the development of assays for the diagnosis of active infection caused by the HTLV-1 virus. From a previous characterization, after sequencing, it was possible to select 3 clones of VHHs (anti-p24/C11, anti-p24/C52 and anti-p24/C86). However, only clones VHH-11 and VHH-52 were effectively recombined in pET-22b(+) vector and then subjected to expression standardization in Escherichia coli strain BL21(DE3) and SHuffle® T7 Express. Of the expression conditions evaluated to obtain VHH C52 in a soluble form, the one that proved to be more effective was with induction at 30ºC and 0.3 mM of IPTG in the SHuffle® T7 Express strain. After purification by immobilized metal ion affinity chromatography (IMAC), Western blot and ELISA results confirmed, respectively, the presence of the VHH-histag and the permanence of its reactivity against p24. From the synthesis of gold nanoparticles (AuNPs) by the Turkevich method and characterization by transmission electron microscopy (TEM), it was possible to establish the bioconjugation conditions of VHH-anti-p24/52 at a concentration of 8 μg and pH 9, to AuNPs, in the presence of 75 mM of the acrylic acid stabilizing agent N-hydroxysuccinimide-ester (NHS). The immunoreactivity of biosensors by DOT BLOT assay demonstrated the ability of bioconjugates to recognize different concentrations of recombinant HTLV-1 p24. The results achieved demonstrated the potential of VHH-anti-p24/52, as an input for the constitution of optical immunosensors applicable to the detection of HTLV-1 p24 protein. Still in this work, the application of VHH constituting an electrochemical biosensor was explored, from the coupling of VHH anti p24/52 to gold electrodes. In preliminary tests, it was possible to obtain a sensor capable of detecting up to 1 ng/μL of HTLV-I recombinant p24. The results achieved demonstrate the potential of anti-p24 VHHs, however, additional tests are needed to determine the application of VHH-type antibodies constituting optical and electrochemical biosensors for the construction of different devices for detecting p24 of the HTLV-1 virus.

Culicoides biting midges are vectors of arboviruses, including Oropouche Virus and
Bluetongue Virus. These insects are found in forest areas, but deforestation can contribute to
the adaptation of vectors to anthropogenic environments. Much information about this group
remains unknown, especially in Rondônia, where there are few studies. This study aims to
assess the diversity of biting midges in fragmented forest environments; evaluate the
identification of cryptic species, with emphasis on vectors, through the DNA Barcode tool;
check the blood meals used by female midges. The collections were carried out in the Jamari
National Forest in 2018. In total, 48 species of 1,480 individuals were identified, distributed in
5 subgenera: Anilomyia, Avaritia, Haematomyidium, Hoffmania and Mataemyia, and 13
informal species groups: acotylus, carpenteri, dasyophrus, debilipalpis, eublepharus, fluvialis,
guttatus, hylas, leoni, limai, pachymerus, paraensis and reticulatus. The most abundant species
were Culicoides pseudodiabolicus (457 - 30.88%), C. glabrior (154 - 10.41%), C. debilipalpis
(137 - 9.26%), C. coutinhoi (97 - 6.55% ), C. quasiparaensis (63 - 4.26%) and C. brownei (57
- 3.85%). Fifteen new species occurrence records were signed for the state of Rondônia. The
Santa Maria trail was more diverse (45 species out of 1,007 individuals) compared to the Potosi
trail (35 species out of 473 individuals). DNA was extracted and PCR performed from 455
engorged females, and identified in 186 individuals, with a source blood meals from Tamandua
tetradactyla (Anteater), Pithecia irrorata (Gray’s bald-faced saki), Choloepus hoffmanni
(Hoffmann’s two-toed sloth), Choloepus didactylus (Southern two-toed sloth), Psophia viridis
(Trumpeter), Ramphastos tucanus tucanus (Toucan), Pauxi sp. (Curassow), Ateles chamek
(Spider monkey) and Homo sapiens (human). 186 sequences of 19 Culicoides species were
obtained and submitted to DNA barcoding analysis, directed to the COI gene, and resulted in
an alignment containing 84 haplotypes with a size of 558 bp. For two specimens it was not
possible to identify morphologically at a specific level due to problems in assembling the
individuals, but when analyzed in the phylogenetic tree together with other species, these
individuals grouped with the clades C. pseudodiabolicus and C. debilipalpis, demonstrating
that such specimens belong to such a taxonomic unit. The DNA barcoding tool has been shown
to be capable of rapid and accurate identification of species of organisms using one or a few
short standardized regions of DNA and has been widely used for effective identification of other
groups at a specific level as well. The expansion of knowledge about the diversity of midges,
ecology, feeding sources and molecular identification tools can provide important information
for understanding the ecological, epidemiological and taxonomic profile of these insects in the
Amazon.

Phospholipases A2 (PLA2s) are proteins found in snake venoms that have hemolytic,
anticoagulant, myotoxic, edematogenic, bactericidal and inflammatory actions. In
Bothrops jararacussu snake venom, these proteins make up about 30% of the venom
and two types were isolated, BthTX-I, an enzyme that has a lysine at position 49
(Lys49-PLA2) and BthTX-II, an enzyme that has an aspartate at position 49 (Asp49-
PLA2). These toxins are myotoxic and inflammatory. The NLRP3 inflammasome is an
intracytoplasmic platform that recognizes molecules released when tissue is damaged.
After this recognition, caspase-1 is activated and IL-1β is released, contributing to the
inflammatory response observed in cases of snakebite envenomation. The objective
of this study was to verify the action of these PLA2s, BthTX-I and BthTX-II, in the
activation of the NLRP3 inflammasome, in an experimental envenomation model in
mice and, in vitro, in elicited murine macrophages as well as in C2C12 myoblasts. For
this, the animals received an intramuscular injection of BthTX-I or sterile saline
solution. Serum was collected for measurement of creatine kinase (CK), lactate
dehydrogenase (LDH) and IL-1β. The muscle was removed for Western blot and gene
expression by RTq-PCR for the NLRP3 inflammasome. Leukocyte influx in muscle was
also evaluated by intravital microscopy. In macrophages stimulated with BthTX-I and
BthTX-II and in C2C12 myoblasts, the NLRP3 inflammasome, the P2X7 receptor, the
type 2 (EP2) and type 4 (EP4) prostaglandin receptors and the cyclooxygenase (COX)
pathway were evaluated by RTq-PCR, indirect immunofluorescence, Western blot in
the presence and absence of EP2 and EP4 antagonists. The data obtained showed
that BthTX-I induced leukocyte influx into the muscle of the animals, observed under
microscopy, as well as the release of LDH and CK into the plasma of the animals. The
treatment of the animals with A438079, a P2X7 receptor antagonist, did not change
these parameters. BthTX-I induced the activation of the NLRP3 inflammasome in the
muscle, but the participation of P2X7R in this effect was not observed. Together, the
results showed for the first time that BthTX-I in gastrocnemius muscle induces
inflammation and, consequently, NLRP3 inflammasome activation, caspase-1
activation and IL-1β release. In elicited and stimulated macrophages, the two toxins
induced gene expression of the inflammasome components NLRP3, P2X7, cytokines
(IL-1β, IL-6 and IL18), COX-1 and COX-2, EP2 and EP4. On the other hand, these
findings were not observed in C2C12 myoblasts. Treatment of macrophages with
antagonists for EP2 (PF04418948) and EP4 (GW627368X) abolished this effect. The
two toxins also induced protein expression of NLRP3 inflammasome components and
treatment with the antagonists reduced this effect. Activation of the NLRP3
inflammasome was confirmed by immunofluorescence assay and by quantification of
IL-1β released by macrophages. By treating macrophages with antagonists for EP2
(PF04418948) and EP4 (GW627368X) it was possible to confirm the participation of
PGE2 receptors in this effect. Together, the data obtained show that the muscle
damage caused by BthTX-I in vivo triggers the activation of the NLRP3 inflammasome
complex. In vitro, BthTX-I and BthTX-II induce macrophages to activate the NLRP3
inflammasome with the participation of the COX pathway and PGE2 receptors,
contributing to the harmful effect observed in envenomation.

Hepatitis B is considered an important public health problem worldwide, the chronic form of the disease caused by the hepatitis B virus (HBV) is considered the main cause of cirrhosis and cellular hepatocarcinoma. HBV is a DNA virus, of the Hepadnaviridae family and it is estimated that 257 million people are chronically infected by the virus. Classified into 10 genotypes (A-J) and 24 subgenotypes (A1-A3, B1-B5, C1-C6, D1-D6, and F1-F4) HBV is geographically distributed in a diversified way. In the Amazon basin, genotypes A, D, and F are concentrated, with genotype F considered specific to the Americas with predominance in native populations. Virological aspects such as genotypes, subgenotypes, mutations, and coinfection with hepatitis Delta virus (HDV) are determinants of the complexity of the disease in chronic carriers. To characterize the genotypes and subgenotypes of HBV in indigenous people with chronic HBV and co-infected with HDV analyze epidemiological, sociodemographic, serological, and molecular data of indigenous people living in the cities of Guajara Mirim and Nova Mamore located in the state of Rondônia/ Brazil. All the procedures used in the study followed the terms determined by the National Council of Ethics in Research – CONEP. In this context, the study population consisted of 41 indigenous people of both sexes, over 18 years of age, selected from the reactive results of HBsAg and Total Anti-HBc immunoassays. With the consent of the participants, the biological samples of serum for molecular analysis were collected by the team of the Molecular Virology Laboratory at Fiocruz-RO. The prevalence of HBV found in indigenous people located in the border region between Brazil and Bolivia was 2.8% (168/5908); of these, a total of 41 indigenous people, 27 mono-infected with HBV and 14 coinfected with HDV, were volunteers in the study. It was observed that 49% (20/41) of the participants had a family history of HBV infection and more than half of the population were =or> 40 years old (24/59%). Indigenous males (66% n=41) were more frequent than females (34% n=41). The mean time of HBV infection was 12.6 years (with a minimum of 2 and a maximum of 24 years). A total of 65.9% (27/41) of the Indigenous people were positive for DNA-HBV. Of the positives, 39% (16/41) are mono-infected and 27% (11/41) are co-infected with HDV. In the genotypic characterization, genotypes A, D, and F were observed, with a frequency of 40.7% (11/27) for genotype D (HBV-D), 33.3% (9/27) for genotype F (HBV -F,) and 25.9% (7/27) A (HBV-A); as for the observed subgenotypes, F2, F4, D2, D3, A1, and A2 were found. In addition, an important C69* stop codon mutation associated with the risk of cellular hepatocarcinoma was observed in a single sequence of individuals coinfected with HDV. Thus, there is evidence of genotypic diversity in the population that reveals a predominance of genotype D, which is more specific to the southern region of Brazil. The HBV genotypes and subgenotypes circulating among the Indigenous people of the State of Rondônia demonstrate for the first time the HBV-D genotype with greater frequency among the natives of the region. In conclusion, our findings showed a diversity of HBV genotypes, which is also found in other Brazilian geographic regions. Genomic surveillance in endemic locations for hepatitis B favors preventive public health measures to reduce HBV infection, and consequently HDV in endemic regions for the virus.

The use of sugar baits to deliver insecticides can be an alternative measure for the control of Ae. aegypti, which may include the use of specific devices for its application, i.e., bait stations (EI). The composition of these baits can be quite simple, i.e., sugar solution and insecticide (TSB), or it can also include attractants (ATSB). Although different insecticides and attractants have already been studied, few studies have investigated the bait composition and its relationship with its ingestion by mosquitoes using different models of EIs. That said, the aim of this study was to evaluate the effect of the composition of sugar baits (TSB and ATSB) with ivermectin (IVM) and its use in different bait stations (EI) on Ae. aegypti in the laboratory. Sugar baits with different types (crystal and brown) and concentrations (10%, 50% or 70%) of sugar and IVM (0.312ppm to 1000ppm) containing or not attractants (concentrated juice or guava pulp) were prepared through dilutions simple of stock solutions. The EIs were constructed using bottles, boxes, artificial plants and adapted plant pots. The experiments were carried out in screened cages (containing 20 to 30 mosquitoes) and the baits
were offered on cotton balls, except for the last experiment which was carried out in a tent, containing 100 mosquitoes and the baits were sprayed in EIs. Engorgement and mortality of males and females of Ae. aegypti was recorded using a stereomicroscope after 6 hours. Sugar type and juice source did not affect the engorgement of ATSBs, which was lower in relation to TSB. Higher concentrations of sucrose (50% and 70%) in baits with 100ppm of IVM were more engorged but did not change the mortality rate of males and females. High concentrations of IVM (500-1000ppm) using 70% sucrose reduced engorgement and, in general, caused mortality above 89%. The use of different concentrations of IVM in baits containing 10% and 70% sucrose to calculate the Lethal Concentrations (CLs) resulted in 28% and 63% higher engorgement in males and females, respectively, in baits containing 70% sucrose. compared to those containing 10% sucrose in ATSBs. Using TSBs, regardless of sucrose concentration, the engorgement rate was greater than 84% for males and females. The mortality rate increased with increasing IVM concentrations in the two baits, regardless of the sucrose concentration evaluated. In addition, the use of 70% sucrose caused a significant reduction in the LC50 of the TSB for females and males, however there was a significant increase in the LC90 in both baits only for females. Finally, engorgement was higher in the EIs with the baits containing 70% sucrose. Bait sprayed in Resting Boxes were generally more engorged by males and females. In the present study, the use of TSB, 70% sucrose and the Resting Box model bait station resulted in better mosquito engorgement under the experimental conditions used.

O genoma do HBV é considerado o menor entre os vírus DNA, possuindo tamanho aproximado de 3.2kb e 4 regiões de leitura aberta. O genoma possui diferenças nucleotídicas suficientes para classificação do HBV em 10 genótipos principais (A, B, C, D, E, F, H, I e J) e mais de 30 subgenótipos. O surgimento de mutações e variantes virais tem sido motivo de bastante discussão na literatura em relação a probabilidade de algumas dessas cepas ocasionarem escape ao sistema imunológico do hospedeiro, potencialidade de causar evolução à doença hepática de forma mais grave como cirrose e HCC, além do surgimento de variantes resistente ao tratamento. Esta pesquisa tem como objetivo analisar a frequência de mutações e variantes circulantes do vírus da hepatite B em indivíduos portadores crônicos do Ambulatório Especializado de Hepatites de Porto Velho, Rondônia. Trata-se de um estudo transversal com componente descritivo e analítico. O recrutamento de pacientes foi realizado por conveniência de acordo com os critérios de inclusão e exclusão. Foi adotada a técnica de PCR convencional para a amplificação do genoma completo do HBV, sendo que um total de 4 diferentes finais são originados, os quais dão cobertura de todo o genoma. A genotipagem foi determinada por meio da construção de uma árvore filogenética. A identificação de mutações nos genes (pré-S/S, P, X e pré-C/C) foi realizada por meio da construção dataset’s correspondentes a cada gene. De todas as amostras amplificadas, apenas 34 foram sequenciadas atém o presente momento em pelo menos 1 dos 4 fragmentos do genoma. Os achados do estudo mostram a presença dos subgenótipos A1 (17/34, 50%), D3 (7/34, 20,6%), F2 (5/34, 14,7%), D2 (2/34, 5,9%), A2 (1/34, 2,9%), D1 (1/34, 2,9%) e F4 (1/34, 2,9%). As análises no gene S mostraram a presença das mutações sG130S (2/28, 7,1%), Y100C (1/28, 3,44%), sL109M (1/28, 3,6%), sP120T (1/28, 3,6%), sG130N (1/28, 3,6%), sM133T (1/28, 3,6%), S/T140I (1/28, 3,6%) e sE164G (1/28, 3,6%). Um total de 6 amostras (6/30, 20%) apresentaram alguma deleção na região Pré-S2. Duas amostras continham, respectivamente, as alterações rtQ215H (1/28, 3,6%) e rtM250I (1/28, 3,6%). No gene X, foram identificadas as alterações A1762T/G1764A (4/5, 80%), T1653C (2/5, 40%), T1753C (2/5, 40%), G1613A (1/5, 20%). Os resultados das análises na região pré-C/C mostraram a presença das substituições G1896A (2/7, 28,6%), P5L (1/7, 14,3%) e A131V (1/7, 14,3%). Os achados das variantes genotípicas observadas corroboram com resultados de pesquisas na região. Apesar da relação das mutações no gene S com escape imunológico, não foi identificado nenhum paciente HBsAg negativo. Resultados de pesquisas não relacionam mutações de resistência identificadas ao comprometimento da eficácia da atual terapia preconizada pelo Ministério da Saúde. As mutações nos genes pre-C/C e X podem ter potencial de avanço de doença hepática, no entanto, uma análise experimental mais abrangente é necessária. As comparações de frequências de genótipos e deleções pré-S2 identificadas neste estudo ao estágio da doença hepática dos indivíduos apresentaram valor de p não significativo.

Bioactive molecules, such as peptides, proteins and enzymes are found in the biodiversity showing therapeutic potential in neglected diseases. Snake venom and its fractions are of great interest due to its virucidal potential demonstrated in in vitro studies. The Dengue Virus (DENV) is the most important arbovirus that infect humans - with 400 million cases every year, and with an increasing number of severe cases with dengue hemorrhagic fever or dengue shock syndrome. Although there is no specific medication to treat dengue infection, snake venoms may represent a potential source of antiviral activity. In this perspective, the present study aims to evaluate the effects of different toxins isolated from the Bothrops diporus snake venom on DENV. For the study, cell cultures of the C6/36 and VERO lineages were established for viral cultivation and propagation, from the isolation of DENV from the serum of patients previously characterized by molecular biology. The detection of virus serotype maintained in culture was performed by RT-qPCR using specific oligonucleotides, and viral titration performed by flow cytometry, with confirmation of cell infection by indirect immunofluorescence and the performance of the kinetics of viral infection of DENV serotype 1 (DENV1). The results obtained for viability tests in VERO cells of the different toxins tested for standardization demonstrated that the Crotoxin in 1h and 24h showed viability greater than ± 60% and when incubated for 5 days, the concentration of 50 µg / mL affected 50% of cell viability. The BthTX-II incubated for 4 days, showed viability greater than ± 50%. While PLA2 BthTX-II from Bothrops jararacussu incubated for four days showed viability greater than ± 50%. When tested the B. diporus venom and toxins – the target biomolecules of this study – on the cell cytotoxic assay, the venom (50µg/mL) showed CC50; and PLA2-Asp (12.5 - 50 µg / mL) affected cell viability in the percentage of ± 50 - 65% respectively. PLA2-Lys has been shown to affect 50% of cells at a concentration of 50 µg/mL. The viral serotypes 1 and 3 were detected after the 2nd viral culture of RT-qPCR. Among the serotypes evaluated, when performing DENV1 replication kinetics in C6/36, the virus showed a gradual increase in viral particles which were stabilized from the 5th to 6th day of incubation being detected by indirect immunofluorescence. In conclusion, after propagation stages it was possible to identify and detect by molecular and cellular methods only DENV1. In cytotoxicity analysis, which is one of the first tests of a drug candidate and performed in mammalian cells, the CC50% presented PLA2s ranged between 25-50 µg/mL. The preliminary results obtained allows the continuation of the study, which consists of evaluating the antiviral activity of snake toxins and venoms. Since PLA2s showed antiviral potential, more researches on the same class of biomolecules contributes to a better understanding of the mechanism of action.

The growing development of antimicrobial resistance, and the decrease in the supply of anti-infective drugs, arouse the need to look for therapeutic alternatives that are effective and safe. Therefore, as a strategy, antimicrobial peptides have numerous advantages, from less likely to induce microbial resistance, potent and fast microbicidal action at micromolar concentrations, synergistic capacity, in addition to immunomodulating properties. Among those, the vast amount of peptides from the skin of anurans stand out, such as Ocellatin-KN, constructed from Ocellatin-K1, isolated from the secretion of the skin of Leptodactylus knudseni. In this frame, the objective of this work was to evaluate in vitro the antimicrobial profile of OKN against ATCC strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, and ATCC strain of the yeast Candida albicans, in addition to the evaluation of the cytotoxic and immunomodulatory characteristics related to the production of ROS, IL-6 and OKN phagocytosis stimulation on murine peritoneal macrophages. The physical-chemical and structural characteristics of OKN were determined in silico, using bioinformatics tools. The antimicrobial and biofilm inhibition (MIC and MBIC) potential of OKN was performed by the broth microdilution method, at concentrations from 500 to 3.9 μg/mL. With the same concentrations as related, the cytotoxicity of the peptide was tested against mouse peritoneal macrophages by the MTT method, with incubations of 24 and 48 hours. An assay for the production of ROS was carried out based on a fluorescence reaction, with concentrations of OKN from 500 to 31.2 μg/mL. The supernatant (from the cytotoxicity assay) produced in 6 hours was used to measure Interleukin 6 using the immunoenzymatic method, with concentrations of OKN from 62.5 to 7.8 μg/mL. The phagocytosis assay was conducted using opsonized zymosan particles using OKN concentrations of 125, 62 and 31 μg/ml. The results were analyzed using the GraphPad Prism software version 6.0, performing the analysis of variance (One-Way ANOVA) with the application of Tukey's post-test. It was observed that OKN has mild activity against the bacteria tested, with a MIC of 125 μg/mL for the two gram-negative strains (E. coli and P. aeruginosa) and 15.6 μg/mL for S. aureus; and no inhibitory effect on C. albicans. Biofilm formation of E. coli and P. aeruginosa was discretely inhibited at the highest concentrations (500 and 250 μg/mL) and noticeably inhibited for S. aureus and C. albicans at all concentrations (500 to 3.9 μg/mL). On murine peritoneal macrophages, OKN was cytotoxic at concentrations equal to and greater than 125 μg/mL, and did not stimulate the production of ROS even at the highest concentrations tested, however, an increase in the production of the cytokine IL-6 was observed in the concentration of 62.5 μg/mL, in addition to increasing the rate of phagocytosis by macrophages at all concentrations evaluated. These findings suggest that OKN has mild antimicrobial activity, antibiofilm potential and likely immunostimulating activity on murine macrophages

Snake venom contains compounds that have the potential to be used in bioprospecting in biology and medicine. These proteins, such as L-amino acid oxidases (LAAOs), cause edema, microbicidal activity, cytotoxicity, coagulant and anticoagulant activity, hemorrhagic and pro-inflammatory effects. LAAO isolated from the venom of Calloselasma rhodosthoma (Cr-LAAO) has been shown to be a powerful stimulator of neutrophil activation and inflammatory mediator production. However, the mechanisms underlying Cr-LAAO-induced cell activation are not well understood. Previous research has shown that this enzyme can activate the NADPH oxidase complex and the PKC-  signaling protein, resulting in the production of reactive oxygen species (ROS), as well as stimulate phagocytosis, chemotaxis with the participation of p38 MAPK and PI3K, MPO release, cytokine production (IL-8, IL-6, and TNF-), neutrophil extracellular traps (NETs), and lipid mediators (LTB4 and PGE2). The activation of the cyclooxygenase (COX) pathway can result in the formation of some lipid mediators within lipid droplets (LDs). LDs are cytoplasmic organelles that are highly associated with the inflammatory response. They are found in a variety of cell types and can increase in number and/or size during an infection. Simultaneously, ROS can activate other inflammatory mechanisms such as inflammasomes. NLRP3-type inflammasomes are cytosolic protein complexes that cleave caspase-1, resulting in the proteolytic activation of pro-inflammatory cytokines such as IL-1. Thus, the purpose of this study is to assess the effect of Cr-LAAO on the activation of the NLRP3 inflammasome, as well as the activation of the COX pathway for the production of PGE2 and its involvement in the formation of LDs. The neutrophil transcriptome was examined using the microarray technique, and it was discovered that Cr-LAAO plays a role in upregulating the expression of genes involved in the NADPH oxidase complex and inflammasomes, as well as genes involved in lipid signaling and metabolism. Immunofluorescence assays for NLRP3 revealed protein accumulation and the formation of punctas in the cytosol, indicating that this complex is activated. Furthermore, protein expression analysis in the presence and/or absence of Apocynin, an inhibitor of NADPH oxidase, revealed that when ROS are inhibited, the expression of proteins that comprise the NLRP3 inflammasome is reduced. The release of IL-1β was also detected, and the pharmacological inhibition of NLRP3, caspase 1 and ROS reduced the levels of this released cytokine. Some of the genes positively regulated in the microarray belong to the phospholipase A2 family, specifically cytosolic phospholipases A2- (cPLA2-); and enzymes involved in the synthesis of PGE2, COX-2, and prostaglandin E synthase (PTGES). In addition, the genes perilipins 2 and 3 (PLIN 2 and 3) and diacylglycerol acyltransferase 1 (DGAT1), which are involved in the formation of LDs, were up-regulated. In neutrophils, Cr-LAAO stimulated increased cPLA2-phosphorylation, LD biogenesis, and PGE2 synthesis. cPLA2- (CAY10650) and DGAT-1 (A922500) inhibitors inhibited LD formation and PGE2 secretion. Finally, this is the first study to look at the effects of Cr-LAAO on the regulation of metabolism and lipid signaling in human neutrophils, as well as its ability to stimulate the activation of the NLRP3 inflammasome complex via ROS production

Sand flies are important insects in public health that act as vectors of Leishmania, that cause leishmaniasis. In Rondônia State, studies about sand flies have been carried out since the 1960s which knowledge of local fauna is important for identification of vectors and understanding of the dynamics of disease transmission. Currently, sand fly identification is based on morphology of adults, which can be problematic when species are morphologically similar. The diversity of these insects and occurrence of cryptic species in Rondônia makes it difficult the identification based only on morphological characters, then approaches that help the classical taxonomy can improve the accuracy of the correct species identification. Therefore, this study aimed to implement the use of DNA Barcoding in Amazonian sand flies to optimize the species identification and investigate the existence of cryptic species, associating this approach to classical taxonomy in the description of fauna of the region. For this study, specimens from other projects carried out by Fiocruz Entomology Laboratory were used, in addition to additional collections carried out in four municipalities of Rondônia. A toral of 652 specimens belonging to 69 morphospecies were morphologically identified, then legs or thorax were removed, and their genomic DNA were extracted, and the cytochrome c oxidase subunit I (coI) fragment was amplified and sequenced. Sequences were analyzed by best-match and best close match similarity criteria and six different delimitation methods of single-locus (ABGD, RESL, TCS, GMYC, PTP and mPTP) were used to infer operational taxonomic units (OTUs). Sequences data of coI region produced in this study was evaluated with those previously published, and the database was analyzed using ABGD and TCS species delimitation methods. The gene coI proved to be useful for identification of approximately 80% of analyzed species from Rondônia, not being effective in separation in some species of the genus Evandromyia, Nyssomyia, Trichophoromyia and Trichopygomyia. The analyzes showed the presence of cryptic diversity, in at least four analyzed algorithms, for the species: Bichromomyia flaviscutellata, Evandromyia bacula, Evandromyia tarapacaensis, Evandromyia wilsoni, Nyssomyia fraihai, Psychodopygus carrerai, Psychodopygus davisi and Trichophoromyia ubiquitalis. Also was possible to establish the association between males and their respective females, which have isomorphic process between species. The approach also enabled description of Pintomyia fiocruzi and Psathyromyia pradobarrientosi females. Sequence analyzes of specimens from other locations and Rondônia demonstrate the importance of using a robust database to improve accuracy in delimitation of sand fly species. When barcodes of closely related species or from different geographic regions were added, changes were observed in delimitation of some species, showing events such as a much greater cryptic diversity or grouping closely related species. The results presented reinforce the need for molecular identification as a complement to morphological identification, demonstrating the importance of using this approach to reveal the cryptic diversity of some sand fly species.

Os mosquitoes have a worldwide distribution, being present in urban, rural and forest environments. Urbanization without planning favors the proliferation and survival of mosquito vectors and diseases in cities. In Brazil, the species of urban mosquitoes that stand out are Aedes aegypti, Aedes albopictus and Culex quinquefasciatus. The three species are associated with the transmission of several arboviruses, such as the Dengue, Zika and Chikungunya and Wuchereria bancrofti viruses (etiological agent of lymphatic filariasis) for the latter species. The general objective of the present work was to verify the abundance of the three main species of mosquitoes and to evaluate the possible occurrence of arboviruses in their populations, in the urban area of Porto Velho, Rondônia. Initially, a preliminary study was carried out in the Tancredo Neves neighborhood with household collections, with the purpose of adapting field and laboratory techniques, and subsequently a study was carried out in 27 residences distributed in nine neighborhoods in intra and peridomestic environments. Two collection techniques were used: ovitrampa (without reed, only for larvae collection) and electric aspiration for the capture of adults lasting 15 minutes. The adults, generated in the laboratory from the larvae, were used to verify the vertical transmission of DENV, CHIKV and ZIKV. The specimens captured by the electric aspiration technique had, in addition to the body, saliva collected on FTA® cards for analysis of the presence of these viruses. The samples were submitted to the RNA extraction process, followed by RT-qPCR in real time. The positive pools were also submitted to a semi-nested PCR protocol and subsequently to capillary sequencing. The species abundance data were analyzed in the Free Platform R and the estimate of infection by virus and by species was calculated by the Minimum Infection Rate (MIR) and Maximum Likelihood (MLE). In the preliminary study, 1,701 mosquitoes were collected, of which 1,332 were used for virus detection, resulting in 76 pools divided between: Culex quinquefasciatus (57), Aedes aegypti (18) and Aedes spp. (1), obtaining two positive pools for ZIKV, resulting in an MIR of 1.50 and 1.51 of MLE. Subsequently, a total of 2,342 mosquitoes were obtained in the nine sampled neighborhoods, distributed in five species, being Cx. Quinquefasciatus, Ae. aegypti and Ae. albopictus the most abundant, constituting 272 pools of which one was positive for DENV (MIR and MLE: 2.58). In addition, it was possible to detect DENV-4 from a sample of saliva collected on an FTA card, which belonged to a female of the Ae species. aegypti. The sequences of the positive samples were identified with DENV-4 genotype II. This study demonstrates the first record of DENV and ZIKV transmission in urban mosquitoes in Rondônia, as well as showing the first occurrence of vertical / venereal transmission of DENV-4 and ZIKV in male mosquitoes of the species Ae. aegypti and Cx. quinquefasciatus, respectively.

Hepatitis Delta Virus (HDV) is a defective virus that needs the Hepatitis B Virus (HBV) envelope to complete its infectious process. Infection with this virus is associated with more severe liver disease compared to HBV monoinfection. Chronic patients with hepatitis delta are more likely to progress to fibrosis, cirrhosis, cellular hepatocarcinoma, and liver failure. Its still limited diagnosis, carried out by detection of Anti-HDV in HBV serum, only allows detection of viral infection. Molecular techniques such as real-time PCR have sensitivity and specificity for accurate diagnosis, follow-up of the natural course of infection, and monitoring response to treatment; however there are no internationally accepted standardized assays yet. This proposal aims to meet this demand by developing a real time PCR assay for HDV detection and quantification. Filling the gap in the developed in house tests, this test will use the One-Step technique and two internal controls will be inserted, aiming to produce a test of high precision, sensitivity, specificity and reproducibility, characterizing it as a tool for fast, reliable and useful diagnosis. mainly in areas of greater endemicity, such as the Western Amazon region.

Leishmaniasis presents itself as an endemic of non-contagious infectious characteristics and of anthropozoonotic character. There are about 350 million people exposed to the risk of contracting this infection and 2 million new cases of this endemic disease are registered annually, with a an estimated 20,000 to 30,000,000 deaths per year worldwide. Treatment of patients with cutaneous leishmaniasis in most countries is performed with the antimonial drug pentavalent (Sb + 5), which may be meglumine antimoniate (Glucantime) or stibogluconate sodium (Pentostam). However, it has been observed in recent years that some strains of Leishmania spp. have less sensitivity to Sb + 5 compounds. One of the possible factors associated with lower sensitivity in the Americas is the presence of the endosymbiont “Leishmania Virus RNA ”type 1 (LRV1) in some strains of Leishmania spp. Faced with the problematic presented, the present work seeks to analyze the groups LRV1 positive and LRV1 negative with in vitro susceptibility to the trivalent antimonial - emetic tartar (Sb + 3), seen in some studies that Sb + 5 is a prodrug and through biotransformation it becomes Sb + 3, form active and toxic antimony. For this, 20 strains of Leishmania braziliensis were selected isolated from patients treated at the Center for Tropical Medicine of Rondônia (CEMETRON) diagnosed with LT, 10 of which harbor the LRV1 symbiote. So far, In vitro tests were carried out with Sb + 3 of 15 strains of L. braziliensis, with 8 LRV1 positive and 7 LRV1 negative. RNA was also extracted from these strains for later quantification of viral load of positive strains for LRV1 and analysis of gene expression related to redox metabolism. The results obtained in the in vitro experiments showed two groups: (i) susceptible and (ii) less susceptible. Of the 15 strains analyzed, 12 presented profiles susceptible and 3 less susceptible. In addition, qPCR was standardized for quantify viral load, as well as analyze Leishmania genes involved in metabolism redox. The results obtained in in vitro susceptibility to the compound Sb + 3 will be correlated with the presence of the endosymbiont LRV1, viral load and gene expression involved in Leishmania redox metabolism. Given the above, it is known that it is extremely understanding the in vitro susceptibility of strains isolated from LT patients because these findings can provide answers of great relevance for the monitoring and treatment of LT and parasitic infection harboring endosymbiont LRV1.

BSTRACT A better understanding of the biological processes underlying the progression of disease infection is urgently needed to reduce the morbidity and mortality of malaria. Like many diseases caused by protozoan parasites. Several evidences support the harmful effect of malaria on skeletal and cardiac muscle in animals and humans. Malaria has been shown to cause harmful effects in humans, leading to muscle weakness, muscle fatigue, breathing difficulties, kidney and liver failure, and can lead to cardiac myopathies. Most antimalarial drugs used are active against various stages of the blood parasite. Anti-malarial drugs, widely used, have a short clinical life due to the development of parasite resistance to the drug. In addition to the issue of resistance, another concern is that antimalarials are also associated with the development of adverse reactions, of which the ability to affect skeletal and cardiac muscle stands out. Therefore, the present study intends to experimentally evaluate the effect of treatment with antimalarials on the behavioral responses in a model of muscle pain in male Balb / c mice infected with Plasmodium berghei of the ANKA strain.

Single nucleotide polymorphisms (SNPs) near the locus of interleukin 28B (IL28B; interferon [IFN] -λ-3) have been associated with the virological response and spontaneous clearance of Hepatitis B Virus (HBV). The impact of IL28B SNPs in HBV infected patients is still poorly studied, especially involving indigenous populations. In view of the above, the study aimed at the genotypic identification of the SNPs rs8099917 and rs12980275 of the IL28B gene. This is the first study on the polymorphism of the IL-28B gene in the indigenous population of the state of Rondônia. The study was carried out with the Indian population Warí of the Pole of Guajará-mirim-RO, in which 40 samples were analyzed, including indigenous men and women, with ages ranging from 18 to 70 years and chronic hepatitis B. Samples were analyzed by quantitative Real-Time PCR for DNA-HBV detection and quantification, the genotypic identification of the SNPs was performed by conventional PCR, followed by RFLP technique and sequencing. It was possible to identify the rs8099917 SNP genotypes in 40 samples, the TT genotype was identified in 90% (36/40) of the indigenous, TG genotype in 8% (3/40) and only one indigenous GG homozygote. For SNP rs 12980275 the AA genotype was identified in 67% (27/40) of the indigenous, 13% AG (5/40) and 20% (8/40) for GG. Therefore, the study demonstrated that the indigenous are inactive chronic carriers of HBV, none presents a serological marker of viral replication. For both SNPs the favorable genotype was the most frequent, the genotype TT (rs8099917) and AA (rs12980275), corroborating with the clinic of the indigenous, since there is no evolution of the disease indicating a good prognosis. Taken together, we report that SNPs of IL28B gene may be a candidate for risk factor for progression of hepatitis B and possible influence on HBV clearance. However, the present study genotyped two polymorphisms of the IL28B gene, investigations with other SNPs involved in the immune response are necessary to better understand the influence of the IL28B gene on HBV infection.

Non-tuberculosis mycobacteria (NTM) have been associated with
with a variety of diseases, including lung, lymph node infections,
and nosocomial infections, including outbreaks. The Mycobacterium Complex
abscessus is composed of three subspecies: M. abscessus subsp. abscessus,
M. abscessus subsp. bolletii and M. abscessus subsp. massiliense, which are
pathogenic, fast-growing mycobacteria (MCR)
distribution in the environment. Precise diagnosis of the subspecies of the
M. abscessus complex is of great importance due to
clinics and specific therapeutic conduct for each infectious agent. At the
from 2008 to 2017 were identified by LACEN / RO 170 samples
positive for NTM in the state of Rondônia, of these 62 (35.8%) were
belonging to the M. abscessus complex.
most frequently identified. In order to evaluate characteristics
phenotypic and genotypic isolates from isolates previously identified as
belonging to the M. abscessus complex and to separate them at subspecies level,
phenotypic tests were performed such as: growth time, morphology of the
colonies, pigment presence, chord factor formation, molecular tests
using the RFLP / PRA / hsp65 methodology, sequencing regions of the
16S rRNA, rpoB and hsp65 genes and PCR from erm (41) gene from 16 isolates
from sputum samples from patients with infection
between 2008 and 2017 in the state of Rondônia. Of 16
bacterial isolates analyzed in PRA hsp65, it was possible to confirm that 15
belonged to the M. abscessus complex, but one isolate showed a pattern of
distinct band from the other mycobacterial isolates. The analysis of
sequencing of the 16S rRNA gene, separated the isolates only at
Partial sequencing of the rpoB and hsp65 genes identified
M. abscessus Complex at the species level, where 18.7% were
identified in M. abscessus subsp. abscessus, 37.5% were identified
as M. abscessus subsp. bolletti, 37.5% were identified as M.
abscessus subsp massiliense and 6.25% identified as M. avium subsp.
hominissuis. PCR amplification of the erm gene (41) helped in the identification
subspecies and confirmed the identification of five M. abscessus isolates
subsp. massiliense, but there was a case of M. abscessus subsp. massiliense
identified by sequencing and not by the erm gene (41). The results of the
analyzes have shown that the phenotypic evidence currently used is not
differentiate mycobacteria from the M. abscessus complex and that the
partial sequencing of the rpoB and hsp65 genes were able to distinguish the
three subspecies, but as there are reports of horizontal gene transfer,
Using these two markers, the need to identify
new targets or combination of targets to overcome the transfer problem
of genes between M. abscessus complex mycobacteria.

Leishmaniasis is a set of diseases caused by protozoa of the genus Leishmania. It presents clínical manifestations in humans that can compromise the skin, mucosa and guts. The available therapeutic options for the treatment of leishmaniasis are based on the use of pentavalent antimonials Pentamidine and Amphotericin B, which have low efficacy, side effects and, more recently, the occurrence of resistance by the parasite to these drugs. The factors presented prompt the search for new compounds with antileishmanial activity and lower toxicity. In this context, the present study aimed to evaluat the in vitro and in vivo leishmanicidal activity of synthetic analogs derived from methoxybenzene obtained commercially from Sigma-Aldrich. The in vitro screening was performed against L. (L.) amazonensis and L. (L.) infantum promastigotes, in which the parasites were treated with serial concentrations of the compounds at 1260- 19.68 μM for 72 hours. Analogs that had a 50% inhibitory concentration of the parasites (IC50) were subsequently selected to evaluate their cytotoxic potential against THP-1, HepG2, VERO and murine macrophages - MØ cells and their hemolytic activity. Cytotoxicity assays were performed using the MTT method. After cytotoxic evaluation on THP-1 cells, the compounds were evaluated at non-cytotoxic concentrations (80- 0.078 μM) to these cells for their in vitro action against L. (L.) amazonensis amastigotes. The assays against promastigotes (IC50) and amastigotes (ICA50) were performed by the Alamarblue method. The in vitro results showed that of the 25 analogs tested, three of them (CCA, S4 and X23) were active against promastigotes of L. (L.) amazonensis and L. (L.) infantum, with IC50 values ranging from 13.84 to 74 μM. Regarding the cytotoxicity, they presented cytotoxic concentration to 50% of the cells (CC50) between 30-302 μM towards THP-1, between 44-1,260 μM for HepG2, between 28-1,260 μM for Vero and between 13-149 μM for MØ. Tests performed against amastigotes revealed that S4 and X23 were even more active against this form, presenting ICA50’s between 10 and 9 μM, respectively, both with selectivity indexes (CC50/ICA50) above 10. The analog CCA did not present activity against amastigotes. No hemolytic activity was observed at the highest concentration for any compound. In the in vivo experiments, mice infected with L. (L.) amazonensis were treated for 10 days, on alternate days, with a total of five days of application with the analog S4 and amphotericin B. Stability of the lesion volume was observed, in addition to reducing the parasite burden for Amphotericin B. For the compound S4, tested at 50 mg/kg, a 90% decrease in the parasite burden was observed, but not flowed by a decrease in footpad swelling. Subsequent to the in vivo antileishmanial evaluation, toxicological tests were performed to evaluate hepatic enzyme production (TGO and TGP) and the concentration of urea and creatinine, which could indicate a possible hepatic and renal toxicity after treatment with S4. After the observation of the potential in vivo antileishmanial activity experiments, action mechanism assays were performed. As a result, the occurrence of morphological and biochemical alterations in L. (L.) amazonensis promastigotes, treated with IC50 and IC90 values of the S4 analogue, was observed, causing the decrease of cellular volume, increase of reactive oxygen species, depolarization of the mitochondrial membrane and autophagic processes, consequently leading to parasite death. In conclusion, this study led to the identification of three analogues (X23, CCA and S4) with antileishmanial activity in vitro and one analog (S4) with in vivo activity against L. (L.) amazonensis and its possible mechanism of action. The results obtained in this study showed unpublished data related to the antileishmanial activity of S4 and allow the continuation of the studies for the search of drugs that act against leishmaniasis.

According to data from the World Health Organization (WHO), about 1 million new cases of Leishmaniasis arise annually. Chemotherapy is based on the drugs ability to interfere in the survival of the parasite presenting several limitations, potentiating this neglected disease as a serious health problem. Approaches in the search for alternatives to treatment using molecular targets of Leishmania spp. are being developed by several research groups. Among these molecular targets are the enzymes involved in essential parasite pathways, such as trypanothione reductase (TR), involved in oxidative stress combating generated by the reactive oxygen species produced by host macrophages during infection. This work aimed to investigate the TR reductase from Leishmania braziliensis as a molecular target for the prospection of new inhibitors and subsequent evaluation of the LAAO of Crotalus atrox with antiparasitic activity. The recombinant protein was expressed in its active homodimeric form, each monomer with estimated molecular mass of 54 kDa and isoelectric point of 6.0. Primary structure analysis of the expressed enzyme revealed a high percentage of identity with the TR's of several trypanosomatids. In order to identify potential inhibitors, the animal poisons biodiversity was prospected assessing inhibitory capacity of the L-amino acid oxidase (LAAO) venom fraction of Crotalus atrox against the recombinant molecular target (IC50: 119 μg/ml). Later, in vitro leishmanicidal activity against L. amazonensis (IC50: 1.8 μg / mL) and L. braziliensis (IC50: 1.3 μg / mL) promastigotes was evaluated and sequentially, the CC50 was determined on cells THP1 (291.38 μg/mL); J774.A1 (24.16 μg/ml); HepG2 (4.05 μg/mL) and VERO (1.95 μg/mL). Molecular modeling by homology was performed for LAAO and TRLb, molecular docking between the two proteins and virtual screening between peptides derived from the primary structure of LAAO against the parasite enzyme. In silico studies allowed the identification and synthesis of 5 peptides which interacted in key regions of the enzyme. In addition, 2 peptides with microbicidal activity were tested to evaluate the inhibitory potential of TR. In this study, the leishmanicidal potential of the LAAO fraction of the C. atrox venom and the identification of the peptide 353RFIYY357, which has in vitro inhibitory activity against TR (IC50: 287,5 μM), was demonstrated. The promising results obtained in this work suggest further research aimed at evaluating the leishmanicidal potential of the peptide and proposing structures derived from this inhibitor capable of adding the antiparasitic effect.