Orientador: Ewerton Garcia de Oliveira Mima / Resumo: Um dos principais mecanismos de resistência microbiana são os sistemas de efluxo, que transportam medicamentos antimicrobiano para fora da célula. A eficácia de alguns agentes de inibição dos sistemas de efluxo tem sido reportada para reverter a resistência microbiana e também para potencializar as terapias antimicrobianas. Além disso, métodos alternativos aos agentes antimicrobianos convencionais têm sido investigados, como a Terapia Fotodinâmica antimicrobiana (aPDT). O objetivo desse estudo foi avaliar in vitro o efeito da aPDT e de dois inibidores de sistemas de efluxo microbiano (curcumina e verapamil) na resistência à inativação de C. albicans. Foram utilizadas duas cepas de C. albicans, uma susceptível (CaS) e outra resistente (CaR) a fluconazol. Os parâmetros de inativação fúngica foram determinados submetendo-se culturas planctônicas de ambas as cepas à curcumina, ao verapamil, ao fluconazol e também à aPDT (mediada pela curcumina 40 μM (14,73 μg/mL) e luz de LED azul de ≅455 nm a 5,28 J/cm2). As duas cepas foram cultivadas e tratadas associando-se os agentes de inibição do efluxo ao fluconazol em concentrações não letais. Os dados de UFC/mL foram analisados pelos testes paramétricos t de Student, ANOVA/Welch e post-hoc de Games-Howell e pelo teste não paramétrico de Mann-Whitney (α=0,05; n=12). Os resultados demostraram que aPDT promoveu uma redução significativa (p<0,001) de 4,5 e 4,42 log10 para CaS e CaR, respectivamente. Para CaS, os valores de Concentrações Ini... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: One of the major mechanisms of microbial resistance is the efflux systems, or efflux pumps, present in the plasma membrane of microorganisms that carry an antimicrobial drug out of the cell. The efficacy of some inhibitors of efflux systems has been reported to reverse microbial resistance, including C. albicans, and also to potentiate antimicrobial therapies. In addition, alternative methods to conventional antimicrobial agents have been investigated, such as antimicrobial Photodynamic Therapy (aPDT). The objective of this study was to evaluate in vitro the effect of aPDT and two inhibitors of microbial efflux systems (curcumina and verapamil) on the resistance to inactivation of C. albicans. For this, two strains of C. albicans, one susceptible (CaS) and another resistant (CaR) to fluconazol were used. Fungal inactivation parameters were determined by subjecting planktonic cultures of both strains to curcumina, verapamil, fluconazol, and also aPDT (mediated by curcumin at 40 μM (14.73 μg/mL) and blue LED light of ≅455 nm and 5.28 J/cm2). These strains were then cultured and treated associating one of the efflux inhibitors with fluconazole using non-lethal concentrations. For the statistical analysis, the normality and the homogeneity of variances were evaluated by the Shapiro-Wilk and Levene tests, respectively. Data were analyzed by Student's t-tests, Welch-corrected ANOVA and Games-Howell post-hoc and Mann-Whitney non-parametric test (α = 0.05) (n = 12). aPDT promoted a s... (Complete abstract click electronic access below) / Mestre

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O objetivo do presente estudo clínico foi compreender o efeito do uso prévio de agentes antimicrobianos na diversidade e a estrutura do microbioma procariótico de saliva, biofilme supragengival e canal radicular de dentes com infecção endodôntica aguda. Realizaram-se coletas microbiológicas de saliva (S), biofilme supragengival (BS) e canal radicular (CR) de pacientes que não utilizaram antibióticos (G1: n=6) e de pacientes que utilizaram antibióticos (G2: n=6). Para a caracterização das comunidades de procariotos por meio de sequenciamento de alto rendimento, foram produzidos pools de seis amostras para cada um dos sítios. A região hipervariável V3-V4 do gene 16S rRNA foi amplificada e a plataforma Illumina MiSeq foi empregada para análise das sequências geradas. Foram determinadas a presença e abundância relativa das unidades taxonômicas operacionais (OTUs) em cada amostra. Procedeu-se a análise de alfa-diversidade para cada amostra, considerando-se as métricas de Simpson, dominância, estimativa de riqueza de Chao-I e o Índice de Shannon. Os valores obtidos foram comparados por meio de testes estatísticos. Para a análise dos índices de beta-diversidade, empregou-se o método de agrupamento UPGMA, com jackknifing e método de comparação UniFrac com peso. A representação gráfica tridimensional da beta-diversidade foi realizada por meio de análise de coordenadas principais. A técnica de PCR gene específico foi empregada para determinar a presença de genes relacionados à resistência bacteriana para agentes beta-lactâmicos (blaTEM, blaZ, ampC, mecA), macrolídeos (ermB, ermC, mefA), tetraciclinas (tetM, tetQ, tetW), lincosamidas (lnuB, lsaB) e vancomicina (vanA, vanD, vanE). A similaridade para a presença/ausência de genes de resistência nas amostras de S, BS e CR em G1 e G2 foi determinada por meio de coeficiente de agrupamento, utilizando-se o método UPGMA com distância Euclidiana. Todos os pacientes apresentavam infecção endodôntica aguda, caracterizada pela presença de dor espontânea, necrose pulpar e dor à percussão vertical. Aumento de volume foi observado em 8/12 pacientes. Os pacientes do Grupo 2 utilizaram beta-lactâmicos previamente à consulta (amoxicilina = 5; cefalexina = 1). Há predomínio de integrantes do domínio Bactéria em todas S, BS e CR. Archaea pertencentes ao gênero Methanobrevibacter foram encontradas apenas em amostras de CR, constituindo menos de 1% do total de OTUs (G1-CR = 0,319%; G2-CR = 0,014%). Há predomínio de bactérias dos filos Firmicutes e Bacteroidetes em amostras de S, BS e CR. Há redução intensa no percentual de OTUs pertencentes ao Filo Firmicutes em amostras de saliva, quando antibiótico foi utilizado (G1-S = 75,371; G2-S = 35,242). Comportamento oposto ocorreu neste ecossistema para integrantes do Filo Bacteroidetes (G1-S = 12,657; G2-S = 33,947). Tanto em G1 quanto em G2, bactérias do gênero Streptococcus predominam em amostras de S e BS. Em canais radiculares, maiores percentuais de OTUs foram observados para os gêneros Porphyromonas e Prevotella. As métricas de alfadiversidade indicam que o uso prévio de antimicrobiano parece oportunizar o estabelecimento de espécies antes menos abundantes, especialmente em saliva (dominância: G1-S>G2-S; índice de Shanon: G1-S<G2-S; índice de Simpson: G1- S<G2-S; índice de Chao-1: G1-S<G2-S). A análise de beta-diversidade mostra proximidade entre G1-BS e G2-BS; há distanciamento entre G1-S e G2-S. As amostras G1-CR e G2-CR estão mais distantes de S e BS, sugerindo maior seleção imposta pelo ecossistema do CR aos procariotos. Os genes de resistência mais frequentemente detectados foram tetM, tetQ, tetM, ermB e mefA. Não foram detectados genes vanA, vanD, vanE, blaZ, mecA, lnuB e ermC. O gene ermB foi frequentemente detectado em amostras de S, BS e CR em ambos os grupos. Em pacientes que não utilizaram antibiótico, o gene tetM e o gene tetQ foram detectados simultaneamente em S, BS e CR (tetM = 4/6; tetQ = 3/6). A análise multivariada não demonstrou nível de agrupamento alto entre amostras de um mesmo paciente, de um mesmo ecossistema, ou de um mesmo grupo. A partir dos resultados do presente estudo, observou-se que a utilização de agente antimicrobiano betalactâmico parece alterar parâmetros composicionais de comunidades de procariotos na cavidade bucal. Entretanto, particularidades relativas a cada ecossistema podem modular a extensão deste efeito, parecendo ser mais intensos em amostras de S do que em BS e CR. / The present clinical study aimed to assess the effect of antibiotics over the diversity and structure in prokaryotic communities of saliva, supragengival biofilm and root canal of teeth with acute primary infections. Samples of saliva (S), supragengival biofilm (BS) and root canal of teeth with acute primary infections (CR) were collected from patients, and were grouped according with the previous use of antibiotic (G1 = no antibiotics; G2 = antibiotics). Pooled samples for each community were evaluated. DNA sequencing was performed with MiSeq (Illumina). The V3-V4 hypervariable region from the 16S rRNA gene was amplified. The presence and relative abundance of the operational taxonomic units (OTUs) was determined for each sample. Alpha-diversity analysis was performed with the Simpson’s index, dominance, Chao-1 richness index and Shannon’s index. Statistical analysis was carried out to compare their values for each community.Beta-diversity was computed through UPGMA clustering and jackknifing. The principal coordinate analysis employed weighted UniFrac. Gene-specific PCR was employed to detect resistance genes to lactamics (blaTEM, blaZ, ampC, mecA), macrolides (ermB, ermC, mefA), tetracyclines (tetM, tetQ, tetW), lincosamides (lnuB, lsaB) e vancomycin (vanA, vanD, vanE). The UPGA clustering analysis with Euclidean distance was applied to investigate the existence of similar groups of samples. A dendrogram was constructed to show the arrangement of the sample groups produced by clustering. All the patients had primary acute endodontic infections, with spontaneous pain, pulp necrosis and tenderness on percussion. Swelling was observed for 8 out of 12 patients. Patients from G2 had lactamics before the urgency appointment (amoxicillin = 5; cephalexin = 1). Bacteria were predominant in S, BS and CR samples. Archaea belonging to the genus Methanobrevibacter were only detected in RC samples and comprised less than 1% of all the OTUs G1-CR = 0.319%; G2-CR = 0.014%). The great majority of the detected OTUs in S, BS, and CR belonged to the phyla Firmicutes and Bacteroidetes. Reduction in the percentage of Firmicutes was observed in G2-S (G1-S = 75.371%; G2-S = 35.242%). A distinct behavior was demonstrated by the Bacteroidetes (G1- S = 12.657%; G2-S = 33.947%). Components belonging to the genus Streptococcus were predominant in S and BS, for both G1 and G2. CR harbored high percentage of species belonging to the genus Porphyromonas and Prevotella. The alpha-diversity indexes demonstrated that the G2-S had an increase in low abundant species (dominance: G1- S>G2-S; Shanon index: G1-S<G2-S; Simpson index: G1-S<G2-S; Chao-1 index: G1-S<G2- S). Beta-diversity showed that G1-BS and G2-BS had close spatial distribution. G1-CR and G2-CR were more distant from S and BS samples. The RC may promote a most intense species selection, due to environmental conditions. The most frequently detected genes associated with resistance to antibiotics were tetM, tetQ, tetM, ermB, and mefA. The genes vanA, vanD, vanE, blaZ, mecA, lnuB, and ermC were not detected in the samples. The gene ermB was frequently detected in S, BS and CR samples. In patients from G1, tetM and tetQ were detected simultaneously in all the three environments (tetM = 4/6; tetQ = 3/6). There was no clustering behavior for samples belonging to different environments in the same patients and between the same environment samples from different groups. An overall analysis from the data allows for suggesting that the use of lactamic agents may alter compositional parameters from prokaryotic communities in the oral cavity to different extents. Specific environmental characteristics from each site may modulate the effect that seemed to be more intense for S than for BS and CR.

FLIP (FLICE inhibitory protein) est une protéine anti-apoptotique qui a des identités de séquence partagées avec la protéine pro-apoptotique caspase-8. FLIP se trouve en compétence avec caspase-8 pour se fixer sur la protéine adaptatrice FADD, ce qui empêche l’activation de caspase-8 bloquant ainsi l'apoptose. Lors du développement des molécules interférant avec des protéines anti-apoptotiques, la recherche d'inhibiteurs de la protéine FLIP qui est surexprimée dans un très grand nombre de cancers, a échoué. Cela s'explique en partie par le fait que peu d'information structurelle de FLIP est actuellement disponible TRAIL est une cytokine de la famille TNFα. Elle est décrite pour activer des voies de signalisation conduisant à la mort cellulaire par apoptose. TRAIL a montré un grand intérêt dans la thérapie anticancéreuse, grâce à sa capacité d’induire la mort des cellules tumorales sans aucun effet sur les cellules normales. Cependant, l’efficacité de TRAIL est limitée par plusieurs mécanismes moléculaires. Un de ces mécanismes est la surexpression de FLIP qui fait compromettre l’utilisation thérapeutique de TRAIL. Le but principal de ce projet est de développer des nouvelles molécules capables d’inhiber la protéine FLIP dans les cellules tumorales, sans aucun effet sur la protéine homologue caspase-8. Après modélisation des protéines FLIP et caspase-8 sur la base de la structure cristallographique de FLIP viral et FADD respectivement, des premières expériences d’ancrage ou “docking” utilisant une banque de composés chimiques du «National Cancer Institute NCI » ont été réalisées. Les 9 molécules les plus intéressantes, étant comme sélectives pour FLIP et non caspase 8, ont été sélectionnées et testées sur des lignées de cancer de poumons surexprimant la protéine FLIP. Une co-administration de chacune des molécules inhibitrices de FLIP avec TRAIL a été faite pour vérifier la restauration de la voie apoptotique dans les cellules cancéreuses. Un test moléculaire de « Pull down assay » a été fait afin de confirmer l’inhibition de l’interaction de FLIP avec FADD. Finalement, l’évaluation de l’activité enzymatique des caspases a été étudiée pour vérifier la réactivation de la voie apoptotique après la combinaison de TRAIL avec les inhibiteurs de c-FLIP. En conclusion, la combinaison de TRAIL avec les inhibiteurs de FLIP aboutit à la restauration de la voie apoptotique dans des cellules cancéreuses. Ces composés nouvellement identifiés, peuvent servir ultérieurement comme des potentiels éléments des stratégies utilisées dans le domaine du traitement du cancer. / FLIP (FLICE Inhibitory Protein) is an anti-apoptotic protein which shares sequencesimilarity with the pro-apoptotic protein caspase-8. FLIP competes with caspase-8 for binding to the adaptor protein FADD (Fas-associated death domain), thus it inhibits caspase-8 activation, thereby blocking apoptosis. During the development of molecules interfering with anti-apoptotic proteins, searching for inhibitors of FLIP protein which is overexpressed in a very large number of cancers, has failed. This is partly due to the fact that little FLIP structural information is available at present. TRAIL is a member of TNFα superfamily. It has been described to activate the apoptotic signaling pathways. TRAIL showed great interest in anti-cancer therapy, due to its ability to induce tumor cell death without any effect on normal cells. However, the efficacy of TRAIL is limited by several molecular mechanisms. One of these mechanisms is the overexpression of FLIP which is able to compromise the therapeutic use of TRAIL. The main goal of this project is to develop novel inhibitory molecules able to interfere with FLIP in tumor cells without any effect on the homologous protein caspase 8. After the construction of FLIP and caspase-8 proteins on the basis of the crystallographic structure of the viral FLIP and FADD respectively, the first docking experiments using a chemical library of the National Cancer Institute NCI have been carried out. The most interesting molecules, being selective for FLIP versus caspase 8, were selected and tested on lung cancer cell lines that overexpress FLIP protein. Co-administration of FLIP inhibitors with TRAIL was performed to verify the restoration of the apoptotic pathway in cancer cells. A molecular test of "Pull down assay" was done in order to confirm the inhibition of the FLIP/FADD interaction. Finally, the evaluation of caspases activity was carried out to confirm the reactivation of the apoptotic machinery after TRAIL/FLIP-inhibitors combination. In conclusion, the combination of TRAIL with FLIP inhibitors resulted in apoptosis restoration in resistant tumor cells. These newly identified compounds may serve later as potential elements in cancer treatment field.

The spread of antibiotic-resistant microorganisms in the environment is recognized widely as an important public health issue, with concerns about future ability to treat infectious diseases. The main risk to public health is that the resistance genes are transferred from environmental bacteria to human pathogens. Safe water is one of the most important needs in public health in the twenty first century. The major health threat posed by drinking unsafe water is the transmission of infectious diseases, which are the leading causes of mortality and morbidity for children under the age of 5 and it is estimated to cause 1.5 million deaths annually in developing countries. In addition to the wide spread cases of water-borne diseases resulting from the contamination of water sources, concerns have been raised when these diseases fail to be cured due to development of resistance to most prescribed antibiotics by the contaminating microorganisms. It is now a well-established fact that E.coli is a significant cause of diarrheal illnesses both in infants and adults in many parts of the world. Data on clinical isolates is plenty while less attention has been given to environmental isolates of these enteric pathogens. Samples from the environment such as water may serve as probable reservoirs of these pathogens; this is compounded by the entry of functional compounds of antibiotics into waterways, through humans and animals that have ingested antibiotics. This is because antibiotics are not completely metabolized and may enter waterways through the waste products of these humans or animals.Studies on antimicrobial resistance is important in order to detect changes in patterns of resistance, implement control measures on the use of antimicrobial agents, and to prevent the spread of multidrug-resistant strains of bacteria. It also provides surveillance data for antibiotic resistances, necessary to define or update guidelines for empirical treatment, as well as a guide for appropriate antibiotic supplies. Study objectives: The objectives of this research were: (i) to determine the total and faecal coliform status of drinking water sources, as an indication of quality; (ii) to determine the bacteriological profile of bacteria flora in the drinking water sources; (iii) to determine prevalence and susceptibility profiles of antibiotic resistant water-borne E.coli; (iv) to investigate the virulence genes associated with multiple antibiotic resistant E. coli isolates; (v) to compare three laboratory based techniques: PCR, API 20E, and Culture based methods used for detection of E.coli and (vi) to determine the association between multiple antibiotic resistance and radiation sensitivity (D10). © University of South Africa 2014 VII Methodology: Four hundred and sixty four (464) water samples were collected for assessment between June 2011 and May 2012. The samples were collected from 57 sampling sites, from six different water sources including: boreholes (10), a canal (1), dams (15), hand-dug wells (15), a river (1), and streams (15). Total coliforms, faecal coliforms, and E. coli analysis were done by the MPN method. Bacteria isolation and identification were done using API 20E, conventional methods, and a PCR based DNA STRIP technology that allows simultaneous detection of virulence genes and confirmation of E. coli isolates. Antibiotic susceptibility testing was also conducted using the Kirby-bauer method. Radiation sensitivity was done using a cobalt 60 source. Results: The results obtained indicated that all the water sources were of poor quality in terms of microbial distributions with total coliform and faecal coliform counts ranging between 0 to 2.4x103 MPN/100ml. E. coli counts ranged between 10 to 7.9x101MPN/100ml. Disease risk assessment of the various water sources indicated that dam water sources presented a high disease risk, while borehole water sources had a low disease risk. A total of five hundred and twenty bacterial isolates (520) were obtained during the period of study. Three hundred and five (305) isolates representing 58.65% of the total were obtained during the dry season, as against (205) representing 41.35% in the rainy season. The most commonly occurring bacteria in the water samples was Klebsiella spp constituting 20%. The next most occurring organism was E. coli (18.7%). This was followed by Pseudomonas aeruginosa (15.61%), Enterobacter spp. (15.4%), Proteus vulgaris (13.1%), and Enterococcus faecalis (9.2%). The least isolated bacteria were Vibrio cholerae (1.2%) and Shigella spp. (1.2%). The prevalence of multi drug resistance E. coli was 49.48 %. E. coli isolates showed a high sistance patterns to the tested antibiotics. They were most resistant to penicillin (32.99%), cefuroxime (28.87%), erythromycin (23.71%), and tetracycline (21.45%). In contrast, they were susceptible to nitrofurantoin (93.8%), cefotaxime and amikacin (91.75%), gentamicin (90.7%), nalidixic acid (89.65%), ciprofloxacin (74.2%), chloramphenicol (69.07%), pipemidic acid (65.97%) and cefuroxime (52.58%). Sixty-three percent (63%) of the multidrug resistant E. coli strains recorded a multiple antibiotic resistance (MAR) index value of >0.2. Six (6%) percent of he multiple antibiotic resistant were eae virulence genes producing however, none of the E. coli isolates produced the stx1 and stx2 virulent gene. The analytical profile index (API) recorded specificity and sensitivity of 99.7% and 98.50 % respectively for the detection of E. coli. The © University of South Africa 2014 VIII culture/ biochemical based methods for detection of E coli recorded specificity of 81.82% and a sensitivity of 96.91%. There was no association (P> 0.05) between radiation sensitivity (D10) and antibiotic resistances. Conclusion: The study has confirmed that majority of the water sources used for drinking and domestic purposes in the study area are highly contaminated with high levels of faecal coliforms above the recommended standards. There were also resence of bacteria of public health importance in the water sources. Both animals and humans could be sources of faecal bacteria contamination of the drinking water sources. The study confirmed a high prevalence of multiple antibiotic resistances in E. coli isolates. The eae virulence gene was associated with some of the multiple resistant E. coli isolates. The study also concludes that API 20E has a high specificity and sensitivity close to that of the PCR. Lastly, There is no association between multiple antibiotic resistant indexes and radiation sensitivity (D10) of antibiotic resistant E. coli. / School of Environmental Sciences / D. Phil. (Environmental Science)

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-04-04T18:19:56Z No. of bitstreams: 1 Filipe Ferreira de Almeida Rego Fatores....pdf: 6473179 bytes, checksum: 008855484cf612885a04f114f51214ba (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-04-04T18:20:12Z (GMT) No. of bitstreams: 1 Filipe Ferreira de Almeida Rego Fatores....pdf: 6473179 bytes, checksum: 008855484cf612885a04f114f51214ba (MD5) / Made available in DSpace on 2016-04-04T18:20:12Z (GMT). No. of bitstreams: 1 Filipe Ferreira de Almeida Rego Fatores....pdf: 6473179 bytes, checksum: 008855484cf612885a04f114f51214ba (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Ba, Brasil / Nesta tese foram realizados três trabalhos distintos sendo que todos envolvem identificar possíveis fatores genéticos ou clínicos relacionados com a infecção pelo HIV-1 ou pelo HTLV-1 ou por ambos. No primeiro trabalho nós objetivamos identificar mutações que poderiam estar relacionadas com o desenvolvimento da TSP/HAM ou carga proviral. Para isto sequenciamos a região LTR5’ do HTLV-1 por Ion Torrent para verificar mutações com baixa frequência. Nós encontramos mutações em 52 posições que estavam presentes em mais de um indivíduo, porém apenas 11 destas estavam presentes em TFBS previamente descritos. Três mutações que não estavam presentes em TFBS previamente descritos foram estatisticamente significantes quando comparadas entre os grupos," sendo" que" estes" sítios" podem" ser" importantes" para" a"mediação" da" transcrição" viral."No" segundo" estudo" nós" objetivamos determinar a prevalência do genótipo selvagem em Hlabisa, Kwazulu-Natal na África do Sul além de identificar possíveis fatores associados a presença deste genótipo em 220 pacientes submetidos a ART. O genótipo selvagem foi detectado em 28 amostras (12,7%). Selecionamos 11 pacientes para realizar o sequenciamento pelo Ion Torrent, nove confirmaram não ter mutações de resistência aos antirretrovirais em alta frequência. Foi encontrada uma alta contagem de CD4+ no início da terapia associado a falha terapêutica assim como uma alta carga viral antes da genotipagem e não foi encontrado associação entre aderência a terapia auto-reportada e a presença do genótipo selvagem. Aproximadamente um em cada oito adultos que falham a terapia possuem o genótipo selvagem sendo este dado confirmado através de sequenciamento de nova geração. Devido ao alto número de genótipos selvagem encontrados, o teste de resistência genotípica deve ser solicitado para se obter um melhor desfecho clinico em níveis individuais e populacionais. No terceiro estudo nós analisamos as diferenças na contagem de linfócitos T CD4+ entre indivíduos infectados apenas com o HIV-1 e indivíduos coinfectados HIV-1/HTLV-1 com falha terapêutica, além de analisarmos a soroprevalência do HTLV-1 em indivíduos infectados pelo HIV-1. Foram encontrados oito pacientes coinfectados (2,1%) dos 381 pacientes analisados. Nós não observamos nenhuma diferença estatística quando analisamos transversalmente os dados clínicos dos pacientes, exceto na primeira contagem de linfócitos T CD4+ após o início do tratamento que estava maior nos indivíduos coinfectados (p=0.03). A análise multivariada longitudinal mostrou que a media de linfócitos T CD4+ ao longo do tratamento, foi estatisticamente maior nos pacientes coinfectados levando em consideração características demográficas, carga viral, fatores relacionados a terapia, entre outros. Nos pacientes coinfectados também não foram encontrados marcadores de HLA relacionados com os supressores de elite do HIV-1. Os dados deste trabalho sugerem que os pacientes coinfectados em terapia antirretroviral deveriam ter um acompanhamento clínico diferenciado dos indivíduos apenas infectados pelo HIV-1, pois a coinfecção poderia estar levando ao aumento do número dos linfócitos T CD4+ sem um possível ganho de resposta imune. / We performed three studies to analyze risk factors associated with retroviruses infections. In the first study we attempted to analyze mutations related to TSP/HAM development or proviral load. For that purpose we have sequenced the LTR 5’ region of HTLV-1 by Ion Torrent. We found that mutations in 52 positions were present in more than one individual, but only 11 were present in the previously described TFBS. Three mutations that were not present in the previously described TFBS were statistically significant comparing groups. Despite the absence of previously described TFBS, these sites might be important for the viral transcription. In the second study we analyzed the prevalence of HIV-1 wild type genotype in adults failing first-line ART. A total of 220 adults were included. The wild type genotype was detected by population sequencing in 28 (12.7%). No major drug resistance mutations were detected by deep sequencing for 81.8% (9/11) of those sampled. Higher baseline CD4+ cell count was associated with a greater likelihood of wild type genotype as was a higher viral load prior to resistance testing but there was no evidence of an association between selfreported adherence and the presence of wild type genotype. Approximately one in eight adults failing first-line ART had wild type genotype and this result was confirmed trough deep sequencing in some samples. Access to genotypic resistance testing may be required in this region to achieve optimal individual-level and population-level outcomes. In the third study we proposed to verify the prevalence of HTLV-1 and to statistically assess differences in CD4+ counts between HTLV-1/HIV-1 co-infected and HIV-1 mono-infected patients living in rural KwaZulu-Natal. The HTLV-1 seroprevalence was 2.1% (8 out 381 patients). The patients were grouped by HTLV-1/HIV-1 co-infected and HIV-1 mono-infected status for the statistical analysis. There were no cross-sectional differences between the groups regarding CD4+ count before therapy, CD4+ count at genotype, age, gender, viral load, duration of ART, immunological failure, ART failure and first ART regimen. However, the first CD4+ count after treatment was higher in the co-infected group (p=0.03). A multivariate, longitudinal model showed that the mean CD4+ count over time for the HTLV-1/HIV-1 coinfected group was significantly higher than the HIV mono-infected group (p<0.05) when adjusting for demographic characteristics, viral load and ART treatment factors. This finding was independent of expression of HLA Class 1 genotypes previously associated with HIV-1 infected elite suppressors. This study suggests that the increase of CD4+ count after therapy suggests a differential clinical management for the HTLV-1/HIV-1 co-infected patients should be implemented.

Made available in DSpace on 2016-12-23T13:49:02Z (GMT). No. of bitstreams: 1 Lilliane Bonella Meireles Baptista.pdf: 1737558 bytes, checksum: f8594f73dd30da9bd59669e3b19d796e (MD5) Previous issue date: 2012-05-31 / As propriedades medicinais de fitoterápicos utilizados tradicionalmente pela população têm sido comprovadas por pesquisas em todo o mundo. Foram avaliadas as atividades antimicrobianas e antioxidantes de oito extratos fitoterápicos produzidos na Pastoral da Saúde de Venda Nova do Imigrante- ES, Brasil, a partir das plantas: Anadenanthera colubrina (Vell.) Brenan, Achillea millefolium L., Aristolochia cymbifera Mart., Casearia sylvestris Sw., Cordia verbenacea DC., Echinodorus grandiflorus (Cham.& Schltdl.) Micheli , Gossypium hirsutum L. e Plantago major L. A atividade antimicrobiana foi avaliada pelo método de microdiluição em caldo determinando a concentração mínima bactericida (CMB) de cada extrato contra 12 espécies bacterianas, incluindo cepas multirresistentes causadoras de infecções hospitalares como MRSA, VRE e K. pneumonie ESBL. Todos os extratos, com exceção do extrato de A. cymbifera cuja CMB > 250 mg/mL, apresentaram atividade antimicrobiana contra as bactérias avaliadas com CMB variando entre 4 e 86 mg/mL para bactérias Gram-positivas e Gram-negativas , com destaque especial para os extratos de A. colubrina e C. verbenacea, para os quais realizou-se o teste de avaliação da cinética de morte bacteriana (time-kill). A atividade antioxidante dos extratos foi avaliada pelo método DPPH, demonstrando que os extratos fitoterápicos apresentam de razoável a ótima ação antioxidante, comparadas ao padrão quercetina. Os resultados obtidos confirmam indicações empíricas atribuídas aos extratos fitoterápicos incluindo a ação antimicrobiana, corroborando para atribuir novos usos aos fitoterápicos avaliados, bem como para incentivar novos estudos com os extratos e as plantas das quais estes são obtidos / The medicinal properties of herbs used traditionally by the population have been proved by researches from all around the world. Antimicrobial and antioxidant activities of eight phytotherapeutic extracts produced in the Pastoral care of health of Venda Nova do Imigrante-ES were assessed, from the plants: Anadenanthera colubrina (Vell.) Brenan, Achillea millefolium L., Aristolochia cymbifera Mart., Casearia sylvestris Sw., Cordia verbenacea DC., Echinodorus grandiflorus (Cham.& Schltdl.) Micheli, Gossypium hirsutum L. and Plantago major L. The antimicrobial activity was evaluated by the microdiluition method for determining the minimal bactericidal concentration (MBC) of each extract against 12 bacterial species, including multi-drug resistant strains causing nosocomial infections like MRSA, VRE and ESBL K.pneumonie. All plant extracts, with the exception of A. cymbifera whose MBC > 250 mg/mL, showed antimicrobial activity against the tested bacteria, with MBC ranging between 4 and 86 mg/mL to Gram-positive bacteria and Gram-negative bacteria, with special highlights for the extracts of A. colubrina and C. verbenacea, for which there was the kinetic evaluation test of bacterial death (time-kill). The antioxidant activity of extracts was evaluated by the DPPH method, demonstrating that the extracts are herbal medicines of regular to great antioxidant action, compared to standard quercetin. The results obtained confirm empirical indications attributed to the phytotherapeutic extracts including antimicrobial action, corroborating to assign new uses for the phytotherapics evaluated, as well as to encourage further studies with the extracts and the plants from which they are derived

O objetivo do presente estudo clínico foi compreender o efeito do uso prévio de agentes antimicrobianos na diversidade e a estrutura do microbioma procariótico de saliva, biofilme supragengival e canal radicular de dentes com infecção endodôntica aguda. Realizaram-se coletas microbiológicas de saliva (S), biofilme supragengival (BS) e canal radicular (CR) de pacientes que não utilizaram antibióticos (G1: n=6) e de pacientes que utilizaram antibióticos (G2: n=6). Para a caracterização das comunidades de procariotos por meio de sequenciamento de alto rendimento, foram produzidos pools de seis amostras para cada um dos sítios. A região hipervariável V3-V4 do gene 16S rRNA foi amplificada e a plataforma Illumina MiSeq foi empregada para análise das sequências geradas. Foram determinadas a presença e abundância relativa das unidades taxonômicas operacionais (OTUs) em cada amostra. Procedeu-se a análise de alfa-diversidade para cada amostra, considerando-se as métricas de Simpson, dominância, estimativa de riqueza de Chao-I e o Índice de Shannon. Os valores obtidos foram comparados por meio de testes estatísticos. Para a análise dos índices de beta-diversidade, empregou-se o método de agrupamento UPGMA, com jackknifing e método de comparação UniFrac com peso. A representação gráfica tridimensional da beta-diversidade foi realizada por meio de análise de coordenadas principais. A técnica de PCR gene específico foi empregada para determinar a presença de genes relacionados à resistência bacteriana para agentes beta-lactâmicos (blaTEM, blaZ, ampC, mecA), macrolídeos (ermB, ermC, mefA), tetraciclinas (tetM, tetQ, tetW), lincosamidas (lnuB, lsaB) e vancomicina (vanA, vanD, vanE). A similaridade para a presença/ausência de genes de resistência nas amostras de S, BS e CR em G1 e G2 foi determinada por meio de coeficiente de agrupamento, utilizando-se o método UPGMA com distância Euclidiana. Todos os pacientes apresentavam infecção endodôntica aguda, caracterizada pela presença de dor espontânea, necrose pulpar e dor à percussão vertical. Aumento de volume foi observado em 8/12 pacientes. Os pacientes do Grupo 2 utilizaram beta-lactâmicos previamente à consulta (amoxicilina = 5; cefalexina = 1). Há predomínio de integrantes do domínio Bactéria em todas S, BS e CR. Archaea pertencentes ao gênero Methanobrevibacter foram encontradas apenas em amostras de CR, constituindo menos de 1% do total de OTUs (G1-CR = 0,319%; G2-CR = 0,014%). Há predomínio de bactérias dos filos Firmicutes e Bacteroidetes em amostras de S, BS e CR. Há redução intensa no percentual de OTUs pertencentes ao Filo Firmicutes em amostras de saliva, quando antibiótico foi utilizado (G1-S = 75,371; G2-S = 35,242). Comportamento oposto ocorreu neste ecossistema para integrantes do Filo Bacteroidetes (G1-S = 12,657; G2-S = 33,947). Tanto em G1 quanto em G2, bactérias do gênero Streptococcus predominam em amostras de S e BS. Em canais radiculares, maiores percentuais de OTUs foram observados para os gêneros Porphyromonas e Prevotella. As métricas de alfadiversidade indicam que o uso prévio de antimicrobiano parece oportunizar o estabelecimento de espécies antes menos abundantes, especialmente em saliva (dominância: G1-S>G2-S; índice de Shanon: G1-S<G2-S; índice de Simpson: G1- S<G2-S; índice de Chao-1: G1-S<G2-S). A análise de beta-diversidade mostra proximidade entre G1-BS e G2-BS; há distanciamento entre G1-S e G2-S. As amostras G1-CR e G2-CR estão mais distantes de S e BS, sugerindo maior seleção imposta pelo ecossistema do CR aos procariotos. Os genes de resistência mais frequentemente detectados foram tetM, tetQ, tetM, ermB e mefA. Não foram detectados genes vanA, vanD, vanE, blaZ, mecA, lnuB e ermC. O gene ermB foi frequentemente detectado em amostras de S, BS e CR em ambos os grupos. Em pacientes que não utilizaram antibiótico, o gene tetM e o gene tetQ foram detectados simultaneamente em S, BS e CR (tetM = 4/6; tetQ = 3/6). A análise multivariada não demonstrou nível de agrupamento alto entre amostras de um mesmo paciente, de um mesmo ecossistema, ou de um mesmo grupo. A partir dos resultados do presente estudo, observou-se que a utilização de agente antimicrobiano betalactâmico parece alterar parâmetros composicionais de comunidades de procariotos na cavidade bucal. Entretanto, particularidades relativas a cada ecossistema podem modular a extensão deste efeito, parecendo ser mais intensos em amostras de S do que em BS e CR. / The present clinical study aimed to assess the effect of antibiotics over the diversity and structure in prokaryotic communities of saliva, supragengival biofilm and root canal of teeth with acute primary infections. Samples of saliva (S), supragengival biofilm (BS) and root canal of teeth with acute primary infections (CR) were collected from patients, and were grouped according with the previous use of antibiotic (G1 = no antibiotics; G2 = antibiotics). Pooled samples for each community were evaluated. DNA sequencing was performed with MiSeq (Illumina). The V3-V4 hypervariable region from the 16S rRNA gene was amplified. The presence and relative abundance of the operational taxonomic units (OTUs) was determined for each sample. Alpha-diversity analysis was performed with the Simpson’s index, dominance, Chao-1 richness index and Shannon’s index. Statistical analysis was carried out to compare their values for each community.Beta-diversity was computed through UPGMA clustering and jackknifing. The principal coordinate analysis employed weighted UniFrac. Gene-specific PCR was employed to detect resistance genes to lactamics (blaTEM, blaZ, ampC, mecA), macrolides (ermB, ermC, mefA), tetracyclines (tetM, tetQ, tetW), lincosamides (lnuB, lsaB) e vancomycin (vanA, vanD, vanE). The UPGA clustering analysis with Euclidean distance was applied to investigate the existence of similar groups of samples. A dendrogram was constructed to show the arrangement of the sample groups produced by clustering. All the patients had primary acute endodontic infections, with spontaneous pain, pulp necrosis and tenderness on percussion. Swelling was observed for 8 out of 12 patients. Patients from G2 had lactamics before the urgency appointment (amoxicillin = 5; cephalexin = 1). Bacteria were predominant in S, BS and CR samples. Archaea belonging to the genus Methanobrevibacter were only detected in RC samples and comprised less than 1% of all the OTUs G1-CR = 0.319%; G2-CR = 0.014%). The great majority of the detected OTUs in S, BS, and CR belonged to the phyla Firmicutes and Bacteroidetes. Reduction in the percentage of Firmicutes was observed in G2-S (G1-S = 75.371%; G2-S = 35.242%). A distinct behavior was demonstrated by the Bacteroidetes (G1- S = 12.657%; G2-S = 33.947%). Components belonging to the genus Streptococcus were predominant in S and BS, for both G1 and G2. CR harbored high percentage of species belonging to the genus Porphyromonas and Prevotella. The alpha-diversity indexes demonstrated that the G2-S had an increase in low abundant species (dominance: G1- S>G2-S; Shanon index: G1-S<G2-S; Simpson index: G1-S<G2-S; Chao-1 index: G1-S<G2- S). Beta-diversity showed that G1-BS and G2-BS had close spatial distribution. G1-CR and G2-CR were more distant from S and BS samples. The RC may promote a most intense species selection, due to environmental conditions. The most frequently detected genes associated with resistance to antibiotics were tetM, tetQ, tetM, ermB, and mefA. The genes vanA, vanD, vanE, blaZ, mecA, lnuB, and ermC were not detected in the samples. The gene ermB was frequently detected in S, BS and CR samples. In patients from G1, tetM and tetQ were detected simultaneously in all the three environments (tetM = 4/6; tetQ = 3/6). There was no clustering behavior for samples belonging to different environments in the same patients and between the same environment samples from different groups. An overall analysis from the data allows for suggesting that the use of lactamic agents may alter compositional parameters from prokaryotic communities in the oral cavity to different extents. Specific environmental characteristics from each site may modulate the effect that seemed to be more intense for S than for BS and CR.

O Sarcoma de Ewing (ES) é um raro tumor de ossos e tecidos moles com uma característica translocação cromossomal, a fusão EWS/FLI-1, que atua sobre diversos processos oncogênicos. O desenvolvimento da resistência à quimioterapia é comum no tumor e continua como uma das principais causas na falha do tratamento. O objetivo desse estudo foi avaliar a expressão de genes após a indução de resistência em linhagens celulares de ES. Foi selecionado um conjunto de genes (CCAR1, TUBA1A, POLDIP2, SMARCA4 e SMARCB1) a partir da mineração da literatura em resistência tumoral para duas drogas utilizadas na terapia de ES, doxorrubicina e vincristina. Descrevemos a expressão de cada gene selecionado antes e após as linhagens SK-ES-1 serem submetidas a um protocolo de indução de resistência para ambos os fármacos, que obteve êxito ao induzir as células à resistência. A expressão relativa dos níveis de mRNA foi avaliada e foi encontrada em maior expressão para os genes SMARCA4, SMARCB1 e POLDIP2, e em menor expressão para os genes TUBA1A e CCAR1, quando comparadas às linhagens de controle não-resistentes de cada quimioterápico. Os resultados sugerem o envolvimento de mecanismos de reparo de dano ao DNA, remodelamento de cromatina via SWI/SNF, atividade de microtúbulos e atividade spliceossomal nos processos de resistência quimioterápica em ES. / Ewing Sarcoma (ES) is a rare bone and soft tissue tumor with a characteristic chromosomal translocation, the fusion protein EWS/FLI-1, that drives several oncogenic processes. The development of resistance to chemotherapy is common and remains as the main cause of treatment failure. The goal of this study was to evaluate the expression of selected genes in ES cell lines after induction of resistance. A set of genes (CCAR1, TUBA1A, POLDIP2, SMARCA4 and SMARCB1) was data mined from tumoral resistance literature for two drugs used in ES therapy, doxorubicin and vincristine. We describe the expression of each selected gene before and after SK-ES-1 cell lines were exposed to a drug resistance inducing protocol for doxorubicin and vincristine. Cell lines were successfully induced to be resistant to doxorubicin and vincristine. The relative mRNA expression levels were upregulated for genes SMARCA4, SMARCB1 and POLDIP2 and downregulated for genes TUBA1A and CCAR1, when comparing resistant and non-resistant ES cell lines for each drug. The results suggest involvement of repair pathways, SWI/SNF chromatin remodeling, microtubule and spliceosomal activity processes in drug resistance mechanisms in ES.

Introdução: a utilização de luvas de látex para procedimentos é essencial nas atividades que envolvem risco biológico no cuidado à saúde, no entanto ainda é controversa a segurança microbiológica das luvas de procedimento, considerando sua exposição ambiental. Objetivos: quantificar as Unidades Formadoras de Colônias (UFC) das luvas de látex para procedimentos em momentos distintos (início, meio e fim das caixas) na situação real e controle de enluvamento; avaliar a contaminação das luvas conforme o tempo de exposição ambiental das caixas; isolar o microrganismo mais freqüente e determinar o perfil de sensibilidade aos antibióticos. Material e Método: trata-se de um estudo comparativo e prospectivo realizado em uma unidade de terapia intensiva de um hospital-escola. Após aprovação do comitê de ética em pesquisa, procedeu-se à coleta das amostras microbiológicas das luvas de látex por meio da digito-pressão em placas de Petri, preparadas com meio de cultura Mueller Hinton (MH). Esse procedimento foi realizado em 31 caixas de luvas de látex para procedimentos em diferentes momentos (início, meio e fim das caixas) e em situação real de enluvamento e controle. O Etest® foi utilizado na determinação do perfil de sensibilidade aos antibióticos (Ciprofloxacina®, Oxacilina®, Cefepime®, Gentamicina®, Amicacina® e Vancomicina®). Os dados foram submetidos a análise estatística por meio do Teste de Cochran e Spearman, considerando nível de significância de a=0,05. Para comparação do número de UFC, realizou-se análise de variância (ANOVA) com medidas repetidas. Resultados: totalizaram-se 372 placas de Petri, das quais 186 foram obtidas na situação real de enluvamento e 186 no controle. A média de UFC foi de 4,7, na situação controle, e de 6,2 na situação real de enluvamento, não demonstrando diferenças estatisticamente significantes (p=0,601). Em relação à avaliação dos pares de luvas no início, meio e fim da caixa na situação real de enluvamento e controle, não foram evidenciadas diferenças significantes no crescimento microbiano (UFC). Na associação entre o tempo de abertura ao término da caixa de luvas e o número de UFC, tanto na situação real de enluvamento quanto no controle, não houve correlação (p=0,63). Quanto aos microrganismos, a bactéria Staphylococcus spp. foi a mais freqüente. Com relação ao perfil de sensibilidade, 13 cepas (24,8%) apresentaram resistência a pelo menos 2 antibióticos; destas, 3 (5,5%) tiveram resistência a vancomicina. Conclusão: os resultados corroboraram a hipótese inicial da pesquisa quanto à segurança microbiológica das luvas de látex para procedimentos considerando os baixos valores de UFC. / Introduction: the use of latex gloves is essential and unquestionable in activities that involve biological risk in the health care. However, it is still controversial the microbiological safety of gloves for procedures considering their environmental exposition. Objectives: quantify the Colony-Forming Units (CFU) of latex gloves for procedures in different moments (beginning, middle and end of boxes) in real situation and gloving control; evaluate the contamination of gloves regarding the boxes time of environmental exposition; isolate the most frequent microorganism and determine the sensibility profile to antibiotics. Material and Method: It is a comparative and prospective study performed in an intensive therapy unit of a school hospital. After approval of the committee of ethics in research the collection of microbiological sample of latex gloves through digital pressure in Petri plaques prepared with culture media Mueller Hinton (MH) was initiated. This procedure was performed in 31 latex gloves boxes for procedures in different moments (beginning, middle and end of boxes) and, in real gloving and control situations. The Etest® was used to determine the sensibility profile of antibiotics (Ciprofloxacin®, Oxacillin®, Cefepime®, Gentamicin®, Amikacin® e Vancomycin®). The data was statistically analyzed through the Cochran and Spearman test considering a level of significance of a=0,05. Analysis of variance (ANOVA) with repetitive measures was used for the comparison of number of UFC. Results: a total of 372 Petri plaques were obtained, 186 in real gloving situation and 186 in the control situation. The CFU average was 4,7 in the control situation, and 6,2 in the real gloving situation, with no statistically significant differences (p=0,601). There was no evidence of significant differences in the microbial growth (UFC) in the evaluation of gloves pairs in the beginning, middle and end of boxes in both the real and control situations. There was no correlation (p=0.63) in the association between time of opening and finishing the box of gloves with the number of CFU, both in the real and control situations. The Staphylococcus spp. was the most frequent microorganism. Regarding the sensibility profile, 13 strains (24.8%) were resistant to at least two antibiotics; from these three (5.5%) resisted Vancomycin®. Conclusion: The results obtained support the initial hypothesis regarding the microbiological safety of latex gloves for procedures considering the low levels of CFU.