Thesis (Ph. D.)--State University of New York at Binghamton, Department of Psychology, 2008. / Includes bibliographical references.

The significant social and economic losses as a result of bovine tuberculosis (bTB) present a continuous challenge to cattle industries in the United Kingdom (UK) and worldwide. Furthermore, as a zoonotic disease, bTB may pose a threat to humans. The potential transmission of bTB in cattle, estimated by the basic reproductive ratio (R0) was found to range between 1.0 and 1.9 in previous studies. In the UK, there has been an overall increase in bTB incidence in the last two decades despite national control and eradication programmes spanning over five decades. Such programmes mainly consist of surveillance based on the administration of skin tests and culling of animals reacting positive to these tests. Animal mobility restrictions are implemented in this case. At the same time, several studies have demonstrated that there is significant host genetic variation in individual cattle susceptibility to bTB, making the disease amenable to improvement with genetic or genomic selection. In addition, genomic analyses enhance the understanding of genetic mechanisms underlying the disease and its dynamics. The overall aim of this PhD thesis was to address existing scientific research gaps on the genetics of bTB resistance in dairy cattle. The following specific objectives were set: 1) to identify genomic regions underlying susceptibility to bTB using novel trait definitions, 2) to quantify the impact of long-term genetic selection for increased resistance to bTB on disease prevalence and dynamics and 3) to determine the consequences of genetically selecting for increased resistance to bTB on other economically important traits in dairy cattle. Genome-wide association studies (GWAS), regional heritability mapping (RHM) and chromosomal association analyses were applied in order to identify genomic regions associated with bTB (objective 1). Phenotypes comprised de-regressed estimated breeding values of 804 Holstein-Friesian sires obtain from the UK national genetic evaluation for bTB. Phenotypes pertained to three bTB trait definitions: i) positive reactors to the skin test with positive post-mortem examination results (phenotype 1); ii) positive reactors to the skin test regardless of post-mortem examination results (phenotype 2) and iii) as in (ii) plus non-reactors and inconclusive reactors to the skin test with positive post-mortem examination results (phenotype 3). In all cases, non-reactors without a subsequent positive post-mortem were considered to be healthy animals with regards to bTB. Genotypes based on a 50K SNP DNA array were available and a total of 34,874 SNPs remained after quality control. The estimated polygenic heritability for susceptibility to bTB was 0.26, 0.37 and 0.34 for phenotypes 1, 2 and 3, respectively. GWAS identified a putative SNP on Bos taurus autosomes (BTA) 2 associated with phenotype 1, and another on BTA 23 associated with phenotype 2. Genomic regions encompassing these SNPs were found to harbour potentially relevant annotated genes. RHM confirmed the effect of these genomic regions and identified new regions on BTA 18 for phenotype 1 and BTA 3 for phenotypes 2 and 3. Heritabilities of the genomic regions ranged between 0.05 and 0.08 across the three phenotypes. Chromosome association analysis indicated a major role of BTA 23 on susceptibility to bTB. A stochastic genetic epidemiological model based on four main disease states, namely susceptible (S), exposed (E), infectious (I) and test-sensitive (T), was developed to address objective 2. Effects of selection for increased resistance to bTB were investigated in a closed, genetically heterogeneous simulated population whose structure reflected the UK national dairy herd. Disease dynamics reflected real bTB data from the UK national genetic evaluation. The proposed genetic epidemiological model was implemented to simulate breakdowns under both absence and presence of selection. Genetic selection was simulated over 20 generations in 50 replicates, while exploring various selection intensities reflecting selection of the 10, 25, 50, 70 and 100% (no selection scenario) most resistant sires. Results indicated that selection significantly reduced the average underlying susceptibility across generations. The risk of breakdown was reduced by half after 4 and 6 generations for high selection intensities (10 or 25% of sires selected) and after 9 and 15 generations for low selection intensities (50 or 70% of sires selected). The average percentage of secondary cases was reduced to less than 1% in 4 and 5 generations for high selection intensities, and in 7 and 11 generations for low selection intensities. The reduction in the number of secondary cases across generations could also be indicative of the possible impact of genetic selection on the basic reproductive ratio (R0) which is defined as the number of secondary cases that results from an infectious individual in a naive population. Genetic selection also reduced severity and duration of breakdowns across generations. Finally, with regards to objective 3, a stochastic simulation was used to investigate the long-term effects of selection for resistance to bTB on other economically important traits in the UK dairy selection programme. Selection was simulated in a genetically heterogeneous population across 10 generations in 50 replicates. Animal genetic values for bTB and other traits were simulated based on variance and genetic correlation estimates obtained from literature. Independent culling levels selection of sires was applied in every generation whereby selection was first based on increasing resistance to bTB, then improving either an overall index, milk fat yield (FY) or milk protein yield (PY). This mimics real life practices regarding the newly released national genetic evaluations for bTB resistance. The overall index comprised several traits of interest such as milk yield (MY), FY, PY, feet and legs (FL), mammary (MAM), milk somatic cell count (SCC), calving interval (CI), non-return to service at 56 days (NR56) and lifespan (LS). A fertility index (FI) consisting of CI and NR56 was also considered in the analyses. Regarding bTB, different levels of selection intensities were explored corresponding to selection of the 10, 25, 50, 70 and 100% (no selection) most resistant sires. Two levels of selection intensity on the overall index, FY or PY were considered corresponding to selecting the best 5 and 10% of sires that were left after first selecting for bTB resistance. Results indicated that selection for increased bTB resistance would generally not have far-reaching consequences on other important traits. As expected, susceptibility to bTB declined with time and increasing selection intensity. Trends for all production traits (MY, FY and PY) in the present study were affected by selection for increased bTB resistance because of their significant genetic correlations with bTB. However, body conformation traits (FL and MAM) were not affected by selection for increased bTB resistance due to zero correlation assumed between these traits and bTB in the present study. Selection on bTB hampered improvement of SCC but enhanced LS because it was correlated unfavourably with SCC but favourably with LS. In all selection scenarios, the overall index improved and was generally not affected by selection for bTB resistance. Similarly, the FI was not affected by selection on bTB in all cases. However, secondary selection on production traits only (FY or PY) led to a decline in FI. Results presented in this thesis add insight into the genetic architecture of bTB and offer a prediction of potential effects of genetic selection for increased resistance to bTB in dairy cattle. The genomic regions and candidate genes identified to be associated with susceptibility to bTB will assist to further elucidate pathways critical to cattle susceptibility to bTB. / Consistent with previous studies of other populations and trait definitions, results from genomic association analyses suggest that susceptibility of cattle to bTB is heritable and likely a polygenic trait, amenable to improvement by genetic and/or genomic selection. Embarking on routine selection for resistance to bTB will reduce future bTB prevalence and severity of breakdowns across selection generations, as manifested by results of this thesis. The results also highlight the importance of considering selection as a complementary strategy to existing interventions. This has the potential to accelerate control and ultimate eradication of bTB. This strategy could assist the UK to achieve the national goal of being officially bTB free by 2038. Furthermore, as indicated by results of this thesis, selection against bTB in the national breeding programme will not adversely affect other economically important traits. Assimilation of bTB into the overall index will better manage possible antagonistic correlations between bTB susceptibility and some of the other traits.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-08-01T21:34:39Z No. of bitstreams: 1 Pablo Rafael Silveira Oliveira Modulação da ativação de macrofagos e agravamento da infecção....pdf: 1352782 bytes, checksum: 6124c322249b9e7b7ed3d1afbbb270a0 (MD5) / Made available in DSpace on 2012-08-01T21:34:39Z (GMT). No. of bitstreams: 1 Pablo Rafael Silveira Oliveira Modulação da ativação de macrofagos e agravamento da infecção....pdf: 1352782 bytes, checksum: 6124c322249b9e7b7ed3d1afbbb270a0 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Em humanos, tanto L. amazonensis quanto L. braziliensis causam a leishmaniose cutânea localizada, mas, no Brasil, o desenvolvimento da leishmaniose cutâneodifusa (LCD) é exclusivamente atribuído às infecções por L. amazonensis. Por outro lado, a leishmaniose cutâneo-mucosa (LCM) está usualmente associada à L. braziliensis. Enquanto a LCD é caracterizada pelo elevado número de parasitos nas lesões e pela falta de resposta celular anti-Leishmania, a LCM é geralmente acompanhada por resposta celular intensa e baixo número de parasitos nas lesões. Portanto, é provável que diferenças espécie-específicas sejam determinantes para o desenvolvimento dos dois polos de responsividade imune nas leishmanioses cutâneas. Experimentos realizados em nosso laboratório mostraram que a imunização de camundongos BALB/c com o extrato de formas amastigotas de L. amazonensis (ELa) confere suscetibilidade à infecção por L. braziliensis. Os principais objetivos do presente estudo foram identificar e elucidar os mecanismos de ação dos fatores do ELa capazes de conferir suscetibilidade à infecção experimental por L. braziliensis. Para isso, o ELa foi fracionado por cromatografia líquida em coluna de troca iônica. As atividades das frações obtidas foram avaliadas quanto às suas capacidades em interferir nas funções normais de macrófagos de BALB/c, tornando-os suscetíveis à infecção cutânea causada por L. braziliensis. Foram preparadas quatro misturas de frações adjacentes, eluídas da coluna, que possuíam proteínas com perfis eletroforéticos na presença de dodecil sulfato de sódio e atividades proteolíticas semelhantes. Estas misturas de frações foram denominadas de frações 1, 2, 3 e 4. As frações 2, 3 e 4, e não a 1, foram capazes de agravar a doença causada por L. braziliensis. Após a ativação dos macrófagos com lipopolissacarídeo bacteriano (LPS), o ELa suprimiu a produção de óxido nítrico e das citocinas inflamatórias avaliadas (TNF-α, IL-12p70 e IL-6). De maneira oposta, o tratamento com o ELa aumentou a produção de IL-10 por macrófagos ativados por LPS. Nenhuma das frações contendo moléculas eluídas da coluna de troca iônica, ou a fração efluente da coluna, foi capaz de suprimir globalmente a produção de citocinas inflamatórias e de NO e aumentar a produção de IL-10 in vitro, como observado com o extrato total de amastigotas de L. amazonensis, após a ativação dos macrófagos por LPS. Apenas o tratamento in vitro com a fração 3 suprimiu a produção de TNF-α por macrófagos. A identificação e a elucidação dos mecanismos de ação dos fatores capazes de agravar a leishmaniose cutânea podem ser determinantes para o desenvolvimento de estratégias para o combate de patógenos intracelulares e também para a imunomodulação terapêutica de doenças autoimunes e alérgicas. / In humans, infections with Leishmania (Viannia) braziliensis usually induces a intense cellular response and few lesions restricted to the skin and/or mucous membrane, while infections with Leishmania (Leishmania) amazonensis can cause also a very serious form of the disease, known as diffuse cutaneous leishmaniasis, in which there is a poor cellular response to the parasite and disseminated lesions on the skin of the host. Most inbred strains of mice, like the BALB/c, are susceptible to L. amazonensis and resistant to L. braziliensis infections. This parasite-related difference could result from the activity of a L. amazonensis-specific virulence factor. In agreement with this hypothesis, we previously demonstrated that the immunization of BALB/c mice with L. amazonensis amastigote extract (LaE), and not with L. braziliensis extract, confers susceptibility to L. braziliensis infection. The main objectives of this study were to identify and elucidate the mechanisms of action of the LaE factors that contribute to the enhancement of experimental cutaneous leishmaniasis. The LaE was fractioned by liquid chromatography on ion-exchange column. The collected fractions were evaluated by their ability to subvert the normal functions of macrophages, making BALB/c mice susceptible to the infection by L. braziliensis. The fractions 2, 3 and 4, but not the fraction 1, were able, as well as the total extract of L. amazonensis, to aggravate the cutaneous infection. The LaE suppressed the inflammatory cytokine production (IL-12p70, IL-6 and TNF-α) in LPSstimulated macrophages. On the other hand, LaE increased the IL-10 production in LPS-stimulated macrophages. None of the fractions obtained by liquid chromatography on ion-exchange column, neither the effluent fraction were able to down-modulate the global production of inflammatory cytokines and NO and increase the IL-10 production in activated macrophages. The in vitro treatment with the fraction 3 suppressed the TNF-α production in macrophages. The identification and elucidation of the action mechanisms of the factors that aggravate cutaneous leishmaniasis may be determinant for the development of novel strategies to combat intracellular pathogens but also for therapeutic immunomodulation of autoimmune and allergic diseases.

Made available in DSpace on 2014-06-11T19:26:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-04Bitstream added on 2014-06-13T18:29:47Z : No. of bitstreams: 1 benessiuti_mf_me_bauru.pdf: 4336666 bytes, checksum: b38250d3f308f9ca935e1e148d3fa7a0 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A região das nascentes do ribeirão Guaratinguetá, localizada no contraforte da Serra da Mantiqueira, Vale do Paraíba, tem sido intensamente modificada por ações antrópicas criando condições que favorecem os movimentos de massa, princiapalmente devido à supressão da mata nativa. Estas ações, somada ao aumento a intensidade de chuvas sobre uma complexa geologia da região, têm potencializado os escorregamentos translacionais, de modo que foram registradas mais de 40 ocorrências, nas chuvas do final de 2008 e início de 2009. Neste contexto, as proposta desta dissertação é de identificar os mecanismos que geram essas instabilidades através da avaliação das características geotécnicas de dois escorregamentos característicos na bacia do ribeirão Guaratinguetá. Para isso, as atividades envolveram ensaios de laboratório e de campo. No laboratório foram realizados ensaios para a caracterização completa das amostras, ensaios de sucção, para determinação das curvas características, e ensaios de cisalhamento direto, para determinação dos parâmetros de resistência. No campo, foram determinadas as propriedades hidráulicas através do permeâmetro de Guelph e a resistência à penetração dos horizontes utilizando o Penetrômetro Dinâmico de Cone. O estudo dos possíveis processos de identificação de instabilidade foi realizado a partir de retro-análises dos dois escorregamentos estudados, através do software SLOPE/W, da GeoSlope, e de simulações de cenários, através do modelo matemático Shalstab. Nestas análises, foram consideradas diferentes hipóteses relativas à presença de água no subsolo, onde foram verificados os respectivos fatores de segurança. Os resultados das retro-análises indicam coerência entre as superfícies previstas e observadas no campo, para a condição de solo saturado acima da superfície de ruptura e solo na umidade residual... / The land degradation process as consequences of deforestation and farming activities at the northem part of the Guaratinguetá creek watershed, Paraíba Valley, is compromising the environmental balance of the region traduced by increase in the numbers of shallow landslides occurring during rain seasons. In summer 2009, more than 40 shallow landslides occurred during an intense rainfall with serious consequence for the local population. In this context, the purpose of this dissertation is to understand the mechanism that triggered the soil movement based on geotechnical investigation of two landslides in the Guaratinguetá creek watershed. The geotechnical profiles of two landslides and the hydraulic parameters were determined by field tests using Dynamic Cone Penetrometer and Guelph Permeameter. In laboratory, physical and mechanical soil properties were determined. The shear strength parameters were obtained by direct shear tests on undisturbed speciments for three initial gravimetric water contents (residual, natural and saturated). The instability process was inferrred by back-analysing these two landslides using the SLOPE/W, GeoSlope, and simulating scenarios using SHALSTAB model. These analyses took into account the physical properties and the shear strength parameters of the solis for different water contents. The predicted slip surfaces were in agreement with the observed ones when the analyses considered the soil parameters above the slip surface as saturated and the soil parameters below the slip surface as in residual water content condition. The susceptibility map using SHALSTAB were then carried out for the soil parameters for satured condition. The influence of the vegetation (root strength) was inserted in the analyses by a scale factor based onn Algebra Map Technique. The validation of the applied methodology was verified by crossing the predicted unstable... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:32:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-04-14Bitstream added on 2014-06-13T19:02:49Z : No. of bitstreams: 1 araujo_pc_dr_rcla.pdf: 6448977 bytes, checksum: a417ef7a09cc4dcb465b4c84dfc411b9 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Os escorregamentos são os principais processos ligados à dinâmica externa, que estão em desenvolvimento nas escarpas da Serra do Mar, o que tem despertado grande interesse científico e motivado o desenvolvimento de trabalhos visando o entendimento de seus mecanismos, pois estão intimamente relacionados a situações de risco. Contemplando as mais variadas formas de abordagens, os trabalhos desenvolvidos nessa região tem um ponto em comum: As análises, em sua grande maioria, são feitas através de modelos analógicos, que utilizam medidas diretas e apresentam resultados qualitativos. No presente trabalho, apresenta-se uma abordagem quantitativa que utiliza recursos da geotecnologia para gerar mapas de suscetibilidade a escorregamentos através do método pesos das evidências (WOFE), baseado em probabilidade condicional ou Bayesiana. Esse método é utilizado para medir as relações espaciais entre eventos já ocorridos e os mapas de evidências associados aos condicionantes desses eventos. Um pressuposto importante desse método é a independência condicional entre as evidências, sendo sua avaliação feita através dos métodos estatísticos qui-quadrado e teste global. O método proposto foi aplicado em uma área do Município de São Sebastião, litoral norte do Estado de São Paulo, utilizando mapas com padrões binários. A obtenção dos mapas binários obedeceu ao critério de contraste máximo, que é a medida de correlação espacial entre os escorregamentos e as evidências. A avaliação dos resultados obtidos indica que a abordagem utilizada apresenta potencial para aplicação em estudos sobre suscetibilidade a escorregamentos e avaliações sobre risco, em função do uso de probabilidade condicional. / Landslide is the main process linked to external dynamic that take place in the Mar Mountain Range (Serra do Mar) escarpments. It has been of great scientific interest and has motivated researches that aim the comprehension of its mechanisms. The approaches have been diversified, but with a common sense: the analyses are mainly made through analogical models, using direct measurements and showing qualitative results. Alternatively, this work shows a quantitative approach, using geotechnology resorts that give raise to landslides susceptibility maps through the weights of evidence method (WOFE). This method, based in the conditional or bayesian probability, is useful to measure the spatial correlations among events and evidence maps, which are selected as factors that influence the occurrence of these events. The conditional independence between the evidence maps is an important purpose of the WOFE method, and the evaluation is made through the overall test and chi-square statistic methods. The method was applied in an area of the São Sebastião County, in the North Coast of the São Paulo State, Brazil, using binary maps. The binary maps were the result of the reclassification using the maximum contrast criterion, which is the measurement of the spatial correlation between landslides and evidences. The evaluation of the results show that the approach presents potential for application in studies on landslide susceptibility and evaluations on risk, based of the use of conditional probability.

Made available in DSpace on 2014-10-09T12:23:58Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:57Z (GMT). No. of bitstreams: 1 01252.pdf: 5489068 bytes, checksum: 2cf58e6491611bf6489c5c3a6ccf4f81 (MD5) / Dissertacao (Mestrado) / IEA/D / Instituto de Fisica, Universidade de Sao Paulo - IF/USP

Orientador: Paulina Setti Riedel / Banca: Ardemirio de Barros Silva / Banca: Arlei Benedito Macedo / Banca: Ricardo Vedovello / Banca: Iandara Alves Mendes / Resumo: Os escorregamentos são os principais processos ligados à dinâmica externa, que estão em desenvolvimento nas escarpas da Serra do Mar, o que tem despertado grande interesse científico e motivado o desenvolvimento de trabalhos visando o entendimento de seus mecanismos, pois estão intimamente relacionados a situações de risco. Contemplando as mais variadas formas de abordagens, os trabalhos desenvolvidos nessa região tem um ponto em comum: As análises, em sua grande maioria, são feitas através de modelos analógicos, que utilizam medidas diretas e apresentam resultados qualitativos. No presente trabalho, apresenta-se uma abordagem quantitativa que utiliza recursos da geotecnologia para gerar mapas de suscetibilidade a escorregamentos através do método "pesos das evidências" (WOFE), baseado em probabilidade condicional ou Bayesiana. Esse método é utilizado para medir as relações espaciais entre eventos já ocorridos e os mapas de evidências associados aos condicionantes desses eventos. Um pressuposto importante desse método é a independência condicional entre as evidências, sendo sua avaliação feita através dos métodos estatísticos qui-quadrado e teste global. O método proposto foi aplicado em uma área do Município de São Sebastião, litoral norte do Estado de São Paulo, utilizando mapas com padrões binários. A obtenção dos mapas binários obedeceu ao critério de contraste máximo, que é a medida de correlação espacial entre os escorregamentos e as evidências. A avaliação dos resultados obtidos indica que a abordagem utilizada apresenta potencial para aplicação em estudos sobre suscetibilidade a escorregamentos e avaliações sobre risco, em função do uso de probabilidade condicional. / Abstract: Landslide is the main process linked to external dynamic that take place in the Mar Mountain Range (Serra do Mar) escarpments. It has been of great scientific interest and has motivated researches that aim the comprehension of its mechanisms. The approaches have been diversified, but with a common sense: the analyses are mainly made through analogical models, using direct measurements and showing qualitative results. Alternatively, this work shows a quantitative approach, using geotechnology resorts that give raise to landslides susceptibility maps through the "weights of evidence" method (WOFE). This method, based in the conditional or bayesian probability, is useful to measure the spatial correlations among events and evidence maps, which are selected as factors that influence the occurrence of these events. The conditional independence between the evidence maps is an important purpose of the WOFE method, and the evaluation is made through the overall test and chi-square statistic methods. The method was applied in an area of the São Sebastião County, in the North Coast of the São Paulo State, Brazil, using binary maps. The binary maps were the result of the reclassification using the maximum contrast criterion, which is the measurement of the spatial correlation between landslides and evidences. The evaluation of the results show that the approach presents potential for application in studies on landslide susceptibility and evaluations on risk, based of the use of conditional probability. / Doutor

Made available in DSpace on 2014-10-09T12:23:58Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:07:57Z (GMT). No. of bitstreams: 1 01252.pdf: 5489068 bytes, checksum: 2cf58e6491611bf6489c5c3a6ccf4f81 (MD5) / Dissertacao (Mestrado) / IEA/D / Instituto de Fisica, Universidade de Sao Paulo - IF/USP

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Nos Ãltimos anos, a melhoria dos mÃtodos de diagnÃstico micolÃgico e as doenÃas imunossupressoras causaram grande impacto na incidÃncia das micoses profundas e oportunistas em todo o mundo, fato que impulsionou a realizaÃÃo de estudos de prospecÃÃo por novas drogas antifÃngicas. A histoplasmose à uma micose sistÃmica, causada pelo fungo Histoplasma capsulatum var. capsulatum que, em pacientes hÃgidos, pode mimetizar a tuberculose quanto aos aspectos clÃnicos e radiolÃgicos. Alguns casos de histoplasmose refratÃria ao tratamento com drogas antifÃngicas convencionais vÃm sendo descritos. O objetivo deste trabalho foi determinar o efeito inibitÃrio, in vitro, das drogas antituberculose: isoniazida (INH), pirazinamida (PZA) e etambutol (EMB); antifÃngicas: anfotericina B (AMB), fluconazol (FLC), itraconazol (ITC) e voriconazol (VRZ) e de anÃlogos quÃmicos da isoniazida frente a cepas de H. capsulatum (n=30), assim como avaliar o emprego de diferentes meios de cultura para a realizaÃÃo dos testes de sensibilidade. Para isso, primeiramente, foram repicadas 18 cepas de H. capsulatum em Ãgar BHI e utilizadas na realizaÃÃo dos testes de sensibilidade frente aos agentes antituberculose citados e anÃlogos quÃmicos da isoniazida, isolados e em combinaÃÃo com os antifÃngicos FLC, ITC e VRZ por meio da tÃcnica de macrodiluiÃÃo em caldo. Cada uma das 12 cepas restantes foi repicada em Ãgar batata dextrose, Ãgar BHI, Ãgar malte a 2% e Ãgar lactrimel e, analisadas ao microscÃpio Ãptico quanto a presenÃa de macroconÃdios tuberculados, sendo quantificados de acordo com os parÃmetros: (0-10); (10-50); (>50) macroconÃdios/campo. As culturas foram empregadas na determinaÃÃo dos testes de sensibilidade frente aos agentes antifÃngicos AMB, FLC, ITC e VRZ, utilizando a tÃcnica de microdiluiÃÃo em caldo. As drogas antituberculose inibiram o crescimento das cepas in vitro com valores de CIM de 0,04 a 0,30 mg/mL para INH, 0,55 a 3,13 mg/mL para PZA e 1,56 a 6,25 mg/mL para EMB. No tocante Ãs drogas antifÃngicas, todas as cepas foram sensÃveis apresentando valores de CIM que variaram de 0,0625 a 0,25 Âg/mL para AMB; 15,62 a 62,5 Âg/mL para FLC; 0,0039 a 0,0312 Âg/mL para ITC e 0,00156 a 0,25 Âg/mL para VRZ. Quanto Ãs combinaÃÃes entre os fÃrmacos antituberculose e os derivados azÃlicos, todas foram capazes de inibir o crescimento in vitro das cepas de H. capsulatum, sendo detectado sinergismo nas nove combinaÃÃes. Os anÃlogos da isoniazida apresentaram valores de CIM 2, 4, 8 e 15 vezes superior a atividade da droga antituberculose padrÃo. A partir da anÃlise micromorfolÃgica do fungo repicados nos quatro meios de cultura foi identificado a menor quantificaÃÃo (0-10 macroconÃdios/campo) para Ãgar batata, Ãgar BHI, Ãgar malte e Ãgar lactrimel, perfazendo um total de 11, 10, 6 e 7 cepas, respectivamente. O meio de cultura Ãgar malte foi o mais adequado para produÃÃo de macroconÃdios (10-50) e (>50), norteando um total de 6 cepas, seguido do meio lactrimel, 5 cepas. Em relaÃÃo a determinaÃÃo da CIM e o meio de cultura utilizado para o procedimento, observou-se que quando o inÃculo era proveniente de cepas em Ãgar malte e Ãgar BHI foi possÃvel a visualizaÃÃo da CIM em 11 cepas. Enquanto repiques feitos em Ãgar batata e lactrimel nÃo foi possÃvel determinar os valores de CIM para 8 e 5 cepas, respectivamente. Os resultados deste estudo fornecem dados adicionais sobre o potencial antifÃngico das drogas antituberculose e suas interaÃÃes com os derivados azÃlicos. Entretanto, novos estudos se fazem necessÃrio, visando determinar os mecanismos de aÃÃo desses compostos no metabolismo celular dos fungos. / In the past years, the improvement of mycological diagnosis methods and immunosuppressive diseases have caused a great impact in the incidence of opportunistic and deep mycoses all around the world, which motivated the performance of new antifungal drugs prospective studies. Histoplasmosis is a systemic mycosis caused by the fungus Histoplasma capsulatum var. capsulatum, which may mimic tuberculosis, in healthy individuals, concerning clinical and radiological aspects.ome cases of histoplasmosis that are refractory to the treatment with conventional antifungal drugs have been described. The aim of this study was to determine the in vitro inhibitory effect of the antituberculosis drugs: isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB); antifungal drugs: amphotericin B (AMB), fluconazole (FLC), itraconazole (ITC) and voriconazole (VRZ) and chemical analogs of isoniazid against strains of H. capsulatum (n=30), as well as to evaluate the use of different culture media for the performance of the susceptibility tests. For such, first, the antituberculosis agents INH, PZA and EMB and the analogs of isoniazid were tested isolatedely, and then, in association with the antifungal drugs FLC, ITC and VRZ, against 18 strains of H. capsulatum, previously grown onto BHI agar, through broth macrodilution technique. Each of the 12 remaining strains grown onto potato agar, BHI agar, 2% malt extract agar and lactritmel agar were microscopically analyzed, concerning the presence of tuberculate macroconidia, which were quantified as follows: 0-10, 10-50 and >50 macroconidia/field. Fungal cultures were used to determine the susceptibility of H. capsulatum to the antifungal agents AMB, FLC, ITC and VRZ, through broth microdilution methodology. The antituberculosis drugs inhibited the in vitro growth of the fungal strains, with MICs ranging from 0.04 to 0.30 mg/mL for INH; 0.55 to 3.13 mg/mL for PZA and 1.56 to 6.25 mg/mL for EMB. Concerning antifungal drugs, all the strains were susceptible, with MIC values ranging from 0.0625 to 0.25 Âg/mL for AMB; 15.62 to 62.5 Âg/mL for FLC; 0.0039 to 0.0312 Âg/mL for ITC, and 0.00156 to 0.25 Âg/mL for VRZ. When associating antituberculosis drugs with azole derivatives, all associations inhibited the in vitro growth of H. capsulatum strains, and synergy was observed for the nine combinations tested. Analogs of isoniazide presented MICs of 2, 4, 8 and 15-fold better than the standard antituberculosis drug. Basing on micromorphological analysis, the lowest quantification of macroconidia/field (0-10) was observed for 11, 10, 6 and 7 strains previously grown onto potato agar, BHI agar, malt agar and lactritmel agar, respectively. Malt agar was the most adequate medium for the production of macroconidia, 10-50 and >50/field, with a total of six strains; followed by lactritmel agar, with 5 strains. Concerning the relationship between MIC and culture medium used during the test, it was observed that inoculum from strains grown onto malt agar and BHI agar allowed the detection of the MIC for 11 strains. On the other hand, for those inocula grown onto potato agar and lactritmel agar, the MIC values were not detected for 8 and 5 strains, respectively. The results of this study provide additional data on the antifungal potential of antituberculosis drugs and their interactions with azole derivatives. However, new studies are necessary in order to determine the mechanism of action of these compounds on fungal cellular metabolism.

Ph.D. / Heat shock proteins (HSP) (in particular hsp70) are increasingly synthesised during and following exposure to stressful insults, playing an important role in protection and adaptation. Protective effects of HSP concerning infection and immunity include self/non-self discrimination, enhancement of the immune response, immune protection, thermotolerance and cytoprotection from inflammatory mediators (reactive oxygen species and cytokines). Considering the general protective role of hsp70 and its specific immunological functions, including antigen processing and presentation, variation in hsp 70 genes may contribute towards differential coping with stress and disease susceptibility. In humans, three members of the hsp70 gene family, hspl0-1, hsp70-2 and hsp70-hom, were mapped to the MHC class Ill region approximately 280 kbp centromeric to the TNFa gene and 92 kbp telomeric to the C2 gene. Polymorphisms in MHC-Iocalized hsp70 genes have been implicated in susceptibility to a number of diseases, independently or in combination with class II polymorphisms due to linkage disequilibrium (LD). MHC alleles are most often associated with immunosuppressive diseases. Tuberculosis (TB) has a strong immunological basis, involving cell-mediated immunity with human leukocyte antigen (HLA) variants implicated in its susceptibility/resistance. In the light of the above, the role of hsp70 polymorphism in TB susceptibility, alone or in combination with MHC class II alleles, was investigated through the following objectives: 1) Typing of hsp70 gene polymorphism (hsp70-1, hsp70-2 and hsp70-hom) in controls and TB cases from the Cape Coloured population of South Africa 2) Comparison between Cape Coloureds and Caucasoid populations with regard to hsp70 allele and genotype distribution 3) Studying linkage disequilibrium between members of MHC class II (HLADRB1) and Ill (hsp70) alleles in the Cape Coloureds 4) Simulation of MHC class II and Ill haplotypes in this particular population Hsp70 polymorphism was studied in controls (n=106) and TB cases (n=107) from the complex hybrid Cape Coloured population inhabiting the Western Cape region of South Africa - a population showing increased susceptibility to TB. PCR-RFLP and PAGE analysis were used to determine the hsp70 allele frequencies and genotype distribution of the individuals studied, while linkage disequilibrium between MHC class II and Ill, and within class Ill alleles, was investigated using the software "Graphical Overview of Linkage Disequilibruim" (GOLD). Haplotypes comprising MHC class II and Ill alleles were simulated using the software PHASE.