Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2008 / Epidemiological reports have shown that South Africa, whilst a developing country, irs overweight and obesity prevalence rates in children is fast approaching those seen in the developed world. This country's population is unique in that it is made up of different ethnic groups with different socia-economic status, partly due to the past and present political environments in the country. South Africa, in particular, is faced with a rapid increasing childhood obesity of 10% among children under the age of two and 5-20% among those less than six years of age. The prevalence of obesity is increasing in children of all ages and represents the complex integration of genetic, behavioural and environmental influences. The Ectonucleotide Pyrophosphatase Phosphodiesterase 1 (ENPP1) gene is located on chromosome 6q22-q23; a locus linked to obesity and diabetes, spans 83 kb of genomic DNA and contains 25 exons. Studies in humans have shown a correlation between overexpression of ENPP1 and insulin resistance, obesity, and type 2 diabetes. ENPP1 has been implicated in up to 20% of Caucasian and 50% of Black communities suffering from obesity. The overall objective of the proposed study is to assess whether ENPP1 polymorphisms contribute to childhood obesity/overweight, and their association with components of metabolic syndrome in a South African Coloured population. Subjects for this study were identified through a screening program that aimed to determine the prevalence of obesity in learners between the ages of 8 - 18 years from the Western Cape Province, South Africa. The first phase of the project was to clearly differentiate between obese subjects and controls. The cut-off points for obesity established by Cole and co-workers in 2000, and adopted by the International Obesity Task Force (IOTF), were used to classify the obese subjects.

Orientadores: Christine Hackel, Juergen K. V. Reichardt, Maricilda P. Mello / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T00:27:44Z (GMT). No. of bitstreams: 1 Ferraz_LucioFabioCaldas_D.pdf: 2604031 bytes, checksum: c313be68a5b9599e035866a8ee12ef8c (MD5) Previous issue date: 2006 / Resumo: O hormônio androgênico di-hidrotestosterona (DHT) possui fundamental importância na diferenciação sexual masculina e no desenvolvimento e manutenção da próstata. Duas enzimas atuam diretamente na concentração deste andrógeno nas células: 1) com uma função anabólica, a enzima 5α-redutase tipo 2 (gene SRD5A2) é responsável pela síntese de DHT ao converter testosterona (T) em 5α-di-hidrotestosterona e 2) com uma função catabólica, a enzima 3β- hidroxi desydrogenase/Δ5-Δ4-isomerase de esteróides tipo 2 (gene HSD3B2) é responsável pela degradação do DHT, além de contribuir para síntese indireta de testosterona por uma via anabólica. Isto exposto, cenários distintos se apresentam considerando as atividades deficientes dessas enzimas: i) a deficiência congênita da enzima 5a-redutase tipo 2 conduz a uma forma específica de pseudohermafroditismo masculino (PHM) no qual a conversão de T em DHT está nula ou defeituosa, inviabilizando a virilização normal da genitália externa em indivíduos com cariótipo 46,XY e ii) em razão das propriedades bifuncionais da enzima 3β-HSD2, tanto na via de síntese quanto de degradação de andrógenos, sua deficiência congênita pode conduzir a quadros clínicos distintos de ambigüidade genital. No adulto, mutações somáticas que afetem sua atividade enzimática podem contribuir para a manifestação do câncer de próstata, pelo acúmulo do DHT. O presente trabalho aborda as duas enzimas esteroidogênicas envolvidas com o metabolismo da DHT, buscando caracterizar mutações germinativas e/ou somáticas que conduzem a deficiências enzimáticas relacionadas a diferentes condições clínicas. Com relação à deficiência em 5a-redutase tipo 2, investigou-se a presença de mutações germinativas no gene SRD5A2 em amostras de DNA 20 pacientes de sexo genético masculino com suspeita de deficiência em 5α-redutase tipo 2, pertencentes a 18 famílias brasileiras, por meio de sequenciamento direto dos produtos de PCR dos cinco exons do gene e de suas regiões flanqueadoras. Foram identificadas alterações moleculares em 18 desses pacientes, compreendendo tanto mutações não anteriormente referidas na literatura (G158R, del642T, 217_218insC e IVS3+1G>A), como mutações recorrentes já descritas em outros grupos étnicos ou em indivíduos de outras regiões geográficas. Os resultados detalhados, bem como a discussão, acham-se apresentados no Capítulo III.1, sob a forma de artigo publicado. (...continua) / Abstract: The androgenic hormone dihydrotestosterone (DHT) has fundamental relevance in normal male sexual differentiation and in prostate development and maintenance. Two enzymes act directly on the regulation of DHT concentration at cellular level: 1) with an anabolic function the steroid 5α-reductase type 2 enzyme (SRD5A2 gene) leads to DHT synthesis by converting testosterone (T) in 5α-dihydrotestosterone and 2) with a catabolic pathway the 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase type 2 enzyme (HSD3B2 gene) is responsible for DHT degradation, besides contributing to indirect synthesis of testosterone in an anabolic pathway. Thus, different scenarios can be considered regarding the deficiencies in the activities of these enzymes: i) congenital steroid 5α-reductase type 2 enzyme deficiency leads to a specific form of male pseudohermaphroditism (MPH), where the conversion of T into DHT is defective or inexistent, preventing normal virilization of the external genitalia in individuals with a 46,XY karyotype; and ii) due to the bi-functional properties of the 3β-HSD2 enzyme, either at synthetic or degradation androgen pathways, its congenital deficiency can lead to distinct manifestations of genital ambiguity. Furthermore, in the adult, somatic mutations that affect 3β-HSD2 enzymatic activities could contribute to prostate cancer manifestation, due to DHT accumulation. The present work approaches these two steroidogenic enzymes involved with the DHT metabolism, aiming to characterize germinal and/or somatic mutations leading to enzymatic deficiencies related to different clinical conditions. Concerning the steroid 5α- reductase type 2 deficiency, we screened for germinal mutations on SRD5A2 gene in DNA samples of 20 patients from 18 Brazilian families with suspected SRD5A2 deficiency, by directly sequencing of the PCR products from the five exons and flanking regions of the gene. Molecular alterations were detected in 18 of these patients, comprising either mutations not previously reported in the literature (G158R, del642T, 217_218insC e IVS3+1G>A) as well as recurring mutations already described in other ethnical groups or in individuals from other geographical regions. The detailed results and corresponding discussion are presented at Chapter III.1, as a published paper. (¿to be continued) / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Orientador: Liana Maria Cardoso Verinaud / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T19:49:25Z (GMT). No. of bitstreams: 1 Souto_PaulaCristinadeSouza_D.pdf: 7669317 bytes, checksum: eeec29689d95e50ddec45928c2c76343 (MD5) Previous issue date: 2006 / Resumo: O Paracoccidioides brasiliensis é um fungo dimórfico que cresce na forma miceliar a temperatura ambiente e na forma de levedura a 35-37ºC. Recentemente, no laboratório de Imunopatologia do Departamento de Microbiologia e Imunologia do Instituto de Biologia da Unicamp, foi observado que uma amostra da cepa virulenta Pb18 crescia de forma diferente das outras. A análise preliminar desta cultura mostrou que o fungo se apresentava, exclusivamente, em formas intermediárias entre levedura e micélio. Assim, este trabalho teve como objetivo a caracterização parcial deste isolado atípico através da comparação com a cepa selvagem. Para tanto, foram analisados: a morfologia do isolado a 25 e 37ºC; a composição de proteínas de parede celular; o desenvolvimento da doença e a resposta imunológica adaptativa em camundongos BALB/c. Os resultados mostraram que este isolado não alterou a sua forma de crescimento, independente da temperatura de cultivo ou da passagem por animal. Através da análise das proteínas de parede celular observou-se ausência de expressão de algumas proteínas e baixa concentração da glicoproteína 43 (gp43) no fungo atípico quando comparado com o selvagem. A infecção causada por este isolado atípico foi mais branda do que aquela observada com a cepa virulenta Pb18, com completa resolução das lesões de disseminação. Entretanto, não foi possível determinar diferenças na avaliação da resposta imune adaptativa dos animais infectados com os diferentes isolados. Apesar da necessidade de mais estudos, estes resultados indicam que o isolado atípico pode vir a ser uma importante ferramenta para o estudo da biologia do fungo e sua relação com o hospedeiro / Abstract: P. brasiliensis is a dimorphic fungus that grows in mycelial phase at room temperature and in yeast form at 35-37°C. Recently, in the Immunopathology Laboratory of the Department of Microbiology and Immunology (Biology Institute/Unicamp), it was observed that a sample of the virulent strain Pb18 grew in a different way from other ones. Preliminary analysis of this culture showed that the isolated exhibited, exclusively, an intermediate form of pseudohyphae. That way, the objective of this work was the partial characterization of the atypical isolated (Pb18PH) through the comparison with the wild strain Pb18. For this, there were analyzed: the morphology of the isolated to 25 and 37ºC; the composition of cell wall proteins; the disease development and the adaptative immune response in BALB/c mice. The results showed that Pb18PH didn't alter their growth form, independently of the cultivation temperature or the passage through animal. By the analysis of the cell wall proteins, it was observed that Pb18PH didn't express some proteins and presented low concentration of the glicoprotein 43 (gp43) when compared with the wild one. The infection caused by this atypical isolated was softer than that observed with the virulent strain Pb18. Besides, it was noted the complete resolution of disseminated lesions. However, it was not possible to determine differences in the adaptative immune response evaluation of the animais infected with the different isolated. To despite the need of more studies about this atypical isolated, our results indicate that Pb18PH would come to be an important tool for the study of the fungus biology and their relationship with the host / Doutorado / Imunologia / Doutor em Genetica e Biologia Molecular

Orientadores: Edi Lucia Sartorato, Andrea Trevas Maciel-Guerra / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T16:54:52Z (GMT). No. of bitstreams: 1 Miranda_PauloMauriciodoAmorDivino_M.pdf: 2620124 bytes, checksum: aacc629866a9ff7880f48f266f4f3743 (MD5) Previous issue date: 2010 / Resumo: Nos seres humanos, a visão é o sentido que retrata com melhor fidelidade o ambiente que os cerca. A ausência do sentido da visão é definida como cegueira. As conseqüências da cegueira são problemas de saúde pública importantes, pois têm um impacto significativamente negativo sobre o desenvolvimento econômico e social dos indivíduos afetados. A perda visual pode ser resultado de lesão nos centros nervosos superiores, nas vias ópticas ou em estruturas do próprio olho. As causas vão desde traumas oculares até doenças congênitas, como: glaucoma congênito, catarata congênita e Neuropatia Óptica Hereditária de Leber (LHON). A LHON é uma doença, causada por alterações no DNA mitocondrial, caracterizada pela perda repentina da visão em ambos os olhos, devido a uma degeneração do nervo óptico. Atualmente, 17 principais mutações associadas à LHON foram registradas, onde três dessas mutações representam 95% dos casos (mutações primárias) e as 14 mutações subseqüentes representam apenas 5% do total (mutações secundárias). Não foram relatados, até o momento, estudos que definam a freqüência das mutações ou estudos populacionais de prevalência no Brasil. Desta forma, o presente estudo teve como principal objetivo definir a freqüência das mutações da LHON em pacientes brasileiros. Foram avaliados 55 pacientes com hipótese diagnóstica de LHON ou neuropatia óptica adquirida de origem desconhecida. Mutações primárias (G11778A, T14484C e G3460A) e mutações secundárias nos genes MT-ND1, MT-ND4, MT-ND4L, MT-ND5, MT-ND6 e MT-CYB foram rastreadas em todos os indivíduos estudados. O rastreamento das mutações primárias foi realizado pelo método de restrição enzimática. Para os pacientes que não apresentaram mutações primárias procedeu-se o seqüenciamento direto para rastrear mutações secundárias. O método de DHPLC foi padronizado para o rastreamento de mutações primárias. Mutações primárias foram encontradas em 19 pacientes, ou seja, em 38,2% dos casos. A freqüência dessas mutações ficou definida como: 67% da mutação G11778A (13 casos), 28% da mutação T14484C (5 casos) e 5% da mutação G3460A (apenas 1 caso). A mutação G11778A apresentou números próximos aos encontrados nas populações estudadas em outras partes do mundo, confirmando ser a principal causa de LHON também em pacientes brasileiros. A mutação T14484C, como na maioria das populações mundiais, mostrou-se como a segunda maior causa da LHON, porém mostrou uma freqüência quase duas vezes maior que o descrito na literatura. Enquanto que a mutação G3460A apresentou freqüência muito baixa se comparada as das demais populações estudadas. Não foram encontradas mutações secundárias. A ausência dessas mutações pode ser atribuída à possibilidade de haver mutações em regiões do DNA mitocondrial não rastreadas no presente estudo. Há também a possibilidade de os pacientes em que não foram localizadas mutações não serem portadores da LHON, apresentando outra anomalia com dados clínicos semelhantes. No presente estudo também foi possível, a partir do teste molecular a confirmação da hipótese diagnóstica de LHON em alguns pacientes / Abstract: The vision is the sense that best expresses the environment surrounding the mankind. The term blindness is used to define the absence of the vision. The consequences of blindness are important problems of public health concern as they cause a negative impact on the economic and social development of the affected individuals). The visual loss is characterized by congenital deprivation and it can be total or partial, permanent or temporary. Furthermore, visual loss may result from superior nerve centers damage, optic pathways lesions or physic damage of the eye itself. Thus, the visual loss causes range from trauma to ocular congenital diseases such as congenital glaucoma, congenital cataract and Leber Hereditary Optic Neuropathy (LHON). LHON is a mitochondrial disease characterized by sudden loss of vision in both eyes, due to an optic nerve degeneration. Currently, 17 main LHON associated mutations were published, three of which account for 95% of the cases (primary mutations) and the subsequent fourteen account for only 5% of the total (secondary mutations). The frequencies of mutations envolved in LHON in Brazilian patients have not been reported so far. Therefore, the aim of this study was to define the LHON mutations frequency in Brazilian patients. We evaluated 55 patients with LHON diagnosis or acquired optic neuropathy of unknown origin. Primary mutations (G11778A, T14484C and G3460A) and secondary mutations in the genes MT-ND1, MT-ND4, MT-ND4L, MT-ND5, MT-ND6 and MT-CYB were screened in all individuals studied. Screening of primary mutations was performed by the method of enzyme restriction. Patients who did not have primary mutations were screened for secondary mutations by direct sequencing. The DHPLC method was used for primary mutations screening. Primary mutations were found in 19 patients, ie 38.2% of cases. The frequency of these mutations was defined as 67% for the G11778A mutation (13 cases), 28% for the T14484C mutation (5 cases) and 5% for the G3460A mutation (only 1 case). The mutation G11778A showed numbers similar to those found in the populations studied in other parts of the world, confirming that the main cause of LHON also in Brazilian patients. The T14484C mutation, as in most world populations, showed up as the second leading cause of LHON, but it showed a frequency almost twice that reported. While the G3460A mutation frequency showed very low compared to the other populations studied. No secondary mutation was found. The absence of these mutations can be attributed to the possibility of mutations in mitochondrial DNA regions not screened in this study. There is also the possibility that patients in whom no mutations were not found to be carriers of LHON, with another anomaly with similar clinical data. This study evaluated the situation of primary and secondary mutations associated with LHON in a sample of Brazilian individuals. It was also possible, from the molecular testing to confirm the diagnosis of LHON in some patients / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Orientador: Edi Lucia Sartorato / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T12:01:33Z (GMT). No. of bitstreams: 1 Silva-Costa_SueliMatildeda_D.pdf: 3629847 bytes, checksum: 5dcc278ff8f0cd9eda6627d011e23a06 (MD5) Previous issue date: 2013 / Resumo: A deficiência auditiva é dos defeitos sensoriais o mais comum, afetando cerca de um em cada quinhentos recém-nascidos. Mais de 60% dos casos de perda auditiva congênita são causadas por fatores genéticos. Apesar da grande heterogeneidade da perda auditiva genética, mais de 50% estão associadas a mutações no locus DFNB1 no cromossomo 13q12. Este locus contém os genes GJB2 e GJB6 que codificam as proteínas conexina 26 e 30 respectivamente. A alta prevalência de indivíduos com perda auditiva apresentando mutações recessivas no locus DFNB1 em apenas um alelo tem dificultado o diagnóstico molecular e consequentemente o aconselhamento genético. Portanto, o principal objetivo deste trabalho foi elucidar a causa genética da perda auditiva em quarenta e oito indivíduos monoalélicos para mutações recessivas no gene GJB2 (46) ou no gene GJB6 (2). Prováveis novas deleções no locus DFNB1 foram encontradas em quatro indivíduos heterozigotos para mutações no gene GJB2. Além disso, entre os quarenta e oito indivíduos estudados foram detectadas duas mutações no gene SLC26A4 (p.V609G, p.C282Y) em três deles e ainda, duas mutações no gene CDH23, p.R1746Q e p.R301Q, em dois indivíduos. As mutações, p.V609G, p.C282Y e p.R1746Q, foram encontradas em heterozigose e, portanto, não é possível afirmar que, de fato, essas alterações sejam a causa da surdez. Contudo, a mutação p.R301Q, encontrada em homozigose em um dos pacientes estudados, trata-se de uma mutação missense e poderia explicar o fenótipo. Entre os quarenta e oito indivíduos estudados foram ainda detectadas oito alterações mitocondriais em dezoito casos. Quatro delas podem estar envolvidas com a perda auditiva justificando assim a surdez. Quanto a análise do cluster miR-183, nenhuma mutação patogênica foi encontrada em nenhum dos quinhentos e vinte e um indivíduos analisados, o que corrobora com dados da literatura onde mutações nos microRNAs, miR-96-183-182 não são uma causa comum de surdez. Concluiu-se que, a pesquisa de mutações em outros genes diminui o número de casos com diagnóstico inconclusivo. Entretanto, devido à grande heterogeneidade genética e clínica da perda auditiva muitos permanecem ainda com diagnóstico indefinido / Abstract: Hearing impairment is the most common sensory impairment, affecting approximately one in five hundred newborns. More than 60% of the congenital hearing loss cases are caused by genetics factors. Despite the enormous heterogeneity of genetic hearing loss, up to 50% of the cases are associated with mutations in the DFNB1 locus on chromosome 13q12. This locus contains the GJB2 and GJB6 genes, which code for the gap junction (GJ) proteins connexin 26 (Cx26) and connexin 30 (Cx30) respectively. A large number of affected individuals carry only a single identified recessive mutation in locus DFNB1, making the molecular diagnostic and genetic counseling difficult. The aim of this study was to elucidate the genetic cause of hearing loss in forty eight individuals monoallelic for recessive mutations in the GJB2 gene (46) or in GJB6 gene (2). Probable new DFNB1 locus deletions were found in four individuals heterozygous for mutations in GJB2 however. Moreover, among the forty eight heterozygous individuals studied, two mutations were detected in the SLC26A4 gene (p.V609G, p.C282Y) in three of them and two mutations in CDH23 gene (p.R1746Q p.R301Q) were identified in two individuals. Mutations, p.V609G, and p.C282Y p.R1746Q were found in heterozygous and therefore could not be considered the cause of deafness in the patients studied. However, the mutation p.R301Q was present as homozygous in one individual. This alteration is a missense mutation, and may explain the deafness in this patient. Among the forty eight subjects studied, we detected eight mitochondrial alterations in eighteen cases. Four of them may be involved with hearing loss, thus justifying deafness. As the analysis of the miR-183 cluster, no pathogenic mutation was found in any of the five hundred twenty-one individuals analyzed, which agrees with literature data where mutations in microRNAs, miR-96-183-182 are not a common cause of deafness. We conclude that the detection of mutations in other genes reduces the number of cases with inconclusive diagnostic. However, due to high clinical and genetic heterogeneity of hearing loss with many still remains undefined diagnosis / Doutorado / Genetica Animal e Evolução / Doutora em Genética e Biologia Molecular

Orientadores: Marcos Antônio Machado, Alessandra Alves de Souza / O último capítulo está em inglês / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T01:07:33Z (GMT). No. of bitstreams: 1 Silva_MarianadeSouzae_D.pdf: 4833630 bytes, checksum: 8baede33597b26761daf544db4af1291 (MD5) Previous issue date: 2012 / Resumo: Xylella fastidiosa é o agente causal da clorose variegada dos citros (CVC), da doença de Pierce em videiras e doenças em muitas outras culturas. Com o seqüenciamento completo do genoma de várias linhagens deste organismo, a função de cada gene , principalmente os ligados à virulência ainda estão pouco caracterizados. A fixação da X. fastidiosa nos vasos do xilema e no canal alimentar dos insetos vetores, cigarrinhas, é necessária para a virulência, formação de biofilme e transmissão desta bactéria. Uma vez que o gene pilC esta envolvido na formação do pili tipo IV e este por sua vez é importante na motilidade e formação do biofilme, neste presente trabalho é mostrado dados fenotípicos obtidos com a interrupção de pilC na linhagem J1a12 de X. fastidiosa. A interrupção do gene pilC na linhagem J1a12 foi obtida por recombinação homóloga utilizando o plasmídeo pUCBM21oriC. Reação de polimerase em cadeia (PCR) e análises por Southern blot dos mutantes indicaram que o gene cromossômico foi substituido pelo gene truncado e com isso a produção da proteína correspondente, PilC, não foi indicada pelo Westen blot. Microscopia eletrônica de varredura transmissão revelou que o tamanho e o número das fímbrias foi reduzido para os mutantes de pilC de X. fastidiosa. Esses mutantes não mostraram capacidade de colonização das regiões acima do ponto de inoculação dos vasos do xilema da planta de laranja Pêra e, além disso, não apresentaram formação de biofilme. No estudo da formação do biofilme, a caracterização da expressão de proteínas foi realizada através do uso da eletroforese de primeira e segunda dimensão e a espectrometria de massa encontramos um total de 456 proteínas expressas na condição de biofilme maduro e 387 proteínas no crescimento planctônico, sendo que, o biofilme apresentou 37% (ou 144 proteínas) diferencialmente expressas em relação ao crescimento planctônico. As alterações encontradas no estado fisiológico das células em biofilme podem ser explicadas pela diferença no padrão de proteínas encontrado. Essas proteínas são produtos correspondentes à 31 genes presentes no biofilme maduro da linhagem isolada de citros 9a5c, esses foram envolvidos na adaptação, no metabolismo e na adesão dessa bactéria. Foi observado superexpressão de genes relacionados ao quorum sensing, comprovando a existência da comunicação entre células e com isso, o desenvolvimento da estruturação do biofilme (biofilme maduro) levando à obstrução dos vasos e desenvolvimento da doença / Abstract: Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), Pierce's disease in grapevines and other diseases in many crops. With the full genome sequencing of several strains of this organism, the function of each gene, particularly those related to virulence are still poorly characterized. The setting of X. fastidiosa in xylem vessels and the alimentary canal of insects, leafhoppers, is required for virulence, biofilm formation and transmission of this bacterium. Once the gene is involved in the formation pilC of type IV pili and this in turn is important in motility and biofilm formation, this work is shown in this phenotypic data obtained with the interruption in the strain of pilC J1a12 X. fastidiosa. The interruption of the gene pilC in the strain J1a12 was obtained by homologous recombination using the plasmid pUCBM21oriC. Polymerase chain reaction (PCR) and Southern blot analysis of mutants indicated that the chromosomal gene was replaced by the truncated gene and thereby producing the corresponding protein, PilC was not indicated by Western blot. Scanning transmission electron microscopy revealed that the size and number of fimbriae was reduced for the mutants of pilC X. fastidiosa. These mutants showed no ability to colonize the region above the inoculation of the plant xylem vessels of Pera and, moreover, did not show biofilm formation. In the study of biofilm formation, the characterization of protein expression was performed by using electrophoresis of first and second dimension and mass spectrometry we found a total of 456 proteins expressed in condition of mature biofilm and 387 proteins in planktonic growth, and , the biofilm showed 37% (or 144 proteins) differentially expressed in relation to planktonic cells. The changes found in the physiological state of cells in biofilm can be explained by the difference in the pattern of proteins found. These proteins are products corresponding to 31 genes present in the mature biofilm of strain isolated from citrus 9a5c, they were involved in the adaptation, metabolism and adhesion of bacteria. We observed overexpression of genes related to quorum sensing, proving the existence of communication between cells and thus, the development of the biofilm structure (mature biofilm) leading to obstruction of vessels and development of disease / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Submitted in fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry, Durban University of Technology, Durban, South Africa, 2017. / In drug discovery and development of anti-tubercular therapeutics, it is necessary to study the physiology and genetics of the molecular mechanisms present in the Mycobacterium tuberculosis. The virulence of M. tuberculosis is attributed to its unique genome, which contains a high frequency of glycine-rich proteins and genes involved in the metabolism of the fatty acids. Consequently, the presence of a diversity of the pathogenic pathways such as acid tolerance and drug resistance mechanisms in M. tuberculosis makes the treatment of Tuberculosis (TB) challenging. However, the molecular basis of the virulence factors involved in the pathogenesis is not fully understood. Accordingly, the current study focuses on better understanding of the pathogenic proteins present in this bacterium using available computational techniques. In South Africa, there is an alarming increase in the drug-resistant TB in HIV co-infected patients, which is one of the biggest challenges to the current anti-tubercular therapies. An extensive literature search showed that the mutations in the virulent proteins of M. tuberculosis resulted in the development of drug tolerance in the pathogen. The molecular and genetic studies identified frequently occurring point mutations associated with the drug resistance in proteins of M. tuberculosis. Despite the efforts, TB infection is still increasing because different pathogenic pathways in the bacterial system are still undiscovered. Therefore, this study involves an in silico approach aimed at the identification of novel drug resistance implicated point mutations. The site- directed mutations leading to the development of resistance against four first-line drugs (Ethambutol, Isoniazid, Rifampicin, and Streptomycin) were studied extensively. In the primary investigation, pathogenic mutational landscapes were classified in the sequences of the studied proteins. The effects of these mutations on the stability of the proteins were studied using diverse computational techniques. The structural basis of the point mutations with the highest destabilizing effects was analyzed using the principles of the Density Functional Theory (DFT), molecular docking and molecular dynamics (MD) simulation studies. The varied conformational behavior resulted from these predicted substitutions were compared with the experimentally derived mutations reported in the literature. The outcome of this study enabled the identification of the novel drug resistance-associated point mutations which were not previously reported. Furthermore, a detailed understanding of the conformational behavior of diverse virulent proteins present in M. tuberculosis was also generated in this study. Literature study showed that inside the host’s macrophage cells, the virulent proteins such as isocitrate lyase, lipase lipF, magnesium transporter MgtC, porin protein OmpATb, a protein of two component systems PhoP, Rv2136c and Rv3671c have an established role in the development of the acid tolerance. On the other hand, information regarding their role in the acid resistance is scarce. Accordingly, the structural basis of their role in acid resistance was analyzed using constant pH based MD simulations. In the studied proteins, the lipF and PhoP showed highest structural stability in highly acidic conditions throughout the course of MD simulations. Therefore, these proteins may play a primary role in the process of resistance. In addition to these pathogenic proteins, there is a need to identify new undiscovered virulent proteins in the genome of M. tuberculosis, which increases the efficiency of the current therapy. The knowledge generated by the analyses of the proteins involved in resistance and pathogenic mechanisms of M. tuberculosis forms the basis for the identification of new virulence factors. Therefore, an in silico protocol was used for the functional annotations and analyses of the virulence characteristics. M. tuberculosis contains 1000 Hypothetical Proteins (HPs), which are functionally uncharacterized proteins and their existence was not validated at the biochemical level. In this study, the sequences of the HPs were extensively analyzed and the functions of 662 HPs were successfully predicted. Furthermore, 483 HPs were classified in the category of the enzymes, 141 HPs were predicted to be involved in the diverse cellular mechanisms and 38 HPs may function as transporters and carriers proteins. The 307 HPs among this group of proteins were less precisely predicted because of the unavailability of the reliable functional homologs. An assessment of the virulence characteristics associated with the 1000 HPs enabled the classification of 28 virulent HPs. The structure of six HPs with highest predicted virulence score was analyzed using molecular modelling techniques. Amongst the predicted virulent HPs, the clone for Rv3906c purchased from the DNASU repository because of the ease of its availability. The gene of Rv3906c was isolated and cloned into a pET-21c expression vector. The analyses of the nucleotide sequence showed that Rv3906c gene (500 bp) encodes a 169 amino acid protein of molecular weight 17.80 kDa (~18.0 kDa). The sequence analyses of Rv3906c showed that the HPs showed high similarities with pullulanase, a thermophilic enzyme. The stability profile at different temperatures for Rv3906c generated using MD simulations showed that Rv3906c maintained its structural identity at higher temperatures. It is expected that this study will result in the design of better therapeutic against the infection of M. tuberculosis, as novel undiscovered virulence factors were classified and analyzed in addition to the conformational profiles of the virulent proteins involved in the resistance mechanisms. / M

In response to compatible rhizobia, leguminous plants develop unique plant organs, root nodules, in which rhizobia fix nitrogen into ammonia. During nodule invasion, the rhizobia gain access to newly divided cells, the nodule primordia, in the root inner cortex through plant-derived cellulose tubes called infection threads. Infection threads begin in curled root hairs and bring rhizobia into the root crossing several cell layers in the process. Ultimately the rhizobia are deposited within nodule primordium cells through a process resembling endocytosis. Plant host mechanisms underlying the formation and regulation of the invasion process are not understood. To identify and clone plant genes required for nodule invasion, recent efforts have focused on Medicago truncatula. In a collaborative effort the nodulation defect in the lin (lumpy infections) mutant was characterized. From an EMS-mutagenized population of M. truncatula, two non-allelic mutants nip (numerous infections with polyphenolics) and sli (sluggish infections) were identified with defects in nodule invasion. Infection threads were found to proliferate abnormally in the nip mutant nodules with only very rare deposition of rhizobia within plant host cells. nip nodules were found to accumulate polyphenolic compounds, indicative of a host defense response. Interestingly, nip was also found to have defective lateral root elongation suggesting that NIP has a role in both nodule and lateral root development. NIP was found to map at the upper arm of chromosome 1. In sli, infection threads were observed to bring rhizobia from infection threads to newly divided nodule primordium cells in the roots inner cortex. Polyphenolic accumulation in sli nodule/bumps was found. Lateral roots in sli were found to be clustered at the top of the root, indicating that sli like nip may be defective in lateral root development.

Familial Hypertrophic cardiomyopathy (HCM) causes ventricle walls to thicken and often leads to sudden death especially in adults. Mutations in the subfragment 2 (S2) of β-cardiac myosin are implicated in the genetic disorder. This S2 region is a coiled-coil rod region resulting from the dimeric form of myosin II. It has been proposed that an elastic quality allows normal S2 to absorb force during the powerstroke according to the sliding filament model. To test the flexibility of single molecules of S2 against levels of physiological force, the Gravitational Force Spectrometer (GFS) is being developed. This novel system employs a standard microscope on an equatorial mount that allows the spectrometer to be rotated freely in space. Stationary glass beads are attached to a microscope slide where the molecule is tethered between the stationary bead and a smaller mobile bead. The GFS is oriented so that the force of gravity can act on the mobile bead and so impart a small force to the tethered subfragment. Additionally, a video system in conjunction with ImageJ software makes a distance measurement of the molecule possible with a resolution of around 11 nm. The S2 can be stretched parallel or perpendicular to the coiled coil to elucidate different structural properties of the rod. This study is the first to show structural evidence that S2 in vertebrate skeletal myosin uncoils proportionally to physiological force loads. Because of this, the usefulness and promise of the novel GFS is highlighted, and the biological role of S2's flexibility can be directly commented on. If the dimer undergoes uncoiling at physiological force loads as shown, then it is reasonable to think that this might occur in nature in response to the stress of the powerstroke on a single molecule. This unwinding could be to absorb force as a mechanism to protect the muscle fiber.

Orientador: Maria Beatriz Puzzi / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T22:49:13Z (GMT). No. of bitstreams: 1 Souto_MariamPatriciaAuada_D.pdf: 7783740 bytes, checksum: ba776e2125f132647d619bed067b9da5 (MD5) Previous issue date: 2006 / Resumo: A síndrome de Sjögren-Larsson (SSL) é uma ictiose congênita com alterações neurológicas decorrentes da deficiência enzimática da enzima aldeído graxo desidrogenase (FALDH). Mutações no gene ALDH3A2 são responsáveis pela doença. A proposta foi estudar os aspectos clínicos, histológicos, estruturais e moleculares de dez pacientes com SSL. Métodos: Foram selecionados dez pacientes com SSL de quatro famílias distintas. Foi realizada ressonância magnética cerebral (RM) em oito pacientes e, em sete destes, também espectroscopia de prótons; os resultados foram comparados com a espectroscopia de sete voluntários sadios. Foram coletadas biópsias de pele de cada paciente e pele sã de 17 voluntários. Os espécimes foram corados com hematoxilina-eosina e imagens digitais foram adquiridas a partir do exame histológico de rotina; a luminância em escala de cinza foi analisada utilizando método quantitativo de análise de textura de imagem digital: transformada rápida de Fourier, calculada a partir dos valores do momento de inércia (função harmônica) das frequências espaciais nas direções vertical e horizontal da camada espinhosa da epiderme. Amostras de pele de nove pacientes com SSL também foram obtidas para cultura de fibroblastos utilizando método padrão para dosagem da enzima FALDH. Para a extração de DNA genômico foi coletado sangue total periférico de nove pacientes. Os fragmentos foram amplificados por PCR. Os resultados obtidos foram analisados e comparados com a seqüência normal de DNA. Resultados: A mutação encontrada foi a 1108-1G?C no intron 7/exon 8. Os pacientes exibiram diferenças na presença e intensidade dos sinais e sintomas da doença (prurido, retardo mental, espasticidade, fotofobia e alterações da retina). No exame de RM dos pacientes com SSL foi observada desmielinização da substância branca profunda; na espectroscopia, presença de pico anormal de lípídios. O estudo da textura da camada espinhosa da epiderme, sob a Transformada de Fourier, mostrou amplitudes de função harmônica mais largas no grupo com SSL na direção vertical e não na direção horizontal, correspondendo a núcleos e nucléolos maiores e ao espessamento focal da carioteca dos ceratinócitos. Conclusões: As manifestações clínicas e histológicas da SLS não são exclusivamente decorrentes da mutação c.1108-1G>C, mas também expressão de outras modificações genéticas e influências ambientais. A mutação descrita pode ser classificada como uma causa comum da doença no Brasil. Os achados de textura podem ser explicados pela maior síntese de DNA na epiderme na SSL, tendo sido descrita, também, uma produção mais rápida da camada córnea e uma renovação celular 3,5 vezes o normal / Abstract: Background: Sjögren-Larsson syndrome (SLS) is an autosomal recessive disorder characterized by ichthyosis, mental retardation, and spasticity. Various mutations in the ALDH3A2 gene that codes for fatty aldehyde dehydrogenase (FALDH) are responsible for the disease but the genotype-phenotype relationships are undefined. Objectives: The purpose of this study was to describe ten individuals with Sjögren-Larsson syndrome from four families representing the first cohort of Brazilian patients defined at the molecular level and investigate whether image analysis of routine H&E skin sections using fast Fourier transformation (FFT) could detect structural alterations in SLS. Patients and methods: A 5-mm punch biopsy for histologic analysis was obtained under local anesthesia from each patient. Similar biopsies were obtained from age- and anatomical site-matched from 17 controls. The samples were stained with haematoxylin and eosin and observed under a light microscope. Digital images of routine histologic sections were taken and their gray scale luminance was analyzed by FFT. The inertia values were determined for different ranges of the spatial frequencies in the vertical and horizontal directions. Skin biopsies were also obtained from nine patients with SLS for establishing fibroblast cultures using standard methods to determine FALDH enzyme activity. MR imaging was performed in eight patients. Singel-voxel proton MR spectra were acquired from cerebral white matter in seven patients with SLS and in seven controls. Total genomic DNA was extracted from peripheral blood of nine patients using standard methods. ALDH3A2 exons and their flanking sequences were amplified by PCR and sequenced. Results: All patients were homozygous for a unique c.1108-1G>C splice-site mutation and displayed a profound reduction in fatty aldehyde dehydrogenase enzyme activity. The patients exhibited a moderate-to-severe form of Sjögren-Larsson syndrome with respect to mental retardation, spastic diplegia and ichthyosis. Within this phenotypic context, patients varied, even within a family, in the presence of pruritus, thickness of the epidermal granular layer, retinal glistening white dots and photophobia. Brain MR imaging revealed retardation of myelination. Proton MR spectroscopy of white matter revealed abnormal high-lipid peak. The study of epidermal spinous layer texture by Fast Fourier Transform showed higher amplitudes in the vertical direction in SLS patients, corresponding to greater nuclei and nucleoli, higher number of nucleoli and focal thickening of keratinocytes¿ carioteca. Conclusion: We concluded that the latter clinical features are not strictly determined by the c.1108-1G>C mutation, but are subject to modification by other genetic/environmental factors. The c.1108-1G>C mutation may be classified as a common cause of Sjögren-Larsson syndrome in Brazil. The textural findings are in keeping with higher DNA synthesis and a 3.5x turn over that were demonstrated in SLS epidermis / Doutorado / Clinica Medica / Doutor em Clínica Médica