Made available in DSpace on 2014-06-11T19:27:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2004Bitstream added on 2014-06-13T20:36:39Z : No. of bitstreams: 1 silva_gn_me_botfm.pdf: 467036 bytes, checksum: 3b9fa526eae390b7ae86bf47c5bcf731 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O linfoma de Hodgkin (LH) tem características clínicas e anátomo-patológicas distintas dos linfomas não-Hodgkin. Seu componente neoplásico, as células células de Hodgkin/Reed-Sternberg (H-RS), corresponde a cerca de 2% do tumor. As células H-RS apresentam imunofenótipo peculiar e sua origem ainda é objeto de estudo. O LH é classificado em LH clássico (que inclui os subtipos celularidade mista, esclerose nodular, depleção linfocítica e LH rico em linfócitos) e LH predominância linfocítica nodular. A associação do vírus de Epstein-Barr (EBV) com LH é conhecida e acredita-se que este vírus desempenha importante papel no desenvolvimento de parcela significativa dos casos de LH. No LH, o acúmulo de células reativas como linfócitos, plasmócitos, eosinófilos, histiócitos e fibroblastos ocorre em resposta a citocinas secretadas pelas células H-RS. A interleucina 10 (IL-10), importante citocina antiinflamatória da resposta imunitária, tem sido encontrada em grande quantidade em indivíduos com LH. É possível que essa elevada expressão de IL-10 esteja vinculada a determinados polimorfismos de nucleotídeos simples (SNP) na seqüência promotora do gene da IL-10, que podem se associar a características anátomoclínicas do LH. O presente trabalho avaliou a freqüência dos polimorfismos da IL-10 nas posições promotoras -1082, -819/-592 e estudou a correlação destes polimorfismos em LH com subtipos histológicos, infecção pelo EBV, faixa etária e, em alguns casos, estadiamento clínico da doença. Os resultados demonstram que os diferentes fenótipos para produção de IL-10, genótipos na posição -1082 e genótipos nas posições -592/-819 não têm relação com subtipos do LH e idade dos pacientes. Por outro lado, verificou-se que o genótipo GG na posição -1082 e a combinação de haplótipos GCC/GCC para alta... / Clinical and pathologic features of Hodgkin’s lymphoma (HL) reflect an abnormal immune response, which is in part due to the elaboration of a variety of cytokines. It was reported that interleukin 10 (IL -10), which may be produced by the malignant Hodgkin/Reed-Sternberg (H-RS) cells, is an important prognostic factor in HL, and it could play a role in the pathogenesis of this neoplasm. It is well established that genetic factors affect protein expression and function. In this regard, polymorphisms in the promoter region of IL-10 gene may cause different phenotypes for IL- 10 synthesis and activity. Three dimorphic single nucleotide polymorphisms (SNPs) have been identified at positions -1082, -819 and -519 within the IL-10 promoter region (SNPs/IL-10). These polymorphisms are in close proximity to several transcription factors binding-sites, and may interfere with IL-10 gene transcription. The aim of this study was to evaluate whether is there a particular distribution of SNPs at positions -1082, -819 and -519 in the IL-10 gene promoter in patients with HL, as well as to access the differences in the SNPs/IL10 frequency regarding HL subtype, patient age, EBV infection status, and clinical staging of the disease. For these purposes, sixty-five cases of HL and fifty cases of reactive follicular lymphoid hyperplasia (RFLH) were evaluated for SNPs/IL-10 by polymerase chain reaction amplification plus restriction fragment length polymorphism analysis (PCR-RFLP). Compared to HL EBV-negative cases, in HL EBV-positive cases it was observed a significant increase in the frequency of GG genotype at position -1082 of IL-10 gene promoter region, which is associated to high level of IL -10 production. No differences were observed in the frequency of different SNPs/IL-10 concerning patient age, HL subtype, and clinical stage of the disease. These results... (Complete abstract, click electronic address below)

Submitted in fulfillment of the requirements for the degree of Master of Technology: Health Sciences, Durban University of Technology, Durban, South Africa, 2013. / Several genes are associated with an increased susceptibility to respiratory diseases, including asthma, which may be exacerbated by ambient air pollution. These genes include the Tumour Necrosis Factor alpha (TNFα) gene and the Cluster of Differentiation 14 (CD14) gene A total of 104 schoolchildren from seven primary schools in a heavily industrialized region of south Durban participated in the study. For the purpose of this study, DNA was extracted from whole blood using the GENTRA Puregene kit. Genotyping for the TNF-308α G/A polymorphism was conducted using restriction fragment length polymorphism (RFLP) polymerase chain reaction (PCR). The CD14 (-159) C/T genotype was determined using real time PCR and Taqman probes (Applied Biosystems). Multiple logistic models and Pearson’s chi-squared tests were used to evaluate the association between any asthma, BHR, atopy, persistent asthma and genotype. Covariate-adjusted generalised estimating equations (Martin et al.) with lags of 1-5 days were used to evaluate genotype effect modification of exposure-response. The TNF-308α variant A allele was quite common in the population, it was detected in more than forty percent of the population and with an allelic frequency of 0.24. Similarly almost 38% of the population carried the variant CD14 (-159) T allele, with an allelic frequency of 0.24. TNF-308α G/A and CD14 (-159) C/T polymorphisms were not associated significantly with asthma, and its related respiratory phenotypes. In addition there was no association detected between any of the gene polymorphisms and the levels of their respective cytokine proteins. Increased TNFα levels were associated with persistent asthma. On the other hand lower sCD14 levels were associated with atopy in children. There was a significant relationship between TNF- α levels and acute asthma (p=0.03) and sCD14 levels and atopy (p=0.04) GEE models showed that the TNF- 308-α A allele carriers had a greater deterioration of lung function post pollution exposure to SO2 (intraday variability FEV1 readings lag 2) β= 2.62, CI (0.51, 4.71) p= 0.02 and p (interaction=0.03). There was a statistically significant gene environment interaction with NO in individuals who were carriers of the TNF- A allele (Nadir of PF readings lag 2: β= -12.3, CI (-22.09, -2.51), p=0.01 p (interaction) =0.03.and 5 day average β= - 42.83, CI (-70.11,-15.55), p≤0.005 and p (interaction) =0.01). With analysis of the CD14 gene polymorphism gene environment interaction, adverse effects of SO2 were limited to individuals carrying the C allele of this polymorphism, β= - 1.50, CI (-0.36, 3.37), p=0.01, p (interaction) =0.01. Carriers of the T allele seemed to have a protective effect with NO2 and NO exposure. Intraday variability of FEV1 improved 5 days post exposure to NO2 β= -4.02, CI (-6.52,- 1.53), p=0, p (interact) =0.05. There was also improvement five days post exposure to NO β= -9.42, CI (-12.45, -6.03), p= p≤0.005, p (interact) ≤0.005 There was no association of co-inheritance of the 2 gene polymorphisms, CD14 (-159) C/T and TNF-308α G/A, and protein expression or respiratory phenotype. The GEE model results were consistent with modification of air pollutant-pulmonary function relationships by proinflammatory cytokine associated genotypes. Results indicate that genetic susceptibility combined with exposure to pollutants causes adverse respiratory effects. This study supports the importance of further investigation on these and other genotype variants involved in inflammation and respiratory linked phenotypes in larger cohorts. / M

Orientador: Carmen Silvia Bertuzzo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T05:02:17Z (GMT). No. of bitstreams: 1 Faria_IsabelCristinaJacintode_D.pdf: 2884064 bytes, checksum: ef1354c3f8b573c2a7b4dc0801a2f254 (MD5) Previous issue date: 2007 / Resumo: A Asma é uma doença inflamatória crônica de vias aéreas, na qual várias células têm papel fundamental, tais como os eosinófilos, mastócitos e linfócitos T. O processo inflamatório está associado à hiper-responsividade brônquica a uma variedade de estímulos. A asma é uma doença genética complexa e multifatorial que não acompanha os padrões monogênicos de hereditariedade. Estudos de ligação sugerem que múltiplos genes estão envolvidos na patogenia dessa doença. O objetivo desse trabalho é verificar em uma amostra de pacientes com asma leve, moderada e grave, uma possível associação entre os polimorfismos dos genes TGF-'beta'1, CD14, IL-4, IL-4Ra, ADAM33 e a gravidade da asma. A análise do polimorfismo T869C do gene TGF-'beta'1 foi realizada pela técnica de PCR + ARMS. Os outros polimorfismos C-509T do gene TGF-'beta'1, C-159T do gene CD14, C-590T da IL-4, Ile50Val da IL-4Ra , S2 do gene ADAM33 foram detectados por PCR + enzima de restrição. Em relação ao polimorfismo T869C (TGF-?1), em nossa amostra observou-se uma associação entre o genótipo CC e os pacientes portadores de asma grave. Quanto ao polimorfismo C-509T(TGF-'beta'1), nenhuma associação foi encontrada. Quando se comparou a distribuição da frequência genotípica do polimorfismo C-159T(CD14) na asma grave com o grupo controle foi observado um resultado significativo com genótipos TT e CT. Não encontramos associação entre C-590T (IL4) e asma. Houve significância entre o genótipo Val/Val (IL-4Ra) e a asma leve. O heterozigoto GC do polimorfismo S_2 (ADAM33) com a asma leve e grave. O homozigoto CC com a asma leve. Nossos resultados indicam que os polimorfismos T869C(TGF-?1) e C-159T(CD14) podem estar envolvidos na modulação da gravidade da asma / Abstract: Asthma is a chronic inflammatory disease affecting air pathways, in which several cells, such as eosinophils, mastocytes and T lymphocytes, play fundamental roles. The inflammatory process is associated with bronchial hyperresponsiveness to a variety of stimulus types. Asthma is a complex and multifactorial genetic disease that does not follow the monogenic line of hereditary patterns. Linkage studies suggest that multiple genes are involved in its pathogenesis. The objective of this work was to verify, in a sample of patients with mild, moderate and acute asthma, a possible association between TGF-'beta'1, CD14, IL-4, IL-4Ra, ADAM33 gene polymorphisms and asthma severity. The T869C polymorphism of the TGF-'beta'1 gene was analyzed using the ARMS-PCR technique. The other polymorphisms C-509T of the TGF-'beta'1 gene, C-159T of the CD14 gene, C-590T of the IL-4 gene, Ile50Val of the IL-4Ra gene , S2 of the ADAM33 gene were be detected by PCR + restriction analysis. The T869C polymorphism (TGF-'beta'1) in our sample showed an association between the CC genotype and the patients who presented acute asthma. No association was found concerning the C-509T (TGF-'beta'1) polymorphism. Comparing the genotype distribution of the C-159T (CD14) polymorphism on the control group of acute asthma, a significant result with TT and CT genotypes was observed. There was no association between C-590T (IL4) and asthma. There was a significant association between Val/Val (IL-4Ra) genotype and mild asthma. The GC heterozygote of the S_2 polymorphism (ADAM33) with acute and mild asthma. The CC homozygote and mild asthma. The results indicated that T869C (TGF-'beta'1) and C-159T (CD14) polymorphisms can modulate asthma severity / Doutorado / Genetica Medica / Mestre em Ciências Médicas

Orientador: Carmen Silvia Bertuzzo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T08:44:23Z (GMT). No. of bitstreams: 1 Paiva_AnaCarolinaZimianide_M.pdf: 1547364 bytes, checksum: 2eadb25c166c68e374144663ac431576 (MD5) Previous issue date: 2007 / Resumo: A asma atópica é uma doença com prevalência média na população brasileira de 20%. Estima-se que 60% dos casos de asma sejam intermitentes ou persistentes leves, 25% a 30% moderados e 5% a 10% graves. O receptor beta-2-adrenérgico, quando ativado, provoca o relaxamento da musculatura lisa das vias aéreas. Os polimorfismos Arg -> Gly 16 (46 A->G) e Gln -> Glu27 (79C->G) do gene ADRB2 provocam uma mudança nos aminoácidos situados ao lado do sítio ligante do receptor. Estes receptores não são responsáveis pela presença da asma, mas agem como moduladores de gravidade da doença. Desse modo, o estudo dos polimorfismos do gene ADRB2 é muito importante para prever a resposta ao uso de broncodilatadores beta-adrenérgicos no tratamento da asma atópica. Os objetivos do trabalho foram: 1) Determinar o genótipo de pacientes asmáticos e voluntários sadios quanto aos polimorfismos Gln27, Glu27, Arg16 e Gly16. 2) Verificar se existe correlação entre o genótipo estudado e a gravidade da asma. Foram genotipados 87 pacientes com asma atópica, de acordo com o critério GINA, e 141 voluntários sadios. Após a extração de DNA do sangue periférico foi efetuada PCR-alelo específica (ARMS). No método de ARMS-PCR é utilizado um primer comum a todas as reações e outro alelo específico,isto é, apenas uma base nitrogenada é diferente. Além disso é utilizado um par de primers como controle interno da reação. Os resultados encontrados foram analisados através do teste de qui-quadrado. Amostra de pacientes quanto ao códon 16 não está em equilíbrio de Hardy-Weinberg, entretanto a amostra controle está. No códon 27, ambas as amostras estão em desequilíbrio. A diferença na freqüência dos alelos no códon 16 entre os pacientes e o grupo controle foi significativa, enquanto no códon 27 não. Na comparação entre os diferentes genótipos no códon 16 dos asmáticos e do grupo controle foi observada diferença significativa, modtrando que o alelo Arg 16 pode estar envolvido na etiologia da asma. Comparando-se os pacientes com asma grave e grupo controle, foi encontrada uma diferença significativa, assim como quando comparados os pacientes com moderada, também sendo encontrada diferença significativa quando comparados os pacientes leve. No códon 27 verificou-se diferença significativa entre os genótipos dos pacientes e voluntários sadios. Comparando-se os pacientes com asma grave e grupo controle foi encontrado uma diferença significativa, assim como quando comparados os pacientes com moderada, também sendo encontrada diferença significativa quando comparados os pacientes leve. Na verificação dos genótipos do grupo controle e do total de pacientes temos uma diferença significativa. Na comparação entre os grupos de asmáticos só tivemos diferença significativa entre o grupo de asma grave e leve. Na comparação de asma grave e moderada não tivemos diferença significativa assim como moderada e leve. Os genótipo homozigotos Arg 16 parece estar relacionada à presença da asma e homozigotos Gln 27 parecem estar associados com a gravidade da asma / Abstract: The atopic asthma is an illness with 20% of average prevalence in the Brazilian population. It?s estimated that 60% of the asthma cases are intermittent or persistent light, 25% to 30% moderate and 5% to 10% serious. The receptor beta-2-adrenergic, when activated, causes the relaxation of the aerial ways' smooth muscle. The polymorphisms Arg -> Gly 16 (46 A->G) and Gln -> Glu27 (79C->G) from the ADRB2 gene cause a change on amino acids situated beside the receptor?s linking place. These receptors are not responsible for the asthma?s occurrence, but they act as modulators of the illness? severity. In this way, the study of the ADRB2 gene's polymorphisms is very important to foresee the reply of using beta-adrenergic bronchodilators in the treatment of the atopic asthma. The objectives of the proceeding were: 1) Determinate the genotype of healthy and asthmatic voluntary patients in respect of the polymorphisms Gln27, Glu27, Arg16 and Gly16. 2) Verify if there is a correlation between the studied genotype and asthma?s severity. 87 patients with atopic asthma were genotyped, in accordance with the healthy criterion GINA, and 141 volunteers. After the extraction of peripheral blood's DNA, an specific PCR-allele (ARMS) was made. In the ARMS-PCR method, it is used a common primer for all the reactions and another specific allele, that means, only one base is different. Moreover, a pair of primers is used as internal control of the reaction. The achieved results were analyzed through the qui-square test. The patients' sample, according to codon 16, is not in Hardy-Weinberg equilibrium, while the control sample is. In codon 27, both of the samples are in disequilibrium. The difference in the frequency of alleles in codon 16 between the patients and the control group was significant, while in codon 27 not. In the comparison between the different genotypes in codon 16 of the asthmatic and the control group, significant difference was observed, showing that the Arg 16 allele can be involved in the asthma's etiology. Comparing the patients with serious asthma and the control group, significant difference was found, as well as when compared the patients with moderate, also being found significant difference when compared the patients with light. In codon 27, significant difference was verified between the genotypes of patients and healthy volunteers. Verifying the genotypes of the control group and the total of patients, we have a significant difference. In the comparison between the asthmatic groups, we only had significant difference between the serious asthma group and the light one. In the comparison between serious and moderate asthma, we didn?t have significant difference, as well as between moderate and light. The genotype homozygote Arg 16 seems to be related to the asthma's occurrence and homozygote Gln 27 seems to be associated with the asthma?s severity / Mestrado / Mestre em Farmacologia

Os tumores do sistema nervoso central representam aproximadamente 2% de todos os tipos de cânceres. Embora a incidência dos tumores do SNC seja pequena, comparada com outras neoplasias, estes tumores estão entre as mais graves malignidades humanas, pois afetam o órgão responsável pela coordenação e integração de todas as atividades orgânicas. Os gliomas são os tumores mais comuns do SNC. Apesar do progresso marcante na caracterização da patogênese molecular dos gliomas, esses tumores permanecem incuráveis e, na maioria dos casos, refratários aos tratamentos, devido à sua heterogeneidade molecular. O aparecimento desses tumores ocorrem a partir do acúmulo de alterações genéticas nas células. Para entender o mecanismo molecular de formação e progressão tumoral é indispensável identificar os genes que acumulam essas alterações. Um polimorfismo de base única (SNP Single Nucleotide Polymorphism) é geralmente definido como uma substituição estável de apenas uma base na molécula de DNA com frequência maior que 1%, em pelo menos uma população Os SNPs são reconhecidos como importantes ferramentas na genética humana e médica e têm sido amplamente utilizados nos estudos de associação genética de várias doenças complexas, como por exemplo: distúrbios cardiovasculares, psiquiátricos e autoimunes, obesidade, osteoporose, diabetes e câncer Sendo assim, este trabalho teve como objetivo analisar polimorfismos entre populações caso e controle na intenção de identificar associações destes genótipos na suscetibilidade aos tumores. A técnica utilizada para a análise de polimorfismos foi de PCR-RFLP onde observamos diferenças nas distribuições genotípicas entre pacientes e controles nos SNPs EGF+61, GSTP-1Ile 105 Val, XRCC1 Arg 194 Trp, Pro 206 Pro, Arg 280 His, Arg 399 Gln, Gln 632 Gln, XRCC2 Arg 188 His, XRCC3 Thr 241 Met e XRCC4 G1394T, onde as variantes EGF G61, Trp194, Val105, Pro206, His280, Gln632, His188, Met241 e XRCC4 T1394 foram observados com maior freqüência entre os portadores de gliomas. Dessa forma, estas variantes podem ser fatores de susceptibilidade para o desenvolvimento dos tumores. / The Central nervous system tumors represent about 2% of all cancers. Although the incidence of CNS tumors is small compared with other cancers, these tumors are among the most serious human malignancies, because they affect the body responsible for coordination and integration of all organic activities. Gliomas are the most common tumors of the CNS. Despite remarkable progress in characterizing the molecular pathogenesis of gliomas, these tumors remain incurable and, in most cases, refractory to treatment, due to its molecular heterogeneity. The appearance of these tumors occurs from the accumulation of genetic changes in cells. To understand the molecular mechanism of tumor formation and progression is essential to identify genes that accumulate these changes. A single base polymorphism (SNP Single Nucleotide Polymorphism) is generally defined as a stable replacement of only one base in the DNA molecule often greater than 1% in at least one population SNPs are recognized as important tools in human genetics and medical and have been widely used in genetic association studies of various complex diseases, such as: cardiovascular, psychiatric and autoimmune diseases, obesity, osteoporosis, diabetes and cancer. Thereby, the objective of this study was to analyze the polymorphisms between cases and control the intention to identify associations of these genotypes in susceptibility to tumors. The technique used for the analysis of polymorphisms were PCR-RFLP where we observe differences in genotype between patients and controls in the EGF +61, GSTP-1 Ile105Val, XRCC1 Arg 194 Trp, Pro 206 Pro, Arg 280 His, Arg 399 Gln, Gln 632 Gln, XRCC2 Arg 188 His, XRCC3 Thr 241 Met and XRCC4 G1394T, where the variants EGF G61, Trp194, val105, Pro206, His280, Gln632, His188, Met241 and XRCC4 T1394 were observed more frequently among patients with gliomas. Thus, these variants can be important factors of susceptibility to the tumor development.

Arsenic is the most pervasive environmental toxic substance. As a consequence of its ubiquity, nearly every organism has genes for resistance to inorganic arsenic. In one project I examined the role of glutaredoxin 2 (Grx2) in reduction of arsenate to arsenite. I demonstrated that Grx2 has both glutaredoxin thiol transfer activity and glutathione S-transferase (GST) activity. In a second project investigated arsenic resistance in a microbiome organism. I discovered that the human gut microflora B. vulgatus has eight continuous genes in its genome and these genes form an arsenical-inducible transcriptional unit. In two other projects I investigated the properties of two As(III) S-adenosylmethionine (SAM) methyltransferase (ArsM in microbes and AS3MT in animals). In this project we demonstrate that most fungal species have ArsM orthologs with only three conserved cysteine residues, and AfArsM from Aspergillus fumigatus methylates only MAs(III) and not As(III). For human, arsenic methylation process is thought to be protective from acute high-level arsenic exposure. However, with long term low-level exposure, hAS3MT is thought to produce intracellular methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)), which are considerably more toxic than inorganic As(III) and may contribute to arsenic-related diseases. Several single nucleotide polymorphisms (SNPs) in putative regulatory elements of the hAS3MT gene have been shown to be protective. In contrast, three previously identified exonic SNPs (R173W, M287T and T306I) may be deleterious. I identified five additional intragenic variants in hAS3MT (H51R, C61W, I136T, W203C and R251H). I purified the eight polymorphic hAS3MT proteins and characterized their enzymatic properties. Each enzyme had low methylation activity through decreased affinity for substrate, lower overall rates of catalysis and/or lower stability. I propose that amino acid substitutions in hAS3MT with decreased catalytic activity lead to detrimental responses to environmental arsenic and may increase the risk of arsenic-related diseases.

HLA (Human Leukocyte Antigen) molecules provide a framework for T-cell recognition of antigenic peptides and thus play an important role in the immune system and defence against pathogens. HLA molecules are encoded for by genes on the short arm of chromosome six in the human genome, a region of over 4000 kilo bases (kb) known as the human major histocompatibility complex (MHC) or HLA complex. According to structural and functional characteristics, the genes of this region have been classified into three families, namely classes ƒ¹, ƒ¹ƒ¹ and ƒ¹ƒ¹ƒ¹. The HLA genes are highly polymorphic and because of this characteristic, it increases the functional range of recognition of different antigens and contributes to immunological specificity. Previous studies on population groups such as Indians and Cambodians, have identified an association between HLA polymorphisms and susceptibility to TB. A large number of different population groups with diverse gene pools reside in South Africa, of which the Cape Coloured population is one. This anthropologically distinct population group¡¦s diverse gene pool originated from founder individuals of the colonizing population, which came from various nations and cultural backgrounds, including Europe, Africa (such as Khoi, San Xhosa, Sotho and East African populations), Madagascar and the Far East. Unusual MHC allele frequencies and haplotypes have been identified in the Cape Coloured population. The Cape Coloureds are highly susceptible to TB and reside in the Western Cape, which has a TB incidence rate that is higher than that of any other province in South Africa. The recently admixed Coloured population is a valuable candidate population for the identification of genes/mutations underlying complex diseases. This study focussed on polymorphisms of class ƒ¹ƒ¹ genes, specifically HLA-DRB, and their possible contribution to disease susceptibility in populations of South Africa. Two questions have been formulated: 1) What knowledge can we gain from the current literature on HLA variants in the different population groups of South Africa and disease susceptibility? 2) Is there an association between alleles of the most polymorphic class ƒ¹ƒ¹ MHC gene, HLA-DRB, and susceptibility or resistance to TB in the Cape Coloured population? These two primary questions have been addressed by: 1) Reviewing the literature concerning HLA alleles in diverse population groups in South Africa and their contribution to disease susceptibility. Chapter 2 addresses this objective and is written in the format of a review article to facilitate its future publication, 2) Conducting a TB case-control study, typing HLA-DRB in Cape Coloured individuals residing in the Western Cape to investigate the possible association between TB susceptibility or resistance and specific DRB alleles. The HLA-DRB1, DRB3 and DRB4 typing by means of PCR-SSP (polymerase chain reaction ¡V sequence specific primers) was done on DNA isolated from 106 TB patients and 107 controls from the Cape Coloured population. The results obtained for this experimental investigation is presented (also in publication format) in Chapter 3. Summary of main findings: 1) The literature overview of publications describing HLA related disease association in the different population groups in South Africa (Chapter 2), revealed that unique alleles contributing to susceptibility of various diseases have been identified in some South African populations. The large number of ethnic groups in South Africa and unique populations such as the Cape Coloureds provide a genetic resource, which has the potential to be utilized for candidate gene hunting. 2) A weak association between susceptibility for TB and HLA-DRB1*0301-0302 (DR3) and HLA-DRB3*0101-0301 (DR52) exists in the Cape Coloured population (Chapter 3). Since the South African population consists of a large number of different population groups with diverse gene pools, a unique opportunity to study disease predispositions among specific population groups with diverse genetic make-up is presented. With these different populations, often residing in a common environment, the interplay of environmental and genetic factors in disease development could be studied. For example, HLA typing of these populations could clarify the extent to which inter-population HLA variation contributes towards disease susceptibility. The identification of certain HLA-DRB alleles potentially contributing to TB susceptibility, could lead to an understanding of the differential factors involved in disease susceptibility and in turn lead to the understanding of the fundamental mechanisms involved. This could result in therapeutic approaches and treatments that will be advantageous to the population concerned. / Prof. L. Bornman

>Magister Scientiae - MSc / Autism or autism spectrum disorder (ASD) is a very diverse neurological disorder that manifests specifically in children and infants between the ages of two to three years of age. An individual suffering is deemed as autistic and individuals suffering would be classed under the banner of ASD. It is observed that sufferers have impairment in their social and interactive skills. It has both genetic and environmental factors that contribute to its diversity and although the primary cause of autism is still unclear, scientist are investigating both factors. In this study we aimed to investigate the molecular genetics of autism in the South African (SA) population. This was done in two parts, a genetic association study and afunctional genomics (proteomic study). An association study of the 2 single nucleotide polymorphisms (SNPs) of the Cadherin 10 type II gene (CDH10) (rs4307059 and rs4327572) was investigated in the SA healthy and autistic population. The proteomic approach was used to determine the differential expression of genes of the healthy population and compared to the autistic population of African descent. In both parts of the project, objectives were achieved. The SNPs were successfully genotyped however no association was determined for autism in the SA population. The urine protein profiles with 1 dimensional (1D) and 2dimensional (2D) Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis (SDSPAGE)generated in this study has revealed the following proteins, Uromodulin, Vitelline membrane outer layer protein homologue, kinninogen-1, Alpha-1-Antitrypsin, Ig Kappa chain region C, and CD59 glycoprotein that require further investigation. The results indicated that six of the identified proteins were expressed in both groups but were found to be either quantitatively or statistically significant. However, a statistically significant difference was observed in the expression of one protein (Uromodulin) which was observed to be expressed in the healthy group but absent in the experimental group. However further investigation is required validation of these findings.

Introduction: The two subtypes of Antineutrophil Cytoplasmic autoantibody associated systemic vasculitis (AASV) cANCA and pANCA associated vasculitis are the commonest causes of rapidly progressive glomerulonephritis. In spite of recent advances in the pathogenesis and development of new therapeutic agents, long term outcomes are still poor with five year mortality of 25%. There are epidemiological, histological, clinical and outcome related differences between these two conditions. This strongly suggests that there must be differences between genetic factors and pathogenesis of these two conditions. There was also a perception amongst the clinicians that AASV is more common in the Greater Manchester area. Hence in this study I calculated the incidence of pauciimmune glomerulonephritis in Greater Manchester and analysed the genetic differences between cANCA and pANCA associated vasculitis. Methods: Five year incidence of pauciimmune glomerulonephritis was calculated in Greater Manchester between 1/1/1999 to 31/12/2003. I recruited 147 patients with ANCA associated vasculitis. Clinical data was collected. I studied single nucleotide polymorphisms (SNPs) of tumour necrosis factor alpha(TNFα), interleukin 8 (IL-8), transforming growth factor beta (TGFβ), platelet endothelial cell adhesion molecule 1 (PECAM-1), Chemokine (CC motif) ligand-5 C chemokine (CCL-5), interleukin 10 (IL-10) and interleukin 18 (IL-18) genes and compared the frequencies of genotypes and alleles in patients with cANCA and pANCA associated small vessel vasculitis and healthy volunteers. I also studied circulating cytokine profiles of IL-10 and IL-18. Results of IL-18 SNPs were validated in AASV cohort from South-East USA. Further I studied the gene expression patterns of active and remission state of AASV and metabolomics profile of cANCA and pANCA positive patients during active and remission state of vasculitis. Clinical outcomes (relapses and renal survival) were correlated with the genotypes. Results: I found a significantly higher incidence (9.8/million population) of pauciimmune glomerulonephritis in Greater Manchester compared to the previously published data from UK and USA (2.73 to 4.6/million). Renal function at the time of diagnosis predicted the long term renal survival. I also found a novel genetic association of increased frequency of high producer IL-18 SNPs 113T, 127C and 137G in pANCA positive patients compared to normal volunteers (p=0.04) and cANCA positive patients (trend- p=0.08). This was associated with increased levels of circulating IL-18 levels in these patients. This association was further confirmed in an independent cohort of AASV from USA. I also found a lower frequency of low producer GG genotype of IL-10 -1082 SNP (p=0.05) and this was associated with lower levels of circulating IL-10 in these patients compared to pANCA positive patients. I found significant difference in the metabolomics profiles of cANCA andpANCA positive patients. In paired plasma samples, levels of some metabolites were high during remission state compared to active vasculitis. Conclusions: These findings strongly support the hypothesis that there is an increased incidence of pauciimmune glomerulonephritis in Greater Manchester. There are genetic differences in cANCA and pANCA positive patients which may explain the different observed outcomes. Genome wide association study would strengthen these findings and should guide the vasculitis community to reclassify, assess and perhaps treat these two conditions separately.

Indiana University-Purdue University Indianapolis (IUPUI) / Vincristine is metabolized by the cytochrome P450 3A subfamily of enzymes possibly including CYP3A5, a genetically polymorphic enzyme. The contribution of CYP3A5 to the metabolism of vincristine was quantified by various in vitro models: cDNA-expressed enzymes, human liver microsomes, and human hepatocytes. With these models, the major CYP metabolite of vincristine, M1, was identified and extensively characterized. The rates of M1 formation in the cDNA-expressed enzyme models were at least 7-fold higher with CYP3A5 than CYP3A4; approximately 90% of the hepatic metabolism was predicted to be CYP3A5-mediated. For human liver microsomes with high CYP3A5 expression, the CYP3A5 contribution was substantial, approximately 80%. Human hepatocytes with at least one CYP3A5*1 allele also metabolized vincristine, albeit at a slower rate (10-fold) than human liver microsomes. The CYP3A5 low-expressing hepatocytes did not metabolize vincristine. We conclude that for high CYP3A5 expressers, the majority of the CYP metabolism is mediated by CYP3A5. By in vitro/in vivo scaling with microsomes, the hepatic clearances of high CYP3A5 expressers are predicted to have a 5-fold higher hepatic clearance than low expressers. However, the role of metabolism in the systemic clearance of vincristine is unknown. To study the disposition of vincristine in vivo, a sensitive and selective LC/MS/MS assay was validated for the quantification of vincristine and M1 quantification in human plasma. Vincristine and M1 were identified and quantified in select pediatric plasma and urine samples. For future large-scale clinical studies, the vincristine and M1 concentrations in plasma will be quantified to understand the role of CYP3A5 genotype in vincristine pharmacokinetics. For patients that are CYP3A5 high expressers, the systemic clearance of vincristine may be higher than that of low CYP3A5 expressers. Thus, CYP3A5 genotype may be an important determinant of inter-individual variability in clinical outcomes.