Orientador: Carmen Sílvia Bertuzzo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T19:39:30Z (GMT). No. of bitstreams: 1 Marcelino_AlineRobertaBariani_M.pdf: 1077026 bytes, checksum: b7315e705aba4125ea2a4e2574a78b6b (MD5) Previous issue date: 2013 / Resumo: A Fibrose Cística (FC) é uma doença autossômica recessiva causada por mutações no gene CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) que acarretam em defeito ou na ausência da proteína por ele sintetizada. A CFTR, produto deste gene é uma proteína canal, localizada na membrana apical das células, responsável pela condução de íons cloreto. As mutações levam à ausência da proteína CFTR ou a alteração qualitativa/quantitativa da proteína, que acarreta no desequilíbrio osmótico entre os meios intra e extracelular. Como consequência há a ocorrência de muco viscoso e de difícil excreção nos pulmões e obstrução dos ductos pancreáticos, afetando desta forma o sistema respiratório e digestório. São conhecidas mais de 1.900 mutações no gene CFTR, sendo que mesmo em pacientes com mutações iguais como a F508del - com alta prevalência na população brasileira - há divergência entre os fenótipos observados. Dessa forma, o genótipo CFTR parece não ser determinante na modulação da gravidade clínica, uma vez que, indivíduos com mesmo genótipo CFTR apresentam manifestações clínicas diferentes. Outros genes, diferentes do CFTR foram associados à gravidade clínica dos pacientes, revelando que os produtos por eles expressos exercem algum tipo de ação modificadora do fenótipo da FC. Tais genes foram denominados modificadores e atuam em fatores secundários relacionados à evolução do quadro clínico, como a articulação do sistema imune. Os genes COX2 e IFRD1, com ação importante no sistema imune e no recrutamento de células de defesa foram identificados como modificadores da FC em estudos prévios realizados em uma população diferente da brasileira. No presente estudo, os polimorfismos -765G>C e 8473T>C no gene COX2 e 57460C>T no gene IFRD1, candidatos a modificadores, foram identificados nos pacientes e um estudo de associação genótipo-fenótipo foi conduzido a fim de verificar a ação moduladora de tais polimorfismos nos pacientes estudados. Nenhuma associação foi encontrada, exceto para o íleo meconial (p=0,028 - em pacientes com duas mutações identificadas no gene CFTR pertencentes à classe I, II e III) e para a polipose nasal (p=0,022 - em pacientes sem considerar o genótipo CFTR) para o polimorfismo 8473T>C no gene COX2 / Abstract: Cystic fibrosis (CF) is an autosomal recessive disease caused by CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene mutations that lead to defective polypeptide or lack of the protein CFTR. The CFTR is a channel protein located in the apical cells membrane, responsible for chloride ions conductance. The mutations lead to an osmotic disequilibrium between intra and extracellular mediums, which causes viscous mucus production that is hard to be eliminated from lungs and pancreatic ducts, affecting, this way, respiratory and digestive systems. More than 1,900 CFTR different mutations are known, and even patients that carries identical mutations as F508del - the most common one in Brazilian population - shows a great discrepancy between the phenotypes that are observed. Thus, CFTR genotype seems not to be crucial in disease clinical course modulation, once different subjects carrying the same CFTR mutations reveal distinctive clinical manifestations. Genes besides CFTR were associated to CF patients clinical manifestation, revealing that the molecules they express have some kind of modifier activity in CF phenotype. Such genes were labeled as modifier genes and they act in secondary factors related to clinical course evolution as immune system response. The genes COX2 and IFRD1 have an important role in immune system and defense cell recruitment and they were identified as CF modifiers in previous studies that analyzed different population from the Brazilian one. In this current study, the polymorphisms -765G>C, 8473T>C and 57460C>T located in these genes were identified in our patients and association genotype-phenotype were carried out in order to verify the modulator activity of such variants in the studied casuistic. There was not found association, except for meconium ileus (p=0,028 - in patients with two CFTR mutations from class I, II and III) and for nasal polyposis (p=0,022 - in patients whose CFTR genotype was not considered) to 8473T>C polymorphism in COX2 gene / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Orientador: Laura Sterian Ward / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T14:43:40Z (GMT). No. of bitstreams: 1 Bufalo_NatassiaElena_D.pdf: 3667340 bytes, checksum: 4934d4d9652dfe1e6d10650634ac9866 (MD5) Previous issue date: 2012 / Resumo: Tanto a Doença de Graves (DG) como o Carcinoma Diferenciado da Tiroide (CDT) são patologias de etiologia multifatorial e envolvem uma interação entre meio ambiente e fatores genéticos de predisposição. Ambas apresentam nítida preferência pelo sexo feminino. Por isso, variações no metabolismo do estrógeno poderiam estar associadas a esta preferência, pois, seus metabólitos podem causar dano ao material genético. Os objetivos foram determinar a influência dos polimorfismos dos genes CYP17A1, HSD-17?1, CYP1A1, CYP1A2, CYP1B1, COMT e SULT1E1 no risco para a DG e para o CDT. Para tanto, foi estudado 282 pacientes com DG (234 mulheres e 48 homens; 39,80±11,69 anos), 292 pacientes com CDT (248 mulheres e 44 homens; 42,23±14,81 anos), comparados com 308 controles (246 mulheres e 62 homens; 36,86±12,95 anos). Para o estudo dos polimorfismos, utilizou-se a técnica TaqMan SNP Genotyping. Alterações polimórficas nos genes CYP17A1 (p=0,0421), CYP1A1 m1 (p=0,0328), CYP1A2*1F (p=0,0085), CYP1B1 códon 119 (p<0,0001) e CYP1B1 códon 432 (p=0,0059) aumentam a suscetibilidade ao CDT. Já as alterações polimórficas nos genes CYP1A1 m1 (p<0,0001), CYP1B1 códon 119 (p<0,0001) e CYP1B1 códon 432 (p=0,0208) aumentam a suscetibilidade à DG. Entre os pacientes com DG, mulheres heterozigotas para CYP1A1 m1 apresentam maior número de gestações (p=0,0071), paridade (p=0,0204) e abortos (p=0,0012), além de bócio mais pesado (gramas) (p=0,0082). Já entre os pacientes com CDT, mulheres heterozigotas para CYP17A1 apresentam maior número de abortos (p=0,0150). A herança heterozigota para o gene CYP1A2*F está relacionada com idade mais precoce (p=0,0073) para o surgimento do CDT. A herança do polimorfismo do gene CYP1B1 códon 119 está associada ao hábito tabagista (p=0,0269), uso de reposição hormonal (p=0,0197) e presença de menopausa (p=0,0317) para pacientes com CDT. Já para pacientes com DG, este polimorfismo se correlaciona com concentrações mais elevadas de T4livre (p=0,0409) e TRAb (p=0,0465). A herança em heterozigose do gene CYP1B1 códon 432 foi mais frequente em pacientes com carcinoma papilífero do que nos pacientes com carcinoma folicular (p=0,0243), além da herança em homozigose se correlacionar com maior idade (p=0,0204) e com sobrepeso (p=0,0392) em pacientes com DG. Mulheres com o genótipo homozigoto polimórfico para CYP1B1 códon 453 se correlacionam com uso de anticoncepcional (p=0,0332) no CDT. Maior número de gestações (p=0,0256) e de paridade (p=0,0141) se correlacionam com mulheres homozigotas para o gene HSD-17?1 na DG. Concluímos que os fatores exógenos e endógenos ligados aos hormônios sexuais possuem considerável variabilidade individual, devido aos polimorfismos da via de metabolização do estrógeno. As diferentes heranças polimórficas individuais, que são atribuídas aos polimorfismos dos genes codificadores de enzimas envolvidas na produção, metabolização e eliminação do estrógeno devem definir subpopulações de mulheres que são afetadas pela maior exposição aos estrógenos e aos seus metabólitos, os quais afetam o crescimento celular e podem induzir danos celulares carcinogênicos a ativar a resposta imune / Abstract: Graves' disease (GD) and Differentiated Thyroid Carcinoma (DTC) are multifactorial diseases and involved an interaction between environmental factors and genetic predisposition. Both diseases have preference for female. Therefore, variations in estrogen metabolism could be associated with that preference, because its metabolites can cause damage to genetic material. The objectives were to determine the influence of polymorphisms of genes CYP17A1, HSD-17?1, CYP1A1, CYP1A2, CYP1B1, COMT and SULT1E1 at the susceptibility for GD and DTC. We studied 282 patients with GD (234 females and 48 males, 39.80±11.69 years old), 292 patients with DTC (248 women and 44 men, 42.23±14.81 years old), compared with 308 controls (246 women and 62 men, 36.86±12.95 years old). TaqMan SNP genotyping technique was used to study the polymorphisms. Polymorphisms in CYP17A1 (p=0.0421), CYP1A1 m1 (p=0.0328), CYP1A2*1F (p=0.0085), CYP1B1 codon 119 (p<0.0001) and CYP1B1 codon 432 (p=0.0059) increases the susceptibility to DTC. The polymorphisms of CYP1A1 m1 (p<0.0001), CYP1B1 codon 119 (p<0.0001) and CYP1B1 codon 432 (p=0.0208) increases the susceptibility for GD. Women with GD have a higher number of pregnancies (p=0.0071), parity (p=0.0204), abortions (p=0.0012) and goiter heavier (grams) (p=0.0082) when heterozygous for CYP1A1 m1. Among patients with DTC, women heterozygous for CYP17A1 highest number of abortions (p=0.0150). The inheritance in heterozygous for the CYP1A2*F are correlated to younger age (p=0.0073) for the susceptibility to DTC. The inheritance of polymorphism of the CYP1B1 codon 119 gene is associated with smoking (p=0.0269), use of hormone replacement therapy (p = 0.0197) and menopausal status (p=0.0317) for patients with DTC. However, for patients with GD, this polymorphism correlated with higher concentrations of FT4 (p=0.0409) and TRAb (p=0.0465). The inheritance in heterozygous for CYP1B1 codon 432 gene was more frequent in patients with papillary carcinoma than in patients with follicular carcinoma (p=0.0243) and the homozygous inheritance correlate with older (p=0.0204) and overweight (p=0.0392) in patients with GD. Women with the inheritance for polymorphic homozygous for CYP1B1 codon 453 correlated with contraceptive use (p=0.0332) in the DTC. Increased number of pregnancies (p=0.0256) and parity (p=0.0141) correlated with women homozygous for the HSD -17?1 gene in GD. We conclude that exogenous and endogenous factors related to sex hormones have considerable individual variability due to polymorphisms of the estrogen metabolic pathway. The different polymorphic individual heritages, which are attributed to the polymorphisms of genes encoding enzymes involved in the production, metabolism and excretion of estrogen should define subpopulations of women who are affected by increased exposure to estrogens and their metabolites, which affect cell growth and can induce cellular damage carcinogens activate the immune response / Doutorado / Ciencias Basicas / Doutor em Clínica Médica

Os agentes infecciosos têm sido considerados iniciadores da desestabilização da placa de ateroma. Este mecanismo pode estar relacionado a uma intensificação do processo inflamatório através da interação dos receptores de membrana CD14 e TLR com os microorganismos. Para avaliar esta hipótese, estudou-se a participação da resposta inflamatória induzida por Chlamydophila pneumoniae (Cp) e Mycoplasma pneumoniae (Mp) em indivíduos com infarto agudo do miocárdio (IAM). Avaliou-se também, a possível associação entre polimorfismos dos genes CD14, TLR2, TLR4 e TNFA e a expressão dos genes IL6, TLR2, TLR4 e TNFA em leucócitos do sangue periférico, assim como a sua associação com o IAM. Para isso, foi realizado um estudo caso-controle constituído por pacientes com IAM e por indivíduos sem evidência de doença cardiovascular (grupo controle). As imunoglobulinas IgM e IgG séricas anti-Cp foram detectadas por imunofluorescência indireta. O DNA dos agentes infecciosos foi detectado no sangue periférico pela PCR em tempo real. A genotipagem dos polimorfismos TNFA -308G>A, IL6 -174G>C, CD14 -260C>T, TLR4 (Asp299Gli e Thr39911e) e TLR2 Arg753Gln e a quantificação relativa da expressão gênica nas células sanguíneas foram analisados pela PCR em tempo real. A porcentagem de positividade para DNA de Cp foi de 18,0% e 8,1% nos grupos IAM e controle (p=0,071), respectivamente, (p=0,071). Foram positivos para DNA de Mp, 5,0% e 11,2% dos indivíduos nos grupos IAM e controle, respectivamente (p=0,318). Sete indivíduos (7,1%) do grupo IAM tiveram títulos anti-Cp IgG positivos (1:512) e 3,9% dos indivíduos do grupo controle (p=0,718). A expressão do TLR4 foi significantemente menor no grupo IAM (0,00113±0,00102) comparado ao grupo controle (0,00144±0,000806; p=0,003). As frequências genotípicas e alelicas dos polimorfismos TNFA -308G>A, CD14 -260C>T, TLR4 (Asp299GIi e Thr39911e) e TLR2 Arg753Gln foram similares entre os grupos estudados (p>0,05) sugerindo que esses polimorfismos não estão associados com IAM nesta amostra populacional. No grupo IAM, houve associação entre o genótipo -260CT+TI CD14 com títulos IgG anti-Cp detectados na diluição 1:16 (p=0,042). Da mesma forma, o alelo A do polimorfismo -308G>A TNF-&#945; foi associado com títulos positivos de IgG anti-Cp na diluição 1:512 (p=0,0058). No grupo IAM, pacientes positivos para DNA de Cp tiveram maiores concentrações de fibrinogênio do que pacientes negativos para este agente infeccioso (541,8±161,5mg/dL e 450,5±196,8mg/dL, respectivamente; p=0.043). Os agentes infecciosos Chlamydophila pneumoniae e Mycoplasma pneumoniae não foram significantemente mais frequentes em indivíduos que tiveram infarto agudo do miocárdio em relação ao grupo controle, porém houve uma associação, no grupo IAM, entre positividade para DNA de C. pneumoniae e concentrações mais elevadas de fibrinogênio. / Atheroma plaque instability has been attributed to the presence of some infectious agents. This mechanism may be related with increased stimulus of inflammatory process through interactions of CD14 and TLR with infectious agents. In this present study, it was evaluated the association of the presence of Chlamydophila pneumoniae and Mycoplasma pneumonia with acute myocardial infarction (AMI). A case-control study was conducted with AMI patients and non-AMI individuais as controls. Immunoglobulin G (lgG) and IgM antibodies anti-Chlamydophila pneumoniae were detected by indirect immunifluorescent assay and the Cp DNA and Mp DNA were detected by real time PCR (RT-PCR) in peripheral blood cells. Using the same method, the individuals were genotyped and the gene expressions of TLR2, TLR4, IL-6 e TNF-&#945; were evaluated by RT-qPCR. In AMI patients, Cp DNA and Mp DNA were positive in 18,0% and 5,0% samples, respectively. In controls, 8,1% and 11,2% were positive for Cp DNA and Mp DNA, respectively. TLR4 expression was significantly decreased in AMI patients (0.00113±0.001 02) compared with controls (0.00144±0.000S06; p=0.003). The frequencies of -308G>A TNF-&#945;., -260C>T CD14, Asp299Gli TLR4, Thr39911e TLR4e Arg753Gln TLR2 SNPs in AMI group were similar to those found in controls. On the other hand, In AMI group, the -260CT+TT CD14 genotype was associated with anti-CP IgG antibody titer of 1/16. Likewise, the rare allele of -308G>A TNF-&#945; was associated with anti-CP IgG antibody titer of 1/16. Cp DNA positive patients had high concentration of fibrinogen when compared with negative patients. In conclusion, Cp DNA and Mp DNA positivity were not associated with AMI.

The Constraint Satisfaction Problem (CSP) is a fundamental NP-complete problem with many applications in artificial intelligence. This problem has enjoyed considerable scientific attention in the past decades due to its practical usefulness and the deep theoretical questions it relates to. However, there is a wide gap between practitioners, who develop solving techniques that are efficient for industrial instances but exponential in the worst case, and theorists who design sophisticated polynomial-time algorithms for restrictions of CSP defined by certain algebraic properties. In this thesis we attempt to bridge this gap by providing polynomial-time algorithms to test for membership in a selection of major tractable classes. Even if the instance does not belong to one of these classes, we investigate the possibility of decomposing efficiently a CSP instance into tractable subproblems through the lens of parameterized complexity. Finally, we propose a general framework to adapt the concept of kernelization, central to parameterized complexity but hitherto rarely used in practice, to the context of constraint reasoning. Preliminary experiments on this last contribution show promising results.

Abstract The cholesteryl ester transfer protein (CETP) is a plasma protein that transfers cholesteryl esters and triglycerides between plasma lipoproteins. Humans with a genetic CETP deficiency have high plasma high density lipopoprotein cholesterol (HDL-C) levels, whereas the CETP transgene lowers plasma HDL-C levels in mice. The role of CETP in the development of atherosclerosis is unclear due to the controversial results of many human and animal studies. The present research was designed to investigate the CETP gene as a candidate gene in the regulation of plasma HDL-C levels and the development of atherosclerosis in humans. The CETP gene was screened for mutations and polymorphisms associated with these traits in a well-characterized, homogenous population sample of 515 men and women and in a sample of 115 men with low HDL-C levels and coronary heart disease (CHD). Using polymerase chain reaction and single-strand conformation polymorphism analysis (PCR-SSCP), three polymorphic sites were found (A373P, I405V, R451Q) in the exons of the CETP gene, one in intron 9 and one in the 3'untranslated region of the CETP gene. In addition, the genotypes of a functional promoter polymorphism were determined. The V405 allele was associated with lower plasma CETP activity in the whole population sample, and the Q451 allele and the P373 allele were associated with higher plasma CETP activity in men, whereas the genotypes of the promoter polymorphism were not significantly associated with plasma CETP activity. The genotypes of the CETP gene explained about 20 % of the variation of plasma CETP activity in men. The CETP gene polymorphisms were found to be a minor regulator of plasma HDL-C levels, and these associations interacted with alcohol consumption, sex and triglyceride levels. The strongest association was detected between the promoter polymorphism and HDL-C levels in women. The variation at the CETP gene locus explained about 8 % of the variation in plasma HDL-C levels in women, but less than 1 % in men. CETP gene polymorphisms (A373P, I405V and R451Q) were associated with carotid intima-media thickness, explaining about 6 % of the variation in men and 4 % in women. However, none of the polymorphisms were associated significantly with the CHD risk. In conclusion, the CETP gene was found to be polymorphic and a minor regulator of plasma HDL-C levels and the development of atherosclerosis.

Abstract Successful pregnancy is characterized by a shift toward Th2 type immune response and suppression of adaptive immune responses to ensure acceptance of the semi-allogenic fetal graft. Also the innate immune system plays a major role during pregnancy. Recurrent miscarriage is defined as three or more consecutive pregnancy losses. About 1% of all women will suffer recurrent miscarriage. The causes of recurrent miscarriage remain unexplained in half (50%) of the cases. Susceptibility to recurrent miscarriage is probably mediated by Th1 type immune response with pronounced expression and secretion of pro-inflammatory cytokines (e.g. TNFα and IFNγ) paralleled with decreased production of anti-inflammatory cytokines (e.g. IL-10). Factors that regulate immune response during pregnancy include hormonal factors (e.g. hCG and progesterone). Immunogenetic factors also contribute to this regulation. Several functionally important polymorphisms in various immunomodulatory genes have been identified during recent years. Some of these polymorphisms may be important in regulating the Th1/Th2 balance during pregnancy. Putative immune dysregulation caused by these polymorphisms has been researched intensively. Conflicting results have been published about associations between several of these polymorphisms and recurrent miscarriage. In this study, HLA-G (exon 2 and 3), IL-10 (-1082A/G), IL-1RA (intron 2 VNTR) and CD14 (-159C/T) polymorphisms were studied in 38 Finnish women with RM. All of these polymorphisms have been associated with altered gene expression. Distribution of HLA-G*I, II, III and IV were 0.577, 0.375, 0 and 0.048 respectively in the studied Finnish population. According to the present classification the G*I allele group mostly consists of the allele 010101, while G*II covers the combination of 010102, 010401 and 0105N, as well as some other rare alleles. There were no associations between recurrent miscarriage and the HLA-G, IL-10 and CD14 polymorphisms. However, in IL-1RA polymorphism, the rare IL1RN*3 allele was increased in women with recurrent miscarriage. It is not known, if this particular allele is associated with differences in IL-1RA or IL-1 production. Although the study population was small, it may be supposed that quantitative differences in the production of single immunomodulatory molecules due to normal genetic variation may not be grossly harmful to the fetal allograft. This indicates the robustness and flexibility of the reproduction system. For survival, it is essential that minor variations are tolerated. Thus, large-scale studies focusing on the effect of a pro-inflammatory genetic profile based on the presence of several pro/anti-inflammatory genetic markers are needed to discover if immunogenetic factors predispose women to recurrent miscarriage.

Abstract Respiratory distress syndrome (RDS) is a disease characterized by neonatal respiratory failure. It is principally caused by a deficiency of pulmonary surfactant, which is a lipoprotein mixture essential for reducing surface tension at the air-liquid interface of the alveolus. Prematurity is the major risk factor predisposing to RDS. Several pieces of evidence suggest the role of genetic factors in the susceptibility to this multifactorial disease. The present study was performed to determine whether polymorphisms of the surfactant protein SP-A1, SP-A2 and SP-B genes associate with RDS and to evaluate the relative contributions of genetic and environmental factors to the disease etiology. Allelic associations between the candidate genes and RDS were investigated using a matched and unmatched case-control and family-based study design. Disease concordance in monozygotic vs. dizygotic twin pairs was determined to measure the impact of heredity in RDS. SP-A and SP-B genes were shown to play a significant role in susceptibility to RDS. In very premature singleton infants born before 32 weeks of gestation, SP-A1 and SP-A2 allelic variations were associated with RDS, whereas the SP-B gene showed no direct association. Instead, the association between the high-risk (6A2, 1A0) or low-risk (6A3, 1A1/1A2) SP-A alleles and RDS was dependent on SP-B Ile131Thr variation, being restricted to a subset of infants carrying the homozygous genotype Thr/Thr. No allelic associations were evident in premature infants born after 32 weeks of gestation. RDS concordance was not significantly higher in monozygotic than in dizygotic twin pairs, implying a non-genetic disease etiology. However, the present study suggests that the concordance difference underestimates the extent of heredity. Twin pregnancies include intrauterine environmental factors that complicate the interpretation of the hereditary impact. SP-B Ile131Thr variation was associated with RDS in the first-born, but not in the second-born twins. The present results indicate that susceptibility to RDS is highly heterogeneous, involving complex environmental and genetic interactions. The degree of prematurity, singleton vs. multiple pregnancy, and birth order in a multiple birth are environmental confounders that determine disease subgroups. Genetic variations in the SP-A and SP-B genes account for part of the genetic component of RDS.

Abstract Neurogenic factors are known to be important in the development of hypertension. Our current knowledge of the role of autonomic nervous system in chronic hypertension is, however, limited. The purpose of the present study was to evaluate the possible abnormalities in heart rate variability (HRV) and baroreflex sensitivity (BRS) in patients with long standing systemic hypertension compared to subjects without evidence of cardiovascular disease. A particular aim was also to examine whether genetic variation in the renin-angiotensin-aldosterone system (RAS) genes have an influence on cardiovascular autonomic regulation. Case-control studies were carried out on a total of 280 normotensive and 214 hypertensive subjects drawn from a random middle-aged population originally recruited for an epidemiologic study of cardiovascular risk factors. The possible association of BRS with the genetic polymorphisms of renin-angiotensin-aldosterone system genes was studied in a cross-sectional study of 315 healthy controls. Genetic associations were also tested in a younger, independent population sample of 66 subjects. The effects of intensified antihypertensive treatment on autonomic cardiovascular control were evaluated in 33 hypertensive patients with poor blood pressure control. Wide interindividual variation in both HRV and BRS was observed in normotensive as well as hypertensive subjects. Overall HRV and autonomic responses to a change in body posture were blunted in long-standing hypertension. Decreased HRV was mainly related to elevated blood pressure and obesity. For the first time in a population-based study, it was confirmed that BRS is impaired in patients with long-standing hypertension despite adequate antihypertensive treatment. In contrast to HRV, BRS was reduced in hypertensive subjects also after adjustment for blood pressure and obesity. BRS also varied widely both between healthy and hypertensive individuals. The wide interindividual variation in the markers of autonomic cardiovascular regulation was not, however, completely explained by demographic variables, cardiovascular risk factors or lifestyle, suggesting a genetic component contributing to HRV and BRS. The polymorphism in the aldosterone synthase (CYP11B2) gene was found to strongly associate with BRS in two independent random populations of apparently healthy subjects. The association was even stronger in the younger population. On the basis of the observations made in the older population, it seems possible that women are protected against the effect of age and blood pressure on BRS and tend to maintain the genomic influence longer. Intensified antihypertensive combination therapy improved blood pressure control and caused regression of left ventricular hypertrophy, and resulted in significant improvements of HRV and BRS. The present study shows that HRV and BRS are altered in long-standing systemic hypertension. Together with age, blood pressure and obesity, genetic factors seem to be important determinants of BRS. However, abnormal autonomic cardiovascular regulation does not seem to be an irreversible phenomenon, but can be partly restored by modern combination antihypertensive therapy.

The majority of human proteins are post-translationally modified by covalent addition of one or more complex oligosaccharides (glycans). Alterations in glycosylation processing are associated with numerous diseases and glycans are attracting increasing attention both as disease biomarkers and as targets for novel therapeutic approaches. Using a recently developed high performance liquid chromatography (HPLC) method for high-throughput glycan analysis, genome-wide association studies (GWAS) of 33 directly measured and 13 derived N-glycan features were performed in 3533 individuals from four European isolated populations. Polymorphisms at six loci were found to show genome-wide significant association with plasma concentrations of N-glycans. Several of these gene products have well characterised roles in glycosylation, however, SLC9A9 and HNF1A were two of the novel findings. Subsequent work performed by collaborators found HNF1A to be a “master regulator” of genes involved in the fucosylation of plasma N-glycans. Additionally, this work led to the discovery that N-glycans could act as biomarkers to discriminate HNF1A-MODY from type 1 and type 2 diabetes mellitus (T1D, T2D) patients. After the success of the total plasma N-glycan GWAS, it was thought that stronger and more biologically interpretable associations may be found from the investigation of N-glycans isolated from a single protein. Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic networks that govern IgG glycosylation, N-linked IgG glycans were quantitated using ultra performance liquid chromatography (UPLC) in 2247 individuals from the same four European populations from the previous study. GWAS of the 77 N-glycan measures identified 15 loci with a p-value < 5x10-08. Four loci contained genes encoding glycosyltransferases, while the remaining loci contained genes that have not previously been implicated in protein glycosylation. However, most have been associated with autoimmune and inflammatory conditions and/or hematological cancers. Several high-throughput methods for the analysis of N-glycans have been developed in the past few years but thorough validation and standardization of these methods is required before significant resources are invested in large-scale studies. To this end, four of these methods were compared, UPLC, multiplexed capillary gel electrophoresis (xCGE), and two mass spectrometric (MS) methods, for quantitative profiling of N-glycosylation of plasma IgG in a subset of 1201 individuals recruited from two of the cohorts used in the previous GWAS studies. A “minimal” dataset was compiled of N-glycan structures able to be measured by all four methods. To evaluate their accuracy, correlations were calculated for each structure in the minimal dataset. Additionally, GWAS was performed to test if the same associations would be observed across methodologies. Chromatographic methods with either fluorescent or MS-detection yielded slightly stronger associations than MS-only and xCGE, but at the expense of lower levels of throughput. Advantages and disadvantages of each method were identified, which should aid in the selection of the most appropriate method for future studies. This work shows that it is possible to identify new loci that control glycosylation of plasma proteins using GWAS and the potential of N-glycans for biomarker development. It also provides some guidelines for methodology selection for future studies of N-glycans.

Magister Scientiae - MSc / The aim of this study was to investigate the forensic usefulness of various Y-chromosome short tandem repeat loci among South African sub-populations. Three different sets of Y-chromosome short tandem repeat loci were chosen for investigation. / South Africa