The aim of the thesis – to investigate the role of the inflammatory bowel disease associated genetic variants in a subset of Crohn’s disease and ulcerative colitis patients from Lithuania and Latvia and to test the relation of genetic markers to disease phenotype. The following objectives were accomplished: the associations of the inflammatory bowel disease associated single nucleotide polymorphisms in the subset of Crohn’s disease and ulcerative colitis patients were determined; the associations of the single nucleotide polymorphisms with the phenotype of the inflammatory bowel disease were evaluated; the interactions of single nucleotide polymorphisms (SNP-SNP) and their association with inflammatory bowel disease were determined; the significance of the combinations of disease associated single nucleotide polymorphisms for diagnosis of inflammatory bowel disease was evaluated. TaqMan and SNPlex genotyping methods were used in this work. The statistical data analysis consisted of: statistical study power and data quality evaluations; single marker case-control association analysis using χ2 or Fisher's exact tests, Breslow-Day test, Cochran-Mantel-Haenzsel test and post-hoc Bonferroni correction; genotype–phenotype association analysis using χ2 test and post-hoc Bonferroni correction; SNP-SNP epistasis for case-control sample using logistic regression test and post-hoc Bonferroni correction; In sicilo prediction of gene interactive network; genetic risk profile construction... [to full text] / Disertacijos tikslas – ištirti su uždegiminėmis žarnyno ligomis siejamų ge¬netinių žymenų reikšmę Krono liga bei opiniu kolitu sergantiems Lietuvos ir Latvijos ligoniams bei nustatyti žymenų ryšį su ligos fenotipu. Tikslui pasiekti, įgyvendinti šie uždaviniai: ištirta su uždegiminėmis žarnyno ligomis siejamų vieno nukleotido polimorfizmų sąsaja su Krono liga bei opiniu kolitu; nustatyta vieno nukleotido polimorfizmų sąsaja su uždegiminių žarny¬no ligų fenotipu; ištirtos vieno nukleotido polimorfizmų tarpusavio epistazinės sąvei¬kos (VNP-VNP) bei jų ryšys su uždegiminėmis žarnyno ligomis; nustatytas su liga siejamų vieno nukleotido polimorfizmų derinių reikšmingumas uždegiminių žarnyno ligų diagnostikai. Darbe taikyti SNPlex ir TaqMan genotipavimo metodai. Statistinę duomenų analizę sudarė: studijos galios bei duomenų kokybės įvertinimas; atvejo-kontrolės genų sąsajos tyrimas, taikant χ2 arba Fišerio kriterijus, Breslow-Day testą, Cochran-Mantel-Haenzsel testą bei post-hoc Bonferroni kriterijų; genų sąsajų su Krono ligos ir opinio kolito fenotipu tyrimai, naudojant χ2 bei post-hoc Bonferroni kriterijų; VNP-VNP epistazinės sąveikos įvertinimas, taikant logistinės regresijos testą bei post-hoc Bonferroni kriterijų; genų tarpusavio sąveikos tinklo in silico tyrimas; genetinės rizikos profilio sudarymas, naudojant logistinę regresinę analizę.

The genes that regulate pubertal timing in the general population are not well understood. Recently, genome-wide association studies have demonstrated that genetic variants near LIN28B associate with variation in pubertal timing in humans. To investigate where within the hypothalamic-pituitary-ovarian (HPO) axis Lin28b, and its homologue Lin28a, regulate pubertal timing, expression of these genes was assessed across the pubertal transition. The finding that Lin28a/b expression decreases only in the ovary suggests that the Lin28 pathway may exert its regulatory effects with respect to puberty in the ovary. Another aim of this thesis was to examine the effect of estrogen on Lin28b expression in immortalized GnRH neuronal cells, but the data remains equivocal and detailed future studies are needed to make definitive conclusions. The ovarian expression data lay the foundation for further studies using conditional knockout mice to verify the importance of the tissue and age specific developmental pattern that was identified.

The genes that regulate pubertal timing in the general population are not well understood. Recently, genome-wide association studies have demonstrated that genetic variants near LIN28B associate with variation in pubertal timing in humans. To investigate where within the hypothalamic-pituitary-ovarian (HPO) axis Lin28b, and its homologue Lin28a, regulate pubertal timing, expression of these genes was assessed across the pubertal transition. The finding that Lin28a/b expression decreases only in the ovary suggests that the Lin28 pathway may exert its regulatory effects with respect to puberty in the ovary. Another aim of this thesis was to examine the effect of estrogen on Lin28b expression in immortalized GnRH neuronal cells, but the data remains equivocal and detailed future studies are needed to make definitive conclusions. The ovarian expression data lay the foundation for further studies using conditional knockout mice to verify the importance of the tissue and age specific developmental pattern that was identified.

[Truncated abstract] The low density lipoprotein receptor-related protein (LRP), a member of the low-density lipoprotein (LDL) receptor gene family is involved in numerous biological processes including lipoprotein metabolism. This thesis concerns investigations into some aspects of LRP metabolism/regulation and possible roles in coronary artery disease (CAD). Specific aims were: to investigate the association between polymorphisms in the LRP gene and in its associated protein, the lipoprotein receptor-associated protein (RAP), with the risk of CAD; to extensively examine the influence of the LRP exon 22 C200T polymorphism on lipid metabolism; to develop and characterise assays for the mRNA expression of LRP and 2 other genes relevant to lipid metabolism, the LDL receptor (LDLR), and HMG CoA reductase (HMGCR); and finally, to apply the latter techniques to studies on the influence of genetic variation in LRP, and dietary and drug interventions, on LRP, LDLR and HMGCR mRNA expression in nucleated blood cells from healthy human subjects. Six hundred CAD subjects and 700 similarly aged controls were genotyped for 8 LRP gene polymorphisms as well as for the RAP V311M polymorphism. ... In the final phase of my studies, I examined the influence of 4 weeks therapy with a cholesterol lowering drug, an HMGCR inhibitor, atorvastatin (20mg daily), on the mRNA expression of LDLR, LRP and HMGCR in human nucleated blood cells. Twelve normal Caucasian male subjects aged 49 ? 5 (SD) years were studied. Plasma total cholesterol and LDL-C decreased by averages of 29 % and 41 % after the 4 week period. This was accompanied by an elevation in LDLR mRNA expression by approximately 30 35 %. In contrast, there was no significant effect on LRP and HMGCR mRNA expression. In conclusion, the original findings in this thesis included: demonstration of a strong influence of the LRP exon 22 C200T polymorphism on coronary artery disease and LDLR expression, but without a clear effect on fasting or postprandial lipid levels; data on the biological variation in LDLR and LRP gene expression in nucleated blood cells from normal subjects; the influence of an oral fat load on the expression viii of these genes, finding that LDLR was significantly depressed; and finally, the observation that statin therapy upregulated LDLR in nucleated blood cells.

Η ενδοθηλίνη-1 (ΕΤ-1) είναι ένα ισχυρό αγγειοσυσπαστικό και βρογχοσυσπαστικό μόριο το οποίο παρουσιάζει και προφλεγμονώδεις ιδιότητες. Η ικανότητά του να ελκύει φλεγμονώδη κύτταρα στην εστία παραγωγής της, να προκαλεί την παραγωγή μορίων προσκόλλησης στην επιφάνειά τους αλλά και κυτταροκινών αποτελεί απόδειξη της δράσης αυτής. Από την άλλη οι κυτταροκίνες είναι σε θέση να επάγουν την σύνθεση και την έκκριση αυτού του μορίου, δημιουργώντας έτσι ένα μοριακό φαύλο κύκλο ενίσχυσης της φλεγμονής των αεραγωγών που λαμβάνει χώρα στη Χρόνια Αποφρακτική Πνευμονοπάθεια (ΧΑΠ). Με το τρόπο αυτό η φλεγμονή συνεχίζει να υφίσταται ακόμα και μετά την διακοπή του ερεθιστικού παράγοντα που προκάλεσε την έκλυσή της όπως είναι π.χ. το κάπνισμα. Έτσι, οι λειτουργικοί πολυμορφισμοί που αυξάνουν την παραγωγή της ΕΤ-1 δυνατό να αυξάνουν και το κίνδυνο ανάπτυξης ΧΑΠ. Υλικά-μέθοδοι: Σε αυτή την προοπτική μελέτη ερευνήθηκε η επίδραση στην αναπνευστική λειτουργία δυο λειτουργικών πολυμορφισμών του γονιδίου της ΕΤ-1 σε ένα πληθυσμό 190 καπνιστών (95 υγιείς καπνιστές και 95 καπνιστές που νοσούσαν από ΧΑΠ). Οι δυο πολυμορφισμοί αφορούσαν μια ένθεση αδενίνης στο 5’-άκρο στη θέση +138 (εξώνιο 1, 138/ex1ins/delA) και την αντικατάσταση μιας γουανίνης με θυμίνη στη θέση +5665 (εξώνιο 5) που αλλάζει το αμινοξύ λυσίνη της θέσης 198 σε ασπαραγίνη (Lys198Asn). Στα άτομα αυτά διενεργήθηκε λειτουργικός έλεγχος της αναπνοής σε ετήσια βάση για συνολικό χρονικό διάστημα τριών ετών. Αποτελέσματα: Η μέση ετήσια μείωση στον δυναμικά εκπνεόμενο όγκο στο πρώτο δευτερόλεπτο (ΔFEV1) ήταν η παράμετρος που μελετήθηκε. Αυτή ήταν μεγαλύτερη για όσους έφεραν το μεταλλαγμένο γόνο 138/ex1ins/delA σε σχέση με τα άτομα που έφεραν το φυσιολογικό αλληλόμορφο. Οι ετεροζυγώτες για το μεταλλαγμένο αλληλόμορφο που ανήκαν στην ομάδα των καπνιστών δίχως ΧΑΠ παρουσίαζαν μια ΔFEV1 μεγαλύτερη κατά 19,4 ml σε σχέση με τα άτομα που ήταν ομόζυγα για το φυσιολογικό αλληλόμορφο (p=0.004). Η αντίστοιχη διαφορά για τους ετεροζυγώτες του μεταλλαγμένου αλληλόμορφου που ανήκαν στην ομάδα των καπνιστών που νοσούσαν από ΧΑΠ ήταν 11,15 ml (p=0.003). Αντιθέτως, όσοι έφεραν τον πολυμορφισμό Lys198Asn παρουσίαζαν μια μικρότερη ΔFEV1 σε σχέση με τα άτομα που έφεραν το φυσιολογικό αλληλόμορφο. Έτσι, οι ετεροζυγώτες για το μεταλλαγμένο αλληλόμορφο που ανήκαν στην ομάδα των καπνιστών δίχως ΧΑΠ παρουσίαζαν μια ΔFEV1 μεγαλύτερη κατά 11,24 ml (p<0.001) ενώ η αντίστοιχη διαφορά για τους ετεροζυγώτες του μεταλλαγμένου αλληλόμορφου της ομάδα των καπνιστών που νοσούσαν από ΧΑΠ ήταν 11,42 ml (p=0.002). Τα παραπάνω συνηγορούν υπέρ μιας προστατευτικής δράσης του πολυμορφισμού Lys198Asn στην αναπνευστική λειτουργία. Συμπεράσματα: Τα δεδομένα αυτής της μελέτης δείχνουν πως τόσο η ΕΤ-1 όσο και οι λειτουργικοί πολυμορφισμοί του γονιδίου της δύνανται να ενέχονται στο τελικό φαινότυπο της ΧΑΠ και στη σοβαρότητα αυτής. / Background: Endothelin-1 (ET-1) is a potent vasoconstrictor and bronchoconstrictor but it has been shown to have also proinflammatory properties. Its ability to attract inflammatory cells in its site of production, upregulates the synthesis of adhesion molecules and stimulates the release of cytokines. The fact that cytokines have the ability to induce its synthesis and release, creates a dynamic loop for self-preservation and augmentation of the airway inflammation in COPD, even after the ceasing of the noxious stimulus i.e. cigarette smoke. Therefore, functional polymorphisms that may lead to increased levels of ET-1 may also cause an increased susceptibility to COPD development. Materials and Methods: We analyzed the longitudinal effect on lung function of two ET-1 gene polymorphisms in a population of 190 smokers (95 non-COPD and 95 COPD smokers). The two polymorphisms involved an insertion polymorphism (+138 adenine insertion 3A/4A, 138bp downstream from the transcription start site, exon 1) and a single nucleotide transversion polymorphism on exon 5 (G/T, Lys198Asn). A total of 190 subjects were enrolled in the study for each polymorphism and were followed for 3 years by annual spirometry sessions. Results: The adjusted annual decline of forced expiratory volume in 1 second (dFEV1) was greater for those having at least one copy of the mutated gene ins/delA compared to those with the wild type allele both in the non-COPD smokers group (mean difference in dFEV1 of 19.4 ml/year, p=0.004) and COPD smokers (mean difference in dFEV1 of 11.15 ml/year, p=0.003). On the contrary, those heterozygous for the Lys198Asn polymorphism were found to have a slower decline in FEV1 compared to those homozygous for the wild type allele. The non-COPD smokers group had a gain-in-loss of 11,24 ml/year (p<0.001) while the COPD-smokers group had a slower decline of 11,42 ml/year (p=0.002). Those homozygous for the polymorphisms examined show an even greater deviation from those with the wild type allele but due to the small number comprising their group, the results don’t have enough statistical power. Though, they still show the trend of the effect the polymorphisms have on annual FEV1 decline. Conclusions: The present data shows that ET-1 and its functional polymorphisms may be implicated in COPD phenotype and severity.

Thesis (MSc (Genetics))—University of Stellenbosch, 2007. / No obvious explanations exist for the development of abnormal involuntary movements (AIM), but several hypotheses have been proposed for tardive dyskinesia (TD) development. Since TD seems to have a genetic basis, several genetic variants have been investigated in TD development in various populations. Few studies have focused on African populations. This study focused on genetic variants (previously investigated in other populations) and the development and severity of AIM and TD in a Xhosa schizophrenia population. Genotype and allele frequencies determined were compared to those described in the literature for other populations. Following a report of an association between Ala-9Val and schizophrenia in a Turkish population, this study subsequently investigated this association in the Xhosa population. MnSOD Ala-9Val was genotyped using HEX-SSCP analysis and the DRD3 Ser9Gly variant was genotyped using restriction enzyme digestion by MscI. Genotyping was followed by statistical comparisons of the various groups, as well as association analyses between the variant and schizophrenia (only for MnSOD), AIM, or TD development and severity. The groups included a Xhosa schizophrenia group, a subgroup of the Xhosa schizophrenia group that had AIM (AIM+) and did not have AIM (AIM-), a subgroup of the AIM+ group that had TD (TD+), and a healthy Xhosa control group. A possible interaction between Ala-9Val and Ser9Gly in the development of AIM and TD was also investigated. Lastly, it was attempted to genotype CYP2D6*4, CYP2D6*10 and CYP2D6*17 using various PCR methods followed by restriction enzyme analysis. MnSOD Ala-9Val genotype and allele frequencies were similar to those of the Turkish population, but differed to those of the Asian populations. No association between Ala-9Val and the development and severity of schizophrenia was found. However, a relationship between genotype and AIM or TD development was observed, as well as an association between TD severity and Ala- 9Val genotype. DRD3 Ser9Gly genotype and allele frequencies were similar to those of the African American population, but differed from other populations. No significant association between Ser9Gly and the development and severity of AIM or TD was detected, nor was an interactive effect between Ala-9Val and Ser9Gly in AIM or TD development observed. The genotyping of CYP2D6 proved difficult and these variants could therefore not be analysed. The CYP2D6*4 genotype and allele frequencies that could be determined from some samples, were similar to the frequencies described previously for African populations. While we did not find an association between Ser9Gly in TD or AIM development and severity, nor an interaction between Ala-9Val and Ser9Gly, we did observe a relationship between Ala-9Val and AIM or TD development and TD severity. The effect of this variant is probably small and other variants, specifically those in genes involved in free radical removal should be investigated in combination with Ala-9Val. With regard to CYP2D6 it is suggested that high-throughput genotyping methods (e.g. microarray technology) should be used in the future. This will enable simultaneous genotyping of several variants and can be used in various populations. This study is the first of its kind by focusing on the unique South African Xhosa population and TD or AIM development.

Constraint satisfaction, graphs and algebras Jakub Bulín Abstract This thesis consists of three papers in the area of algebraic approach to the constraint satisfaction problem. In the first paper, a joint work with Deli'c, Jackson and Niven, we study the reduction of fixed template CSPs to digraphs. We construct, for every relational structure A, a digraph D(A) such that CSP(A) and CSP(D(A)) are logspace equivalent and most rele- vant properties carry over from A to D(A). As a consequence, the algebraic conjectures characterizing CSPs solvable in P, NL and L are equivalent to their restrictions to digraphs. In the second paper we prove that, given a core relational structure A of bounded width and B ⊆ A, it is decidable whether B is an absorbing subuniverse of the algebra of polymorphisms of A. As a by-product, we show that Jónsson absorption coincides with the usual absorption in this case. In the third paper we establish, using modern algebraic tools (e.g. absorption theory and pointing operations), the CSP dichotomy conjecture for so-called special oriented trees and in particular prove that all tractable core special trees have bounded width. Keywords: constraint satisfaction problem, algebra of polymorphisms, absorbing subuniverse, bounded width, oriented tree.

The presented dissertation consists of both a quantitative part and a qualitative part. The quantitative part deals with the relationship between 5-HTTLPR S/L, MAOA H/L, and COMT Val158Met polymorphisms, the stress reaction of new-born infants after a heel stick blood draw (measured by determining salivary cortisol at three time points) and temperament assessed at age three months by Rothbarth's Infant Behavior Questionnaire-Revised in a sample of 84 infants. Observed polymorphisms were related both to the course of the stress reaction and to temperament. The short allele of serotonin transporter polymorphism was connected to higher scores in the secondary scale of Negative Affect and lower scores in the secondary scale of Attention/Regulation. Homozygotes for the more active allele of MAOA polymorphism (HH) had the lowest scores in Negative Affect compared to both of the remaining groups, they also had higher scores in the secondary scale of Extraversion and Attention/Regulation and a greater decrease of cortisol in comparison to HL heterozygotes. The presence of low-active L allele predisposed their carriers to higher scores in Negative Affect and lower scores in Attention/Regulation. LL homozygotes had the highest increase of cortisol after a heel stick blood draw. The Met allele of COMT Val158Met polymorphism was connected to higher Extraversion and Attention/Regulation and a greater cortisol decrease. It was possible to predict all three secondary scales of IBQ-R from the stress reaction after the heel stick blood draw. Negative Affect was predicted by a higher increase and a lower decrease of cortisol. Extraversion and Attention/Regulation were predicted by a greater cortisol decrease. The magnitude of cortisol decrease partially mediated the influence of COMT Val158Met polymorphism on Extraversion. The qualitative part of the dissertation is a multi-casuistic study of six couples parenting infants with difficult temperaments. It is based on semi-structured interviews that were analysed in accordance with qualitative procedures. The most difficult infant displays to manage were unsoothable crying in the first six months and early sleeplessness and a later escalation of sleeping problems. Mothers were esentially not able to gain control over the amount of crying, but some of them managed to influence their experience to achieve a greater acceptance of it. To do this, it was necessary for them to eliminate their feelings of failure in the parental role. The parents' biggest dilemma concerning their infants' sleeping problems was whether to use the "cry it out" strategy or not to manage them. For some parents parenting a difficult infant was an opportunity to re-evaluate their approach to parenting and the parental role, significantly broadening the concept of both. Caring for a difficult infant significantly strained the marital relationship; four couples experienced marital crisis during the care of their child. The father's involvement in infant care seemed very important in this respect. Insufficient involvement led to dissatisfaction in the mother, the way the mother communicated her demands further influenced the marital relationship. Particular behaviour that the mother understood as the father's involvement in infant care emerged.

A pubarca precoce (PP) é definida como o desenvolvimento de pêlos pubianos antes dos 8 anos de idade em meninas e 9 anos de idade em meninos. Embora a PP não interfira diretamente com os eventos da puberdade, algumas evidências sugerem que estas meninas tenham maior risco para desenvolver, mais tarde, a Síndrome dos Ovários Policísticos (PCOS). A 17ß-hidroxiesteróide desidrogenase tipo 5 (17ßHSD5) é a principal responsável pela conversão de androstenediona em testosterona. Variações no gene que codifica para essa enzima, em especial os polimorfismos de nucleotídeo único (SNPs), podem estar relacionados com hiperandrogenismo e PCOS. A vitamina D, além dos efeitos sobre metabolismo ósseo, parece modular outras ações extra-esqueléticas, incluindo secreção e sensibilidade tecidual à insulina. A Vitamina D vem sendo associada com resistência insulínica e variantes do gene do receptor da vitamina D (VDR), vem sendo estudadas em populações de risco como no diabetes. No entanto, pouco se sabe sobre o envolvimento destes polimorfismos na PP. Os objetivos do presente trabalho foram: Avaliar os níveis de 25-hidroxivitamina D; determinar a frequência dos polimorfismos FokI, BsmI, ApaI e TaqI do gene do VDR e do SNP-71AG do gene da 17ßHSD5; verificar se existe associação entre esses polimorfismos com variáveis antropométricas, metabólicas e hormonais em uma amostra de pacientes com PP e controles do sul do Brasil. Foram arroladas 36 meninas com PP e 197 controles saudáveis. As genotipagens foram realizadas por PCR em tempo real para os SNPs -71AG, BsmI e FokI e por PCR-RFLP para os SNPs ApaI e TaqI. O SNP -71 AG do gene da 17ßHSD5 apresentou distribuição genotípica de 52,4% AA, 39,1% AG e 8,6% GG, sendo a frequência dos alelos A:G de 0,72:0,28. Analisando os dois grupos, verificamos uma maior freqüência do alelo variante (G) no grupo de meninas com PP quando comparadas aos controles (0,37 e 0,26, respectivamente), no entanto sem diferença estatística (p=0,054); não foram verificadas associações do polimorfismo com os dados clínicos e hormonais. As meninas com PP apresentaram níveis séricos de 25(OH)D inferiores aos das meninas controles (18,08±8,32 versus 21,27±7,03; p=0,032). Na análise dos polimorfismos, observou-se que o genótipo polimórfico GG do SNP ApaI TG, apresentou uma frequência maior em PP (30,6%) do que nas controles (16,2%) (Odds Ratio: 2,269; 95% Intervalo de Confiança: 1,015 – 5,076; p=0,042). Este genótipo foi também associado com níveis mais baixos de estradiol (35,30 (14,80 – 50,48) versus 12,22 (6,49 – 23,69); p=0,030) e testosterona total (0,52 (0,39 – 0,84) versus 0,20 (0,11 – 0,47); p=0,009) nas meninas com PP, mas não foi associado com os níveis de 25(OH)D. Por outro lado, verificou-se associação entre a presença dos polimorfismos TaqI TC (genótipo TC+CC) e BsmI GA (genótipo GA+AA) e níveis séricos de 25(OH)D mais elevados no grupo de meninas saudáveis (19,86±7,16 versus 22,55±6,69, p=0,007; 19,53±6,94 versus 22,88±6,76, p=0,001, respectivamente). Em conclusão, os dados deste estudo indicam que: 1) houve maior freqüência do alelo variante G SNP -71 AG do gene da 17βHSD5, com uma associação limítrofe desse alelo com o diagnóstico clínico de PP; 2) o polimorfismo ApaI TG associou-se com PP e parece estar modulando os processos esteroidogênicos nas meninas com PP; 3) houve uma interação entre os polimorfismos TaqI TC e BsmI GA e concentrações circulantes de vitamina D em meninas do sul do Brasil. / Precocious pubarche (PP) is usually defined as the development of pubic hair before the age of 8 in girls and age of 9 in boys. Although the PP does not interfere directly in puberty events, some evidence suggests that these girls have higher risk for the development of Polycystic Ovary Syndrome (PCOS) at later ages. The Type 5 17β-Hydroxysteroid Dehydrogenase (17ßHSD5) is the principal responsible for the conversion of androstenedione to testosterone. Variations in the gene encoding for this enzyme, especially Single Nucleotide polymorphisms (SNPs), may be related with hyperandrogenism, and PCOS. Besides the effects on bone metabolism, vitamin D appears to modulate other extra-skeletal actions, including secretions and tissue sensitivity to insulin. Vitamin D has been associated with insulin resistance and variants in the vitamin D receptor (VDR) gene, have been studied in populations at risk of Diabetes. However, little is known about these polymorphisms in the PP. The aims of this work were: to evaluate the levels of the 25-hydroxyvitamin D; to determine the polymorphisms FokI, BsmI, ApaI and TaqI in VDR gene and SNP -71AG in 17ßHSD5 gene frequencies; to asses if exist association between this SNPs and anthropometric, metabolic and hormonal characteristics in patients with PP and controls of the southern Brazil. Were enrolled 36 girls with PP and 197 healthy controls. Genotypic analyzes were evaluated by Real Time for the SNPs -71AG, BsmI and FokI and by PCR-RFLP for the ApaI e TaqI polymorphisms. Genotype frequency for SNP -71 AG of the 17ßHSD5 gene was 52.4% AA, 39.1% AG and 8.6% GG, A:G allelic frequency was 0.72:0.28. Analyzing both groups, higher frequency of the variant allele (G) in patient PP than controls (0.37 e 0.26, respectively) was found but without statistical difference (p=0.054); there were no associations between this polymorphism and clinical and hormonal features. PP girls have serum levels of 25(OH)D lower than those from control group (18.08±8.32 versus 21.27±7.03; p=0.032). The polymorphism analyze was observed that genotype GG of the SNP ApaI TG showed a higher frequency in PP (30.6%) than controls (16.2%) (Odds Ratio: 2.269; 95% confidence interval: 1.015 – 5.076; p=0.042). The same genotype was associated with lower estradiol (35.30 (14.80 – 50.48) versus 12.22 (6.49 – 23.69); p=0.030) and total testosterone levels (0.52 (0.39 – 0.84) versus 0.20 (0.11 – 0.47); p=0.009), in girls with PP. There were no association between this polymorphism and serum 25(OH)D. On the other hand, there was association between the presence of the polymorphisms TaqI TC (TC + CC genotype) and BsmI GA (GA + AA genotype) and higher serum 25(OH)D in the group of healthy girls (19.86 ± 7.16 versus 6.69 ± 22:55 , p = 0.007; 19:53 ± 6.94 versus 22.88 ± 6.76, p = 0.001, respectively). In conclusion, data from this study indicate that: 1) there was a higher frequency of the variant allele G of the SNP -71 AG of the 17ßHSD5 gene, with a borderline association of this allele with the clinical diagnosis of PP; 2) ApaI TG polymorphism is associated with PP and seems to modulate the processes steroidogenesis in girls with PP; 3) there was an interaction between the polymorphisms TaqI TC and BsmI GA and vitamin D concentrations in girls from southern Brazil.

A Síndrome dos Ovários Policísticos (PCOS) é a endocrinopatia mais frequente em mulheres na idade reprodutiva, além de ser a causa mais comum de hiperandrogenismo e anovulação crônica. Diferentes fenótipos da PCOS foram identificados, e um melhor entendimento dessas diferentes apresentações clínicas se faz necessário para o reconhecimento de riscos, medidas preventivas e terapêuticas específicas para cada fenótipo. Associações entre polimorfismos de substituição de um único nucleotídeo (SNPs) em genes que codificam enzimas responsáveis pelo metabolismo androgênico e PCOS foram descritos. O anticoncepcional oral (ACO) é utilizado para o tratamento de mulheres com PCOS por seu efeito supressivo na secreção de androgênios ovarianos e melhora do hirsutismo. Entretanto, os dados na literatura são conflitantes quanto aos efeitos do ACO nos parâmetros metabólicos de mulheres com PCOS. Além disso, não está bem estabelecido se a presença de alelos polimórficos está associada com diferenças nos fenótipos da PCOS e se pode influenciar na resposta ao tratamento com ACO. Os objetivos desta tese foram investigar a influência dos SNPs -71 AG no gene AKR1C3 e SNP50 no gene CYP19 gene (substituição de G por A) na resposta de mulheres com PCOS ao tratamento com ACO e verificar se o SNP50 está associado com fenótipos da PCOS. Cento e sessenta e duas mulheres com PCOS foram estratificadas em PCOS clássicas (hiperandrogenismo e disfunção ovulatória, c-PCOS) e PCOS ovulatórias (hiperandrogenismo, ciclos ovulatórios, aparência policística dos ovários, ov-PCOS) e uma subamostra de 51 mulheres (que não apresentavam resistência insulínica evidente) completaram 6 meses de tratamento com ACO (20 ug etinilestradiol e 75 ug gestodeno, 21/28 dias por ciclo). A presença ou ausência dos alelos polimórficos foram consideradas para expressar os resultados que avaliaram os SNPs -71 AG e SNP50. O escore de hirsutismo foi similar em c-PCOS e ov-PCOS, e as diferenças nos parâmetros hormonais e metabólicos observadas foram independentes da presença do alelo A do SNP50. Após os 6 meses de tratamento com ACO, como era esperado, os níveis de testosterona total e o escore clínico de hirsutismo diminuíram, enquanto os níveis da globulina carreadora de hormônios sexuais aumentaram. Houve uma pequena redução da pressão arterial sistólica e do hormônio luteinizante. As medidas de insulina e do índice HOMA permaneceram inalteradas após o tratamento. Houve um aumento dos níveis de lipídios, mas os valores permaneceram dentro dos limites da normalidade. Nenhuma das alterações observadas esteve associada com a presença dos alelos polimórficos dos SNPs -71 AG ou SNP50. As conclusões são de que o ACO é uma alternativa eficaz para o tratamento dos sintomas do hiperandrogenismo, sem comprometimento dos parâmetros metabólicos, pelo menos naquelas mulheres sem resistência insulínica prévia ao tratamento. Os SNPs -71AG no gene AKR1C3 e SNP50 no gene CYP19 não contribuem para as melhoras observadas com o uso do ACO. Além disso, o SNP50 parece não estar associado com as diferenças existentes entre os fenótipos clássico e ovulatório da PCOS. / Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder in women at reproductive age, and also the most common cause of hyperandrogenism and chronic anovulation. Different phenotypes of PCOS have been identified, and a better knowledge of these clinical symptoms is necessary to recognize risks, prevention, and treatment strategies for each phenotype. Associations between single nucleotide polymorphisms (SNPs) in genes that codify enzymes responsible for the androgenic metabolism and PCOS have been described. The oral contraceptive pill (OCP) is used to treat women with PCOS due to the suppressive effect on ovarian androgen secretion, with consequent amelioration of hirsutism. However, data are conflicting in literature regarding the effects of OCP on metabolic variables in PCOS. Besides that, it is not well established whether the presence of polymorphic alleles is associated with PCOS phenotypes and whether can influence on the response to OCP treatment. The aims of this thesis were to investigate the influence of the SNPs -71 AG at AKR1C3 gene and SNP50 of CYP19 gene (G to A substitution) on the response of PCOS to treatment with oral contraceptive pills and to assess whether the SNP50 is associated with PCOS phenotypes. A hundred sixty two hirsute women were stratified into a classic PCOS group (hyperandrogenism and ovulatory dysfunction, c-PCOS) and an ovulatory PCOS group (hyperandrogenism, ovulatory cycles, polycystic ovaries, ov-PCOS), and a subsample of 51 women (without evidences of insulin resistance) completed a 6-month OCP trial (20 ug ethinylestradiol plus 75 ug gestodene, 21/28 days per cycle). We considered the presence or absence of the polymorphic alleles to express results and to perform the comparisons regarding the SNPs -71 AG and SNP50. Hirsutism score was similar in c-PCOS and ov-PCOS, and the differences in hormone and metabolic variables between phenotypes were independent of the presence of allele A for SNP50. After 6 months of OCP treatment, as expected, total testosterone and hirsutism score declined, while sex hormone binding globulin increased. There was a slight reduction in systolic blood pressure and luteinizing hormone levels. Insulin and homeostasis model assessment remained unchanged after treatment. There was an increase in lipids, but the values remained at the normal range. None of these changes were associated with the presence of polymorphic alleles for -71 AG or SNP50 polymorphisms. We conclude that OCP is an alternative to ameliorate androgenic symptoms without compromising metabolic parameters, at least in women without insulin resistance before treatment. The -71AG SNP of AKR1C3 gene and the SNP50 of CYP19 gene did not contribute to the improvements observed. Besides that, SNP50 may not be associated to the existing differences between classic and ovulatory PCOS phenotypes.