The common human GNB3 825C > T variant, which is present in 50% of the world’s chromosomes, has previously been shown to predispose individuals to hypertension, cardiac and neural disorders. This variant causes the production of a stable and gain of function protein Gβ_3S- This thesis describes the discovery of a novel D153del mutation that produces an unstable, loss of function, protein Gβ_3D in the recessively inherited, retinopathy globe enlarged (rge) chickens. This thesis also demonstrates that the normal Gβ₃ downstream phosphorylation signalling pathways are significantly altered in a tissue specific manner in rge chicken organs and in a human GNB3 825TT lymphoblast cell line. In rge tissues expressing Gβ_3D protein, the cAMP induced GRK2 phosphorylation activity is significantly altered. Moreover MAPK1 (ERK2) phosphorylation is significantly decreased compared to normal tissues. In contrast human 825TT cell lines expressing the Gβ_3S protein, showed enhanced cAMP induced GRK2 and MAPK (ERK1 and ERK2) phosphorylation activity. These results confirm previous findings of 825C > T Gβ₃ studies, that Gβ_3S is indeed a hyper-activating structural variant, in contrast to the D153del Gp3D is a classical recessively inherited non-functional mutation.

Chalcogen-containing materials are an area of increasing interest for spintronic applications. The synthesis, structures and reactivity of these novel compounds are normally studied by solution-state nuclear magnetic resonance (NMR) spectroscopy, density functional theory (DFT) calculations and single-crystal X-ray diffraction. In this thesis, a range of chalcogen-containing heterocycles has been explored, focussing on the solid-state nature and exploring the bulk samples. Therefore, all materials were studied by powder X-ray diffraction and solid-state NMR, in addition to conventional solution-state NMR and single-crystal X-ray diffraction. DFT calculations were also used to interpret the solid-state NMR spectra and to gain insight into the NMR parameters. In the first chapter of results, a series of mixed Te, Se acenaphthenes is investigated. 77Se and 125Te NMR parameters are explored to determine whether changes in the Te aryl-group have an impact on the local environments of both nuclei. Dynamics and the requirement to consider relativistic effects for calculations of NMR parameters of heavy atoms is discussed. In the second results chapter, a series of novel P-S and P-Se six-membered heterocycles are described in terms of their synthesis, reactivity, and 31P and 77Se local environments. We observed and measured some unusual “through-space” couplings that occur between molecules and which mechanism and pathways are supported by DFT calculations. In the third results chapter, these heterocycles are oxidised with O, S and Se and the NMR parameters are discussed in terms of the structure. Polymorphism, phase transitions and weak interactions are some of the phenomena present in these novel compounds. This thesis demonstrated that solid-state NMR is a very good probe to study Se- and Te-containing materials.

BACKGROUND & AIMS - Metabolic syndrome (MetS), characterized by abdominal obesity, atherogenic dyslipidemia, elevated blood pressure, and insulin resistance is a major public health concern in the United States. The effect of Apolipoprotein E (Apo E) polymorphism has been relatively well studied in relation to cardiovascular disease; however, its effects on MetS are not well established. METHODS - We conducted a cross-sectional study consisting of 1,551 participants from the National Heart, Lung, and Blood Institute (NHLBI) Family Heart Study to assess the relation of Apo E polymorphism with the prevalence of MetS. Information on the different Apo E genotypes was extracted from the database and we defined MetS according to the AHA-NHLBI-IDF-WHO Harmonized Criteria. We used generalized estimating equations to estimate adjusted odds ratios for prevalent MetS and the Bonferroni correction to account for multiple testing in the secondary analysis. RESULTS – Our study population had a mean age (SD) of 56.5 (11.0) years and 49.7% had MetS. There was no association between the Apo E genotypes and MetS. The multivariable adjusted ORs (95% CI) were 1.00 (reference), 1.26 (0.31-5.21), 0.89 (0.62- 1.29), 1.13 (0.61-2.10), 1.13 (0.88-1.47) and 1.87 (0.91-3.85) for the *e3/e3, *e2/e2, *e2/e3, *e2/e4, *e3/e4 and *e4/e4 genotype respectively. In a secondary analysis, the *e2/e3 genotype was associated with lower HDL levels, with the multivariable adjusted ORs (95% CI) of 0.59 (0.36-0.95) when compared to the reference *e3/e3 genotype. CONCLUSIONS - Our findings do not support an association between Apo E polymorphism and MetS in a multi-center population based study of predominantly white US men and women. The *e2/e3 genotype was associated with lower HDL levels as compared to the *e3/e3 genotype. KEY WORDS: Apolipoprotein E (Apo E) polymorphism, metabolic syndrome, blood pressure, glucose, waist circumference, triglycerides, high-density lipoprotein cholesterol

Made available in DSpace on 2016-08-15T20:27:55Z (GMT). No. of bitstreams: 1 AnaCDSB_TESE.pdf: 11862899 bytes, checksum: db329299185e2c022af1f916b746aae8 (MD5) Previous issue date: 2014-11-14 / The increase in anthelmintic resistance in gastrointestinal nematodes on small ruminants became a worldwide issue on the development of livestock. In order to assess the resistance scenario in that context in the Rio Grande do Norte region, in vivo and in vitro resistance tests were carried out in 30 rural properties using chemicals in the avermectin, salicylanides, benzimidazoles and imidatiazols groups. For the in vivo test, group I was used as control and four other experimental groups received oral medication, being group II given albendazole (10 mg/kg), group III levamisole (7.5 mg/kg), group IV closantel (5.0 mg/kg) and group V ivermectin (0.2 mg/kg). The nematode infection was determined using fecal egg count (FEC) essays and the efficiency of each treatment was analyzed by the fecal egg count reduction test (FECRT). For the in vivo e in vitro tests, using tiabendazole, the necessary concentration to inhibit 50% of egg hatching (EC50) and assays with a 50% inhibition of larval development (LD50) were assessed, however 50% of larval development and migration inhibition assays (LD50) essays were carried out using ivermectin. For the molecular analysis Haemonchus contortus/property were isolated for the calculation of the frequency of resistant and sensitive alleles for single nucleotide polymorphisms (SNPs): F220Y, F167Y and E198A. The results obtained from in vivo tests showed that resistance was detected for all of the drugs, with leavamisole and closantel appearing as the most effective treatments with FECRT results of 84.3% and 74.6%, respectively. In the in vitro assays, tiabendazole had a result in the hatching test of EC50 0.598 µg/ml (95% IC: 0.323-0.818), while exhibiting in the larval development test a LD50 and LD99 0.518 µg/ml(95% IC: 0.432-0,887) and 4.359 µg/ml (95% IC: 3.047-8.178), respectively. For ivermectin in the larval development test, DL50 0.637 µg/ml(95% IC: 0.382 0.903 µg/ml) e DL99 19.56 µg/ml(95% IC: 11.44 46.55 µg/ml) was observed, while in the migration essay the LD50 value was 16.337 µM (95% IC: 12.7-21.3 µM). In the molecular analysis a higher frequency of the resistant allele for SNP F200Y was observed, presenting higher percentage values of 85.81% and 73.11%, while the F167Y resistant alleles had lower values ranging from 2.93% to 43.73%. In the E198A SNP, only sensitive alleles were detected. Assessing the scenario of anthelmintic resistance in the region, one notices that it s established for the main existing drugs on the market / O aumento na resistência anti-helmíntica em nematóides gastrintestinais de pequenos ruminantes tornou-se um problema global para o desenvolvimento da pecuária. Nesse contexto, a fim de avaliar o cenário da resistência na região do Rio Grande do Norte foram realizados em 30 propriedades rurais testes de resistência in vivo e in vitro utilizando produtos químicos dos grupos das avermectina, salicilanilidas, benzimidazóis e imidatiazóis. Para o teste in vivo utilizou-se o grupo I controle quatro grupos experimentais, sendo grupo II albendazol (10 mg/kg), grupo III levamisol (7,5 mg/kg), grupo IV closantel (5,0 mg/kg) e grupo V ivermectina (0,2 mg/kg). A infecção por nematóides foi determinada utilizando ensaios de ovos por grama de fezes (OPG) e a eficiência de cada tratamento pelo teste de redução da contagem de ovos nas fezes (FECRT). Para os teste in vitro, foi utilizando tiabendazol, com concentrações necessária para inibir 50% de eclosão dos ovos (EC50) e ensaios com inibição de 50% do desenvolvimento larval, porém com a ivermectina foram realizados ensaios de inibição de 50% do desenvolvimento e migração larval (DL50). Para análise molecular foram isolados Haemonchus contortus em cada propriedade avaliada para cálculo da frequência de alelos resistentes e sensíveis para os polimorfismos de nucleotídeo único (SNPs): F200Y, F167Y e E198A. Os resultados obtidos com os testes in vivo e in vitro mostraram que a resistência foi detectado em todas as drogas testadas, com o levamisol e closantel apresentando-se como os tratamentos mais eficientes, com resultados FECRT de 84,3% e 74,6%, respectivamente. Nas análises in vitro o resultado do tiabendazol no teste de eclosão foi EC50 0,598 µg/ml (IC95%: 0,323-0,818), enquanto que no desenvolvimento larval uma DL50 e DL99 de 0,518 µg/ml(IC95%: 0,432-0,887) e 4,359 µg/ml(IC95%: 3,047-8,178), respectivamente. Para ivermectina no teste de desenvolvimento larval foi observado DL50 0,637 µg/ml(CI95%: 0,382 0,903 µg/ml) e DL99 19,56 µg/ml(CI95%: 11,44 46,55 µg/ml), enquanto que no de migração o valor da DL50 foi 16,337 µM (CI95%: 12,7-21,3 µM). Na análise molecular observou-se uma frequência maior do alelo resistente para o SNP F200Y apresentando porcentagens mais altas com valores de 85,81% e 73,11%, enquanto que o F167Y apresentou valores menores de alelos resistentes variando entre 43,73% e 2,93%. No SNP E198A foram detectados alelos apenas sensíveis. Avaliando o cenário da resistência anti-helmíntica na região, percebe-se que está estabelecida para as principais drogas existentes no mercado

>Magister Scientiae - MSc / Reference single nucleotide polymorphism (refSNP) identifiers are used to earmark SNPs in the human genome. These identifiers are often found in variant call format (VCF) files. RefSNPs can be useful to include as terms submitted to search engines when sourcing biomedical literature. In this thesis, the development of a bioinformatics software package is motivated, planned and implemented as a web application (http://sniphunter.sanbi.ac.za) with an application programming interface (API). The purpose is to allow scientists searching for relevant literature to query a database using refSNP identifiers and potential keywords assigned to scientific literature by the authors. Multiple queries can be simultaneously launched using either the web interface or the API. In addition, a VCF file parser was developed and packaged with the application to allow users to upload, extract and write information from VCF files to a file format that can be interpreted by the novel search engine created during this project. The parsing feature is seamlessly integrated with the web application's user interface, meaning there is no expectation on the user to learn a scripting language. This multi-faceted software system, called SNiPhunter, envisions saving researchers time during life sciences literature procurement, by suggesting articles based on the amount of times a reference SNP identifier has been mentioned in an article. This will allow the user to make a quantitative estimate as to the relevance of an article. A second novel feature is the inclusion of the email address of a correspondence author in the results returned to the user, which promotes communication between scientists. Moreover, links to external functional information are provided to allow researchers to examine annotations associated with their reference SNP identifier of interest. Standard information such as digital object identifiers and publishing dates, that are typically provided by other search engines, are also included in the results returned to the user. / National Research Foundation (NRF) /The South African Research Chairs Initiative (SARChI)

L'objectif de notre première étude était d'identifier les déterminants génétiques et environnementaux du risque de maladie thromboembolique veineuse (MTEV) chez les femmes sous contraceptifs oraux combinés (COC). Après ajustement pour les facteurs confondants, les principaux déterminants environnementaux de la MTEV étaient le tabagisme (OR = 1,65) et un indice de masse corporelle supérieur à 35 kg.m2 (OR = 3,46). En outre, la thrombophilie héréditaire sévère (OR = 2,13) et les groupes sanguins non-O (OR = 1,98). Nous avons confirmé que l’histoire familiale au premier degré de MTEV prédit mal la thrombophilie. En conclusion, cette étude confirme l'influence du tabagisme et de l'obésité et pour la première fois l'impact du groupe sanguin ABO sur le risque de MTEV chez les femmes sous COC. Elle confirme également la faible sensibilité de l'histoire familiale de MTEV pour dépister les thrombophilies héréditaires.Le but de la deuxième étude était d'étudier, chez les utilisatrices de COC, l'impact des polymorphismes génétiques nouvellement identifiés par les études pangénomiques associés au risque de MTEV dans la population générale. Neuf polymorphismes situés sur les gènes KNG1, F11, F5, F2, PROCR, FGG, TSPAN15 et SLC44A2 ont été génotypés dans un échantillon de 766 cas et 464 témoins dans le cadre de l’étude PILGRIM. Seul le polymorphisme rs2289252 situé sur le F11 était significativement associé au risque de MTEV. La présence de l’allèle rs2289252-A du F11 était associée à un risque accru de MTEV (OR =1,6). En outre, la combinaison de l’allèle rs2289252-A et du groupe sanguin non-O, était associée à un risque d’OR de 4. / The aim of our first study was to identify the genetic and environmental determinants of venous thromboembolism (VTE) risk in a large sample of women using combined oral contraceptives (COC). A total of 968 women with a personal history of VTE during COC use were compared with 874 women under COC, but no personal history of VTE. After adjustment for confounding factors, the main environmental determinants of VTE were smoking odds ratio (OR = 1.65) and a body mass index greater than 35 kg.m-2 (OR = 3.46). In addition, severe hereditary thrombophilia (OR = 2.13) and non-O blood groups (OR = 1.98) have been shown to be important genetic risk factors for VTE under COC. First-degree family history of VTE predicts thrombophilia poorly. In conclusion, this study confirms the influence of smoking and obesity and for the first time the impact of ABO blood group on the risk of VTE in women under COC It also confirms the low sensitivity of the family history of VTE to detect hereditary thrombophilia. The purpose of the second study was to study, in COC users, the impact of newly identified genetic polymorphisms by genome-wide as associated with the risk of VTE in the general population. Nine polymorphisms on the KNG1, F11, F5, F2, PROCR, FGG, TSPAN15 and SLC44A2 genes were genotyped in a sample of 766 patients and 464 controls in the PILGRIM study. Only the rs2289252 polymorphism on the F11 was significantly associated with the risk of VTE. The presence of the F11 rs2289252-A allele was associated with an increased risk of VTE (OR = 1.6). In addition, the combination of the rs2289252-A allele and the non-O blood group was associated with an OR risk of 4.

Magister Scientiae - MSc (Medical BioSciences) / Despite attempts to develop new drugs to reduce the worldwide mortality rate attributable to tuberculosis (TB), the illness remains a threat. Isoniazid (INH) has been used as a frontline drug for decades. However, several resistant strains of the organism - Mycobacterium tuberculosis (M. tuberculosis) - still emerge. The drug is mainly metabolised by a family of enzymes, arylamine N-acetyltransferases (NAT). The three human NAT genes - NAT1, NAT2 and the pseudogene, NATP - are found on chromosome 18p22. NAT1 and NAT2 are isoenzymes which differ at certain amino acid positions. Subsequently, the differences affect substrate specificity. NAT1 shows specificity to p-aminobenzoic acid (PABA) and paminosalicylate (PAS). Previously, computer algorithms were used to predict the efficacy of the enzyme with regard to the acetylation phenotype it confers. The two which were focused on, Sorting Intolerant From Tolerant (SIFT) program and Polymorphism phenotyping version 2 program (PolyPhen-2), showed conflicting results for the effect of SNPs on the acetylation rate and subsequent enzyme function. Further structural prediction methods were used to test the effect of V231G on the structure and consequent function of the native protein, NAT1.

Introdução: Existem fortes evidências de um fator genético estar envolvido na etiologia do transtorno bipolar (TB), contudo a interação entre polimorfismos genéticos e alterações bioquímicas permanecem desconhecidas. O fator neurotrófico derivado do cérebro (BDNF) parece exercer um papel importante na patofisiologia do TB. Objetivos: O presente estudo tem por objetivo avaliar a associação do polimorfismo localizado no gene do fator neurotrófico derivado do cérebro (BDNF) e os níveis séricos desta substância em pacientes com transtorno bipolar. Material e Métodos: Foram selecionados 114 pacientes com TB tipo I de acordo com critério do DSMIV e 137 controles pareados por sexo, idade e anos de estudo para a análise do polimorfismo val66met do BDNF e do BDNF sérico. Suas associações foram medidas através da análise de variância (ANOVA).Resultados: Não houve diferenças significativas na freqüência dos genótipos do polimorfismo val66met do BDNF entre pacientes e controles (p>0.05; teste Qui-quadrado). Não foi encontrada associação entre o polimorfismo do gene do BDNF e o diagnósticode transtorno bipolar(eutímicos) nos níveis séricos de BDNF (p=0.34; ANOVA Fatorial) Conclusão: O polimorfismo do BDNF val66met parece não interferir no nível sérico de BDNF em pacientes bipolares em tratamento e controles sem TB, sugerindo que a variante BDNFMet não diminui a secreção constitutiva; possivelmente este polimorfismo do BDNF exerça alguma influencia nos níveis séricos do BDNF durante os episódios agudos da doença. / Introduction: There is strong evidence demonstrating that genetic inheritance is associated with higher susceptibility to bipolar disorder (BD) but the interaction between gene polymorphisms and biochemical changes remains largely unknown. The brainderived neurotrophic factor (BDNF) may play a role in the pathophysiology of BD. Objectives: The aim of the present study was to evaluate the association between BDNF polymorphism val66met and its serum levels in bipolar patients. Methods: One hundred and seven Caucasian type-I bipolar patients were recruited from the Bipolar Disorders Program and underwent Structured Clinical Interview for DSMIV- Axis I for diagnosis. The subjects were matched by age, gender and education with137 controls without BD. The association between BDNF serum levels and polymorphism was analysed by ANOVA. Results: No significant differences were found in the frequency of the BDNF val66met genotype or allele distribution between patients and controls (p>0.05; Chi-square test). We have found no significant interaction between BDNF polymorphism anddiagnostic status (bipolar disorder and controls) on serum BDNF levels (p=0.34; Factorial ANOVA) Conclusion: BDNF val66met polymorphism does not affect serum BDNF levels in a sample of mostly euthymic BD subjects currently on medication. Considering that the BDNFMet variant decreases only the activity-dependent but not the constitutive BDNF secretion, it is conceivable that BDNF polymorphism may exert some influence on serum BDNF levels during acute mood episodes.

The aim of this thesis was to analyze the polymorphism in position -371bp (related to ATG start codone) of MSTN gene, SNP in position 1984bp of MYF5 gene and influence of these polymorphisms on tenderness, water holding capacity, pH and color of meat. Samples (241) of bull meat of Czech pied cattle were used for analysis. PCR-RFLP method was applied to genotype MSTN and MYF5 genes. For polymorphisms detection was used restriction endonucleases DraI (for MSTN gene) and TaqI (for MYF5 gene). From established genotypes were computed their frequencies and alleles frequencies. The frequencies of genotypes in MSTN gene were AA=0,729, AB=0,258, BB=0,013 and alleles frequencies were A=0,858, B=0,142. The frequencies of genotypes in MYF5 gene were AA=0,181, AB=0,542, BB=0,278 and alleles frequencies were A=0,452 a B=0,548. Using statistical analysis, the influence of genotypes of MYF5 gene on the water holding capacity, pH and the color of meat was determined.

The aim of the thesis is to study the influence of the milk protein beta-lactoglobulin on the milk renneting properties. Based on the polymorphisms in -lactoglobulin gene (BLG), the genotypes AA, AB and BB were defined by the method PCR-RFLP in the case of 730 dairy cows. The results of the genotypes analysis show that in the studied population the heterozygotes AB occur approximately eleven times more often than homozygotes BB and thirty times more often than homozygotes AA. The allele A frequency was 0.4732 and the allele B 0.5268. The genotype BLG influence on production traits of milk was analysed in the aggregate of 429 milk samples. The statistically demonstrative allele B influence on the percentage of proteins representation was found only in one group of dairy cattle from farm Sedlec. The alleles BB were associated with lower values of these parameters. The statistically significant difference influenced by the genotypes or the gene BLG alleles was not found in any other measurement. In the thesis the testing of milk renneting properties by the rennet coagulation time (RCT) and the determination of titratable acidity in milk was made on 51 samples. The results show that the BLG genotypes had no influence on the RCT. In the determination of titratable acidity in milk results the genotype influence appeared and the p-value is lower than the given significance level = 0.05. It could mean that the AB genotype is connected with the higher milk acidity. However, the results can be distorted by the low numbers of the population.