Cytochrome P4502E1 (CYP2E1), the ethanol-inducible isoform of cytochrome P450 superfamily, catalyzes many low molecular weight endogenous and exogenous compounds, including ethanol, acetone, drugs like acetaminophen and chlorzoxazone, and industrial solvents like benzene and styrene, most of which are carcinogenic. Besides, it has a high capacity to produce reactive oxygen species. CYP2E1 is induced by ethanol and isoniazid, as well by some pathophysiological conditions like diabetes and starvation. CYP2E1 gene shows genetic polymorphisms which are thought to play a major role in interindividual variability in drug response and in susceptibility to chemical-induced diseases, like several types of cancers. It is well established that CYP2E1 polymorphisms vary markedly in frequency among different ethnic and racial groups. Therefore, in this study, the frequency of two important CYP2E1 polymorphisms / the single nucleotide polymorphisms C-1019T / G-1259C in 5&rsquo / -flanking region and T7678A poymorphism in intron 6, in Turkish population was investigated. For this purpose, whole blood samples were collected from 132 healthy volunteers representing Turkish population and genomic DNA for each subject was isolated in intact form. The genotypes were determined by PCR amplification of corresponding regions followed by restriction endonuclease RsaI, PstI (for C-1019T / G-1259C SNPs) and DraI (for T7678A SNP) digestions. The genotype frequencies, for C-1019T / G-1259C SNPs, which are in complete linkage disequilibrium, were investigated on 116 DNA samples, and determined as 97.4% for homozygous wild type (c1/c1), 2.6% for heterozygotes (c1/c2) and 0.0% for homozygous mutants (c2c2). The allele frequency of wild type allele (c1) was calculated as 98.7% and that of mutated allele (c2) as 1.3%. The genotype frequencies for T7678A SNP, investigated in 108 DNA samples were determined as 80.6% for homozygous wild type (DD), 19.4% for heterozygotes (CD) and 0.0% for homozygous mutants (CC). The corresponding allele frequencies were 90.3% for wild type allele (D), and 9.7% for mutated allele (C). Genotype frequencies of both polymorphisms fit Hardy-Weinberg equation and showed no significant difference with respect to gender. The genotype distributions of both polymorphisms showed similarity when compared to other Caucasian populations like French, Swedish, German, and Italian populations, while both polymorphisms studied differed significantly from Chilean, Japanese, Taiwanese and Chinese populations, as compared with Chi-Square test.

Made available in DSpace on 2014-08-20T13:32:47Z (GMT). No. of bitstreams: 1 dissertacao_lucas_teixeira_hax.pdf: 388762 bytes, checksum: c93c16af033ccd2aa4ff52ec81723de9 (MD5) Previous issue date: 2013-02-27 / The genes of the somatotropic axis, which act regulating the metabolism and physiology of the mammals, present polymorphism associated to some characteristics of economical interest, such as reproductive performance and milk production. Such factors may be influenced by the mutation on only one nucleotide in the base sequence of the gene of the growth hormone receptor (GHR), which may alter the density of GHR on the hepatic tissue. Changes in the coupling of the growth hormone (GH) in the hepatic tissue alter the serum concentration of the insulin-like growth factor 1 (IGF-I), as IGF-I is produced mainly by the liver when it is stimulated by the growth hormone. Different studies have evaluated the effect of polymorphisms in the gene responsible for encoding IGF-I on the reproductive performance and milk production of high production dairy cows. Among other functions, the IGF-I mediates the effects of gonadotropins on the follicular cells, stimulating the growth and differentiation of theca and granulosa follicular cells, playing also a significant role on the final growth and maturation of the dominant follicle. Furthermore, high serum IGF-I concentrations are associated with a earlier return to cyclicity post partum in high yield dairy cows. Thus, the objective of this study was to evaluated the relevance of the mutations in GHR and IGF-I on the calving conception interval, number of inseminations per pregnancy and milk production in Holstein cows. One hundred and fifty five Holstein cows, submitted to a semi extensive management system, subjected to fixed-time artificial insemination (TAI) that got pregnant up to 250 days in milk in 2011, were selected. Among the animals tested, 29% presented GHR AluI (+ / +), 57.5% AluI (+ / -) and 13.5% AluI (- / -) genotype. 34.9% presented IGF-I SnaBI (+ / +), 45.8% SnaBI (+ / -) and 19.3% SnaBI (- / -) genotype. No association was observed between GHR AluI and IGF-I SnaBI genotypes and calving conception interval, number of inseminations per pregnancy and milk yield (P> 0.05). Likewise, there was no association between the interaction of GHR AluI and IGF-I SnaBI genotypes and calving conception interval, number of inseminations per pregnancy and milk yield (P> 0.05). Finally, further studies are necessary to better understand the relevance of GHR AluI and IGF-I SnaBI genotypes to the calving conception interval number of inseminations per pregnancy and milk production in Holstein cows. / Os genes do eixo somatotrópico, que atuam na regulação do metabolismo e fisiologia dos mamíferos, apresentam polimorfismos associados a algumas características de interesse econômico, como desempenho reprodutivo e produção de leite. Tais fatores podem ser influenciados por mutações de apenas um nucleotídeo na sequência de bases do gene do receptor do hormônio do crescimento (GHR), que podem alterar a expressão do GHR no tecido hepático. Mudanças no acoplamento do hormônio do crescimento (GH) no tecido hepático alteram a concentração sérica de fator de crescimento semelhante à insulina tipo1 (IGF-I), visto que o IGF-I tem sua produção endócrina principalmente no fígado mediante estimulação do hormônio do crescimento. Diversos trabalhos têm estudado o efeito de polimorfismos no gene que codifica para IGF-I no desempenho reprodutivo e produção de leite de vacas leiteiras de alta produção. Entre outras funções, o IGF-I atua como mediador dos efeitos das gonadotrofinas nas células foliculares, estimulando o crescimento e diferenciação das células da teca e da granulosa foliculares, apresentando também um importante papel no crescimento final e na maturação do folículo dominante. As altas concentrações sanguíneas de IGF-I estão também associadas a um retorno à ciclicidade mais precoce de vacas leiteiras pós-parto de alta produção. Dessa forma, o objetivo deste estudo foi avaliar a importância de mutações no GHR e IGF-I no desempenho zootécnico, IPC, número de inseminações por prenhez e produção de leite em vacas da raça Holandês. Foram avaliadas 155 vacas da raça Holandês em sistema semi extensivo submetidas à inseminação artificial em tempo fixo (IATF) e que conceberam até 250 dias em lactação no ano de 2011. Entre os animais analisados, 29% apresentaram o genótipo GHR AluI, (+/+), 57,5% AluI (+/-) e 13,5% AluI (-/-). Já para o IGF-I SnaBI 34,9% apresentaram o genótipo IGF-I SnaBI (+/+), 45,8% SnaBI (+/-) e 19,3% SnaBI (-/-). Não foi observada associação entre os genótipos GHR AluI e IGF-I SnaBI e o intervalo parto-concepção, número de inseminações por prenhez e produção de leite (P>0,05). Da mesma forma, não houve associação entre a interação dos genótipos de GHR AluI e IGF-I SnaBI e o intervalo parto-concepção, número de inseminações por prenhez e produção de leite (P>0,05). Finalmente, novos estudos avaliando uma maior população de animais são necessários para elucidar a importância dos genótipos de GHR AluI e IGF-I SnaBI no intervalo parto-concepção, número de inseminações por prenhez e produção de leite.

Les études d'association pangénomiques ont permis d'identifier de nouveaux loci, dont le gène CR1 comme associé au risque de maladie d'Alzheimer (MA). Le récepteur du complément de type 1 (CR1) est une glycoprotéine transmembranaire, présente notamment à la surface des érythrocytes (CR1E), mais également dans le plasma sous forme soluble (CR1s). Le CR1 peut prendre des formes fonctionnelles différentes, qui pourraient conférer des niveaux de risque différents, voire suggérer des mécanismes physiopathologiques de la MA. Si la relation entre CR1 et MA est aujourd'hui connue, son mécanisme reste énigmatique.L'objectif principal de cette thèse était de corréler aux données génétiques (single nucleotide polymorphisms, polymorphismes de longueur, polymorphismes de densité), des éléments phénotypiques acquis comme la densité du CR1E ou le CR1S. D'une part, notre étude a montré grâce à deux méthodes différentes, que la MA était associée à une densité basse de l'isoforme long de CR1 (CR1*2) et suggérait l'existence d'allèle silencieux de CR1. D'autre part, nous avons montré que même si les critères génétiques étaient respectés, certains phénotypes pourraient être acquis au cours de la maladie. Nos résultats suggèrent que la MA résulterait plus d'une insuffisance d'épuration des dépôts amyloïdes, que d'une réponse excessive dont la réaction inflammatoire serait délétère. Bien que cette recherche génotypique et phénotypique, à potentiel physiopathologique, nécessite des investigations à plus grande échelle, elle pourra ouvrir la voie à des nouvelles pistes thérapeutiques qui ne peuvent être envisagées aujourd'hui faute de vue claire du ou des mécanismes en cause. / Genome-wide association studies have identified new loci, including the CR1 gene, as being associated with Alzheimer's disease (AD) risk. The complement receptor type 1 (CR1) is a transmembrane glycoprotein found on the surface of erythrocytes (CR1E), and also in the plasma in soluble form (CR1s). CR1 can have different functional forms that may confer different risk levels, or even suggest pathophysiological mechanisms of AD. Indeed, the relation between CR1 and AD is now well established, the mechanism of this association remains to be elucidated.The main objective of this thesis was to correlate acquired phenotype elements, such as density of CR1E (number of CR1 antigenic sites per erythrocyte) or CR1s with genetic data (single nucleotide polymorphisms, length and density polymorphisms). Firstly, our study showed using two different methods that AD is associated with low density of the long CR1 isoform (CR1*2) and suggested the possible existence of silent CR1 alleles. Secondly, we showed that although genetic criteria were met, some phenotypes could be acquired during the course of the disease. Our findings suggest that AD stems more from insufficient clearance of amyloid deposits than from excessive response whose inflammatory reaction might be deleterious. Although this genetic and phenotypic study with pathophysiological potential still require further investigation on a larger scale, she could pave the way towards new therapeutic avenues that currently remain elusive in the absence of a clear overview of the mechanisms involved.

Master of Science / Department of Animal Sciences and Industry / Twig T. Marston / One hundred ninety-three crossbred steers from two herds were used to determine the association of leptin gene polymorphisms and effects of feedlot management of lean and fat steers on carcass performance. Steers were sorted into FAT and LEAN groups by ultrasound backfat at weaning and randomly assigned to a finishing phase. Steers were assigned to a backgrounding phase (BACK) and were fed a forage-based diet for 90 days or directly entered a feedlot phase (FEED). Genotypes were determined by IGENITY® (Atlanta, GA) for a panel of nine single nucleotide polymorphisms (SNP) in the leptin gene (UASMS1, UASMS2, C963T, E2FB, A1457G, and A252T), leptin receptor (T945M), growth hormone receptor (G200A), and fat metabolism enzyme (K232A). Initial backfat (BF) means for the FAT and LEAN group were 3.4 mm and 1.8 mm, respectively. Mean on-test weight was heavier for FAT (306.5 kg) than LEAN (292.9 kg). Age-adjusted hot carcass weights (HCWT) were heavier for LEAN/BACK when compared to FAT/FEED and FAT/BACK (P<0.05). Dressing percent for the FAT/FEED group tended to be higher (P<0.10) over all groups except LEAN/BACK. Steers that went directly to the feedlot had higher marbling scores than backgrounded groups. FAT/FEED had higher 12th rib BF than the other contemporaries. None of the SNPs were useful for predicting ultrasound BF at weaning. Some association was detected with UASMS2 and HCWT (P<0.10) resulting in an 11 kg difference between genotype CC and CT (P<0.05). Five of the leptin polymorphisms (UASMS1, UASMS2, A1457G, C963T, and E2FB) were associated with adjusted carcass BF (P=0.01, 0.06, 0.01, 0.01, and 0.01, respectively) and calculated yield grade (P<0.01). A252T was associated with REA, and genotype TT was larger than AA and AT (P<0.05). This study suggests that segregation by initial fatness estimates and feedlot management strategies has the opportunity to increase HCWT by 35 kg. Sorting cattle upon feedlot entry by ultrasound BF and segregation using genetic markers are useful tools that can assist in the estimation of carcass composition in the live animal. With additional research, the possibility exists to incorporate genetic markers into feedlot selection to assist in marketing decisions.

BACKGROUND:General, breed- and diet-dependent associations between feed efficiency in beef cattle and single nucleotide polymorphisms (SNPs) or haplotypes were identified on a population of 1321 steers using a 50K SNP panel. Genomic associations with traditional two-step indicators of feed efficiency - residual feed intake (RFI), residual average daily gain (RADG), and residual intake gain (RIG) - were compared to associations with two complementary one-step indicators of feed efficiency: efficiency of intake (EI) and efficiency of gain (EG). Associations uncovered in a training data set were evaluated on independent validation data set. A multi-SNP model was developed to predict feed efficiency. Functional analysis of genes harboring SNPs significantly associated with feed efficiency and network visualization aided in the interpretation of the results.RESULTS:For the five feed efficiency indicators, the numbers of general, breed-dependent, and diet-dependent associations with SNPs (P-value<0.0001) were 31, 40, and 25, and with haplotypes were six, ten, and nine, respectively. Of these, 20 SNP and six haplotype associations overlapped between RFI and EI, and five SNP and one haplotype associations overlapped between RADG and EG. This result confirms the complementary value of the one and two-step indicators. The multi-SNP models included 89 SNPs and offered a precise prediction of the five feed efficiency indicators. The associations of 17 SNPs and 7 haplotypes with feed efficiency were confirmed on the validation data set. Nine clusters of Gene Ontology and KEGG pathway categories (mean P-value<0.001) including, 9nucleotide binding / ion transport, phosphorous metabolic process, and the MAPK signaling pathway were overrepresented among the genes harboring the SNPs associated with feed efficiency.CONCLUSIONS:The general SNP associations suggest that a single panel of genomic variants can be used regardless of breed and diet. The breed- and diet-dependent associations between SNPs and feed efficiency suggest that further refinement of variant panels require the consideration of the breed and management practices. The unique genomic variants associated with the one- and two-step indicators suggest that both types of indicators offer complementary description of feed efficiency that can be exploited for genome-enabled selection purposes.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Reproduction and robustness traits are integral in ensuring sustainable, efficient and profitable sheep farming. Increases in genetic gain of reproduction and robustness traits are however, hampered by low heritability coupled with the difficulty in quantification of these traits for traditional selective breeding strategies. The aim of the current study was therefore to identify genomic regions underlying variation in reproduction traits and elucidate quantitative trait loci (QTL) and/or genes associated with reproductive traits. The Elsenburg Merino flock has been divergently selected for the ability to raise multiple offspring and has resulted in a High and a Low line that differ markedly with regard to reproductive output and other robustness traits. The flock thus served as an ideal platform to identify genomic regions subject to selection for reproductive traits. To pinpoint genomic regions subject to selection, a whole-genome genotyping platform, the OvineSNP50 chip, was selected to determine the genotype of more than 50 000 SNPs spread evenly across the ovine genome. The utility of the OvineSNP50 chip was determined for the Elsenburg Merino flock as well as additional South African Merino samples and three other important South African sheep breeds, the Blackheaded Dorper, South African Mutton Merino (SAMM) and the Namaqua Afrikaner. Although genotyping analysis of the Elsenburg Merino flock indicated some signs of poor genotype quality, the overall utility of the genotype data were successfully demonstrated for the South African Merino and the other two commercial breeds, the Dorper and SAMM. Genotyping results of the Namaqua Afrikaner and possibly other indigenous African breeds may be influenced by SNP ascertainment bias due to the limited number of indigenous African breeds used during SNP discovery. Analysis of pedigree, phenotypic records and SNP genotype data of the Elsenburg Merino cohort used in the current study, confirmed that the lines are phenotypically as well as genetically distinct. Numerous putative genomic regions subject to selection were identified by either an FST outlier approach or a genomic scan for regions of homozygosity (ROH) in the High and Low lines. Although annotated genes with putative roles in reproduction were identified, the exact mechanism of involvement with variation in reproduction traits could not be determined for all regions and genes. Putative ROH overlapped with QTL for several reproduction, milk, production and parasite resistance traits, and sheds some light on the possible function of these regions. The overlap between QTL for production and parasite resistance with putative ROH may indicate that several, seemingly unrelated traits add to the net-reproduction and may have been indirectly selected in the Elsenburg Merino flock. A SNP genotyping panel based solely on reproduction traits may therefore be ineffective to capture the variation in all traits influencing reproduction and robustness traits. A holistic selection strategy taking several important traits, such as robustness, reproduction and production into account may as such be a more effective strategy to breed animals with the ability to produce and reproduce more efficiently and thereby ensure profitable and sustainable sheep farming in South Africa. / AFRIKAANSE OPSOMMING: Reproduksie- en gehardheids-eienskappe is noodsaaklik om volhoubare, doeltreffende en winsgewende skaapboerdery te verseker. ‘n Toename in genetiese vordering in reproduksie- en gehardheids-eienskappe word egter bemoeilik deur lae oorerflikhede tesame met die probleme in kwantifisering van hierdie eienskappe vir tradisionele selektiewe diereteelt strategieë. Die doel van die huidige studie was dus om gebiede in die genoom onderliggend tot variasie in reproduksie-eienskappe te identifiseer en die rol van verwante kwantitatiewe eienskap loki (KEL) en/of gene met reproduktiewe eienskappe te bepaal. Die Elsenburg Merinokudde is uiteenlopend geselekteer vir die vermoë om meerlinge groot te maak en het gelei tot 'n Hoë en 'n Lae lyn wat merkbaar verskil ten opsigte van reproduksie-uitsette en ander gehardheids-eienskappe. Die kudde het dus gedien as 'n ideale platform om genomiese areas onderhewig aan seleksie vir reproduksie-eienskappe te identifiseer. Om vas te stel waar genomiese areas onderhewig aan seleksie gevind kan word, is ‘n heel-genoom genotiperingsplatform, die OvineSNP50 skyfie, gekies om die genotipes van meer as 50 000 enkel nukleotied polimorfismes (ENPs) eweredig versprei oor die skaap genoom, te bepaal. Die nut van die OvineSNP50 skyfie is bepaal vir die Elsenburg Merinokudde sowel as addisionele Suid-Afrikaanse Merinos en drie ander belangrike Suid-Afrikaanse skaaprasse, die Swartkop Dorper, Suid-Afrikaanse Vleismerino (SAVM) en die Namakwa Afrikaner. Hoewel genotipe resultate van die Elsenburg Merino kudde sommige tekens van swak genotipe gehalte getoon het, kon die algehele nut van die genotipering resultate vir die Suid-Afrikaanse Merino en die ander twee kommersiële rasse, die Dorper en SAVM, bevestig word. Genotipering resultate van die Namakwa Afrikaner en moontlik ook ander inheemse Afrika rasse kan deur ENP vasstellingspartydigheid beïnvloed word as gevolg van die beperkte aantal inheemse Afrika rasse gebruik tydens ENP ontdekking. Ontleding van stamboom inligting, fenotipe rekords en ENP genotipe data van die Elsenburg Merino-kohort gebruik in die huidige studie, het bevestig dat die lyne fenotipies asook geneties verskil. Talle vermeende genomiese areas onderhewig aan seleksie is geïdentifiseer deur 'n FST uitskieter benadering of deur ‘n genomiese skandering vir gebiede van homogositeit (GVH) in die Hoë en Lae lyne. Hoewel geannoteerde gene met potensiële rolle in reproduksie geïdentifiseer is, kan die presiese meganisme van betrokkenheid by variasie in reproduksie-eienskappe nie bevestig word vir al die gebiede en gene nie. Vermeende GVH oorvleuel met KEL vir 'n paar reproduksie-, melk-, produksie- en parasietweerstand-eienskappe, en werp daarom lig op die moontlike funksie van hierdie gebiede. Die oorvleueling tussen KEL vir produksie en parasietweerstand met vermeende GVH kan daarop dui dat 'n hele paar, skynbaar onverwante, eienskappe bydrae tot net-reproduksie, wat indirek geselekteer mag wees in die Elsenburg Merino-kudde. ‘n ENP genotiperingspaneel uitsluitlik gebaseer op reproduksie-eienskappe mag daarom onvoldoende wees om die variasie in alle eienskappe wat betrekking het op reproduksie- en gehardheids-eienskappe, in te sluit. ‘n Holistiese seleksie strategie wat verskeie belangrike eienskappe, soos gehardheid, reproduksie en produksie in ag neem, mag ‘n meer effektiewe strategie wees om diere te teel met die vermoë om in 'n meer doeltreffende manier te produseer en reproduseer en om daardeur winsgewende en volhoubare skaapboerdery in Suid-Afrika te verseker.

<p>Background: The reasons why some individuals become IgE-sensitised and allergic are largely unknown, though genetic- and early life environmental factors seem to be of importance.</p><p>Objective: The overall aim of this thesis was to investigate the relationship between IgE-sensitisation and allergic disease, viral infections, genetic markers and early life cytokines.</p><p>Results: IgE-sensitised children were found to have reduced numbers of IL-12 producing cord blood mononuclear cells (CBMC), whereas children diagnosed with eczema were found to have reduced numbers of IFN-γ producing CBMC. When dividing the children into early onset of IgE-sensitisation and late onset of IgE-sensitisation we found that the children with an early onset had low numbers of PHA-induced IL-4, IL-12 and IFN-γ secreting CBMC. At the age of two there was a general exacerbation of cytokine responses in the IgE-sensitised children, and the results were similar for the children with early onset IgE-sensitisation. Children with a late onset IgE-sensitisation were more similar to the non-sensitised children, but with a specific increase in the response to cat allergen (IL-4 and IFN-γ). The mothers of IgE-sensitised children, were just as their children, found to have an exaggerated cytokine response as compared to mothers of non-sensitised children. Maternal responses correlated well to the responses seen in the child, though the samples were taken two years after delivery.</p><p>Cytomegalovirus (CMV) infection in early life was associated to reduced numbers of IL-4, and increased numbers of IFN-γ producing cells at the age of two. No association between CMV seropositivity and IgE-sensitisation was seen. Epstein-Barr virus (EBV) infection, on the other hand, was inversely correlated with IgE –sensitisation, whereas no statistically significant association to cytokine production could be seen.</p><p>We also showed that the IL12B 1188 C-allele was associated to having a positive skin prick test at the age of two. The rare alleles of the three SNPs investigated (IL12B 1188C, IL12RB1132C and IRF1 1688A) were all associated to low IL-12 production at birth.</p><p>Conclusions: Our results indicate that allergic diseases are complex traits, and that both the genetic and the cytokine background differ between the different allergic diseases. We can also conclude that the time of onset seem to play a role when investigating IgE-sensitisation, and that perhaps early and late onset IgE-sensitisation have partly different causes. CMV and EBV infection early in life are associated to a protective cytokine profile and to protection from IgE-sensitisation, respectively, again indicating the heterogeneity and the complexity of allergic diseases.</p>

Thesis (MSC)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: South Africa is shouldering the highest burden of HIV-infection. Inter-individual differences in response to antiretroviral treatment (ART) and the limited availability of second and third-line ART regimens call for optimising first-line ART in South African populations. Measuring antiretroviral drug levels in patients may be of clinical value as an intermediate indicator of treatment response and may moreover serve to assess the genetic variation underlying differential drug exposure. This study aimed to determine the effect of SNPs in the CYP2B6 gene and efavirenz (EFV) levels measured in hair on ART outcomes in females of two South African populations. Female Xhosa (XH) (n = 81) and Mixed Ancestry (MA) (n = 53) patients receiving the first-line regimen component EFV for at least three months donated saliva for genomic DNA extraction and 20 strands of hair for determination of EFV concentrations by high performance liquid chromatography. Regulatory and exonic regions in the CYP2B6 gene, which codes for the major metabolising enzyme of EFV, were subjected to bi-directional sequence analysis in 15 XH and 15 MA individuals to assess common genetic variation in these populations. Out of 45 single nucleotide polymorphisms (SNPs) identified, 17 SNPs of known or predicted functional importance in EFV metabolism, including four novel SNPs, were genotyped in the entire patient cohort by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis. All SNPs were tested for Hardy-Weinberg equilibrium (HWE) and maximum likelihood haplotypes and assessed for an association with EFV levels measured in hair, likelihood of developing adverse drug reactions (ADRs) and virological response to EFV-based treatment. After correcting for age and ethnicity, homozygous carriers of c.516G>T (CYP2B6*6) had significantly increased EFV levels (p = 0.0021; mean: 12.0 ng/mg; IQR: 3.95 – 6.99 ng/mg; n = 12), as did heterozygotes of c.983T>C (CYP2B6*18) (p = 0.0005; mean: 7.315 ng/mg; IQR: 6.59 – 15.10 ng/mg; n = 10). No CYP2B6*18 homozygotes were detected. No association between EFV levels and virological response was evident (p = 0.8467), but CYP2B6*6 predicted increased odds of virological failure (VL > 80 copies/ml) after correcting for adherence, race, age, weight, time on treatment, baseline CD4, smoking, alcohol and WHO disease stage (p = 0.0328). Carriers of the CYP2B6*1 allele had increased odds (OR = 5) of favourable treatment outcome (VL < 80 copies/ml). In accordance with other studies, this study provides evidence that genetically predisposed poor metabolisers of EFV may be at increased risk of virological failure, possibly following non-adherence. Concurrently, these patients may be more vulnerable to adverse drug reactions and are more frequent in the XH (13%) than MA (4%). These results should be verified in larger patient cohorts, but contribute to a better understanding of the effect of genetic factors on EFV exposure and ART outcome in two South African populations. The outcomes of this study may thus provide recommendations for prospective studies and impact future clinical decisions. / AFRIKAANSE OPSOMMING: Suid-Afrika dra die grootste las van MIV-infeksies. Inter-individuele verskille in reaksie op anti-retrovirale terapie (ART) en die beperkte beskikbaarheid van tweede- en derde-linie ART-reekse regverdig die optimisering van eerste-linie ART in Suid-Afrikaanse bevolkings. Meting van antiretrovirale middel-vlakke in pasiënte, as ‘n intermediêre aanduiding van reaksie op behandeling, kan van kliniese belang wees en kan ook die waarde van die bepaling van genetiese variasie, onderliggend aan differensiële blootstelling aan middels, bepaal. Die doel van hierdie studie is om die effek van enkel-nukleotied polimorfismes (SNPs) in die CYP2B6-geen en efavirenz (EFV)-vlakke in hare op ART-uitkoms te bepaal in vroue van twee Suid-Afrikaanse bevolkingsgroepe. Vroulike Xhosa (XH) (n = 81) en Gemengde Herkoms (GH) (n = 53) pasiënte wat EFV as deel van eerste-linie ART vir ten minste drie maande ontvang het, het speekselmonsters vir genomiese DNA-ekstraksie en 20 hare vir die bepaling van EFV-konsentrasies deur hoë werkverrigting vloeistofchromatografie (“HPLC”) geskenk. Regulatoriese en eksoniese areas in die CYP2B6-geen, wat vir die vernaamste metaboliserende ensiem van EFV kodeer, is deur middel van tweerigting-volgordebepalings-analise in 15 XH en 15 GH individue ondersoek om gemeenskaplike genetiese variasie in hierdie bevolkings te bepaal. Uit ‘n totaal van 45 SNPs wat geïdentifiseer is, is 17 SNPs wat bekende of voorspelde belangrike rolle in EFV-metabolisme speel, insluitend vier nuwe SNPs, ondersoek. Hierdie SNPs is in die volledige pasiënt-kohort gegenotipeer deur polimerase-ketting reaksie gebaseerde restriksie fragment lengte-polimorfisme (PKR-RFLP) analise. Alle SNPs is getoets vir Hardy-Weinberg-ewewig (HWE) en maksimum waarskynlikheidshaplotipes en is geassesseer vir assosiasie met EFV-vlakke gemeet in hare, die waarskynlikheid om ongunstige reaksies tot die middel te ontwikkel en virologiese reaksie op EFV-gebaseerde behandeling. Nadat vir ouderdom en herkoms gekorrigeer is, het homosigotiese draers van c.516G>T (CYP2B6*6) beduidend verhoogde EFV-vlakke (p = 0.0021; gemiddeld: 12.0 ng/mg; IQR: 3.95 – 6.99; n=12) getoon, so ook heterosigote vir c.983T>C (CYP2B6*18) (p = 0.0005; gemiddeld: 7.315 ng/mg; IQR: 6.59 – 15.10 ng/mg; n = 10). Geen CYP2B6*18 homosigote is gevind nie. Daarbenewens is geen duidelike assosiasie tussen EFV-vlakke en virologiese reaksie gevind nie (p = 0.8467), maar CYP2B6*6 het verhoogde waarskynlikheid op virologiese mislukking (VL > 80 kopieë/ml) getoon nadat daar vir mddel-getrouheid, ras, ouderdom, gewig, tydsduur van behandeling, basis-CD4, rook, alkohol en Wêreld Gesondheids Organisasie siekte-fase gekorrigeer is (p = 0.0328). Draers van die CYP2B6*1-alleel het verhoogde waarskynlikheid (OR = 5) op gunstige behandelingsuitkomste getoon (VL < 80 kopieë/ml). In ooreenstemming met ander studies verskaf hierdie studie bewyse dat pasiënte wat geneties geneig is tot stadige metabolisme van EFV ‘n hoër risiko kan hê vir virologiese mislukking, wat moontlik ‘n gevolg is van middel-ontrouheid. Hierdie pasiënte kan ook meer geneig wees tot vatbaarheid vir ongunstige middel-reaksie en kom meer voor in die XH (13%) as in die MA (4%). Hierdie resultate moet in groter pasiënt-kohorte gestaaf word, maar dra by tot ‘n beter begrip van die effek van genetiese faktore op blootstelling aan EFV en ART-uitkoms in twee Suid-Afrikaanse bevolkings. Die uitkomste van hierdie studie kan dus as aanbevelings gebruik word vir voornemende studies en ook toekomstige kliniese besluite beïnvloed. / The Medical Research Fund (MRC) for funding this project. The University Centre for Studies in Namibia (TUCSIN) and Deutscher Akademischer Austausch-Dienst (DAAD) for financial support

Introduction: Despite many advances in therapeutic and surgical techniques for glioblastoma multiforme (GBM), this form of brain cancer still remains incurable. A hallmark feature of GBM is the ability of the glioma cells to infiltrate surrounding brain tissue. The invasive nature of glioma cells is a key challenge in considering treatment for patients with GBM. Certain members of the matrix metalloproteinase (MMP) family play a role in tumor cell invasion and metastasis (Coussens, et al., 2002). A functional SNP resulting from an additional guanine at position -1607 in the MMP-1 promoter creates an erythroblastosis twenty six transcription factor protein (ETS) DNA consensus binding site, which results in significantly higher transcriptional activity of MMP-1 (Rutter et al., 1998). Several published studies show the incidence of this 2G allele is significantly higher in aggressive and metastatic tumors. Binding of an adjacent transcription factor DNA consensus site, activator protein -1 (AP1) site at -1607 has been shown to cooperate with ETS binding to activate transcription of the MMP-1 gene. We have reported a significant increase in the 2G/2G MMP-1 genotype in glioblastomas (pPurpose: To determine if a novel SNP decoy can inhibit the 2G genotype-dependent increase in MMP-1 transcriptional activity, three specific aims were tested: one, to verify specificity of binding of a transcription factor decoy designed to mimic the -1607 SNP site within the MMP-1 promoter; two, to determine the effect of transcription factor decoy ODN on transcriptional activity of an MMP-1 promoter containing the 2G SNP at -1607; and three, to assess the effect of the transcription factor decoy ODN on MMP-1 mRNA and protein expression in treated glioma cells. Methods: Modified and unmodified decoys were designed to mimic position -1607 to -1593 of the MMP-1 promoter. The SNP decoy contains both ETS and AP1 DNA consensus sites and MMP-1 flanking sequences. We first determined optimal binding conditions with electromobility shift assays (EMSAs). The EMSA assays were used to determine the presence of Ets-1 and AP1 DNA binding activity within the glioma cell lines, T98 and U87. EMSAs were also used to determine if these transcription factors could bind to the MMP-1 promoters with and without the SNP. Lastly, EMSAs were done to determine the binding characteristics of the two modified SNP decoys (LNA-locked nucleic acid, and a PS-phosphothioate modification). The effect of the decoy on MMP-1 transcriptional activity was assessed using a Dual-Luciferase Reporter Assay. The effect of the SNP decoys on mRNA was assessed using quantitative RT-PCR, and on protein expression using a sandwich enzyme-linked immunoassay (ELISAs). Statistical analysis was done using a two-way ANOVA to evaluate the effect of the decoy on MMP-1 transcriptional activity, and protein expression. Results: EMSA results indicate that Ets-1 and AP1 probes, and MMP-1 promoter probes effectively bind proteins from glioma cell nuclear extracts. Addition of excess decoy was able to inhibit protein interactions with the 2G MMP-1 promoter probe and to a lesser extent the 1G promoter probe. The scrambled decoy had no effect. Promoter studies showed a significant increase in transcriptional activity of the 2G promoter and addition of 5 mm PS-SNP decoy could effectively prevent the increase in activity (pConclusions: U87 and T98 cell lines contain DNA binding activity of the transcription factors of interest, namely ETS-1 and AP1. The candidate transcription factors can bind to the MMP-1 promoter in the presence or absence of the 2G. Both the LNA and PS-SNP modified decoys can inhibit nuclear proteins from binding to the MMP-1 2G promoter. The PS-SNP decoy was able to inhibit MMP-1 (2G) gene transcription in a dose dependent manner, whereas the control decoy showed a consistent non-specific effect. The PS-SNP decoy inhibited MMP-1 mRNA and protein expression in glioma cells containing the 2G genotype, and to lesser extent in glioma cells containing the 1G genotype. The results presented here support the conclusion that the chimeric SNP decoy can selectively inhibit the MMP-1 promoter containing the 2G genotype.

La maladie d'Alzheimer (MA) est une maladie neurodégénérative irréversible et progressive entraînant des troubles cognitifs et comportementaux. L'inflammation est caractéristique de la MA. L'interleukine-10 (IL-10), une cytokine anti-inflammatoire a été associée à un risque plus faible de développer la MA. Cependant le lien entre l'IL-10 et la progression de la MA n'a jamais été étudié. Le but de cette thèse a été d'étudier le rôle de l'IL-10 dans le développement ainsi que dans la progression de la MA. Pour mener à bien cette étude, 31 sujets atteints par la MA et 20 sujets contrôles cognitivement intacts ont été recrutés. En fonction de la vitesse de diminution du test de Mini-Mental State Examination et de l’évolution des troubles cognitifs sur deux ans, les patients souffrant de la MA ont été divisés en deux sous-groupes: les patients avec une progression lente (MA lent) et ceux avec une progression rapide (MA rapide). Les analyses se sont portées sur la concentration d’IL-10 en périphérie (plasma, production par les cellules mononuclées du sang périphérique (PBMCs) après stimulation par les peptides Aβ) ainsi que le polymorphisme de son promoteur en position -592, -819 et -1082. En complément, d’autres cytokines impliquées dans l’inflammation ont été étudiées : l’IL-6 (sa concentration plasmatique, sa production par les PBMCs à la suite d’une stimulation par les peptides Aβ et son polymorphisme en position -174) et les polymorphismes du TGF-β1 (-10 et - 25), de l’IFN-γ (-874) et du TNF-α (-308) ainsi que le gène de l'apolipoprotéine E (ApoE). Une étude de la longueur des télomères, liée à l’inflammation, a été aussi réalisée. Les résultats ont montré une association entre le génotype AA et l’allèle A du polymorphisme de l’IFN-γ en position -874 avec la progression rapide de la MA. Une augmentation statistiquement significative de la production d'IL-10 après stimulation par les peptides Aβ a été montrée chez les patients atteints avec une progression lente (MA lent). Une longueur significativement plus courte des télomères a été aussi associée aux patients MA lent. L’ensemble de ces travaux suggère qu’un profil de forte production de l’IL-10 ainsi qu’un profil génétique d’IFN-γ (TT -874) pourrait ralentir la progression de la MA. Il est aussi apparu que la longueur des télomères pourrait être un marqueur du déficit cognitif. Il est clair que ces résultats préliminaires ont besoin d’être confirmés par une étude de plus grande envergure, avec un nombre de patients plus élevé. / Alzheimer’s disease (AD) is an irreversible and progressive neurodegenerative disorder leading to cognitive and behavioral impairment. Inflammation is hallmark of AD although the exact mechanisms involved and the roles of the different inflammatory components are far less clear. Interleukin-10 (IL-10) is a key anti-inflammatory cytokine and IL-10 -1082 A > G polymorphism has been associated with a lower risk of developing AD although the link between IL-10 and the AD progression have never been studied. The aim of this study is to study the role of IL-10 in the risk of developing AD and its role in AD progression. In order to complete successfully this study, 31 AD patients and 20 cognitively intact controls were recruited. Depending of the rate of decrease of mini-mental state test examination (MMSE) and evolution of cognitive disorders, AD patients were divided in two subgroups: patients with slow progression (AD slow) and those with fast progression (AD fast). Analysis were focused on periphery concentration of IL-10 (plasma and and its production by peripheral blood mononuclear cells (PBMCs) after Aβ peptides stimulation) as well as its promoter polymorphism in position -592, -819 and -1082. In addition, other cytokines involved in inflammation were studied: IL-6 (its plasma concentration, its production by PBMCs following stimulation with Aβ peptides and its polymorphism at position -174) and polymorphisms of TGF-β1 (-10 to - 25), IFN-γ (-874) and TNF-α (-308) as well as the gene polymorphisms of Apolipoprotein E (ApoE). A study of telomere length, link to inflammation, was also performed. Results showed IFNγ -874AA genotype and -874A allele was associated with AD fast progression. A statistically significant increase of IL-10 production by PBMCs stimulated with Aβ peptides was shown in AD slow patients. A significantly shorter telomere length was also associated with AD slow patients. All of this work suggests that a profile with high IL-10 production and high IFN-γ (-874 TT) genotype could confer a slower AD progression. It was also found that telomere length may be a marker of cognitive impairment. It is clear that these preliminary results need to be confirmed in a larger study with a larger number of patients.