The emerging field of personalized medicine and the prediction of side effects experienced due to pharmaceutical drugs is being studied intensively in the post genomic era. The molecular basis of inheritance and disease susceptibility is being unravelled, especially through the use of rapidly evolving new technologies. This in turn facilitates analyses of individual variations in the whole genome of both single subjects and large groups of subjects. Genetic variation is a common occurrence and although most genetic variations do not have any apparent effect on the gene product some do exhibit effects, such as an altered ability to detoxify xenobiotics. The human body has a highly effective detoxification system that detoxifies and excretes endogenous as well as exogenous toxins. Numerous studies have proved that specific genetic variations have an influence on the efficacy of the metabolism of pharmaceutical drugs and consequently the dosage administered. The primary aim of this project was the local implementation and assessment of two different genotyping approaches namely: the Applied Biosystems SNaPshot technique and Affymetrix DMET microarray. A secondary aim was to investigate if links could be found between the genetic data and the biochemical detoxification profile of participants. I investigated the approaches and gained insight into which method would be better for specific local applications, taking into consideration the robustness and ease of implementation as well as cost effectiveness in terms of data generated. The final study cohort comprised of 18 participants whose detoxification profiles were known. Genotyping was performed using the DMET microarray and SNaPshot techniques. The SNaPshot technique was used to genotype 11 SNPs relating to DNA repair and detoxification and was performed locally. Each DMET microarray delivers significantly more data in that it genotypes 1931 genetic markers relating to drug metabolism and transport. Due to the absence of a local service supplier, the DMET - microarrays were outsourced to DNALink in South Korea. DNALink generated raw data which was analysed locally. I experienced many problems with the implementation of the SNaPshot technique. Numerous avenues of troubleshooting were explored with varying degrees of success. I concluded that SNaPshot technology is not the best suited approach for genotyping. Data obtained from the DMET microarray was fed into the DMET console software to obtain genotypes and subsequently analysed with the help of the NWU statistical consultation services. Two approaches were followed: firstly, clustering the data and, secondly, a targeted gene approach. Neither of the two methods was able to establish a relationship between the DMET genotyping data and the detoxification profiling. For future studies to successfully correlate SNPs or SNP groups and a specific detoxification profile, two key issues should be addressed: i) The procedure for determining the detoxification profile following substrate loading should be further refined by more frequent sampling after substrate loading. ii) The number of participants should be increased to provide statistical power that will enable a true representation of the particular genetic markers in the specific population. The statistical analyses, such as latent class analyses to cluster the participants will also be of much more use for data analyses and interpretation if the study is not underpowered. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

Orientador: Domingos Alves Meira / Banca: Rogério de Jesus Pedro / Banca: David Salomão Lewi / Banca: Alexandrina Sartori / Banca: Maria Inês de Moura Campos Pardini / Resumo: Iniciativas como a "WHO/UNAIDS '3 by 5' permitiram que se atingisse, no ano de 2007, a marca de 3 milhões de pessoas com acesso à terapia antiretroviral (TARV) em países de baixa e média renda. O aumento da cobertura nestes países demanda custos importantes com anti-retrovirais, porém também levanta outro problema, que é o monitoramento da terapia em localidades de poucos recursos. Há consenso no fato de que devem ser pesquisados marcadores de eficácia da TARV mais acessíveis. Considerando o comportamento da β-2 microglobulina sérica e das citocinas séricas TNF-α, IFN-γ, IL-2, IL-4 e IL-10 com relação à atividade inflamatória induzida pela replicação do HIV-1, o objetivo deste estudo foi o de verificar o comportamento destas substâncias como indicadores da presença, ou não, de falha terapêutica à HAART. Entre agosto de 2004 e novembro de 2005, 89 indivíduos infectados pelo HIV-1, atendidos pela Área de Doenças Tropicais da Faculdade de Medicina de Botucatu-UNESP, e 20 indivíduos normais, doadores de sangue do Hemocentro de Botucatu [43 mulheres e 66 homens; idade média = 39,7 anos (22 - 66 anos)] foram divididos em 4 grupos: G1- 15 indivíduos infectados pelo HIV-1, virgens de tratamento ou sem HAART há pelo menos seis meses e com contagens de linfócitos T CD4 + menores que 350 células/mm3; G2- 31 indivíduos infectados pelo HIV-1, em uso de HAART e sem falha terapêutica virológica (FT); G3- formado por 43 indivíduos infectados pelo HIV-1, em uso de HAART e com FT, e GC- formado por 20 indivíduos normais, não infectados pelo HIV-1, que serviram de controles para as citocinas séricas. Foram revisados os dados demográficos, clínicos e de HAART e realizados os exames β-2 microglobulina sérica, citocinas séricas (TNF-α, IFN-γ, IL-2, IL-4 e IL-10), genotipagem do HIV-1, carga viral plasmática (CV) e linfócitos T CD4 + e T CD8 +. Para... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Initiatives such as WHO/UNAIDS '3 by 5' made it possible to achieve the figure of 3 million people with access to antiretroviral therapy (ART) in middle- and low-income countries in 2007. The increase in these countries' coverage leads to important expenditure on antiretroviral drugs; however, it also raises another problem, which is therapy monitoring in low-income locations. There is agreement on the fact that more accessible ART efficacy markers must be studied. By considering the behavior of serum β-2 microglobulin and serum cytokines TNF-α, IFN-γ, IL-2, IL-4 and IL-10 in relation to inflammatory activity induced by HIV-1 replication, the objective of this study was to assess the behavior of such substances as indicators of the presence, or not, of antiretroviral therapeutic failure (TF). From August 2004 to November 2005, 89 HIV-1-infected individuals assisted by the Tropical Diseases Sector of the Botucatu School of Medicine - UNESP and 20 normal blood donors at the Blood Transfusion Center of Botucatu [43 female and 66 male; mean age = 39.7 years (22 - 66 years)] were divided into 4 groups: G1- 15 HIV-1-infected individuals, previously untreated or without HAART for at least six months and CD4 + < 350 cells/mm3; G2- 31 HIV-1-infected individuals undergoing HAART without virological therapeutic failure (TF), G3- 43 HIV-1-infected individuals undergoing HAART with TF, and CG- 20 normal individuals who served as controls for serum cytokines. Demographic, clinical and HAART data were reviewed, and serum β-2 microglobulin, serum cytokines (TNFα, IFN-γ, IL-2, IL-4 and IL-10), HIV-1 genotyping, plasma viral load (VL) and T CD4 + and T CD8 + lymphocytes tests were performed. The Mann-Whitney test for independent samples was used for between-group comparison in the case of numeric variables, and Fisher's exact test was applied for category variables. Statistical difference... (Complete abstract click electronic access below) / Doutor

The prevalence and molecular characterization of Cryptosporidium spp. in slaughtered cattle 6 months and older was performed. Three species of Cryptosporidium were identified. A subtype of C. parvum was obtained.

Magister Scientiae - MSc / Autism, Aspergers Syndrome and Pervasive Developmental Delay-Not Otherwise Specified (PDD-NOS), among others, fall under an umbrella of disorders known as Autism Spectrum Disorder. Twin studies show that autism is a highly heritable disorder. More than 100 genes have been implicated in the aetiology of autism, each of which is involved in numerous biological processes and a variety of molecular interactions. William-Beuren syndrome is a multisystem developmental disorder caused by the deletion of contiguous genes at the 7q11.23 position. The aims of this study were (i) to genotype three SNPs (rs10279013, rs2293484 and rs17060) in the ABHD11 and ABCA13 genes, respectively, using Taqman® SNP Genotyping assays to detect association with autism in three distinct South African (SA) ethnic groups (Black, Caucasian and Mixed), and (ii) to ascertain common pathways or regulating transcription factors for genes on chromosome 7 that may attribute to it being an “autism hotspot”. Chapter 3 objectives were to identify potential candidate genes using STRING analysis and the Gene Cards database. The Taqman® study indicated significant association for SNP rs2293484 in the South African Caucasian group, as well as for the G allele in the South African Mixed group, where p<0.001. STRING analysis yielded 2 new candidate genes, FZD1 and FZD9. It was also found that the Wnt pathway in mammals plays a significant role in both ASDs and cancer, and there is a definite link between genes regulating cancer, and genes implicated in autism. The study provides evidence for not only the association of the investigated SNP in a South African population, but also provides evidence for the co-morbidity of several neurological and psychological disorders such as depression and bipolar disorder with autism.

Philosophiae Doctor - PhD / Solute carrier transporters belonging to the major facilitator family of membrane transporter are increasingly being recognized as a possible mechanism to explain inter-individual variation in drug efficacy and response. Genetic factors are estimated to be responsible for approximately 15-30% of inter-individual variation in drug disposition and response. The aims of this study were to determine the minor allele frequencies of 78 previously identified single nucleotide polymorphisms in the pharmacogenetically relevant SLC22A1-3 and SLC47A1 genes in the indigenous African population of South Africa. Secondly, to determine whether allele and genotype frequencies for these SNP were different from that reported for other African, Caucasian, and Asian populations. Thirdly, to infer haplotypes from the genetic information which can potentially be used in future to design and interpret results of pharmacogenetics association studies involving these genes and their substrate drugs. Finally, to determine whether the Xhosa population harbour novel SNPs in the SLC22A2 gene, that encodes the kidney-specific hOCT2. SNaPshot™ multiplex single base minisequencing systems were developed and optimized for each of SLC22A1, SLC22A2, SLC22A3, and SLC47A1 covering the previously identified 78 SNPs. These systems were then used to genotype the alleles of 148 healthy Xhosa subjects residing in Cape Town, South Africa. In addition, the proximal promoter region and all 11 exons and flanking regions of the SLC22A2 gene of 96 of the participants were screened for novel SNPs by direct sequencing. The Xhosa subjects investigated lacked heterozygosity and were monomorphic for 91% of the SNPs screened. None of the SLC22A3 and SLC47A1 SNPs investigated was observed in this study. Sequencing of the SLC22A2 gene revealed 28 SNPs, including seven novel polymorphic sites, in the 96 Xhosa subjects that were screened. The minor allele frequencies of the seven previously identified variant SNPs observed in this study were different compared to that observed for American and European Caucasian, and Asian populations. Moreover, the allele frequencies for these SNPs differed amongst African populations themselves. Eight and seven haplotypes were inferred for SLC22A1 and SLC22A2, respectively, for the Xhosa population from the information gathered with SNaPshot™ genotyping. This study highlights the fact that African populations do not have the same allele frequencies for SNPs in pharmacogenetically relevant genes. Furthermore, the Xhosa and other African populations do not share all reduced function variants of the SLC22A1-3 and SLC47A1 genes with Caucasian and Asian populations. Moreover, this study has demonstrated that the Xhosa population harbours novel and rare genetic polymorphisms in the key pharmacogene SLC22A2. This study lays the foundation for the design and interpretation of future pharmacogenetic association studies between the variant alleles of the SLC22A1-3 and SLC47A1 genes in the Xhosa population and drug disposition and efficacy.

Magister Public Health - MPH / HIV/AIDS is a major health problem in Namibia with HIV prevalence estimated at 18.2% among pregnant women. Antiretroviral therapy (ART) was introduced in the public sector in 2003 and ART roll out was expanded throughout the country in the subsequent years. There are 221 ART sites in Namibia which include 34 district hospitals and 187 outreach service points. Currently there are 127,486 patients registered on ART in Namibia. However, there have been cases of patients experiencing treatment failure. The treatment failure can give rise to the emergence of HIV drug resistance. Genotyping information from patients with treatment failure can be valuable for tracking the dominant mutations conferring HIV drug resistance. However, HIV genotyping is not routinely available in Namibia due to cost. It is essential to determine the risk factors associated with development of HIV drug resistance so that these factors can be addressed. The aim of the current study was to describe HIV drug resistance mutations and the risk factors associated with HIV drug resistance among patients failing first- line ART regimen in Oshikoto and Oshana regions in Namibia. The case-control study design was used to collect data from cases who were being suspected of treatment failure to the first–line regimen in Oshikoto and Oshana regions in Namibia. The demographic, clinical and genotype information was collected from patient records. Out of 168 cases, 97 cases were eligible for this study and were matched with 105 controls. The mean age was 44.8 (±13.2) years for controls and 43.3 (±13.3) years for cases. Cases from Oshana and Oshikoto regions harboured 63% and 71% respectively for nucleoside reverse transcriptase inhibitors mutations with the dominant mutation being M184V/I. Sixty-eight percent (68%) and 76% respectively harboured mutations for non-nucleoside reverse transcriptase inhibitors with dominant mutation being K103N. Missed appointments, initiating inappropriate first-line regimen and adverse events or side effects were identified as risk factors for virological failure with odd ratios (OR) of 21.58 (95% CI 6.50 -71.59); 11.70 (95% CI 1.69 - 80.99) and 7.17 (95% CI 1.89 -27.22) respectively. Patients failing the first-line regimen need to be genotyped to assess the development of HIV drug resistance. The patients initiating ART should be educated on impacts of missing clinical appointments and adverse events of the drugs in order to prevent the emergence of drug resistance.

No description available.

Sexual reproduction is an important process amongst eukaryotic organisms, with one function being to maintain genetic variation. The idea that complex eukaryotic species can persist for millions of years in the absence of sex defies fundamental evolutionary dogma, yet a group of organisms known as ancient asexuals were thought to have evolved clonally under deep evolutionary time. Prominent among these are the arbuscular mycorrhizal fungi (AMF), which are obligate plant symbionts that colonize the root cells of plants and extend their hyphae into the soil assisting the plant in acquiring key nutrients. Unlike most eukaryotes, AMF cells are multinucleate with thousands of nuclei moving through a continuous cytoplasm. Genomic analyses have identified a putative mating-type (MAT) locus within the nuclear genomes of model AMF Rhizophagus irregularis, a region that in other fungi dictates the process of sexual reproduction. Additional findings demonstrated that AMF strains carry one of two nuclear organizations. They can be either homokaryotic (AMF homokaryons), where all nuclei within the cytoplasm are virtually identical, or heterokaryotic (AMF dikaryons), where two MAT-locus variants co-exist within the cytoplasm. Despite a lack of observable traits indicative of sex, this homo/heterokaryotic dichotomy is reminiscent of the nuclear organization of sexual fungi. My research aims to build on these findings to investigate the actual role of the MAT-locus in driving AMF reproduction. To address this, I build my thesis into three main chapters. The first chapter reviews our current understanding of AMF genetics and what drives genome evolution in these organisms. The second chapter establishes a relatively easy, inexpensive, and reproducible approach to genotype known MAT variants of R. irregularis in natural and experimental conditions. The last chapter uses experimental crossings between strains to assess cytoplasmic compatibility and nuclear exchange. I demonstrate that dikaryotic spore progenies can be formed after co-culturing two distinct AMF homokaryotic strains. Further analyses of various genomic regions also reveal possible recombination in homokaryotic spore progenies from co-cultures. Overall, this research provides new experimental insights into the origin of genetic diversity in AMF. These findings open avenues to produce genetically new AMF strains in the lab using conventional crossing procedures and provide a glimpse of the mechanisms that generate AMF genetic diversity in the field.

Because tuberculosis (TB) is a public health threat that continues to elude elimination in the United States, there is a need to identify contributing factors that may have implications for targeted control measures. Molecular studies of genetic clustering are crucial for pinpointing these contributing factors. It is for this reason this study was conducted. This was a non-experimental, cross-sectional population-based molecular epidemiological study of TB in SC from 2005 to 2011. Its purpose was to estimate the proportion of TB that may be due to recently acquired infection and to determine the risk factors associated with the genetic clustering of identical M. tuberculosis isolates from TB patients in South Carolina from 2005-2011. The analysis sample included 627 confirmed pulmonary and/or pleural cases of TB, for which complete data on all covariates and a valid genotype were available. The results strongly suggested that about 50% of TB in South Carolina is recently transmitted. The study also revealed that being born in the United States and Black race were independently and significantly associated with being part of a TB genotype cluster. The key messages of this study were as follows: a substantial portion of TB in South Carolina is due to recent transmission, not reactivation or importation, and transmission of TB in South Carolina occurs in groups often defined by American birth and Black race. These important findings indicate that most TB in South Carolina is preventable and that enhanced TB control efforts should be explored. The implication for positive social change is that employing targeted contact investigation informed by these findings could lead to decreased disease transmission. Future studies should explore pilot programs that investigate alternatives to the traditional TB contact investigation.

This thesis is about analyzing genetic differences among isolates of Francisella tularensis – the tularemia-causing bacterium. To elucidate how these bacterial isolates are related, and their geographical and genetic origins, I have developed typing assays for Francisella and used them to study the epidemiology of tularemia. Tularemia is an infectious disease of humans and other mammals found throughout the Northern Hemisphere. The severity of the disease depends on the type of F. tularensis causing the infection. In Sweden, as in other countries of Europe and Eurasia, tularemia is caused by F. tularensis subsp. holarctica, while other varieties of the bacterium occur in Middle Asia and North America. It is important to identify a tularemia infection promptly in order to initiate the correct antibiotic treatment. A rapid identification of the causative F. tularensis variety gives additional clinical information. In recent years, several genomes of various Francisella strains have been sequenced, and in this thesis, I have utilized these genomes to identify genetic markers. In studies reported in the first paper (I) appended to the thesis, we identified and analyzed insertion/deletion mutations (INDELs) inferred to have resulted from a sequence repeat-mediated excision mechanism. We found eight new Regions of Difference (RDs) among Francisella strains. Using RDs together with single nucleotide polymorphisms (SNPs), we were able to predict an evolutionary scenario for F. tularensis in which Francisella novicida was the oldest variety while F. tularensis subsp. holarctica was the youngest. We also found that all virulence-attenuated isolates analyzed had deletions at two specific genetic regions - denoted RD18 and RD19 – suggesting that repeat-mediated excision is a mechanism of attenuation in F. tularensis. In subsequent studies (presented in paper II), we developed a combined analysis of INDELs lacking flanking repeats and variable number of tandem repeats (VNTRs). Both markers could be assayed using the same analytical equipment. The inclusion of INDELs provided increased phylogenetic robustness compared with the use of VNTRs alone, while still maintaining a high level of genetic resolution. In analyses described in the next paper (III), we selected INDELs from paper (II) and discovered novel SNPs by DNA comparisons of multiple Francisella strains. Thirty-four phylogenetically informative genetic markers were included in a hierarchical real-time PCR array for rapid and robust characterization of Francisella. We successfully used the assay to genotype 14 F. tularensis isolates from tularemia patients and DNA in six clinical ulcer specimens. Finally, in paper (IV) we demonstrated a strategy to enhance epidemiological investigations of tularemia by combining GIS-mapping of disease-transmission place collected from patient interviews, with high-resolution genotyping of F. tularensis subsp. holarctica isolates recovered from tularemia patients. We found the geographic distributions of specific F. tularensis subsp. holarctica sub-populations to be highly localized during outbreaks (infections by some genotypes being restricted to areas as small as 2 km2), indicative of a landscape epidemiology of tularemia with distinct point sources of infection. In conclusion, the results acquired during the studies underlying this thesis contribute to our understanding of the genetic genealogy of tularemia at both global and local outbreak scales.