The identification of disease genes (genes that when mutated cause human diseases) is an important and challenging problem. Proper diagnosis, prevention, as well as care for patients require an understanding of disease pathophysiology, which is best understood when the underlying causative gene(s) or genetic element(s) are identified. While the availability of the sequenced human genome helped to lead to the discovery of more than 1,900 disease genes, the rate of disease gene discovery is still occurring at a slow pace. The use of genetic linkage methods have successfully led to the identification of numerous disease genes. However, linkage studies are ultimately restricted by available meioses (clinical samples) which result in numerous candidate disease genes. This thesis addresses candidate gene prioritizations in disease gene discovery as applied toward a genetically heterogeneous disease known as Bardet-Biedl Syndrome (BBS). Specifically, the integration of various functional information and the development of a novel comparative genomic approach (Computational Orthologous Prioritization - COP) that led to the identification of BBS3 and BBS11. Functional data integration and application of the COP method may be helpful toward the identification of other disease genes.

Schizophrenia is a complex mental disorder that is commonly characterized as deterioration of intellectual process and emotional responses and affects 1% of any given population. SNPs are single nucleotide changes that take place in DNA sequences and establish the major percentage of genomic variations. In this study, our goal was to identify SNPs as genomic markers that are related with schizophrenia and investigate the genes and pathways that are identified through the analysis of SNPs. Genome wide association studies (GWAS) analyse the whole genome of case and control groups to identify genetic variations and search for related markers, like SNPs. GWASs are the most common method to investigate genetic causes of a complex disease such as v schizophrenia because regular linkage studies are not sufficient. Out of 909,622 SNPs analysis of the dbGAP Schizophrenia genotyping data identified 25,555 SNPs with a p-value 5x10-5. Next, combined p-value approach to identify associated genes and pathways and AHP based prioritization to select biologically relevant SNPs with high statistical association are used through METU-SNP software. 6,000 SNPs had an AHP score above 0.4, which mapped to 2,500 genes suggested to be associated with schizophrenia and related conditions. In addition to previously described neurological pathways, pathway and network analysis showed enrichment of two pathways. Melanogenesis and vascular smooth muscle contraction pathways were found to be highly associated with schizophrenia. We have also shown that these pathways can be organized in one biological network, which might have a role in the molecular etiology of schizophrenia. Overall analysis results revealed two novel candidate genes SOS1 and GUCY1B3 that have a possible relation with schizophrenia.

Physicians frequently face challenges in predicting which bacterial subpopulations are likely to cause severe infections. A more accurate prediction of virulence would improve diagnostics and limit the extent of antibiotic resistance. Nowadays, bacterial pathogens can be typed with high accuracy with advanced genotyping technologies. However, effective translation of bacterial genotyping data into assessments of clinical risk remains largely unexplored. The discovery of unknown virulence genes is another key determinant of successful prediction of infectious disease outcomes. The trial-and-error method for virulence gene discovery is time-consuming and resource-intensive. Selecting candidate genes with higher precision can thus reduce the number of futile trials. Several in silico candidate gene prioritisation (CGP) methods have been proposed to aid the search for genes responsible for inherited diseases in human. It remains uninvestigated as to how the CGP concept can assist with virulence gene discovery in bacterial pathogens. The main contribution of this thesis is to demonstrate the value of translational bioinformatics methods to address challenges in virulence prediction and virulence gene discovery. This thesis studied an important perinatal bacterial pathogen, group B streptococcus (GBS), the leading cause of neonatal sepsis and meningitis in developed countries. While several antibiotic prophylactic programs have successfully reduced the number of early-onset neonatal diseases (infections that occur within 7 days of life), the prevalence of late-onset infections (infections that occur between 7??30 days of life) remained constant. In addition, the widespread use of intrapartum prophylactic antibiotics may introduce undue risk of penicillin allergy and may trigger the development of antibiotic-resistant microorganisms. To minimising such potential harm, a more targeted approach of antibiotic use is required. Distinguish virulent GBS strains from colonising counterparts thus lays the cornerstone of achieving the goal of tailored therapy. There are three aims of this thesis: 1. Prediction of virulence by analysis of bacterial genotype data: To identify markers that may be associated with GBS virulence, statistical analysis was performed on GBS genotype data consisting of 780 invasive and 132 colonising S. agalactiae isolates. From a panel of 18 molecular markers studied, only alp3 gene (which encodes a surface protein antigen commonly associated with serotype V) showed an increased association with invasive diseases (OR=2.93, p=0.0003, Fisher??s exact test). Molecular serotype II (OR=10.0, p=0.0007) was found to have a significant association with early-onset neonatal disease when compared with late-onset diseases. To investigate whether clinical outcomes can be predicted by the panel of genotype markers, logistic regression and machine learning algorithms were applied to distinguish invasive isolates from colonising isolates. Nevertheless, the predictive analysis only yielded weak predictive power (area under ROC curve, AUC: 0.56??0.71, stratified 10-fold cross-validation). It was concluded that a definitive predictive relationship between the molecular markers and clinical outcomes may be lacking, and more discriminative markers of GBS virulence are needed to be investigated. 2. Development of two computational CGP methods to assist with functional discovery of prokaryotic genes: Two in silico CGP methods were developed based on comparative genomics: statistical CGP exploits the differences in gene frequency against phenotypic groups, while inductive CGP applies supervised machine learning to identify genes with similar occurrence patterns across a range of bacterial genomes. Three rediscovery experiments were carried out to evaluate the CGP methods: a) Rediscovery of peptidoglycan genes was attempted with 417 published bacterial genome sequences. Both CGP methods achieved their best AUC >0.911 in Escherichia coli K-12 and >0.978 Streptococcus agalactiae 2603 (SA-2603) genomes, with an average improvement in precision of >3.2-fold and a maximum of >27-fold using statistical CGP. A median AUC of >0.95 could still be achieved with as few as 10 genome examples in each group in the rediscovery of the peptidoglycan metabolism genes. b) A maximum of 109-fold improvement in precision was achieved in the rediscovery of anaerobic fermentation genes. c) In the rediscovery experiment with genes of 31 metabolic pathways in SA-2603, 14 pathways achieved an AUC >0.9 and 28 pathways achieved AUC >0.8 with the best inductive CGP algorithms. The results from the rediscovery experiments demonstrated that the two CGP methods can assist with the study of functionally uncategorised genomic regions and the discovery of bacterial gene-function relationships. 3. Application of the CGP methods to discover GBS virulence genes: Both statistical and inductive CGP were applied to assist with the discovery of unknown GBS virulence factors. Among a list of hypothetical protein genes, several highly-ranked genes were plausibly involved in molecular mechanisms in GBS pathogenesis, including several genes encoding family 8 glycosyltransferase, family 1 and family 2 glycosyltransferase, multiple adhesins, streptococcal neuraminidase, staphylokinase, and other factors that may have roles in contributing to GBS virulence. Such genes may be candidates for further biological validation. In addition, the co-occurrence of these genes with currently known virulence factors suggested that the virulence mechanisms of GBS in causing perinatal diseases are multifactorial. The procedure demonstrated in this prioritisation task should assist with the discovery of virulence genes in other pathogenic bacteria.

The congenital myasthenic syndromes (CMS) are a group of rare genetic diseases affecting the neuromuscular junction (NMJ). These syndromes affect signal transmission and result in fatigable muscle weakness. In this study we performed exome analysis of 2 CMS patient cohorts and identified SYT11, a synaptotagmin inhibitor of clathrin mediated endocytosis (CME), and MGAT5B, a glycosylation protein, as potential novel CMS genes using bioinformatic analysis on the RD-Connect Genome Phenome Analysis Platform (GPAP). To validate them, we utilized morpholino knockdown models of zebrafish orthologues syt11a, syt11b, and mgat5b and conducted functional assays measuring chorion activity and escape response. Our results show that co-knockdown of syt11a/b or syt11b alone (and not mgat5b) results in a substantial neuromuscular deficit, with ablation of chorion activity and severely reduced escape response. Immunofluorescent studies showed both motor neuron growth and NMJ formation was inhibited by syt11a/b knockdown. In conclusion, syt11b causes a severe neuromuscular phenotype in zebrafish which supports SYT11 as a novel CMS-causing gene.

Os carrapatos são artrópodes hematófagos, vetores de doenças. Vacinas são uma alternativa para o seu controle, já que esses parasitas durante a infestação, estimulam as respostas imunes do hospedeiro, as quais são implicadas em sua rejeição. As glândulas salivares do parasita são importantes para permitir a alimentação e para mediar os mecanismos de escape às defesas do hospedeiro. Diversas evidências indicam que ocorre reatividade cruzada entre espécies de carrapatos e que reações de hipersensibilidade cutânea tardia (DTH) são correlacionadas com resistência ao carrapato. A possibilidade de vacinar cachorros que são parasitados pelo R. sanguineus com antígenos do carrapato do boi, o R microplus, foi investigada por meio da análise in silico de 45 GIs de R microplus clonados em vetor plasmidial (TOPO VR2001), dentre os quais 14 Gls de R microplus se revelaram mais similares a sequências do R sanguineus, e foram empregados para avaliar: 1) A capacidade em elicitar reações cutâneas tardias em cobaias imunes a carrapatos por meio de infestações prévias com R sanguineus. 2) A capacidade de vacinas contendo GIs individuais em afetar infestações de camundongos com adultos de R sanguineus. 3) a capacidade do GI induzir anticorpos específicos após vacinação em camundongos. Dos 14 GIs testados, apenas dois não induziram reações cutâneas, quatro não afetaram nenhum parâmetro parasitológico da infestação, e três não induziram a produção de anticorpos nesses animais. Dentre os GIs, sete foram escolhidos para compor uma vacina multigênica contra o carrapato do cão R sanguineus. A vacina foi capaz de induzir resistência á infestação por R sanguineus em camundongos e em cachorros vacinados evidenciadoa pela diminuição do número de fêmeas que conseguiram colocar ovos, do peso médio da massa de ovos produzidos por essas fêmeas, do índice reprodutivo dessas fêmeas, e da taxa de eclosão das larvas, demonstrando que GIs de R microplus podem ser alvos para formulação de uma vacina contra o carrapato R sanguineus. / Ticks are arthropod vectors of disease. Vaccines are an alternative to chemicals for controlling ticks because during infestations these parasites stimulate host immune responses such as delayed hypersensitivity skin reactions (DTH), which are involved in their rejection and are correlated with resistance to ticks. Tick salivary glands are important for the parasite to acquire blood meals because their products mediate escape mechanisms from host defenses. The possibility of vaccinating dogs against infestations with the brown dog tick, Rhipicephalus sanguineus, with antigens derived from salivary glands of the cattle tick, R. microplus, was investigated by in silica analysis of 45 genes from R. microplus. These genes were targeted because of their putative biological function and had been cloned into the plasmid vector TOPO VR2001. Of them, 14 were chosen to be evaluated in a vaccine because their sequences were the most similar to several genes expressed in salivary glands of R. sanguineus. The plasmids containing the genes of interest (GIs) were used to assess: 1) The ability of the product of the genes to elicit delayed skin reactions in guinea pigs immune to ticks by previous infestations with R sanguineus. 2) The ability of individual GIs delivered as DNA vaccines to affect infestations of mice with adult R sanguineus. 3) The ability of the genes to induce specific antibodies after vaccination in mice. Only two of the 14 genes delivered to guinea pigs via intradermal injection of DNA did not elicit delayed skin reaction, four used in a vaccine did not affect any parameter of tick infestations, and three did not induce production of antibodies in these animals after DNA vaccination. Of the 14 genes, seven were chosen to formulate a multigene vaccine against the dog tick R. sanguineus. The vaccine was able to significantly affect several parameters of infestations by R sanguineus in vaccinated dogs and mice. This was reflected in the reduction of the number of females that were able to lay eggs, of the average weight of the egg mass produced by these females, of the reproductive rate of these females, and of hatching rate of larvae, demonstrating that GIs from R microplus may be targets for development of a vaccine against the tick R sanguineus.

Thesis (MSc (Genetics. Institute of Plant Biotechnology))--Stellenbosch University, 2008. / Traditional methods used for the discovery of novel genes have previously relied upon the ability to culture the relevant microbes and then demonstrate the activity of a specific enzyme. Although these methods have proved successful in the past, they severely limit our access to the genomes of organisms which are not able to be cultured under laboratory conditions. It was therefore the aim of this project to use metagenomic strategies for the identification of novel polymer-producing genes with the prospect of commercial exploitation. In this study, soil-derived metagenomic libraries were functionally screened for potential -glucan producing clones using aniline blue staining. Positive reacting clones were selected and sequenced. Initial sequencing revealed a gene with high homology to previously described glucan synthases, the products of these genes all having significant industrial value. The clone was transformed into a suitable bacterial host, cultured and allowed to produce the polymer of interest. The polysaccharide was purified and subjected to various chemical analyses so as to confirm its monosaccharide composition. Data suggests that this polymer is composed mainly of glucose units and that it may be secreted out of the cell. Purification of the active enzyme was attempted using classical protein purification methods with faint activity being detected using Native polyacrylamide gel electrophoresis (PAGE). Further attempts to demonstrate activity were made through the construction of a GST (glutathione S-transferase) tagged fusion protein. The second part of this study focuses on the construction and screening of a metagenomic DNA library from whey, a by-product of the cheese manufacturing process. It was envisaged that this could provide a resource for the identification of high value polymers when lactose is provided as a sole carbon source. The library was screened for function using Congo Red for the detection of extra-cellular polysaccharides.

Os carrapatos são artrópodes hematófagos, vetores de doenças. Vacinas são uma alternativa para o seu controle, já que esses parasitas durante a infestação, estimulam as respostas imunes do hospedeiro, as quais são implicadas em sua rejeição. As glândulas salivares do parasita são importantes para permitir a alimentação e para mediar os mecanismos de escape às defesas do hospedeiro. Diversas evidências indicam que ocorre reatividade cruzada entre espécies de carrapatos e que reações de hipersensibilidade cutânea tardia (DTH) são correlacionadas com resistência ao carrapato. A possibilidade de vacinar cachorros que são parasitados pelo R. sanguineus com antígenos do carrapato do boi, o R microplus, foi investigada por meio da análise in silico de 45 GIs de R microplus clonados em vetor plasmidial (TOPO VR2001), dentre os quais 14 Gls de R microplus se revelaram mais similares a sequências do R sanguineus, e foram empregados para avaliar: 1) A capacidade em elicitar reações cutâneas tardias em cobaias imunes a carrapatos por meio de infestações prévias com R sanguineus. 2) A capacidade de vacinas contendo GIs individuais em afetar infestações de camundongos com adultos de R sanguineus. 3) a capacidade do GI induzir anticorpos específicos após vacinação em camundongos. Dos 14 GIs testados, apenas dois não induziram reações cutâneas, quatro não afetaram nenhum parâmetro parasitológico da infestação, e três não induziram a produção de anticorpos nesses animais. Dentre os GIs, sete foram escolhidos para compor uma vacina multigênica contra o carrapato do cão R sanguineus. A vacina foi capaz de induzir resistência á infestação por R sanguineus em camundongos e em cachorros vacinados evidenciadoa pela diminuição do número de fêmeas que conseguiram colocar ovos, do peso médio da massa de ovos produzidos por essas fêmeas, do índice reprodutivo dessas fêmeas, e da taxa de eclosão das larvas, demonstrando que GIs de R microplus podem ser alvos para formulação de uma vacina contra o carrapato R sanguineus. / Ticks are arthropod vectors of disease. Vaccines are an alternative to chemicals for controlling ticks because during infestations these parasites stimulate host immune responses such as delayed hypersensitivity skin reactions (DTH), which are involved in their rejection and are correlated with resistance to ticks. Tick salivary glands are important for the parasite to acquire blood meals because their products mediate escape mechanisms from host defenses. The possibility of vaccinating dogs against infestations with the brown dog tick, Rhipicephalus sanguineus, with antigens derived from salivary glands of the cattle tick, R. microplus, was investigated by in silica analysis of 45 genes from R. microplus. These genes were targeted because of their putative biological function and had been cloned into the plasmid vector TOPO VR2001. Of them, 14 were chosen to be evaluated in a vaccine because their sequences were the most similar to several genes expressed in salivary glands of R. sanguineus. The plasmids containing the genes of interest (GIs) were used to assess: 1) The ability of the product of the genes to elicit delayed skin reactions in guinea pigs immune to ticks by previous infestations with R sanguineus. 2) The ability of individual GIs delivered as DNA vaccines to affect infestations of mice with adult R sanguineus. 3) The ability of the genes to induce specific antibodies after vaccination in mice. Only two of the 14 genes delivered to guinea pigs via intradermal injection of DNA did not elicit delayed skin reaction, four used in a vaccine did not affect any parameter of tick infestations, and three did not induce production of antibodies in these animals after DNA vaccination. Of the 14 genes, seven were chosen to formulate a multigene vaccine against the dog tick R. sanguineus. The vaccine was able to significantly affect several parameters of infestations by R sanguineus in vaccinated dogs and mice. This was reflected in the reduction of the number of females that were able to lay eggs, of the average weight of the egg mass produced by these females, of the reproductive rate of these females, and of hatching rate of larvae, demonstrating that GIs from R microplus may be targets for development of a vaccine against the tick R sanguineus.

<p><i>Trypanosoma cruzi</i>, the causative agent of Chagas´ disease, is an evolutionarily ancient species with distinct biological and immunological characteristics. A fundamental understanding of the basic biology of the parasite is necessary in order to develop reliable therapeutic and prophylactic agents against <i>T. cruzi</i>. We have, as a part of the <i>T. cruzi</i> genome project launched by the WHO, generated ESTs corresponding to about one third of the functional genes in the parasite. Only about 1/3 of the unique ESTs could be assigned a function upon sequence comparison to all publicly available data. Comparative analysis of the ESTs to functional genes in <i>S.</i> <i>cerevisiae</i> and <i>C. elegans</i> as well as to sequence data from all other kinetoplastids provided primary insights into the evolutionary divergence of <i>T. cruzi.</i> </p><p>A novel dispersed gene family (<i>DGC3</i>) was identified and shown to be present specifically on chromosome 3 and its homologue. Sequence analysis of ten isolated <i>DGC3</i> genes revealed a high sequence similarity of almost 98% among copies. The <i>DGC3</i> genes were transcribed, <i>trans</i>-spliced with the spliced leader and polyadenylated, but did not seem to have any protein-coding property. These data preliminary suggest that it encodes a novel family of functional RNA. </p><p>In the <i>T. cruzi</i> CL Brener strain, the two alleles of a single copy gene encoding the trypanothione synthetase (TcTRS) enzyme appeared to be highly polymorphic. The divergence of the deduced protein sequence was 4%, almost ten-fold higher than another protein, trypanothione reductase, involved in the same pathway. The observed allelic divergence might influence the TcTRS activity thereby having implications for drug design. Moreover, the <i>TcTRS</i> gene was found to be flanked by a number of genes involved in diverse functions and located to a pair of homologous chromosomes with a size difference of about 2 Mbp. </p><p>A gene potentially encoding the polypyrimidine-binding protein (TcPTB) was identified and characterised regarding its organisation and function. The deduced amino acid sequence was shown to comprise four RRM domains generally present in other PTBs. Interestingly, the <i>TcPTB</i> gene appeared to be expressed in a stage-specific manner implicating different functions during parasite development.</p>

Trypanosoma cruzi, the causative agent of Chagas´ disease, is an evolutionarily ancient species with distinct biological and immunological characteristics. A fundamental understanding of the basic biology of the parasite is necessary in order to develop reliable therapeutic and prophylactic agents against T. cruzi. We have, as a part of the T. cruzi genome project launched by the WHO, generated ESTs corresponding to about one third of the functional genes in the parasite. Only about 1/3 of the unique ESTs could be assigned a function upon sequence comparison to all publicly available data. Comparative analysis of the ESTs to functional genes in S. cerevisiae and C. elegans as well as to sequence data from all other kinetoplastids provided primary insights into the evolutionary divergence of T. cruzi. A novel dispersed gene family (DGC3) was identified and shown to be present specifically on chromosome 3 and its homologue. Sequence analysis of ten isolated DGC3 genes revealed a high sequence similarity of almost 98% among copies. The DGC3 genes were transcribed, trans-spliced with the spliced leader and polyadenylated, but did not seem to have any protein-coding property. These data preliminary suggest that it encodes a novel family of functional RNA. In the T. cruzi CL Brener strain, the two alleles of a single copy gene encoding the trypanothione synthetase (TcTRS) enzyme appeared to be highly polymorphic. The divergence of the deduced protein sequence was 4%, almost ten-fold higher than another protein, trypanothione reductase, involved in the same pathway. The observed allelic divergence might influence the TcTRS activity thereby having implications for drug design. Moreover, the TcTRS gene was found to be flanked by a number of genes involved in diverse functions and located to a pair of homologous chromosomes with a size difference of about 2 Mbp. A gene potentially encoding the polypyrimidine-binding protein (TcPTB) was identified and characterised regarding its organisation and function. The deduced amino acid sequence was shown to comprise four RRM domains generally present in other PTBs. Interestingly, the TcPTB gene appeared to be expressed in a stage-specific manner implicating different functions during parasite development.