[ Truncated abstract ] Atopic diseases such as asthma are thought to be driven to a significant extent by T helper memory cells which are programmed to respond in a harmful way to environmental allergens (e.g. house dust mite). Previous studies in humans and in animal models have established that activation of TH2 cytokine genes in T memory responses to allergens is central to the disease process. However, only a subset of atopics harbouring a TH2-memory response phenotype manifests clinical symptoms of disease. Moreover, clinical trials with TH2 antagonists in atopic patients have proven disappointing, suggesting underlying complexities in disease pathogenesis which escape regulation via these approaches. It was thus hypothesised that additional genes involved in the activation program of allergen-specific T memory cells which are central to disease pathogenesis remain unidentified. The aim of the current study was to identify such novel genes by applying microarray technology to survey genome-wide expression patterns in an in vitro model of allergen-driven human T cell activation. In contrast to previous human microarray studies in this area focusing on mitogen activated T cell lines and clones, the current study avoided the use of strong activation stimuli which have the potential to distort patterns of gene expression, and reports for the first time the findings of microarray analysis of house dust mite allergen-driven acute gene activation in recirculating T memory cells harvested from the peripheral blood of human atopics. ... Finally, methodology was established to investigate the function of the novel atopy-associated genes. In loss-of-function experiments, expression of DACT1 and CAMK2D was silenced in primary T cell responses driven by bacterial superantigens, a model system for studying T cell responses under conditions which mimic antigen-specific activation. Whilst silencing DACT1 and CAMK2D expression did not influence classical readouts of T cell function including proliferation and cytokine production, microarray profiling was employed to identify putative downstream transcriptional targets of each gene. The experimental strategy and optimised methodology presented herein can now be employed to investigate the molecular circuitry linking the novel atopy-associated genes to the T cell activation process. In conclusion, several novel genes associated with allergen-driven T memory responses in atopics have been first described in this thesis and represent logical candidates for more detailed immunological and genetic studies related to the pathogenesis of atopic diseases.

[Truncated abstract] Acute lymphoblastic leukaemia (ALL) is the most common form of cancer that affects children and the leading cause of child cancer-related death. There have been dramatic improvements in the 5-year event free survival (EFS) for childhood ALL in recent years, with EFS reaching 75-90% for some forms of the disease. Despite this success, treatment for the disease is aggressive with numerous long and short-term side effects. Many cases of ALL are characterised by chromosomal defects including translocations, variations in chromosome number and the deletion of the tumour suppressor genes. Although these gross chromosomal changes have been extensively studied in childhood ALL, the cascade of altered gene expression that results from these changes has not. Further improvements in survival and the quality of life of survivors relies on a better understanding of the underlying biology of ALL. The primary aim of this study was to determine the gene expression profile of pre-B ALL specimens and normal, or non-malignant, control cells using microarrays in order to further examine the underlying biology of childhood ALL. ... Analysis of the ALL profile with two normal haematopoietic populations demonstrated that ALL specimens have a profile similar to that of CD34+ cells. Specifically, specimens of the MLL subtype had a profile that uniformly resembled that of CD34+ cells. Other subgroups contained specimens with profiles that ranged in similarity to that of CD34+ cells, however, the gene expression profile of all ALL specimens analysed more closely resembled the CD34+ cells than the more differentiated CD19+IgM- cells. This study identified exceptionally high expression of connective tissue growth factor (CTGF/CCN2) in ALL specimens compared to control cells. CTGF expression was v restricted to B-lineage ALL specimens, however, specimens containing the E2A-PBX1 translocation showed low or no expression. Protein studies by Western blot analysis demonstrated the presence of CTGF in ALL cell-conditioned media. The study presented here provides insight into the biology of ALL including the observation that ALL cells have an immature gene expression profile similar to that of CD34+ cells and the possible existence of an autocrine loop involving CTGF. The findings may also have clinical application in the future treatment of ALL, such as the use of metabolic inhibitors or the blocking of CTGF expression. This study provides an important insight into many aspects of ALL disease biology and may offer potential new therapeutic targets for the treatment of ALL.

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Please read the abstract in Chapter 6: Summary, of this document / Dissertation (MSc (Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

In malaria-endemic regions, Plasmodium falciparum (P. falciparum) infection is characterized by extensive genetic/antigenic diversity. Describing this diversity provides important information about the local molecular epidemiology of infecting P. falciparum parasites. Intriguingly, one of the major obstacles to the development of an effective malaria vaccine has been the genetic polymorphisms exhibited by P. falciparum genes encoding targets of human immune system. This situation has necessitated the development of polyvalent vaccines with wide antigenic coverage that would increase the likelihood of vaccine efficacy that covers wide geographical areas of malaria endemic countries. Limited reports are available on the population genetic diversity and structure of P. falciparum in Malawi, and this is of particular concern as the country has put in place several interventions to combat the disease. The primary aim of the research project was to determine the genetic diversity and population structure of P. falciparum isolates and comparing complexity from four different epidemiological settings in Malawi using msp-2 gene polymorphisms. Samples were collected from four epidemiological locations in the north, centre and southern regions of Malawi. The diversity and genetic differentiation of P. falciparum populations were analyzed based on the highly polymorphic block 3 msp-2 gene. One hundred and twenty patient samples who presented with signs and symptoms of malaria and who had microscopically confirmed P. falciparum infection were enrolled in the study after they had satisfied the inclusion criteria. Parasite DNA was extracted from the blood spot on to filter paper and analyzed by genotyping the msp-2 gene using allele-specific nested PCR. A total of 28 msp-2 block 3 fragments, defined by the size and the allelic types were detected in the 102 patients. The length variants of the PCR product ranged from 240basepairs (bp) to 450bp for the K1/FC and 410bp to 780bp for the 3D7/IC allelic families. Isolates of the 3D7 alleles were predominant in the population (59 percent), compared to isolates of the K1/ FC27 alleles (41 percent) and for 3D7 and K1 most of the isolates were monoclonal infections. In comparisons between the sites, we observed the highest prevalence of mixed infection in Mwanza (46.7 percent) followed by Dwangwa (23.3 percent) compared to Bolero (16.7 percent) and Mitundu (16.7 percent). The difference in prevalence of mixed infections between Mwanza and the other sites was statistically significant (p=0.041). There was also a non-significant trend towards a higher mean genotype number per isolate in the children aged >5 years compared to those below 5 years of age. These data suggest differences in prevalence rates of mixed infections in different geographical/epidemiological settings in Malawi. Further studies are needed to confirm, with larger sample sizes, the observation of a non-significant trend towards higher multiclonality of infection in older children in malaria endemic areas of Malawi.

Heterozygous mutations in BRCA1 or BRCA2 predispose carriers to an increased risk for breast or ovarian cancer. Both BRCA1 and BRCA2 (BRCA1/2) play an integral role in promoting genomic stability through their respective actions during homologous recombination (HR) mediated repair and stalled replication fork protection from nucleolytic degradation. SMARCAD1 (SD1) is a SWI/SNF chromatin remodeler that has been implicated in promoting long-range end resection and contributes to HR. Using human cell lines, we show that SMARCAD1 promotes nucleolytic degradation in BRCA1/2-deficient cells dependent on its chromatin remodeling activity. Moreover, SMARCAD1 prevents DNA break formation and promotes fork restart at stalled replication forks. These studies identify a new role for SMARCAD1 at the replication fork. In addition to the work presented here, I discuss a method for introducing stop codons (nonsense mutations) into genes using CRISPR-mediated base editing, called iSTOP, and provide an online resource for accessing the sequence of iSTOP sgRNASs (sgSTOPs) for five base editor variants (VQR-BE3, EQR-BE3, VRER-BE3, SaBE3, and SaKKH-BE3) in humans and over 3 million targetable gene coordinates for eight eukaryotic species. Ultimately, with improvements to CRISPR base editors this method can help model and study nonsense mutations in human disease.

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