Bibliography: pages 128-163. / Prior to this study, a eDNA library of dehydrated Xerophyta viscosa was differentially screened and several genes were found to be upregulated during dehydration. One of these cDNAs was found to share a high degree of sequence identity with the ER-Iocated Hsp90 or Grp94 family of proteins (hereafter referred to as XVGrp94) and forms the basis of this work. The XVGrp94 eDNA was found to be truncated at the 5· terminus and a full length eDNA was isolated using SMART-RACE™ (§witching Mechanism gt 5' end of RNA Iranscript- Random ~mplification of Complementary .!;rids). This eDNA was sequenced and appeared to be a representative of the Hsp90 family of genes. The putative gene contained an ORF (Open Reading frame) potentially coding for an 812 amino acid protein with a calculated size of 92.83 kDa. It shares 85% homology with other Hsp90s from plants and it contains several characteristic features of these proteins. Additionally, it contains the ER (endoplasmic reticulum) targeting and retention signals. Southern blot analysis confirmed the presence of the gene in the X. viscosa genome possibly as a member of a family of closely related genes. Northern blot analysis revealed a transcript size of 2.8 kb, however, expression patterns of the transcript could not be established. Western blot analysis showed that the XVGrp94 concentration increased significantly in response to heat and dehydration, and a slight increase was observed in response to conditions of high salt, but no response was seen in response to high light, cold or exogenous ABA (abscisic acid) application. The XVGrp94 open reading frame was cloned into the pProEX HTa expression vector and expressed in E. coli, but purification of the recombinant protein was not successful.

Sleep problems and suppressed melatonin production commonly presents with core Autism Spectrum Disorder (ASD) traits. Aralkylamine N-acetyltransferase (AANAT) acts as the penultimate and rate-limiting enzyme in the melatonin biosynthetic pathway, and a study by Hu et al. (2009) reported that AANAT expression was suppressed in an ASD population with severe language impairments. The mechanism responsible for this suppressed expression is unknown. Therefore, the aim of the study was to investigate the genetic and epigenetic features of AANAT in a cohort of South African children with ASD versus children with typical development in combination with a melatonin production study to explore melatonin's contribution to ASD symptomatology. It was expected that meeting this aim would reveal DNA methylation (DNAme) modifications were statistically significant different between case and control participants. Alternatively, that DNAme features would correlate with distinct ASD traits or sleep problems and/or altered melatonin production in case participants. Biological samples and phenotypic data were collected from boys, aged between 6 and 14 years old who were assessed with the Autism Diagnostic Observation Schedule (ADOS-2). The promoter region and gene body of AANAT was sequenced (case n=26, control n=26) and DNAme analysis was performed with the Epityper massARRAY system (case n=19, control n=20). Urinary 6-hydroxymelatonin sulphate (6-OHMS) was quantified with an enzyme-linked immunosorbent assay (case n=4, control n=4). The 6-OHMS investigation was complemented with actigraphy data and a description of sleep behaviour as determined by an abbreviated version of the Children's Sleep Questionnaire. Sequence analysis found no novel single nucleotide polymorphisms and no significant differences between case and control participants. In contrast, a difference (p=0.014) in DNAme at the third CpG site in the promoter region (CpG 3) was identified in case participants assessed with ADOS-2 Module 1 in comparison to case participants assessed with ADOS-2 Modules 2 - and 3. In particular, hypomethylation was more common in participants assessed with Module 1 which is the module used to assess participants with little or no speech abilities. The transcription factor (TF) binding motifs for ZID (zinc finger protein with interaction domain), MEIS1 (Meis homeobox 1) and ZIC1 (zinc finger protein of the cerebellum 1) were identified at or near to CpG 3. These three TFs have known gene ontology terms that relate to neurodevelopment. The age of participants did not correlate with DNAme, and no further statistical significant differences were identified between the DNAme features of case and control participants, nor the correlation analysis of DNAme and ASD traits in case participants. No Module 1 participants volunteered for the 6-OHMS study, and it was therefore not possible to confirm whether DNAme features at CpG 3 correlated with altered melatonin production. The data from the study suggest that hypomethylation at the promoter region of AANAT may be related to speech impairment in ASD, and that epigenetic investigations can uncover molecular underpinnings that correlate to ASD symptomatology. Furthermore, the current study addresses the paucity of molecular information on ASD in Sub-Saharan Africa and thereby contributes to a comprehensive understanding of disease biology. It remains unknown if hypomethylation at AANAT also correlates with suppressed melatonin synthesis in ASD individuals with speech impairments and this need further investigation.

Includes bibliographical references (leaves 103-126). / This study relates to the development of an alkaliphilic, thermo-tolerant, Gram-positive isolate, Bacillus halodurans Alk36, for the over-production and surface display of chimeric gene products. This bacterium harbors the endogenous genetic background to over-produce flagellin protein continuously. In order to harness this ability, key genetic tools, such as gene targeted inactivation, were developed for this strain. The hag gene which codes for flagellin was inactivated on the chromosome giving rise to the B. halodurans BhFC0l mutant. This strain was non-motile as determined on motility plates and confirmed by PCR analysis. Motility was, however, restored through complementation of the expression vector carrying a functional hag gene. Polylinkers were inserted as in-frame, chimeric, flagellin sandwich fusions in order to identify the permissive insertion sites corresponding to the variable regions of the flagellin protein. Flagellin expression and motility were evaluated for these constructs. Two sites were identified for possible peptide insertion in the flagellin gene, one of which produced functional flagella and was able to restore the motility phenotype to a non-motile mutant. Peptides encoding a poly-histidine peptide and the HIV-l clade C gpl20 epitope were respectively incorporated into both of the permissive sites as in-frame fusions and found to be successfully displayed on the cell surface. The poly-His peptide was shown to be functional through metal binding and affinity purification studies. The display of the HIV-1 subtype C gp 120 V3 loop was also shown to be functional through immunological studies using peptide specific antibodies. Surface display of the poly-His and HIV-l epitope was shown to have improved metal binding and enhanced expression levels of the chimeric flagellin when the peptides were insel1ed at amino acid position 180 (pSECNC6). This specific site is the only insertion point that falls within the re-defined variable domain of the FliC protein from B. halodurans Alk36.

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