Thesis (Ph.D.)--University of California, San Francisco, 2009. / Source: Dissertation Abstracts International, Volume: 70-10, Section: B, page: 5972. Adviser: Joanne N. Engel.

The highly conserved eukaryotic elongation factor 1A (eEF1A) plays a canonical role in translation elongation, where it is responsible for delivering the aminoacylated tRNA to the A-site of the 80S ribosome. Further to this essential role it is reported to be involved in a plethora of moonlighting functions that are not fully characterised or understood. One of the human isoforms, eEF1A2, is known to induce cancer when expressed in non-native tissues, although the mechanism by which it promotes tumour growth is not yet known. In this study we have characterised eEF1A overexpression in yeast and provided evidence to suggest that elevated levels of eEF1A result spindle defects which lead to chromosomal abnormalities that have the potential to induce uncontrolled cell growth in human cells. Moreover, we have confirmed conservation of this chromosomal abnormality in human cell lines suggesting that the mechanism that eEF1A utilises to induce these effects are highly conserved. We have also observed that in yeast, eEF1A overexpression results in increased metabolic activity, a hallmark of cancer cells. We hypothesise that eEF1A interacts with the dynactin complex, a regulator of spindle dynamics, resulting in aberrant spindle formation. This in turn leads to chromosomal abnormalities that appear toxic to the cell. Cells appear to overcome the toxicity induced by eEF1A by suppressing plasmid copy number to the lowest levels possible. This however, brings its own problems and appears to result in synthetic effects together with eEF1A overexpression.

Philosophiae Doctor - PhD / This study was carried out with the assumption that practical work does contribute to the teaching and learning of cell biology at Eduardo Mondlane University in Mozambique. In this regard, the main purpose of this study was to investigate the impact of practical work in the teaching and learning of cell biology concepts, specifically focussing on cell divisions concepts. It also aimed at determining the students' perceptions of the role of practical work in the learning of cell biology. On the other hand, the study sought also to understand the lecturers' practical work teaching experiences and views regarding the cell biology practical work. / South Africa

Gastrointestinal secretions are controlled mainly by the gut hormones and by the neurotransmitters released by the autonomic nervous system. The hormones and neurotransmitters (secretagogues) utilise different intracellular mediators to elicit enzyme and fluid secretion. One particular mediator is the second messenger calcium (Ca2+) and there is now much evidence that the abundant divalent cation magnesium (Mg 25 may play an important physiological role in regulating the mobilisation of cellular Ca 2t Thus, the present study was designed to investigate (a) the effect of a modification of extracellular Mg2 on secretagogue-evoked enzyme and hydrochloric acid (HCI) secretion and Ca 2 homeostasis in the pancreatic acinar cells and parietal cells and (b) to characterise the molecular mechanism of Mg 2 transport using fluorimetric and spectroscopic studies. The results have shown that application of either cholecystokinin-octapeptide (CCK-8) or acetylcholine (ACh) to rat pancreatic segments can result in marked increases in amylase and trypsinogen output in normal (1.1 mlvi) extracellular magnesium [Mg 2 0. When [Mg2 0 was elevated to 10 mlvi, there was a significant (P C 0.05) decrease in secretagogue-evoked pancreatic enzyme secretion. On the other hand, in low (0 mM) [Mg2 0 both CCK-8 and ACh elicited marked increases in enzyme secretion similar to the responses obtained in the presence of normal [Mg 2 o . The effects closely correlate with the concurrent reductions and increases in intracellular free calcium concentrations 2 +-. [Ca j ifrom studies performed with fljra-2-AM loaded pancreatic acmi dunng perturbation of [Mg2 0 These findings indicate that Mg 24 can influence enzyme secretion by regulating Ca24 mobilisation. The possible site of action of Mg2 in controlling Ca24 appears to be at the level of Ca 24 influx, since experiments, using thapsigargin or ionomycin, agents which release Ca2+ from intracellular Ca2+ stores, were not affected by a variation in [Mg 2 0 . This study also employed the technique of microspectrofluoritnetry to fi.irther characterise the mechanism of Mg2 transport using mouse pancreatic acinar cells loaded with magfisra-2-AM. Stimulation of acini with CCK-8 evoked an initial sharp rise in [Mg 2 1 followed by a decrease to a new steady-state level (Mg 24 efflux). On removal of CCK-8 [Mg2 1 returned to the pre-stimulated basal level (Mg 24 reuptake). In contrast, CCK-8 gave rise to Ca24 oscillations. When acinar cells were co-loaded with 1 ,2-bis (2- aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) (10 piM) and either magflira-2-AM or fijra-2-AM, CCK-8 evoked only the normal decrease in [Mg 2 j and a slight [Ca2 j+ ,e levation. (10 nM). This . results suggests that the in. i.t ial ns. e in. [Mg 2+-. ji seen with CCK-8 in normal conditions may be due to the Ca 24 interfering with the magfura-2-AM signal. Both thapsigargin (0.5 pM) and ionomycin (5 piM) evoked a marked decrease in [Mg 2+,. . . . 2-i-,jj in magfiira-2-AM loaded acmar cells and an elevation in [Ca j' from fi.ira-2- VAM loaded acinar cells. The results indicate that cytosolic Ca 2 is associated with secretagogue-induced decrease in [Mg 24]1. When acinar cells were pre-treated with either forskolin (10 pM), N Nitro-L-arginine (N-NLA; 2 mM), 8 -Bromo guanosine guanosine cyclic mono-phosphate (Br cGMP; 100 pM) or staurosporine (1 pM), CCK-8 elicited only a small decrease in [Mg24]j compared to a much larger response with CCK-8 alone. In contrast, genistein (10 pM) and 12-0-tetradecanoyl phorbol 13 a acetate (TPA; 1 pM) augmented the decrease in [Mg 24]1 evoked by CCK-8. The results indicate that Ca2 mobilising secretagogues can stimulate Mg 2 efflux which is also mediated by a number of intracellular mediators. This study also investigated the mechanism of Mg 2 transport from the rat pancreas. Permeabilised pancreatic acini were loaded with Mg 2 ' by employing a high Mg2 (12 mM) buffer containing the tonophore A23 187 (6 .dv1). Net Mg 2+ efflux was measured by using the technique of atomic absorption spectrophotometry. Incubation of pre-loaded acini in a buffer deficient in Mg2 resulted in a large and time-dependent release of Mg 2 with maximal efflux occuning within 40 min. Pre-treatment of loaded acini with either bumetadine, SITS or ouabain had no significant effect on Mg2 efflux. In contrast, when acini were pre-treated with either 10 mM dinitrophenol (DNP), io M amiloride, 1 mM lidocaine or I mM quinidine there were significant (P < 0.05) decreases in net Mg 2 efflux. Replacement of extracellular sodium [Na4]0 with either N -methyl-D-glucamine (NMDCI), or choline chloride resulted in a significant (P C 0.05) inhibition of Mg2 effiux. The results of this study indicate that Mg 2 transport (efflux) in rat pancreatic acinar cells may not be associated with the N atKtATPase, the NatKtCl cotransporter or the anion exchanger, but with a Na+ -sensitive Mg2+ transport system. However, this Na+ sensitivity was found to be species dependent as in mouse pancreatic acinar cells Mg2 transport occurred in the absence of [NC] 0 . Studies performed on rat gastric panetal cells have demonstrated that elevated [Mg 2 10 has the same inhibitory effects on secretagogue-evoked acid secretion and cellular Ca 2 transport as that observed with pancreatic acinar cells whereas low [Mg 2 0 had the opposite effect. In conclusion, the results of this study have demonstrated marked interactions between the two divalent cations Mg2 and Ca2 in epithelial secretory cells of the gastrointestinal tract. Mg2+ seems to regulate secretagogue-evoked secretion by controlling cellular Ca2+ mobilisation.

Includes bibliographical references (leaves 129-146). / Human papillomavirus (HPV) infection, specifically with oncogenic types, has been implicated in effectively all cervical cancer cases. Cervical cancer is a global health burden, especially in the developing world. Up to 18 types of HPV are considered oncogenic, of which HPV -16 and -18 cause 70% of cervical cancer cases worldwide. Two vaccines are available on the market: Gardasil(R), targeted against HPV -16, -18; -6 and -11, and Cervarix(TM), against -16 and -18. Both vaccines are based on the L1 capsid proteins of the types they are targeted to and are efficient, pro- phylactic, typespecific vaccines. However, two problems remain: they do not protect against nonvaccine types, that may cause a significant proportion of cancers specifically in African and HIV- positive populations, and they cannot be used to treat existing infections. We designed eight different chimaeric vaccines.

Includes abstract. Includes bibliographical references (leaves 109-129). / Cervical cancer is the second most common cancer affecting women and in most developing countries it remains the leading cause of cancer deaths. In South Africa, more than 3 400 women succumb to the disease every year and 1 in 31 women develop cervical cancer. The causative agent for cervical cancer is the Human papillomavirus (HPV). High-risk (carcinogenic) HPV types have been linked with 99% of the incidences of cervical cancer. The most common types identified in almost 70% of cervical cancer cases worldwide are HPV 16 and 18. HPV infection is very common in young healthy women and most immunocompetent individuals can clear HPV infection. However, in immunosuppresed women, clearance by host immune system is impaired. In addition, multiple HPV infections are quite common in women with Human immunodeficiency virus (HIV) infections. The objectives of this study were to identify HPV types in South African women who also had HIV infection, and secondarily, to determine if recombination of HPV genomes occurs. Determining the HPV types circulating in this country is important to enable identification of most common HPV types, in order to guide the development of vaccines against HPV infection. HPV genotyping was performed by the commercial Roche Linear Array HPV Genotyping Test.

Includes bibliographical references. / Nitrilases catalyse the conversion of a nitrile to its corresponding acid and ammonia by the addition of two water molecules. Cyanide dihydratases, a subgroup of nitrilases, specifically hydrolyse cyanide to formic acid and ammonia. Nitrilases are found in a diverse collection organisms that includes plants, bacteria and fungi. They form one branch a superfamily of structurally related enzymes that are believed to have in common a unique cys-glu-Iys catalytic triad. Many nitrilases exiat as a large molecular weight oligomers of more than 300kDa. In the current study the structures of two cyanide dihydratases, from Pseudomonas stutzeri AK61 and Bacillus pumilus Cl, have solved at a resolution 2.9nm and 32nm respectively by single particle reconstruction from electron micrographs of enzyme particles stained in uranyl acetate. Each enzyme consists of a spiral structure of well-defined length. It is proposed that this arrangement of subunits occurs in many other nitrilases and that a number of unexplained observations in the literature can reconciled by this model.

Malaria is a leading cause of morbidity and mortality worldwide, and results in approximately 600,000 deaths annually. The life cycle of the parasite is complex, and has several distinct stages of development. The transitions between these stages are brought about through tightly controlled and highly synchronized changes in gene expression. Plasmodium falciparum causes the most lethal form of malaria in humans. The parasite is particularly virulent as it is able to evade immune detection by the infected host. This virulence is directly related to the expression of variable antigens on the surface of infected red blood cells. The control of gene expression is known to be largely regulated via RNA Polymerase II (RNAPII) transcription initiation, but in P. falciparum the underlying mechanisms have not been determined. This primarily because very little is known about both the key protein factors and DNA elements which guide the assembly of RNAPII components into the transcription initiation complex. Bioinformatics studies have shown that there is very little amino acid sequence conservation between human and Plasmodium RNAPII transcription initiation components. Together with the observation that the Plasmodium genome has an extremely high A+T content, this suggests that Plasmodium may have specific mechanisms to initiate transcription, which could be targeted by novel anti-malarials. The general transcription factor TFIIB and the TBP-like protein (TLP) are key proteins involved in the recognition of the core promoter, and the initiation of RNAPII transcription initiation complex assembly. TFIIB stabilises DNA binding of the primary promoter recognition factor, TATA-box binding protein (TBP), and is involved in promoter recognition through interactions with specific DNA sequences up- and downstream of the TBP DNA binding site. TBP-like protein is a member of the TBP protein family that has been implicated in life cycle stage specific gene transcription initiation in various eukaryotic model organisms. This research study reports the first successful expression and purification of recombinant epitope-tagged Plasmodium falciparum TFIIB and TLP proteins. Preliminary assays demonstrate DNA-binding activity for the recombinant Plasmodium TBP-like protein, and suggest DNA-binding activity in Plasmodium TFIIB protein, which has not been demonstrated before in eukaryotic TFIIB.

Plasmodium falciparum is the causative agent of the most severe form of malaria and continues to pose challenges to international healthcare, with high mortality rates and emergence of drug-resistant strains. Plasmodium falciparum has multiple sexual and asexual lifecycle stages within its Anopheline mosquito and human hosts, accompanied by distinct morphological changes. The complex lifecycle, along with the ability to adjust rapidly to different environmental niches, is governed by highly regulated and tightly synchronised changes in stage-specific gene expression. In eukaryotes, regulation of RNA Polymerase II transcription initiation is one of the main mechanism of gene expression control. Past research has revealed the presence of crucial elements of RNA Polymerase II (RNAPII) transcription machinery in P. falciparum, however, the precise transcription initiation mechanisms in P. falciparum remain to date undescribed. Bioinformatics studies have found very little homology between human and P. falciparum transcription factors. Furthermore, because of the extreme bias toward A/T in the Plasmodium genome, TATA-box and other core promoter elements that direct transcription initiation, cannot be determined with confidence in bioinformatics studies. Functional characterisation of the key protein factors involved in transcription initiation is a first important step towards the identification of core promoter elements and could reveal currently unknown eukaryotic transcription initiation mechanisms or new anti-malarial targets. In eukaryotes, TATA-binding protein (TBP) and transcription factor IIB (TFIIB) are the key protein factors involved in the core promoter recognition and RNAPII preinitiation complex (PIC) assembly. TBP nucleates PIC assembly by binding the TATA box, thereafter the TBP-TATA complex is further stabilised by TFIIB. In addition, TFIIB has a crucial role in RNAPII recruitment and transcription start site selection and is therefore deemed indispensable in eukaryotic transcription. P. falciparum TBP is the only PIC component in P. falciparum that has been functionally characterised to date, albeit to a very limited extent. This research study reports the successful expression and purification, as well as initial characterisation of DNA-binding activity of P. falciparum TBP and TFIIB. We observe PfTBP binding at multiple locations on putative P. falciparum promoters. We further report PfTBP-independent binding activity of PfTFIIB, that has not been previously observed under the conditions and has implications for novel DNA-binding mechanisms of PfTFIIB. Furthermore, we conclusively demonstrate the formation of a PfTBP-PfTFIIB-promoter ternary complex.

Bibliography: pages 182-223. / B. fibrisolvens H17c is a Gram-positive obligate anaerobe which has been found in the rumen of most ruminants. Strains of B. fibrisol vens have been reported to exhibit activity toward cellulosic and hemicellulosic substrates. The aim of this thesis was to screen a genebank of B. fibrisolvens H17c DNA and to isolate genes expressing cellulase and xylanase activity. Two genes encoding β-1-4- glucosidase (BglA) and endo-β-1-4-xylanase (XynB) were cloned in E. coli.