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Caulobacter crescentus goes through a classic dimorphic cell division cycle to adapt to the stringent environment and reduce intraspecific competition. Caulobacter mother cell gives rise to two progenies with distinct morphology - a motile swarmer cell equipped with a flagellum and a sessile stalked cell equipped with a stalk. Because of the nature of dimorphic lifestyle, Caulobacter becomes a model bacterium to study the cell differentiation, signalling transduction, stress response, and asymmetry development of prokaryotes. The dimorphic cell cycle of Caulobacter is driven by the elaborate spatiotemporal organization of regulatory molecules through regulations of synthesis, degradation, phosphorelay, and localization. There is a wealth of experimental observations about gene/protein interactions and localizations accumulated in recent decades, while several mathematical models have been proposed to study the cell cycle progression in Caulobacter. However, the specific control mechanisms of stress response and spatial asymmetry establishment are yet clearly elucidated, while these mechanisms are of fundamental importance to understanding the bacterial survival strategy and developing the microbial industry. Here we utilize mathematical modeling to study the regulatory network of cell cycle control in C. crescentus, focusing on the stress response and asymmetry development. First, we investigate the starvation response of Caulobacter through the connection of phosphotransferase systems (PTS) and guanine nucleotide-based second messenger system. We have developed a mathematical model to capture the temporal dynamics of vital regulatory second messengers, c-di-GMP (cdG) and guanosine pentaphosphate or tetraphosphate (pppGpp or ppGpp), under normal and stressful conditions. This research suggests that the RelA-SpoT homolog enzymes have the potential to effectively influence the cell cycle in response to nutrition changes by regulating cdG and (p)ppGpp levels. We further integrate the second messenger network into a temporal cell cycle model to investigate molecular mechanisms underlying responses of Caulobacter to nutrition starvation. Our model suggests that the cdG-relevant starvation signal is essential but not sufficient to robustly arrest the cell cycle of Caulobacter. We also demonstrate that there may be unknown pathway(s) reducing CtrA under starvation conditions, which results in delayed cytokinesis in starved stalked cells. The cell cycle development of Caulobacter is determined by the periodical activation and deactivation of the master regulator CtrA. cdG is an essential component of the ClpXP pro- tease complex, which is specifically responsible for the degradation of CtrA. We propose a mathematical model for the hierarchical assembly of ClpXP complexes, together with modeling DNA replication, transcription, and protein interactions, to characterize the Caulobacter cell cycle. Our model suggests that the ClpXP-based proteolysis system contributes to the timing and robustness of the cell cycle progression. Furthermore, we construct a spatiotemporal model with Turing-pattern mechanism to study the morphogenesis and asymmetry establishment during the cell cycle of Caulobacter. We apply reaction-diffusion equations to capture the spatial dynamics of scaffolding proteins PodJ, PopZ, and SpmX, which organize two distinct poles of Caulobacter. The spatial regulations influence the activity and distribution of key cell cycle regulators, governing the dimorphic lifestyle of Caulobacter. Our model captures major spatiotemporal experimental observations of wild-type and mutant cells. It provides predictions of novel mutant strains and explains the spatial regulatory mechanisms of bacterial cell cycle progression. / Doctor of Philosophy / Cell is the basic unit of life that undergoes a process called 'cell cycle' consisting of DNA replication and cell division to exhibit various functions, abilities, and behaviors. The cell cycle is well organized by complex regulations in time and space that determine when and where changes take place. The regulations behind cell cycle development play important roles for living organisms but are not fully understood. In this dissertation, we utilize mathematical models and focus on a model bacterium, Caulobacter crescentus, to capture characteristics of cell cycle and study the underlying regulations. Caulobacter is widely distributed in freshwater, including environments with poor nutrients. It divides asymmetrically, generating a pair of daughter cells with different appearances and replicative potentials. Therefore, Caulobacter population has the flexibility to save energy by halting DNA replication and to reduce the competition with siblings by settling into different places. We utilize the nature of the asymmetrical division of Caulobacter to quantitatively investigate the control mechanisms of cell cycle development, including how cells detect and respond to external cues and develop different organelles at specific times and locations.

To improve the process stability of wastewater treatment plants, the construction of a whole-cell bacterial biosensor is explored to harness the natural stress response of the bacterial cells. The stress response selected in this work is the glutathione-gated potassium efflux (GGKE) system, which responds to electrophilic stress by effluxing potassium from the interior to the exterior of the cell. Thus, the bulk potassium in solution can be monitored as an indicator of bacterial stress. By utilizing this stress response in a biosensor, the efflux of potassium can be correlated to the stress response of the immobilized culture, providing an early warning system for electrophilic shock. This type of shock is a causative factor in many process upset events in wastewater treatment plants, so the application of the sensor would be an early warning device for such plants. The research conducted here focused on the biological element of the biosensor under development. Three immobilization matrices were explored to determine the cell viability and potassium efflux potential from immobilized cells: a calcium alginate, a photopolymer, and a thermally reversible gel. The calcium alginate was unstable, and dissolved after five days, such that the long-term impact of immobilization on the cells could not be determined in the matrix. The photopolymer resulted in very low actvity and viability of immobilized cellsOf the three matrices tested, indicating that the composition of the polymer was toxic to the cells. Of the matrices tested, the thermally-reversible gel showed the best response for further study, in that the matrix did not inhibit cell activity or potassium efflux. / Master of Science

The ability to accurately predict danger and implement appropriate protective responses is critical for survival. Environmental fluctuations can cause damage at the cellular level, leading to the misfolding and aggregation of proteins. Such damage is toxic to cells: in age-related neurodegenerative diseases like ALS, Parkinson’s, Alzheimer’s and Huntington’s Diseases, the accumulation of damaged proteins in the brain ultimately leads to neuronal cell death and disease onset. To date, there is still no cure to combat the progressive degeneration and cell death seen in the brains of patients. Cells within an animal possess defense programs to minimize protein damage. One such defense mechanism is the activation of a program called the Heat Shock Response, which increases production of protective proteins known as heat shock proteins (HSPs). These HSPs act as molecular chaperones to assist with the clearing out of damaged proteins. This program is implemented by a conserved transcription factor, Heat Shock Factor 1 (HSF-1). However, in brains of patients with degenerative diseases, this protective mechanism, for reasons yet unknown, is not constantly activated. My thesis has involved the discovery of innate mechanisms that exist in organisms to activate this cellular protective mechanism against protein misfolding. My research, using the model organism Caenorhabditis elegans, has shown that the protective heat shock response in the cells of the animal can be triggered through neurohormonal signaling. The neurohormonal signaling that I am studying is one that is highly conserved across all organisms from plants to insects to mammals – serotonergic signaling. The stimulation of serotonergic signaling appears sufficient to activate the Heat Shock Response, even in the absence of real damage. In fact, the neuronal release of serotonin facilitates a pre-emptive upregulation of protective genes in the animal, which we have observed to be able to reduce the accumulation of damaged proteins in a C. elegans model of Huntington’s Disease. Additionally, I have seen that anticipating danger can enhance the animal’s stress response in a serotonin-dependent manner, thus facilitating better survival against a subsequent insult that can cause protein damage. Together, these studies present the novel possibility of protection against neurodegenerative disease via modulation of neurotransmission and/or neurosecretion. They also allow for understanding how sensory inputs are coupled to gene expression under stressful conditions. I hope to understand the mechanism by which animals adapt to changes in their environment by coordinating their sensory input with changes in behavior and gene expression.

The Tend and Befriend stress response model suggests that women have, through natural selection, evolved a different stress response reaction to that of men. It thus offers a collective, gender stereotypical reality of women’s responses to stress. In this research the Tend and Befriend model is thus viewed as a dominant public discourse which informs or influences the private narratives or stories of women. It is this interaction between public (dominant) discourses and private narratives which are investigated through using the Tend and Befriend model as a discursive landscape. If gender or gender roles are flexible, there is a concern that individual women might be misrepresented and not given a voice by the dominant discourse which supports gender stereotypical models like the Tend and Befriend model. This qualitative exploration was done by exploring the socially constructed stress responses of five professional women. To investigate this, as researcher I explored the narratives of these women in face-to-face individual interviews. The constructions explored include: How these women understand the way they respond to stress; how they view the Tend and Befriend model; and the influence of the model on them. Through the lenses of social constructionism a broader insight into the stress responses of women may be obtained. From the data analysis, I uncovered very little ‘evidence’ for tending or befriending behaviour as described by Taylor, Klein, Lewis, Gruenewald, Gurung and Updegraff (2000), with the participants. In the exploration the closest response to the model which the participants reported was befriending, however in their construction of befriending they employed it as a workplace strategy. The only form of tending co-constructed in the interview process was a secondary response to stress and a unique outcome to this study: Self-tending. Additionally, as social constructionist research predicts, these participants illustrated that for them stress responses are not concrete, as models would like to suggest, rather they employed an alternate multifaceted stress response approach which was another significant unique outcome to this study. / Dissertation (MA)--University of Pretoria, 2010. / Psychology / unrestricted

Haa1 is a transcription factor that adapts Saccharomyces cerevisiae cells to weak organic acid stresses by activating the expression of various genes. How Haa1 is activated by weak acids is not clear. This study proposes that Hrr25 is an important regulator of cellular adaptation to weak acid stress by inhibiting Haa1 through phosphorylation. YRO2, one of the targets of Haa1, shows increase in expression during stationary phase. This increase is due to basal activity of Haa1 and another, unknown, transcription factor. This study proposes that Gsm1 is another transcription factor that regulates YRO2 expression in the stationary phase. Finally, the mechanism of regulation of YRO2 by Haa1 is largely unknown. This study identifies two possible Haa1-medated cis-acting elements in the YRO2 promoter.

Pathogenic Yersinia cause a manifold of diseases in humans ranging from mild gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica) to pneumonic and bubonic plague (Y. pestis), while all three have a common virulence strategy that relies on a well-studied type III secretion system and its effector proteins to colonize the host and evade immune responses. However, the role of other protein secretion and/or translocation systems in virulence of Yersinia species is not well known. In this thesis, we sought to investigate the contribution of twin-arginine translocation (Tat) pathway and its secreted substrates to the physiology and virulence of Y. pseudotuberculosis. Tat pathway uniquely exports folded proteins including virulence factors across the cytoplasmic membranes of bacteria. The proteins exported by Tat pathway contain a highly conserved twin-arginine motif in the N-terminal signal peptide. We found that the loss of Tat pathway causes a drastic change of the transcriptome of Y. pseudotuberculosis in stationary phase at environmental temperature with differential regulation of genes involved in virulence, carbon metabolism and stress responses. Phenotypic analysis revealed novel phenotypes of the Tat-deficient strain with defects in iron acquisition, acid resistance, copper oxidation and envelope integrity, which we were partly able to associate with the related Tat substrates. Moreover, increased glucose consumption and accumulation of intracellular fumarate were observed in response to inactivation of Tat pathway implicating a generic effect in cellular physiology. We evaluated the direct role of 22 in silico predicted Tat substrate mutants in the mouse infection model and found only one strain, ΔsufI, exhibited a similar degree of attenuation as Tat-deficient strain. Comparative in vivo characterization studies demonstrated a minor defect for ΔsufI in colonization of intestinal tissues compared to the Tat-deficient strain during early infection, whereas both SufI and TatC were required for dissemination from mesenteric lymph nodes and further systemic spread during late infection. This verifies that SufI has a major role in attenuation seen for the Tat deficient strain both during late infection and initial colonization. It is possible that other Tat substrates such as those involved in iron acquisition and copper resistance also has a role in establishing infection. Further phenotypic analysis indicated that SufI function is required for cell division and stress-survival. Transcriptomic analysis revealed that the highest number of differentially regulated genes in response to loss of Tat and SufI were involved in metabolism and transport. Taken together, this thesis presents a thorough analysis of the involvement of Tat pathway in the overall physiology and virulence strategies of Y. pseudotuberculosis. Finally, we propose that strong effects in virulence render TatC and SufI as potential targets for development of novel antimicrobial compounds

Cancer cells survive the harsh oxygen and nutrient deprivation of the tumour microenvironment through the selection of apoptosis-resistant and glycolytic clones (Cairns et al., 2011; Graeber et al., 1996). In particular, the integrated stress response (ISR) has been shown to be pivotal in cancer cell survival in vivo and the resistance of cancer cells to therapy (Harding et al., 2003). In recent years, it has become apparent that increased autophagy is one mechanism by which the ISR can confer resistance to stress (Kroemer et al., 2010). ATF4 is a major transcriptional effector of the integrated stress response in severe hypoxia (<0.01% O₂). ATF4 is a well-established regulator of genes involved in oxidative stress, amino acid synthesis and uptake, lipid metabolism, protein folding, metastasis, and angiogenesis. Recent work has demonstrated an important role of ATF4 in promoting resistance to severe hypoxia through the transcriptional upregulation of MAP1LC3B and ATG5, essential components of the autophagy machinery (Rouschop et al., 2009b; Rzyski et al., 2010). In this work, the author describes several novel ATF4 target genes, and examines their role in the regulation of autophagy and the resistance of cancer cells to severe hypoxia. In the first part of this thesis, the author shows that three BH3-only members of the BCL-2 family of proteins--HRK, PUMA, and NOXA--are upregulated in response to severe hypoxia in an ATF4-dependent manner. In particular, the author shows that the poorly described BH3-only protein HRK is a direct target of transcriptional activation by ATF4, and that HRK induces autophagy in severe hypoxia, thereby providing the first evidence that the integrated stress response can transcriptionally trigger the autophagy process. In contrast to the previously described role of HRK in apoptosis, this thesis demonstrates that HRK can play a pro-survival role in the context of breast cancer cells. In the latter part of this thesis, the author identifies the essential autophagy gene ULK1 as an ISR target. The author shows that ULK1 expression in severe hypoxia is transcriptionally upregulated through direct activation by ATF4. The author identifies ULK1 as a crucial regulator of autophagy and mitophagy in both normoxia and severe hypoxia and shows that ULK1 plays a pivotal role in cancer cell survival. Furthermore, it is shown that human breast cancer patients with high levels of ULK1 relapse earlier than those with low levels of ULK1, thereby identifying ULK1 as a potential target for cancer therapy.