Quiescent adult stem cells are important for both tissue maintenance and for responding to stress. C. elegans provides an ideal context to dissect pathways involved in the maintenance of long-term cellular quiescence. In favorable environmental conditions, larvae develop continuously into reproductive adults. In adverse environmental conditions, larvae can undergo an alternative life history and enter dauer diapause, a long-lived state with characteristics that promote survival and dispersal. Animals that enter dauer can survive for months, many times the normal life span of an animal that develops continuously. Entry into dauer interrupts the development of the vulva and is associated with a reprogramming-like event that ensures that Vulval Precursor Cells (VPCs) remain multipotent and quiescent until conditions improve. In this work, I aim to understand how the pathways that regulate dauer entry dictate cellular outcomes that oppose VPC fate acquisition and cell division.VPC specification is initiated when an inductive EGF signal from the somatic gonad activates EGFR-Ras-ERK signaling in the nearest VPC, P6.p. Using an ERK activity biosensor, we found that EGFR signal transduction is activated in P6.p prior to dauer entry. However, during the molt into dauer, EGFR signal transduction itself is downregulated and ERK activity remains low in P6.p throughout dauer. To understand how the VPCs are maintained as multipotent precursors in dauer larvae, we investigated the level at which negative regulation of EGFR signaling occurs. We found that dauer VPCs are desensitized to both endogenous and ectopic expression of EGF despite the presence and correct localization of the EGFR. A constitutively active allele of Ras, but not the EGFR, was sufficient to increase ERK activity in the VPCs. This suggests that during dauer, regulation of EGFR signal transduction occurs at or above the level of Ras. We conclude that EGFR signaling is opposed within the VPCs themselves at the level of membrane associated events. Entry into dauer is regulated by Insulin (IIS), TGF-β and Nuclear Hormone Receptor (NHR) signaling pathways. To understand how these pathways might act to block VPC specification and cell division, we characterized mutants acting in either the IIS or NHR pathway that show inappropriate VPC developmental progression in dauer larvae. We found that the phosphatase DAF-18/PTEN, a modulator of IIS, is required to maintain VPC quiescence during dauer. We created an endogenously floxed daf-18 allele and used tissue-specific Cre recombinase drivers to determine the cellular focus of DAF-18/PTEN in regulating VPCs. Our data is consistent with DAF-18/PTEN acting nonautonomously to prevent VPC division and to maintain competence in dauer. DAF-16/FOXO, the major downstream effector of IIS, and DIN-1S/SHARP, which acts in NHR signaling, were previously implicated in regulation of the VPCs during dauer. We looked at null mutants over time in dauer life history and found that each transcription factor opposes VPC division during distinct stages in dauer development. While DIN-1S/SHARP appears to be required to maintain quiescence at the end of the L2d-dauer molt, DAF-16/FOXO is required to maintain quiescence in dauer itself. This suggests that regulation of the VPCs during dauer life history is dynamic and occurs in phases with each stage having distinct regulatory mechanisms, which is like what has been described for dauer exit. Our research provides insights into robust protective mechanisms that maintain multipotency and quiescence over long periods of time. While the pathways required to enact the dauer program are well defined, the downstream consequences of these pathways on individual or groups of cells are less understood. Future work will aim to link dauer regulating pathways to the downregulation of EGFR signaling in the VPCs.

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Mathematical modeling of biological systems can be useful to reveal new insights into biological observations. Here we apply mathematical modeling to study the underlying molecular networks driving observed behaviors of two systems. First, we apply systems biology and dynamic systems theory techniques to reveal new insights into the process of hematopoiesis. More specifically, we search the literature to deduce the underlying molecular mechanism that drives cell fate determination in granulocyte-monocyte progenitor (GMP) cells that are exposed to various cytokines. By converting this molecular mechanism into a set of ordinary differential equations (ODEs), we acquired new insights into the behavior of differentiating GMP cells. Next, we explore the cell cycle of the model prokaryotic organism, Caulobacter crescentus. Caulobacter is a uniquely successful oligotrophic bacterium, found abundantly in freshwater systems. While it is not a pathogenic species, Caulobacter is extremely well studied due to its distinguishable asymmetrical morphology and the ability to synchronize populations by cell cycle stage. We built a detailed mathematical model of the molecular mechanism driving the cell cycle. This research suggests a previously unknown role for the unknown form of the master regulator, CtrA, in regulating the G1-S transition. Furthermore, we incorporate a nutrient signaling model into the cell cycle model to investigate how Caulobacter responds to nutrient deprivation. We find that regulation of DivK phosphorylation is an essential component of the nutrient signaling pathway and demonstrate how starvation signals work together in synergy to manifest in observed cell cycle response. / Doctor of Philosophy / Every cell in the human body has the same DNA, yet there are cells of all kinds with different jobs, appearances and behaviors. This simple concept is a consequence of complex regulatory systems within cells that dictate what genes are expressed and when. This dissertation breaks down the molecular mechanisms that regulate gene expression in cells and how these mechanisms result in the interesting behaviors and morphologies that have been observed experimentally. By deriving mathematical equations to describe the molecular mechanisms, we simulate how cell behavior might change under different conditions to make novel discoveries. More specifically, we utilize these techniques to study the freshwater bacterium, Caulobacter crescentus, and human cells of the white blood cell lineage. We utilize our models to identify previously unknown aspects of the molecular mechanisms, develop explanations for mysterious cell behaviors and provide interesting predictions that have not been explored experimentally.

Mycobacterium tuberculosis (Mtb) is one of the most virulent and prevalent bacterial pathogens across the world. As Mtb infects millions of people a year, it remains essential to study its physiology with the goal of developing new therapeutic interventions. A critical part of the bacteria’s ability to propagate is through successful cell division. Although the process of bacterial cell division and the key proteins therein are well understood in Escherichia coli, much remains to be understood about division in mycobacteria. Genetic and cell biological approaches have recently begun to identify key divisome components in Mycobacterium smegmatis. However, questions remain regarding the role and function of one divisome protein in particular, the DNA translocase FtsK. In this dissertation, I investigated the necessity of FtsK for the growth of mycobacteria. Using an inducible knockdown of FtsK, I present evidence that complete loss of FtsK is required to inhibit growth in both Mtb and M. smegmatis, and that these orthologs share a homologous function. Additional work suggests extended loss of FtsK may be lethal to bacteria. These observations support that FtsK is an essential member of the divisome in mycobacteria, facilitating the processes of growth and division.

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The Mycobacterium genus contains major human pathogens, like Mycobacterium tuberculosis and Mycobacterium leprae, which are the causative agents of Tuberculosis and Leprosy, respectively. They have evolved as successful human pathogens by adapting to the adverse conditions prevailing inside the host, which include host immune activation, nutrient depletion, hypoxia, and so on. During such adaptation for the survival and establishment of persistent infection inside the host, the pathogen, like M. tuberculosis, regulates its cell division. It is known that M. tuberculosis enters a state of non-replicating persistence (NRP) inside the host, to establish latent infection, which helps the survival of the pathogen under adverse host conditions such as hypoxia and nutrient depletion. The pathogen can reactivate itself, to come out of the NRP state, and establish active infection at a later stage, when conditions are suitable for its proliferation. The altered physiological state of the latent bacterium makes it tolerant to drugs, which are only effective against proliferating tubercle bacilli. In view of this unique behavioural physiology of tubercle bacilli, it is important to study the process of cell division and how it is regulated in the NRP and actively growing states. The work reported in the thesis is an attempt to understand these aspects of mycobacterial cell division. iii Chapter 1. Introduction: This chapter gives a detailed introduction to bacterial cell division and its regulation in various organisms, like Escherichia coli, Bacillus subtilis, Caulobacter crescentus, and others. In the background of this information, the major studies on mycobacterial cell division and its regulation are presented. Chapter 2. Materials and Methods: This chapter describes in detail all the materials and methods used in the experiments, which are presented in the four data chapters, 3-6. Chapter 3. Ultrastructural Study of the Formation of Septal Partition and Constriction in Mycobacteria and Delineation of its Unique Features: Mycobacteria have triple-layered complex cell wall, playing an important role in its survival under adverse conditions in the host. It is not known how these layers in the mother cell participate during cell division. Therefore, the ultrastructural changes in the different envelope layers of Mycobacterium tuberculosis, Mycobacterium smegmatis, and Mycobacterium xenopi, during the process of septation and septal constriction, were studied, using Transmission and Scanning Electron Microscopy. The unique aspects of mycobacterial septation and constriction were identified and were compared with those of E. coli and Bacillus subtilis septation. Further, based on all these observations, models were proposed for septation in M. tuberculosis and M. smegmatis. Chapter 4. Identification of Asymmetric Septation and Division in Mycobacteria and Its Role in Generating Cell Size Heterogeneity: Bacterial populations are known to harbour phenotypic heterogeneity that helps survival under stress conditions, as this heterogeneity comprises subpopulations that have differential susceptibility to stress conditions. The iv heterogeneity has been known to lead to the requirement for prolonged drug treatment for the elimination of the tolerant subpopulation. Hence, it is important to study the different mechanisms, which operate to generate population heterogeneity. Therefore, in this chapter, studies were carried out to find out whether asymmetric septation and division occur in mycobacteria to generate cell size heterogeneity. Subpopulations of mycobacterial mid-log phase cells of M. tuberculosis, M. smegmatis, and M. xenopi were found to undergo asymmetric division to generate cell size heterogeneity. The asymmetric division and the ultrastructure and growth features of the products of the division were studied. Chapter 5. Study of Mycobacterial Cell Division Using Growth-Synchronised Cells: In this chapter, different stages of cell septation and constriction were studied using growth-synchronised M. smegmatis cells. Phenethyl alcohol (PEA), which has been found to reversibly arrest mycobacterial cells, was used for growth synchronisation. The growth-synchronised mycobacterial cells, which were released from PEA block, were studied at different stages of septation and septal constriction, at the ultrastructural and molecular levels. Chapter 6. Identification of the Stage of Cell Division Arrest in NRP Mycobacteria: The exact stage at which the NRP tubercle bacilli are arrested in cell division is currently unknown. In Wayne’s in vitro model for hypoxia-responsive tubercle bacilli, gradual depletion of oxygen leads to hypoxic stress, inducing the bacilli to enter non-replicating persistence (NRP) state. Using this model, the stage of cell division arrest in M. tuberculosis was characterised at the ultrastructural and molecular levels. Hypoxia-stressed M. smegmatis was used as an experimental system for contrast. The thesis concludes with salient findings, a bibliography, and the list of publications.

When a cell divides into two, it is imperative that each resultant daughter receives a full complement of chromosomes; DNA is ultimately responsible for all cellular processes. Cohesion between sister chromatids from the moment of their generation in S phase is central to ensuring the fidelity of chromosome segregation. Smc1 and Smc3 proteins interact with each other via their hinges and with a bridging kleisin subunit via their heads to form the cohesin ring. It is cohesin, through entrapment of sister chromatid within its ring, that confers sister chromatid cohesion. The process of cohesin’s loading onto DNA is poorly understood. While it is thought to depend on ATP hydrolysis, opening of the ring at one of its three interfaces, and the as yet undefined action of the kollerin complex, comprising Scc2 and Scc4 proteins, the sequence of events as they occur are yet to be defined. A recent screen for suppressors of a thermosensitive scc4 allele in budding yeast revealed a mutation within Smc1’s hinge that could bypass the kollerin subunit. Here, the Smc1 suppressor mutation is investigated. Through targeted mutagenesis, the Smc1D588Y mutant identified in the screen and two additional point mutants, Smc1D588F and Smc1D588W, are herein proven able to bypass Scc4 function completely. Thus we provide the strongest evidence to date to suggest that cohesin’s hinge is a critical factor in its loading. Biochemical evidence shows that isolated Smc1 hinge mutants are defective in their binding to Smc3 hinges. This, together with the genetic link made between the hinge and loading complex, suggests that hinge opening might be a requisite for loading. Through mutagenesis of Scc2 and Scc4 we show that the N-terminus of each protein is responsible for their dimerisation. Furthermore, the N- terminus of Scc2 confers no function other than in its binding to Scc4. Finally, we show that Scc4 is required for the enrichment of both Scc2 and cohesin at centromeres, but not at arm loci. Our results are therefore indicative of there being two different pathways of cohesin loading.

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Dilantin (diphenylhydantoin or DPH) has been given to epileptic mothers to control seizures during pregnancy. Previous research has demonstrated that exposure of human embryos to Dilantin in vivo results in an increased probability of abnormal development and early fetal loss. Preliminary results with cultured 1-cell and 2-cell mouse embryos demonstrated that Dilantin causes mouse embryonic cleavage events to slow during preimplantation development (Chatot et al., unpublished). Dilantin may be responsible for this by inhibiting the rate of DNA synthesis during cleavage or by affecting the expression of proteins that control cell cycle progression. The standard expression pattern of these cell cycle regulatory proteins (cyclins) has not previously been determined in the mouse preimplantation embryo model. In this study, immunolabellingtechniques have been used to determine the expression pattern of cyclins D, A, and B 1 in the first three cell cycles of preimplantation mouse embryo development.This study reveals a unique expression pattern of cyclins D, A, and B1 in the first three cell cycles of preimplantation embryo development. Examination of the beginning of the first cell cycle, or G1, indicated a moderate expression of cyclin B1 and A but no cyclin D expression. During DNA synthesis (S-phase) all cyclin expression was virtually nonexistent. Toward the end of the cell cycle at G2/M, cyclin D expression appeared to be at moderate levels while cyclins A and B 1 exhibited minimal degrees of expression.In G 1 of the second cell cycle, cyclins D and A were minimally to moderately expressed and cyclin B 1 expression was minimal. At S-phase, cyclin D expression dropped to minimal levels whereas cyclins A and B 1 were at minimal to moderate levels of expression. At G2/M of the second cell cycle, cyclin B1 was expressed at minimal to moderate levels and cyclins A and D were both expressed at minimal levels.The third cell cycle began at G 1 with cyclin B 1 being expressed at moderate levels followed by minimal to moderate levels of cyclin D expression and minimal expression for cyclin A. Cyclin D expression increased to moderate levels at S-phase and cyclin A exhibited minimal to moderate levels of expression. Cyclin B 1 was observed at moderate levels of expression at S-phase of the third cell cycle. G2 of the third cell cycle included a drop to minimal levels of expression of cyclin D, while cyclin A expression remained at minimal to moderate levels and cyclin B remained at moderate levels of expression.The cyclin expression pattern for the first three cell cycles in preimplantation mouse embryos is unique compared to known cyclin expression patterns in other species. Cyclin D is expressed in G1 and is known to be necessary for advancement to S-phase in human glioblastoma cell lines (Xiong et al., 1991). Cyclin A is active at S-phase through Win human fibroblasts and xenopus oocytes (Giordino et al., 1991; Minshul et al., 1990). Cyclin B is present at G2 through mitosis in human fibroblasts and xenopus oocytes (Pines and Hunter, 1990; Minshul et al, 1990). / Department of Biology