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How proliferating cells maintain the copy number and overall size of their organelles is not clear. In the budding yeast Saccharomyces cerevisiae the G1 cyclins Cln1,2,3p control initiation of cell division by regulating the activity of the cyclin-dependent kinase (Cdk) Cdc28p. We show that Cln3p controls vacuolar (lysosomal) biogenesis and segregation. First, loss of Cln3p, but not Cln1p or Cln2p, resulted in vacuolar fragmentation. Although the vacuoles of cln3Δ cells were fragmented, together they occupied a large space, which accounted for a significant fraction of the overall cell size increase in cln3Δ cells. Second, cytosol prepared from cells lacking Cln3p had reduced vacuolar homotypic fusion activity in cell-free assays. Third, vacuolar segregation was perturbed in cln3Δ cells. Our findings reveal a novel role for a eukaryotic G1 cyclin in cytoplasmic organelle biogenesis and segregation. Furthermore we show that the scaffold protein Bem1p, a critical regulator of Cdc42p activity, is a downstream effector of Cln3p/Cdc28p complex. The Cdc42p GTPase is known to be required for vacuole fusion. Our results suggest that Ser72 on Bem1p is phosphorylated by Cdc28p in a Cln3p-dependent manner to promote vacuole fusion. Replacing Ser72 with Asp, to mimic phosphorylation at an optimal Cdkconsensus site located in the first SH3 domain of Bem1p, suppressed vacuolar fragmentation in cells lacking Cln3p. Using in vivo and in vitro assays, we found that Cln3p was unable to promote vacuole fusion in the absence of Bem1p or in the presence of a non-phosphorylatable Bem1p-Ser72Ala mutant. Furthermore, activation of Cdc42p also suppressed vacuolar fragmentation in the absence of Cln3p. Our results provide a mechanism that links cyclin-dependent kinase activity with vacuole fusion through Bem1p and the Cdc42p GTPase cycle.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Oct. 30, 2007). Includes bibliographical references (p. 165-174).

Cellular processes critical to sustaining physiology, including growth, division and differentiation, are carefully governed by intricate control systems. Deregulations in these systems often result in complex diseases such as cancer. Hence, it is crucial to understand the interactions between molecular players of these control systems, their emergent network dynamics, and, ultimately, the overall contribution to cellular physiology. In this dissertation, we have developed a mathematical framework to understand two such cellular systems: an early checkpoint (START) in the budding yeast cell cycle (Chapter 1), and the canonical Wnt signaling pathway involved in cell proliferation and differentiation (Chapter 2). START transition is an important decision point where the cell commits to one round DNA replication followed by cell division. Several years of experimental research have gone into uncovering molecular details of this process, but a unified understanding is yet to emerge. In chapter one, we have developed a comprehensive mathematical model of START transition that incorporates several findings including information about the phosphorylation state of key START proteins and their subcellular localization. In the second chapter, we focus on modeling the canonical Wnt signaling pathway, a cellular circuit that plays a key role in cell proliferation and differentiation. The Wnt pathway is often deregulated in colon cancers. Based on some evidence of bistability in the Wnt signaling pathway, we proposed the existence of a positive feedback loop underlying the activation and inactivation of the core protein complex of the pathway. Bistability is a common feature of biological systems that toggle between ON and OFF states because it ensures robust switching back and forth between the two states. To study and explain the behavior of this dynamical system, we developed a mathematical model. Based on experimentally determined interactions, our simple model recapitulates the observed phenomena of bimodality (bistability) and hysteresis under the effects of the physiological signal (Wnt), a Wnt-mimic (LiCl), and a stabilizer of one of the key members of core complex (IWR-1). Overall, we believe that cell biologists and molecular geneticists can benefit from our work by using our model to make novel quantitative predictions for experimental verification. / Ph. D.

Most cellular proteins retain a stable concentration as cells grow and divide, but there are exceptions. Some cell cycle regulators change in concentration with cell size. In fission yeast, Cdc13 (cyclin B), an important activator of the core cell cycle kinase Cdc2 (CDK1), increases in concentration as cells grow. It has been proposed that the concentration of such cell cycle regulators serves as a proxy for cell size and makes cell cycle progression dependent on cell size, thereby contributing to cell size homeostasis. The underlying mechanisms for the size-dependent scaling of these cell cycle regulators are poorly understood. Here, I show that Cdc13 protein concentration, but not mRNA concentration, increases with cell size. Furthermore, only the nuclear, but not the cytoplasmic, fraction of Cdc13 increases in concentration as cell size increases. Computational modeling along with half-life measurements suggests that stabilization of Cdc13 in the nucleus plays an important role in establishing this pattern. Taken together, my results suggest that Cdc13 scales with time, and therefore only indirectly—not directly—with cell size. This leaves open the possibility that Cdc13 contributes to cell size homeostasis, but in a different way than originally proposed. / Master of Science / Cells maintain their size very efficiently, but how they manage to do so is not well characterized. It has been suggested that cells sense their size by the size-dependent concentration changes of cell cycle proteins. I have investigated how cyclin B may serve as such a proxy for cell size in fission yeast. My data suggest that fission yeast cyclin B indirectly scales with cell size through an unknown time-based mechanism.

All animal cells maintain cell size homeostasis, where cell growth (increase in size) is balanced with proliferation (reduction in size via cell division). Yet, different cell types have different sizes and there are physiologically relevant situations where animal cells undergo major cell size changes. So how is cell size regulated? And why is cell size regulated? Are there specific cellular processes that have different functionality in different sized cells? This thesis investigates these questions from the perspective of cellular metabolism. Using a Cyclin dependent kinase 1 knockout mouse model with different degrees of hepatocytes enlargement, gene expression levels were correlated with cell size in vivo. This revealed that the relative expression of mitochondrial and lipid biosynthesis genes are downregulated with increasing cell size. However, mitochondrial content of the liver samples was not decreased, suggesting that cell functions and cell contents scale differently with cell size. To better investigate how mitochondrial functions scale with cell size in non-mutant cells, a novel and high throughput flow cytometry based single-cell analysis method called CoSRA was developed. Using fluorescence mitochondrial probes CoSRA revealed that, while mitochondrial content increases linearly with cell size, mitochondrial membrane potential is decreased in the very smallest and the largest cells. These effects were independent of cell cycle and all animal cell types examined displayed similar effects. Similar nonlinearity was observed in mitochondrial respiration. Furthermore, cell-to-cell variability in mitochondrial membrane potential was minimised in cells which are close to the median cell size of the whole population. The cell size dependence of mitochondrial functions was regulated by mitochondrial dynamics. It was also investigated if mitochondrial functions or lipid biosynthesis are capable of regulating cell size in human cell culture models. Various mitochondrial inhibitions increased cell size by reducing proliferation. Similar results were seen with inhibitions on lipid biosynthesis and especially with inhibitions of mevalonate pathway. Systematic dissection of the mevalonate pathway revealed that protein geranylgeranylation is required for maintaining normal cell size and proliferation ratio. Geranylgeranylation of the recycling endosome regulating protein RAB11 was identified to be at least partially responsible for the cell size regulation by the mevalonate pathway. Furthermore, the link from the mevalonate pathway to RAB11 was found to regulate basal autophagic flux, thus providing a novel connection from lipid biosynthesis to other growth regulating processes. In conclusion, this thesis provides evidence for cell size dependent metabolism, where mitochondrial functions do not increase linearly with cell size. This provides conceptual insights into organelle scaling with cell size and a potential mechanism for maintenance of cell size homeostasis. In addition, mitochondria and lipid synthesis are identified as critical processes for normal cell size homeostasis.

In recent years, mobile communications have become affordable and popular. High cellular capacity in terms of number of users and data-rates is in need. As the available frequency spectrums for mobile communications are limited, the utilization of the radio resources to achieve high capacity without imposing high equipment cost is of utmost importance. Recently, multi-hop cellular networks (MCNs) were introduced. These networks have the potential of enhancing the cell capacity and extending the cell coverage at low extra cost. However, in a cellular network, the cell or system capacity is inversely related to the cell size. In MCNs, the cell size, the network density and topology affect the coverage of source nodes and the total demands that can be served and, thus, the system throughput. Although the cell size is an important factor, it has not been exploited for maximizing throughput. Another major issue in MCNs is the increase in packet delay because multi-hopping is involved. High packet delay affects quality of service provisioning in these networks. In this thesis, we propose the Optimal Cell Size (OCS) and the Optimal Channel Assignment (OCA) schemes to address the cell size and packet delay issues for a time division duplex (TDD) wideband code division multiple access (W-CDMA) MCN. OCS finds the optimal cell sizes to provide an optimal balance of cell capacity and coverage to maximize the system throughput, whereas OCA assigns channels optimally in order to minimize packet relaying delay. Like many optimized schemes, OCS and OCA are computationally expensive and may not be suitable for large real-time problems. Hence, we also propose heuristics for solving the problems. For the cell size problem, we propose two heuristics: Smallest Cell Size First (SCSF) and Highest Throughput Cell Size First (HTCSF). For the channel assignment problem, we propose the Minimum Slot Waiting First (MSWF) heuristic. Simulation results show that OCS achieves high throughput compared to that of conventional (single-hop) cellular networks and OCA achieves low packet delay in MCNs. Results also show that the heuristics, SCSF, HTCSF and MSWF, provide good results compared to the optimal ones provided by OCS and OCA, respectively. / Thesis (Ph.D, Computing) -- Queen's University, 2009-02-02 22:53:41.825

The budding yeast Saccharomyces cerevisiae exhibits exquisite control of cellular size in response to the nutritional composition of its environment. Size control is mediated at the G1/S phase transition, termed Start: passage through Start represents an irreversible commitment to cell division and is contingent on achieving a critical size. When nutrients are plentiful, yeast increase their critical size set-point resulting in larger cells; in contrast, in poor nutrients, yeast pass Start at a smaller size. The genetic basis for nutrient-dependent size control and the means by which yeast sense their size remain elusive. One measure of growth potential is ribosome biogenesis, the rate of which correlates with cell size. I characterized a G-patch domain containing protein, Pfa1, which has been shown to activate the helicase activity of the pre-rRNA processing factor Prp43. Intriguingly, Pfa1 is multiply phosphorylated in response to inhibition of the TOR kinase, the central player in growth regulation. This phosphorylation occurs in a region required for Pfa1 function in ribosome biogenesis, independent of its role as a helicase activator. Consistently, phosphorylation correlates with loss of physical interactions with ribosome biogenesis and altered interactions with the ribosome. Mutation of these phosphorylation sites eliminates TOR-dependent phospho-regulation, and confers sensitivity to TOR inhibition. I propose a model wherein Pfa1 is phosphorylated in response to nutrient stress, leading to relocalization of essential processing factors, and inhibition of both ribosome biogenesis and tRNA maturation. Further, I constructed and verified a non-covalent short oligonucleotide barcode microarray platform, and applied it to genome-scale parallel analyses of both the DNA damage response and cell size control in S. cerevisiae. Through these studies, I uncovered novel connections between size control and numerous cellular processes including: the large subunit of the ribosome; the mitochondrial pH gradient; and proteins involved in oxidant-induced cell cycle arrest.

The budding yeast Saccharomyces cerevisiae exhibits exquisite control of cellular size in response to the nutritional composition of its environment. Size control is mediated at the G1/S phase transition, termed Start: passage through Start represents an irreversible commitment to cell division and is contingent on achieving a critical size. When nutrients are plentiful, yeast increase their critical size set-point resulting in larger cells; in contrast, in poor nutrients, yeast pass Start at a smaller size. The genetic basis for nutrient-dependent size control and the means by which yeast sense their size remain elusive. One measure of growth potential is ribosome biogenesis, the rate of which correlates with cell size. I characterized a G-patch domain containing protein, Pfa1, which has been shown to activate the helicase activity of the pre-rRNA processing factor Prp43. Intriguingly, Pfa1 is multiply phosphorylated in response to inhibition of the TOR kinase, the central player in growth regulation. This phosphorylation occurs in a region required for Pfa1 function in ribosome biogenesis, independent of its role as a helicase activator. Consistently, phosphorylation correlates with loss of physical interactions with ribosome biogenesis and altered interactions with the ribosome. Mutation of these phosphorylation sites eliminates TOR-dependent phospho-regulation, and confers sensitivity to TOR inhibition. I propose a model wherein Pfa1 is phosphorylated in response to nutrient stress, leading to relocalization of essential processing factors, and inhibition of both ribosome biogenesis and tRNA maturation. Further, I constructed and verified a non-covalent short oligonucleotide barcode microarray platform, and applied it to genome-scale parallel analyses of both the DNA damage response and cell size control in S. cerevisiae. Through these studies, I uncovered novel connections between size control and numerous cellular processes including: the large subunit of the ribosome; the mitochondrial pH gradient; and proteins involved in oxidant-induced cell cycle arrest.

Despite increasing computer power, the requirement to upscale 3D geological models for dynamic reservoir simulation purposes is likely to remain in many commercial environments. This study established that there is a relationship between sandbody size, cell size and changes to predictions of reservoir production as grids are upscaled. The concept of a cell width to sandbody width ratio (CSWR) was developed to allow the comparison of changes in reservoir performance as grids are upscaled. A case study of the Flounder Field in the Gippsland Basin resulted in the interpretation of three depositional environments in the intra-Latrobe reservoir interval. The sandbody dimensions associated with these depositional environments were used to build a series of 3D geological models. These were upscaled vertically and horizontally to numerous grid cell sizes. Results from over 1400 dynamic models indicate that if the CSWR is kept below 0.3 there will be a strong correlation between the average production from the upscaled grids compared to those of a much finer grid, and there will be less than 10% variation in average total field production. If the CSWR is between 0.3 and 1, there could be up to 30% difference, and once the CSWR exceeds 1.0 there is only a weak relationship between the results from upscaled grids and those of finer grids. As grids are upscaled the morphology of bodies in facies models changes, the distribution of petrophysical properties is attenuated and the structure is smoothed. All these factors result in a simplification of the fluid flow pathways through a model. Significant loss of morphology occurs when cells are upscaled to more than a half the width of the reservoir body being modelled. A simple rule of thumb is established — if the geological features of a model cannot be recognised when looking at a layer in the upscaled grid, the properties of the upscaled grid are unlikely to be similar to those of the original grid and the predictions of dynamic models may vary significantly from those of a finer grid. This understanding of the influence of sandbody size on the behaviour of upscaled dynamic models can be used in the planning stages of a reservoir modelling project. Two simple charts have been created. The first chart is for calculating the approximate number of cells in a model before it is built. The second chart is for comparing the proposed cell size against the CWSR, so that the predicted discrepancy between the ultimate production from the upscaled grid and one with much smaller cells can be assessed. These two charts enhance discussion between all interested disciplines regarding the potential dimensions of both static and upscaled dynamic models during the planning stage of a modelling project, and how that may influence the results of dynamic modelling. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1375309 / Thesis (Ph.D.) - University of Adelaide, Australian School of Petroleum, 2009