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CDK4/6 inhibitors induce a G1 cell cycle arrest. They have been approved to treat breast cancers and trials are ongoing to test their use in a range of other cancer types. It is important to understand how this G1 arrest corresponds to a cytotoxic response in patients. A prolonged CDK4/6-induced G1 arrest causes a reduction in key replisome components and origin licensing. Upon release from this G1 arrest, DNA replication is impaired and RPE1 cells withdraw from the cell cycle in a p53-dependent manner. In the absence of p53, cells progress through the cell cycle resulting in excessive DNA damage. During this G1 arrest, cells grow excessively which can be rescued by mTOR inhibition, contact inhibition, low serum and MEK inhibition. Rescue of cell growth prevents cell cycle withdrawals and improves long-term proliferation, suggesting that overgrowth is responsible for replication stress defects. This study provides the first link between CDK4/6 inhibitors and genotoxic stress and provides a rationale to predict sensitive tumour types and effective combination strategies.

The renin-angiotensin system (RAS) is a major physiological regulator of body fluid volume, electrolyte balance and blood pressure. The principal effector peptides for this system are the angiotensins, particularly angiotensin II (Ang II), whose biological cations are mediated by specific angiotensin receptors. The use of highly specific nonpeptide angiotensin antagonists has allowed identification and characterisation of two major Ang II receptor subtypes, designated AT1 and AT2. The aims of this thesis were to investigate AT1 and AT2 receptor expression at both mRNA and protein level in freshly isolated and primary cultures of rat proximal tubular (PT) cells, and to determine the effect of culture upon Ang II receptor subtype expression. The possible role of Ang II upon receptor expression was also investigated. This study demonstrated that only the AT1 receptor is expressed in freshly isolated rat PT cells, with no evidence for AT2 receptor expression. Although continuously present throughout the culture period, a significant decrease in AT1 receptor expression was observed with time. Conversely AT2 receptor expression was absent in freshly isolated cells but was observed after 24 hours in culture with expression then remaining stable throughout culture. Clearly Ang II receptor expression is not stable during culture. Primary cultures of rat PT cells exhibit a change in receptor expression similar to those observed in vivo following tissue damage and repair, with an increase in AT2 receptor expression possibly mediated by locally released Ang II. Initial studies however, involving incubation of rat PT cells with exogenous Ang II, have demonstrated no effect upon AT1 receptor mRNA expression.

Neuropeptides, and in particular peptides of the bombesin family, including gastrin releasing peptide, stimulate DNA synthesis and cell proliferation in cultured cells and are implicated as cellular growth factors in a variety of fundamental biological processes including development, tissue regeneration and tumourigenesis. Bombesin elicits multiple signalling pathways in various cell types, including activation of phospholipase C, which leads to activation of protein kinase C and elevation of intracellular calcium; tyrosine phosphorylation of intracellular substrates and reorganisation of the actin cytoskeleton, and cell proliferation. It is not clear however whether these responses are mediated by a single receptor subtype or by different subtypes that couple preferentially to specific pathways. To resolve this the mouse bombesin/GRP receptor was transfected into Rat-1 fibroblasts and the pathways activated by bombesin were investigated. Here it is shown that phospholipase C activation, cell growth and tyrosine phosphorylation emanate from a single class of bombesin receptor. Mitogen Activated Protein Kinase (MAPK) activity is a common point of convergence for mitogenic signals. While the mechanism by which receptor tyrosine kinases activate MAPK is well established, the mechanism by which G protein-coupled receptors activate MAPK is less well understood. The involvement of Ras and Rai in activation of MAPK has not been described in bombesin-induced signalling. This thesis shows that in Rat-1 cells transfected with the bombesin/GRP receptor, bombesin can activate MAPK and Raf-1 in a protein kinase C-independent fashion. Also it is demonstrated for the first time the bombesin stimulates GTP loading of p2lras. Calcium mobilisation from intracellular calcium stores has been observed to be coincident with cell proliferation induced by a variety of growth factors including neuropeptides and especially bombesin. However, a direct role of calcium in this event has not been described. Here it is shown that partial and persistent depletion of intracellular calcium stores, using specific inhibitors of the endoplasmic reticulum calcium pump, thapsigargin and 2,5-di-tert-butylhydroquinone, stimulated reinitiation of DNA synthesis in synergy with either phorbol-12,13-dibutyrate or bombesin in Swiss 3T3 cells. This stimulation of DNA synthesis was dependent on entry of extracellular calcium and hence this suggests a role for calcium in growth control.

The adult human body is made up of trillions of cells and yet began as just one. This could not be possible without careful coordination of cell growth and cell division. The molecular mechanisms that drive and control cell growth as well as its coordination with the cell cycle remain poorly understood. Cell size is the outcome of cell growth. Thus, a decoupling of growth and division would result in cells of increasingly heterogenous sizes. Interestingly, the severity of a cancer is often correlated with increasingly heterogenous size of cells within a tissue. Furthermore, genes that are thought to play important roles in cell growth are often dysregulated in a variety of cancers. This suggests that the misregulation of mechanisms that control cell growth and cell size plays a role in cancer cell development. How cell growth influences cell cycle progression to control cell size is perhaps best understood in budding yeast where key cell cycle transitions are delayed until sufficient growth has occurred. It had been long believed that the predominant cell size checkpoint in budding yeast occurred at cell cycle entry. Recent work, however, suggests that bud growth strongly influences cell cycle progression in mitosis. In this thesis, I present evidence to support a model in which cells influence the extent and duration of bud growth by controlling mitotic exit, a cell cycle transition that seizes bud growth and finalizes daughter cell size.

Cell cycle progression is dependent upon cell growth. Cells must, therefore, translate growth into a proportional signal that can be used to determine when sufficient growth has occurred for cell cycle progression. In budding yeast, a protein kinase called Gin4 is required for normal control of cell growth and undergoes gradual hyperphosphorylation that is dependent upon and proportional to added membrane growth in metaphase within the mitotic phase of the cell cycle. These observation suggest that growth-dependent phosphorylation of Gin4 could play a role in mechanisms that measure cell growth in mitosis. However, the molecular mechanisms that drive growth-dependent hyperphosphorylation of Gin4 are poorly understood. Here, we utilized a combination of biochemical reconstitution and genetic analysis to define the molecular mechanisms that drive growth dependent phosphorylation of Gin4. Our working model is that lipid vesicles that drive plasma membrane growth drive also deliver key signaling molecules that drive Gin4 hyperphosphorylation, which would suggest a simple mechanistic explanation for how growth-dependent Gin4 phosphorylation signals are generated. Gin4 has a KA1 domain that binds phosphatidylserine lipid and is required for growth-dependent phosphorylation of Gin4. Our work rules out a simple model in which phosphatidylserine delivered to sites of membrane growth binds and activates Gin4 to undergo autophosphorylation. Rather, the data suggest that phosphatidylserine recruits Gin4 to sites of membrane growth, where it is phosphorylated and activated by Elm1 in a Yck1-dependent manner. In this manner, Gin4 phosphorylation signals are generated to drive inhibition of the mitotic inhibitor Swe1 to promote the metaphase to anaphase transition, allowing cells to complete division.

Severe defects in cell size are a nearly universal feature of cancer cells. Yet, the mechanisms that drive size defects in cancer cells remain unknown, and it is unclear whether they are a direct consequence of primary oncogenic drivers or a secondary consequence of mutations that accumulate during evolution of cancer cells. Hyperactive mutants of Ras are amongst the most prevalent oncogenic drivers. In budding yeast, previous studies have suggested that hyperactive versions of the Ras homolog (ras2G19V) cause defects in cell size. However, the mechanisms by which ras2G19V drives cell size defects are unknown, and it is unclear whether the size defects are due to accumulation of suppressor mutations. Here, I developed a system for inducible expression of ras2G19V that allows for investigation into the immediate consequences of ras2G19V expression. I found that ras2G19V causes a delay in G1 phase, increased cell size, and aberrant expression of G1 cyclin proteins. Furthermore, ras2G19V appears to inhibit a key step in cell cycle entry, in which an early G1 cyclin induces transcription of late G1 cyclins. The data further suggest that Ras does not influence cell size solely via effects in G1 phase. Finally, we found that expression of oncogenic Ras alone is sufficient to cause cell size defects in NIH 3T3 cells, which suggests that defects in cell size in cancer cells could be a direct consequence of primary oncogenic signals. Together, the data suggest that hyperactive forms of Ras influence cell size in both yeast and mammals. Further analysis will determine whether Ras influences cell size via conserved mechanisms.

The aim of this project is to test the hypothesis that the rate of turnover of newly synthesized proteins plays a major part in the regulation of cell growth. Normal and transformed cells have been used to compare their responses to serum levels. When the cells are cultured in 0.2% serum medium, increase of cell number is slower than in normal cultures. Cell volume is reduced, while cell mass may be maintained or decreased depending on the rate of protein accumulation and cell proliferation, so unbalanced growth may occur in certain circumstances. During serum deprivation, accumulation of newly synthesized proteins slows down due to both aspects of reduced protein synthesis rate and increased degradation of proteins, mainly, medium- and long-lived proteins. After serum restoration, their rate of protein synthesis increased, and the rate of long-lived protein degradation was reduced to a lower level than in cells kept continuously in 10&37 serum, indicating that medium- and long-lived proteins are more important than short-lived proteins in the regulation of cell growth. After serum step-down, untransformed 3T3 cells did not enter a truly G0 state, but showed a very slow increase in cell number; transformed 3T3 and HeLa cells showed 'unbalanced growth', i.e. cell number increased faster than proteins accumulated. Serum deprivation quickly and substantially affected growth of untransformed 3T3 cells, but transformed 3T3 and HeLa cells retain their growth rate higher than that of untransformed 3T3 cells. Contrary to general belief, the transformed 3T3 cells and HeLa cells were quite responsive in many respects to serum withdrawal. This fact indicates that stimulating growth by 'autocrine' mechanism is limited. Investigation of the pathways of protein degration and rate of RNA synthesis were undertaken. Attention was paid to basic problems associated with the serum deprivation, and the serum-deprived cell culture system as a model or paradigm for studies of this nature is discussed.

This thesis describes how using a heterotrophy-recovery-autotrophy route (called the green cycle) for the cultivation of Botryococcus braunii results in high biofuel production. Our experiments show that the addition of citrate to a culture can increase cell numbers by up to 20 times compared with the control in the first week. A metal solution was then added to help cells synthesize chlorophyll, enabling Botryococcus braunii to regenerate the chlorophyll content in the plastids and become autotrophic. In our experiments, high cell numbers were obtained after two weeks and biofuel production was enhanced. We also developed an N-N-A source media which could achieve enhanced cell growth as opposed to using nitrate as the only nitrogen source. This N-N-A source media was able to balance pH variation due to nitrate uptake, so as to maintain the pH level in the media solution without the use of buffer. By applying these techniques to the Botryococcus braunii culture, we found that cell growth could increase by up to 58 times in comparison with the control

Bacterial cell structures are macromolecular features found among all types bacteria. They often serve essential or specialized functions, and are important contributors to the widespread diversity and success of bacteria. I have focused my research on the formation and localization of two of these structures in the plant pathogen Agrobacterium tumefaciens: the type IV secretion system (T4SS) and the bacterial cell envelope. A. tumefaciens is an Alphaproteobacterium uniquely capable of genetically transforming plant host cells during its infection process. DNA and protein substrates are transferred into plant cells via the T4SS, which forms multiple complexes around the bacterial circumference. To understand the mechanisms driving T4SS positioning, I assessed their spatial distribution by quantitative analysis and modeling. My findings indicate that these secretion complexes localize in a non-random periodic pattern along the cell perimeter. These results, along with additional plant infection studies, suggest this spatial organization serves to promote efficient delivery of substrates into host cellsA potential mechanism behind this periodic arrangement is the unipolar cell growth recently described for A. tumefaciens. To better understand this unusual growth mechanism, I carefully scrutinized growing cells, nascent peptidoglycan, and peptidoglycan synthesis components by electron and fluorescence microscopy. My results indicate that the unipolar growth process is surprisingly dynamic, multi-dimensional, and involves both novel and familiar components. These findings thus represent a significant departure from the canonical growth mechanism of E. coli and other well-studied bacilli.

The study was conducted on the fungus Penicillium marneffei. Penicillium marneffei is endemic to south-east Asia and is the only thermally dimorphic species of the Penicillium genus. In its yeast form, it causes a systemic disease called Penicilliosis in immunocompromized individuals. It is currently treated by using moderately effective drugs like Amphotericin B. The dimorphic switch that changes the mold to yeast at 37 °C remains a mystery and could hold the key to designing effective treatments.Dimorphism could be hypothesized to depend on turning on or off certain genes like cell division or cell growth genes. The chief cell growth gene studied for this project is TOR or Target of Rapamycin. It is investigated for differential gene expression in the Mold and Yeast phases of the P. marneffei cells caused by the environmental stress. Also, the cells were treated with rapamycin to inhibit TOR and notice its effect on cell growth. TOR is characterized using bioinformatics analysis. Morphological study is conducted on un-treated and treated cultures using phase contrast microscopy.Cells are collected to extract RNA from the different samples and cDNA is synthesized. To study expression in real time, Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) is performed on the samples using Actin and Calmodulin as controls. The expression of TOR increases in the yeast phase in both untreated and rapamycin treated P. marneffei cells even though the growth is severely stunted. This indicates the presence of a compensatory mechanism by which the cells try to survive the Rapamycin in the medium and remain mostly in filamentous form. Therefore, TOR could be playing a critical role in bringing about the dimorphic change and could aid to devising enhanced treatments for Penicilliosis.

The Na/K-ATPase α1 subunit contains a highly conserved caveolin-1 binding motif (CBM). We have previously shown that the interaction between the α1 CBM and caveolin-1 regulates the formation of caveolae, and intracellular trafficking and distribution of cholesterol. Here we demonstrate an important role of this interaction in control of cell growth. First, we find that knockdown of either the Na/K-ATPase or caveolin-1 inhibits cell proliferation and that this inhibition is greatly enhanced by modest depletion of cellular cholesterol. Second, the expression of wild-type α1, but not the CBM mutant, is capable of restoring normal growth in LLC-PK1 cells. Finally, we find that the expression of N-terminus of the α1 subunit inhibits the interaction between the Na/K-ATPase and caveolin-1. Consequently, it alters normal trafficking of caveolin-1 and cholesterol, and inhibits cell growth. Taken together, our new findings demonstrate that the highly conserved caveolin binding motif of the Na/K-ATPase plays an important role in regulation of cell growth by affecting the trafficking and distribution of caveolin-1 and cholesterol.

This research aims at modeling the cell cycle regulation as a three dimensional Agent Based Model. Agent Based Modeling (ABM) is a computational model for simulating autonomous entities in a network. ABM is emerging as a powerful tool in the field of systems biology. ABM is preferred over traditional modeling methods, as it takes the spatial dynamics of the interactions into account. The system is modeled as a collection of autonomous decision making entities called agents. Each of these agents has its own set of characteristics and behavioral rules that are highly adaptive to its surrounding environment. Since the agents have their own sets of characteristics and rules, they can be implemented using the Object Oriented Programming (OOP) concept. OOP is a powerful programming technique that can be used to model and design applications effectively. Previously our lab implemented an ABM model of the phage lambda virus and used this model to study the behavior of phage lambda under various conditions. Building on our experience, we have developed a more complex agent based model in a Java based ABM platform. The cell cycle model is modeled in MASON, a very robust and efficient ABM platform that supports strong 3D visualization and parallelization. The model shows how the cells interact among each other at an intercellular level. The stages in the cell cycle are implemented and the characteristics of cell growth are studied. This particular model has also been extended to simulate a wound condition and its growth characteristics are again observed. The model is run for certain base parameter values and the system can be simulated for other conditions by changing these parameters. Tissue Engineering is an important and growing field, and computational models such as the one developed in this thesis can be very useful tools for biologists and tissue engineers.

Cancer is a group of diseases characterized by uncontrolled growth and this spread of abnormal cells is caused by various external and internal reasons. This study elucidated the relationship between the growth of cancer cells and concentration of NO and ONOO¯. Nanosensors were applied to measure nitric oxide and peroxynitrite concentration in cancer and normal cells. The ratio between NO/ONOO¯ concentrations was used as an indicator of oxidative stress in cancer cells. ONOO¯ concentration measured from HT1080 cancer cells (fibrosacoma) and MRC-5 normal cells (fibroblasts) was higher than NO concentration. ONOO¯ released from HT1080 cancer and MRC-5 normal cells was similar, while NO concentration in HT1080 cancer cells was significantly higher than in MRC-5 normal cells. Enzyme levels were measured by using Western blotting and correlated with NO and ONOO¯ levels in normal and cancer cells treated with 3-morpholinosydnonimine hydrochloride (SIN-1) (ONOO¯ donor) and sodium nitroprusside (SNP) (NO donor), respectively.After the preincubation with SNP and SIN-1 in MRC-5 cells, a significant decrease in eNOS expression was observed compared to HT1080 cancer cells. This indicates that MRC-5 normal cells were more sensitive to the changes of exogenous NO and ONOO¯. It indicated that exogenous NO or ONOO¯ downregulate eNOS expression in both cancer and normal cells.A treatment of CACO-2 cancer cells with SNP decreased eNOS expression more significantly that a treatment with SIN-1. In this study, we found that NO not only affected eNOS expression but also cell growths. The effect of NO and ONOO¯ on cell growth was investigated by preincubating HT1080 cancer and MRC-5 normal cells with L-arginine, NO substrate and L-NAME, a NOS inhibitor.L-arginine treatment promoted HT1080 cancer cell growth and increased the number of viable cells. HT1080 cancer cells preincubated with L-arginine promoted cell growth and increased the cells number. NO had a greater in promoting effect growth of HT1080 cancer cells. MRC-5 cell growth after treatment, with L-arginine, was slower than the rate observed for HT1080. L-NAME, an eNOS inhibitor, did not effect MRC-5 normal cell growth and cell numbers significantly.The pretreatment of CACO-2 cancer cells with SNP and L-arginine showed higher cell growth than those treated with SIN-1 or L-NAME. SNP and L-arginine inhibited the cell growth, and cell number of CACO-2. SIN-1 treatment inhibited CACO-2 cell growth through increased ONOO¯ level. In CACO-2 cancer cells, NO effects cell growth and further study are needed to identify the level at which NO becomes cytotoxic to CACO-2 cancer cells. NO promotes normal colon epithelial growth, but inhibited CACO-2 colon cancer cell growth. This finding can be potentially useful in determining a strategy for colon cancer treatment.

In this research, environmentally benign carbon dioxide (CO2) was used as a physical blowing agent in the foaming of PS and PS nanocomposites due to the phase-out of chlorofluorocarbons (CFCs) and hydrochlorofluorocarbons (HCFCs). CNFs (carbon nanofibers) and AC (activated carbon) were used as additive/nucleation agents in PS extrusion foaming. Both fillers showed promising application in insulation foams. The typical foaming process includes cell nucleation, cell growth, and cell stabilization. In this study, primary attention was given to the effects of the material related properties, solubility, diffusivity, and shear viscosity, on cell nucleation and cell growth. Two equations of state (EOS), Sanchez-Lacombe (S-L) EOS and perturbed chain statistical associating fluid theory (PC-SAFT), were used to model the solubility and also phase boundaries (binodal and spinodal curves). Nucleation is a very complex phenomenon in physical foam processing. Proposed scaling functions provide a possible way to calculate the energy barrier in the nucleus formation. Cell nucleation rate data were extracted from the literature and also by our experiments. The initial slope of the possible scaling function was calculated by the diffuse interface theory. A scaling function was correlated based on the calculation from experimental data. Shear viscosity of polymers and nanocomposites under high pressure CO2 were studied via unique modified high pressure Couette rheometry. The effects of nanoparticles on shear viscosity of polymer w/ and w/o CO2 were compared. The permeability coefficient, defined as the product of the solubility coefficient and diffusivity, of CO2 and water vapor in polymer nanocomposites and foams were measured near ambient temperature and pressure. The effects of nanoparticle and foam morphology on permeation were studied. To gain more insight on the early stages of the foaming process, in-situ observation of batch foaming and a quenching method were used to study foams with different cell growth times. In addition, the quantitative cell nucleation and growth rates were measured by our novel experimental setup. These cell nucleation rate data provided valuable information for modeling and theoretical correlation. Based on these experimental studies and modeling, a deeper and more comprehensive understanding of polymer and nanocomposite foaming was achieved.

To improve the surface properties of biomaterials, the effects of changes in surface chemistry and morphology of polyethylene terephthalate (PET) films treated with atmospheric pressure plasma were investigated as a function of cellular growth, proliferation, and adhesion. PET films were subjected to helium and helium/oxygen gas plasmas. The contact angle of the treated films decreased due to plasma etching and possible scission indicating that the surfaces become more hydrophilic. Atomic force microscopy results had a large standard error, however the surface visually showed changes in surface micro and nanoscale roughness corresponding to treatment duration. Keratinocytes were plated on the day of plasma treatment and two and five days after plasma treatment and tested half a day, one, two, three, and six days after plating. The same methodology of plating and testing was also applied to hepatocytes. Cell growth, proliferation, and adhesion were characterized via a fluorescent probe based assay and were correlated with surface chemical and nanostructural features. Both the helium and helium/oxygen plasma-treated PET had little or no effect on cell behavior for both keratinocytes and hepatocytes. The nanoscale surface changes due to the plasma surface treatment are believed to be masked by the protein adherence in the media on the surface of the PET.