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.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 3 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2008. / Härtill 4 uppsatser.

Tanto hormônios glicocorticóides (GCs) como o gene supressor de tumor TP53, medeiam a resposta celular a uma diversidade de condições fisiológicas de estresse, sendo reguladores fundamentais do processo de vida/morte de diversos tipos celulares. A interação funcional entre estes fatores vem sendo explorada, recentemente, revelando que GCs exercem um efeito dual sobre p53. O modelo celular ST1/P7 de glioma de rato é particularmente interessante para investigar o papel de p53 na ação de GCs, já que estas linhagens apresentam respostas distintas a GCs. O tratamento com Hidrocortisona (Hy) leva as células ST1 a uma complexa reversão fenotípica tumoral→normal, enquanto as células P7 são altamente resistentes ao tratamento. Foi possível observar que a ativação de p53 por Hy ocorre apenas em células ST1, mas não em P7. Esta ativação é mediada pela indução de fosforilação da Ser15 de p53 e seu acúmulo nuclear, o que resulta no aumento de sua ligação a elementos responsivos a p53 no DNA e na sua capacidade de transativação de p53, levando a um aumento da expressão de alguns de seus genes-alvo. Contudo, o bloqueio de p53 através de siRNA não foi suficiente para alterar a resposta de células ST1 a GCs, indicando que a regulação positiva de p53 por GCs pode ser um evento secundário, mas não essencial, para a resposta anti-tumoral exercida por estes hormônios em células ST1. / Both glucocorticoid hormones (GCs) and the TP53 tumor suppressor gene mediate cellular responses to a diversity of physiological stress conditions, acting as crucial regulators of the life/death process in a wide variety of cell types. The ST1/P7 rat glioma model cell system is particularly interesting to investigate the role of p53 in the action of GCs, since these cell lines display opposite responses to GCs. Treatment with Hydrocortisone (Hy) leads ST1 cells to a complete tumoral→normal phenotypic reversion, while P7 cells are highly resistant to this treatment. It was possible to observe that activation of p53 by Hy occurs only in ST1 cells, but not in GC-resistant P7 cells. This activation is mediated by induction of phosphorylation of the Ser15 residue of p53 and its accumulation in the nucleus, resulting in increased binding of p53 to its responsive elements on the DNA and in activation of its transactivating potential, leading to increased expression of some of its target genes. However, blocking of p53 through siRNA was not sufficient to alter ST1 cells response to GCs, indicating that the positive regulation of p53 by GCs may be a secondary, non-essential, event for the anti-tumor response exerted by these hormones in ST1 cells.

The hair follicle (HF) is a cyclic biological system that progresses through stages of growth, regression and quiescence, each being characterized by unique patterns of gene activation and silencing. MicroRNAs (miRNAs) are critically important for gene silencing and delineating their role in hair cycle may provide new insights into mechanisms of hair growth control and epithelial tissue remodelling. The aims of this study were: 1) To define changes in the miRNA profiles in skin during hair cycle-associated tissue remodelling; 2) To determine the role of individual miRNAs in regulating gene expression programs that drive HF growth, involution and quiescence; 3) and to explore the role of miRNAs in mediating the effects of BMP signalling in the skin. To address Aims 1 & 2, global miRNA expression profiling in the skin was performed and revealed marked changes in miRNAs expression during distinct stages of the murine hair cycle. Specifically, miR-31 markedly increased during anagen and decreased during catagen and telogen. Administration of antisense miR-31 inhibitor into mouse skin during the early- and mid-anagen phases of the hair cycle resulted in accelerated anagen development, and altered differentiation of hair matrix keratinocytes and hair shaft formation. Microarray, qRT-PCR and Western blot analyses revealed that miR-31 negatively regulates expression of Fgf10, the components of Wnt and BMP signalling pathways Sclerostin and BAMBI, and Dlx3 transcription factor, as well as selected keratin genes. Luciferase reporter assay revealed that Krt16, Krt17, Dlx3, and Fgf10 serve as direct miR-31 targets. In addition, miR-214 was identified as a potent inhibitor of the Wnt signalling pathway in the keratinocytes. Mutually exclusive expression patterns of miR-214 and ¿-catenin was observed during HF morphogenesis. MiR-214 decreases the expression of ¿-catenin and other components of Wnt signalling pathways c-myc, cyclin D1, and Pten in the keratinocytes. Luciferase reporter assay proved that ¿-catenin serves as a direct target of miR-214. In addition, miR-214 prevented translocation of ¿-catenin into the nucleus in response to the treatment with an activator of the Wnt signalling pathway lithium chloride, and abrogated the lithium-induced increase of the expression of the Wnt target gene VI Axin2. This suggests that miR-214 may indeed be involved in regulation of skin development and regeneration at least in part, by controlling the expression of ¿-catenin and the activity of the Wnt signalling pathway. To address Aim 3, the role of miRNAs in mediating the effects of the bone morphogenetic protein (BMP) signalling in the skin was explored. MiRNAs were isolated from the primary mouse keratinocytes treated with BMP4 and processed for analysis of global miRNA expression using the microarray approach. Microarray and real-time PCR analysis revealed BMP4-dependent changes in the expression of distinct miRNAs, including miR-21, which expression was strongly decreased in the keratinocytes after BMP4 treatment. In contrast, miR-21 expression was substantially higher in the skin of transgenic mice over-expressing BMP antagonist Noggin. Transfection of the keratinocytes with miR-21 mimic revealed existence of two groups of the BMP target genes, which are differentially regulated by miR-21. Thus, this suggests a novel mechanism controlling the effects of BMP signalling in the keratinocytes. Thus, miRNAs play important roles in regulating gene expression programs in the skin during hair cycle. By targeting a number of growth regulatory molecules, transcription factors and cytoskeletal proteins, miRNAs are involved in establishing an optimal balance of gene expression in the keratinocytes required for the HF and skin homeostasis.

Cancer is a generic term for a large group of diseases where cells of the body lose their normal mechanisms for growth so that they grow in an uncontrolled way. One of the most common treatments of cancer is chemotherapy that aims to kill abnormal proliferating cells; however normal cells and other organs of the patients are also adversely affected. In practice, it¿s often difficult to maintain optimum chemotherapy doses that can maximise the abnormal cell killing as well as reducing side effects. The most chemotherapy drugs used in cancer treatment are toxic agents and usually have narrow therapeutic indices, dose levels in which these drugs significantly kill the cancerous cells are close to the levels which sometime cause harmful toxic side effects. To make the chemotherapeutic treatment effective, optimum drug scheduling is required to balance between the beneficial and toxic side effects of the cancer drugs. Conventional clinical methods very often fail to find drug doses that balance between these two due to their inherent conflicting nature. In this investigation, mathematical models for cancer chemotherapy are used to predict the number of tumour cells and control the tumour growth during treatment. A feedback control method is used so as to maintain certain level of drug concentrations at the tumour sites. Multi-objective Genetic Algorithm (MOGA) is then employed to find suitable solutions where drug resistances and drug concentrations are incorporated with cancer cell killing and toxic effects as design objectives. Several constraints and specific goal values were set for different design objectives in the optimisation process and a wide range of acceptable solutions were obtained trading off among different conflicting objectives. Abstract v In order to develop a multi-objective optimal control model, this study used proportional, integral and derivative (PID) and I-PD (modified PID with Integrator used as series) controllers based on Martin¿s growth model for optimum drug concentration to treat cancer. To the best of our knowledge, this is the first PID/I-PD based optimal chemotherapy control model used to investigate the cancer treatment. It has been observed that some solutions can reduce the cancer cells up to nearly 100% with much lower side effects and drug resistance during the whole period of treatment. The proposed strategy has been extended for more drugs and more design constraints and objectives. / Libyan Ministry of Higher Education

No description available.

No description available.

The development of a multicellular organism, such as a human or an animal, begins with the fertilization of an egg cell. Thereupon the organism grows by repeated cell divisions until the adult size is reached and growth stops. Although it is known that intrinsic mechanisms determine the final size of developing organs and organisms, the basic principles of growth control are still poorly understood. However, there is strong evidence that certain morphogens, which are a special class of signaling molecules, act as growth factors and play a key role in growth control. In this work, growth control is studied from a mainly theoretical viewpoint. A discrete vertex model describing the organization of cells by a network of polygons is used, including a description of the cell cycle and a description of dynamical morphogen distributions. Self-organized growth is studied by introducing growth rules that govern cell divisions based on the local morphogen level. This discrete description is complemented by a continuum theory to gain further insight into the dynamics of self-organized growth processes. The theoretical description is applied to the developing wing of the fruit fly Drosophila melanogaster. In the developing wing, which is an epithelium consisting of single-layered cell sheets, the morphogen Decapentaplegic (Dpp) acts as a key growth factor. Experimental data shows that the Dpp distribution is dynamic and adapts to the size of the developing wing. Two mechanisms that rely on a regulatory molecule species and lead to such a dynamic behaviour of the Dpp distribution are studied. Several growth rules are tested and the resulting growth behaviour is quantitatively compared to experimental data of the developing wing. A particular growth rule, that triggers a cell division when the local morphogen level has increased by a certain relative amount, is found to be consistent with experimental observations under normal and several perturbed conditions. It is shown that mechanical stresses that arise due to spatial growth inhomogeneities can have a stabilizing effect on the growth process.