Browsing by Subject "Cell cycle"
Now showing 1 - 9 of 9
Results Per Page
Sort Options
Item Cell cycle protein interaction with telomerase in a breast cancer culture system(Texas Tech University, 2001-08) Cook, T. KevinBreast cancer causes 40,000 deaths a year and is second only to lung cancer in deaths attributed to neoplastic disease. There is a known age and cell lineage dependence related to the development of the disease. It is vitally important to understand the manner in which this disease develops. One approach is to examine proteins involved in cell cycle regulation. Three such proteins are c-Myc, p53, and pRb. These proteins in conjunction with one another and telomerase perform critical functions within a cell. They have an intertwined pattern of activity. If these functions are not regulated, the cells can proliferate unchecked and give rise to cancer. Our study focused on the differences of protein levels and activity overtime between epithelial and stromal cells of the breast. The data appeared to show that stromal cells exhibit a tighter control over their cell cycle than their epithelial counterparts. Further, it would seem that c-Myc levels are inversely related to telomerase activity. This data points to more studies in the future.Item The cellular and molecular mechanisms underlying ventral midline patterning and morphogenesis in the amniote midbrain(2015-12) Brown, Charmaine Yvonne; Agarwala, Seema; Vokes, Steven; Eberhart, Johann; Ehrlich, Lauren; Gross, JeffThe floorplate (FP) is located at the ventral midline of the developing neural tube, and is involved in patterning and specification of ventral and dorsal cell fates. The FP has long been known to pattern ventral cell fates via secretion of Sonic Hedgehog (SHH). However, the mechanism by which the FP is specified is controversial due to species variations where SHH is differentially required for FP specification in fish and mouse. In Chapter 3, we show that, similar to the fish, the amniote anterior neural plate can be divided into medial (MFP) and lateral (LFP) subdivisions which differentially require SHH and FOXA2 for their specification, and that FOXA2, but not SHH, is sufficient to induce the entire midbrain FP pattern. In addition, we show that all three midbrain signaling centers are physically continuous and interconvertible, with their specification depending on SHH. Prior to the expression of SHH protein, the ventral midline undergoes a morphogenetic event called median hinge point (MHP) formation which buckles the flat neural plate and lifts the neural folds which ultimately fuse into a cylindrical neural tube. Previous studies in the lab have shown that Bone Morphogenetic Proteins (BMP) modulate HP formation. HP formation involves dynamic cell-shape changes, which result in HP cells becoming wedge-shaped. Multiple mechanisms (constriction of the adherens belt via cytoskeletal and junctional remodeling, and polarized endocytosis) have been proposed to explain this shape change. However, they do not explain how reduction in apical area can be achieved in the amniote neural plate where cells are bipolar and only slender processes contact the apical surface in non-mitotic cells. In Chapter 4, we develop an early electroporation technique which is used in Chapter 5 to visualize HP formation in real time as part of a novel 3D explant system. Our results suggest that BMP attenuation regulates cell cycle progression by increasing the duration of G1 and S phases, and causes a subset of cells to prematurely exit the cell cycle and undergo sub-apical G2-M transition, similar to what is seen in the MHP where there is reduced mitotic index and cells undergo mitosis sub-apically.Item Development of imaging-based high-throughput genetic assays and genomic evaluation of yeast gene function in cell cycle progression(2007-12) Niu, Wei; Marcotte, Edward M.Systems biology studies the complex interactions between components of biological systems. One major goal of systems biology is to reconstruct the network of interactions between genes in response to normal and perturbed conditions. In order to accomplish this goal, large-scale data are needed. Accordingly, diverse powerful and high-throughput methods must be developed for this purpose. We have developed novel high-throughput technologies focusing on cellular phenotype profiling and now provide additional genome-scale analysis of gene and protein function. Few high-throughput methods can perform large-scale and high-throughput cellular phenotype profiling. However, analyzing gene expression patterns and protein behaviors in their cellular context will provide insights into important aspects of gene function. To complement current genomic approaches, we developed two technologies, the spotted cell microarray (cell chip) and the yeast spheroplast microarray, which allow high-throughput and highly-parallel cellular phenotype profiling including cell morphology and protein localization. These methods are based on printing collections of cells, combined with automated high-throughput microscopy, allowing systematic cellular phenotypic characterization. We used spotted cell microarrays to identify 15 new genes involved in the response of yeast to mating pheromone, 80 proteins associated with shmoo-tip 'localizome' upon pheromone stimulation and 5 genes involved in regulating the localization pattern of a group II intron encoded reverse transcriptase, LtrA, in Escherichia coli. Furthermore, in addition to morphology assays, yeast spheroplast microarrays were built for high-throughput immunofluorescence microscopy, allowing large-scale protein and RNA localization studies. In order to identify additional cell cycle genes, especially those difficult to identify in loss-of-function studies, we performed a genome-scale screen to identify yeast genes with overexpression-induced defects in cell cycle progression. After measuring the fraction of cells in G1 and G2/M phases of the cell cycle via high-throughput flow cytometry for each of ~5,800 ORFs and performing the validation and secondary assays, we observed that overexpression of 108 genes leads to reproducible and significant delay in the G1 or G2/M phase. Of 108 genes, 82 are newly implicated in the cell cycle and are likely to affect cell cycle progression via a gain-of-function mechanism. The G2/M category consists of 87 genes that showed dramatic enrichment in the regulation of mitotic cell cycle and related biological processes. YPR015C and SHE1 in the G2/M category were further characterized for their roles in cell cycle progression. We found that the G2/M delay caused by the overexpression of YPR015C and SHE1 likely results from the malfunction of spindle and chromosome segregation, which was supported by the observations of highly elevated population of large-budded cells in the pre-M phase, super-sensitivity to nocodazole, and high chromosome loss rates in these two overexpression strains. While the genes in the G2/M category were strongly enriched for cell cycle associated functions, no pathway was significantly enriched in the G1 category that is composed of 21 genes. However, the strongest enrichment for the G1 category consists of the genes involved in negative regulation of transcription. For instance, the overexpression of SKO1, a transcription repressor, resulted in strong cell cycle delay at G1 phase. Moreover, we found that the overexpression of SKO1 results in cell morphology changes that resembles mating yeast cells (shmoos) and activates the mating pheromone response pathway, thus explaining the G1 cell cycle arrest phenotype of SKO1 ORF strains.Item Differential uncoupling of 5' and 3' exonucleolytic activities as determined by mutational analysis of the Saccharomyces cerevisiae exoribonuclease, RAT1(2010-05) Gupton, Leodis Darren; Stevens, Scott W.; Browning, Karen; Johnson, Arlen; Fast, Walter; Yin, WhitneyEukaryotic gene expression requires hundreds of proteins and several RNA factors to facilitate nuclear RNA processing. These RNA processing events include RNA transcription, pre-mRNA splicing, pre-ribosomal RNA (pre-rRNA) processing and trafficking of RNA to its proper location in the cell. As we learn more about the molecular details of the factors governing these highly coordinated processes it is becoming increasingly clear that a subset of factors participate in multiple RNA processing pathways to ensure faithful gene expression. Our work completes the characterization of the Abelson pre-mRNA splicing mutants. We have discovered that the prp27-1 splicing mutant is a severe loss of function allele of RAT1, an essential 5’→3’ exoribonuclease. Several alleles of RAT1 have been previously isolated with each conferring an array of phenotypes thus making the elucidation of its essential in vivo function difficult. We set out to determine how mutations within a specific region determines the RNA processing pathway in which Rat1p has been implicated to function within. In our analysis of Rat1p function we discovered the prp27-1 allele exhibits novel 3’ end processing defects never reported in previous rat1 mutants. We performed mutational analysis to examine the coupling of 5’ and 3’ exonucleolytic activities in nuclear RNA processing events. Through our study we have discovered a means by which the cell coordinately regulates the nuclear RNA degradation complexes to ensure efficient processing of pre-RNAs for the faithful execution of eukaryotic gene expression. Additionally, we offer evidence in support of role for Rat1p in promoting mitotic events in vivo.Item Higher-order generalized singular value decomposition : comparative mathematical framework with applications to genomic signal processing(2010-08) Ponnapalli, Sri Priya; Ghosh, Joydeep; Alter, Orly, 1964-; Beckner, William; Caramanis, Constantine; Evans, Brian L.; Van Loan, CharlesThe number of high-dimensional datasets recording multiple aspects of a single phenomenon is ever increasing in many areas of science. This is accompanied by a fundamental need for mathematical frameworks that can compare data tabulated as multiple large-scale matrices of di erent numbers of rows. The only such framework to date, the generalized singular value decomposition (GSVD), is limited to two matrices. This thesis addresses this limitation and de fines a higher-order GSVD (HO GSVD) of N > 2 datasets, that provides a mathematical framework that can compare multiple high-dimensional datasets tabulated as large-scale matrices of different numbers of rows.Item Murine coronavirus-induced apoptosis and cell cycle dysregulation(2002-05) Chen, Chun-jen; Bose, Henry R.; Makino, ShinjiItem PTIP, a novel BRCT domain-containing apoptotic factor that directly promotes cytochrome c release from mitochondria to cytoplasm(2001-08) Zhang, Yan; Tucker, Philip W.Item Systematic Analysis of Genetic and Pharmaceutical Modulators of the Eukaryotic Cell Cycle(2012-10-19) Hoose, Scott AllenCell replication and division are central to the proliferation of life, and have implications for normal growth and development as well as disease state. Assembly of a complete picture of the systems which control this process requires identification of individual genetic components, but the identity and complete sequence of events that trigger initiation of cell division, at a point called START in yeast, remain unknown. Here, we evaluated panels of non-essential single gene deletion strains and tested the effects of FDA-approved drugs on cell-cycle progression, using flow cytometry to detect altered DNA content. Previous studies relied mainly on cell size changes to systematically identify genes required for the timely completion of START. This analysis revealed that most gene deletions that altered cell-cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell-cycle control mechanisms. We found that cystathionine-beta-synthase (CBS) advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function which does not require that activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Screening chemical libraries to identify compounds that affect overall cell proliferation is common. However, it is generally not known whether the compounds tested alter the timing of particular cell-cycle transitions. Our approach revealed strong cell-cycle effects of several commonly used pharmaceuticals. We show that the antilipemic gemfibrozil delays initiation of DNA replication, while cells treated with the antidepressant fluoxetine severely delay progression through mitosis. We discovered a strong suppressive interaction between gemfibrozil and fluoxetine. The novel interaction between gemfibrozil and fluoxetine suggests that identifying and combining drugs that show cell-cycle effects might streamline identification of drug combinations with a pronounced impact on cell proliferation. Our studies not only transform our view of START, but also expand the repertoire of genetic and chemical means to modulate the eukaryotic cell cycle.