Yes / To develop an efficient nanotechnology fluorescence-based method to track cell proliferation to avoid the limitations of current cell-labeling dyes. Material & methods: Synthesis, PEGylation, bifunctionalization and labeling with a fluorophore (Cy5) of 200 nm polystyrene nanoparticles (NPs) were performed. These NPs were characterized and assessed for in vitro long-term monitoring of cell proliferation. Results: The optimization and validation of this method to track long-term cell proliferation assays have been achieved with high reproducibility, without cell cycle disruption. This method has been successfully applied in several adherent and suspension cells including hard-to-transfect cells and isolated human primary lymphocytes. Conclusion: A novel approach to track efficiently cellular proliferation by flow cytometry using fluorescence labeled NPs has been successfully developed.

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Twenty two methionine-containing di- to octa-peptides were evaluated for their ability to serve as methionine sources to support protein accretion and cell proliferation in C₂C₁₂ myogenic, MAC-T mammary epithelial and ovine myogenic satellite cells. Factors in serum that may be involved in regulating peptide utilization was investigated using MAC-T cells. Growth of MAC-T cells was studied in the presence of methionine-containing dipeptides with 6% desalted adult animal serum from chickens, horses, humans, pigs or rabbits. Serumal peptidase activities on the twenty two methionine-containing peptides were examined in cell-free, methionine-free Dulbecco’s modified Eagle’s medium supplemented with 6% fetal bovine serum. The cell cultures were incubated for 72 h at 37°C in a humidified environment of 90% air : 10% CO₂ for C₂C₁₂ and ovine satellite cells or 95% air : 5% CO₂ for MAC-T cells. The basal medium contained methionine-free Dulbecco’s modified Eagle’s medium supplemented with 6% desalted animal serum or one of the following serumal factors: .4% bovine serum lipids, 1% chemically defined lipid concentrate, bovine insulin (1 ug/mL), or 3% low protein serum replacement (LPSR-1). Treatment media tested included basal medium or basal media supplemented with L-methionine or one of the methionine-containing peptides. Cell cultures incubated with the basal media for 72 h were characterized by decreased cell number and decreased protein content compared with initial cultures. All the methionine-containing peptides (with the exception of glycylmethionine and prolylmethionine for C₂C₁₂ cells), regardles of chain length, were able to support protein accretion with responses ranging from 29 to 123% of that of free L-methionine. The DNA contents of ovine satellite cell cultures indicated that cell proliferation occurred in the presence of all the methionine-containing peptides with responses ranging from 45 to 144% of the L-methionine response. Bovine insulin and lipids were not effective in promoting peptide utilization by MAC-T cells. However, the LPSR-1 facilitated the utilization of methionine-containing peptides in C₂C₁₂ and MAC-T cells. In the cell-free, methionine-free Dulbecco’s modified Eagle’s medium, peptidases could release all the methionine residues from the tetra- to octapeptides during 24 h of incubation and 42 to 70% of the methionine residues from the di- and tripeptides tested. The results demonstrated that cultured animal cells possess the ability to utilize methionine-containing peptides as methionine sources for protein accretion and cell proliferation, but serumal peptidases are at least partially responsible for the observed responses. / Ph. D.

OBJECTIVE:Vascular smooth muscle cell (VSMC) hypertrophy and proliferation occur in response to strain-induced local and systemic inflammatory cytokines and growth factors which may contribute to hypertension, atherosclerosis, and restenosis. We hypothesize VSMC strain, modeling normotensive arterial pressure waveforms in vitro, results in attenuated proliferative and increased hypertrophic responses 48 hrs post-strain.METHODS:Using Flexcell Bioflex Systems we determined the morphological, hyperplastic and hypertrophic responses of non-strained and biomechanically strained cultured rat A7R5 VSMC. We measured secretion of nitric oxide, key cytokine/growth factors and intracellular mediators involved in VSMC proliferation via fluorescence spectroscopy and protein microarrays. We also investigated the potential roles of VEGF on VSMC strain-induced proliferation.RESULTS:Protein microarrays revealed significant increases in VEGF secretion in response to 18 hours mechanical strain, a result that ELISA data corroborated. Apoptosis-inducing nitric oxide (NO) levels also increased 43% 48 hrs post-strain. Non-strained cells incubated with exogenous VEGF did not reproduce the antimitogenic effect. However, anti-VEGF reversed the antimitogenic effect of mechanical strain. Antibody microarrays of strained VSMC lysates revealed MEK1, MEK2, phospo-MEK1T385, T291, T298, phospho-Erk1/2T202+Y204/T185+T187, and PKC isoforms expression were universally increased, suggesting a proliferative/inflammatory signaling state. Conversely, VSMC strain decreased expression levels of Cdk1, Cdk2, Cdk4, and Cdk6 by 25-50% suggesting a partially inhibited proliferative signaling cascade.CONCLUSIONS:Subjecting VSMC to cyclic biomechanical strain in vitro promotes cell hypertrophy while attenuating cellular proliferation. We also report an upregulation of MEK and ERK activation suggestive of a proliferative phenotype. Hhowever, the proliferative response appears to be aborogated by enhanced antimitogenic cytokine VEGF, NO secretion and downregulation of Cdk expression. Although exogenous VEGF alone is not sufficient to promote the quiescent VSMC phenotype, we provide evidence suggesting that strain is a necessary component to induce VSMC response to the antimitogenic effects of VEGF. Taken together these data indicate that VEGF plays a critical role in mechanical strain-induced VSMC proliferation and vessel wall remodeling. Whether VEGF and/or NO inhibit signaling distal to Erk 1/2 is currently under investigation.

The study of the formation of new cardiac muscle cells during postnatal development is a relatively new field. During fetal development, new cells are formed as the heart grows. However, the proliferative ability of postnatal cardiomyocytes is still debated. While several studies have been made on mammals, less is known about the chicken cardiac cells and their postnatal proliferation. As almost all previous studies have used microscopy-based cell counting methods, there has been some limitations on accuracy and amounts of cells that could be counted. The aim of this study is to develop a method for using flow cytometry to analyze proliferative ability of chicken cardiomyocytes and to investigate if any postnatal proliferation exists. For this study, 4 weeks old Red Junglefowl (Gallus gallus) chickens were used for isolating cardiomyocytes. In addition, 19 days old Red Junglefowl embryos were used to asses if a longer incubation time would yield a higher number of proliferative cells. Cells were stained using a commercial EdU imaging kit and analyzed using flow cytometry and imaging flow cytometry. The produced results could not be used for determining the proliferative ability of the cardiomyocytes, but provides crucial information for possible method improvements. In conclusion, this study has laid important groundwork for future studies on the proliferative ability of chicken cardiomyocytes.

A significant consequence of alcohol use disorders (AUDs) is hippocampal neurodegeneration. The hippocampus is responsible for learning and memory, and neurodegeneration in this brain region has been shown to result in cognitive deficits. Interestingly, some alcoholics demonstrate improvements in hippocampus-dependent functions, potentially due the phenomenon termed adult neurogenesis. Adult neurogenesis, the process by which neural stem cells (NSCs) proliferate, differentiate into neurons, migrate into the granule cell layer, and survive, occurs in two brain regions; however, this study examines only neurogenesis occurring in the subgranular zone of the hippocampal dentate gyrus. Four-day binge ethanol exposure in an animal model causes a decrease in neurogenesis during intoxication; however, there is a reactive increase in cell proliferation on day seven of abstinence. The purpose of this study was to determine the timing of increased cell proliferation. Furthermore, most alcoholics also smoke tobacco, and nicotine, the addictive component of tobacco, has also been shown to affect hippocampal neurogenesis. As many people initiate alcohol and tobacco use during adolescence, the second experiment herein examined the effect of nicotine coadministration on alcohol-induced reactive hippocampal cell proliferation.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biochemistry / Master / Master of Philosophy

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy