Cell proliferation is a fundamental biological process, and the ability of human cells to transition from a quiescent to proliferative state is essential for tissue homeostasis. Most cells in eukaryotic organisms are in a quiescent state, but on appropriate physiological or pathological stimuli, many types of somatic cells may exit quiescence, re-enter the cell cycle and begin to proliferate. The ability of cells to remain viable while quiescent, exit quiescence and re-enter into the cell cycle is the basis for varied physiological processes such as wound healing, lymphocyte activation and hepatocyte regeneration, but is also a hallmark of cancer. The transition of mammalian cells from quiescence to proliferation is accompanied by the differential expression of several microRNAs (miRNAs) and transcription factors. Our understanding of miRNA biology has significantly improved, but the miRNA regulatory networks that govern cell proliferation are still largely unknown. We characterized a miR-22 Myc network that mediates proliferation through regulation of the interferon response and multiple cell cycle arrest genes. We identified several cell cycle arrest genes that regulate the effects of the tumor suppressor p53 as direct targets of miR-22, and discovered that miR-22 suppresses interferon gene expression. We go on to show that miR-22 is activated by the transcription factor Myc as quiescent cells enter proliferation, and that miR-22 represses the Myc transcriptional repressor MXD4, mediating a feed forward loop to elevate Myc expression levels. To more effectively determine miRNA targets, we utilized a combination of RNA-induced silencing complex immunoprecipitations and gene expression profiling. Using this approach for miR-191, we constructed an extensive transcriptome wide miR-191 target set. We show that miR-191 regulates proliferation, and targets multiple proto-oncogenes, including CDK9, NOTCH2, and RPS6KA3. Recent advances in determining miRNA targetomes have revealed widespread non-canonical miRNA-target pairing. We experimentally identified the transcriptome wide targets of miR-503, miR-103, and miR-494, and observed evidence of non-canonical target pairing for these miRNAs. We went on to confirm that miR-503 requires pairing outside of the canonical 5' seed region to directly target the oncogene DDHD2. Further bioinformatics analysis implicated miR-503 and DDHD2 in breast cancer tumorigenesis. / text

Functional mesenchymal stem cells (MSCs) derived from human induced pluripotent stem cells (iPSCs) may represent an unlimited cell source with superior therapeutic benefits for tissue regeneration to somatic tissue, such as bone marrow (BM)-derived MSC. In the first part of this project, I investigated whether the differential expression of ion channels in iPSC-MSCs was responsible for their higher proliferation capacity than that of BM-MSCs. The expression of ion channels for K+, Na+, Ca2+ and Cl- currents was assessed by reverse transcription-polymerase chain reaction (RT-PCR). The functional role of these ion channels were then verified by patch clamp experiments to compare the electrophysiological properties of iPSC-MSCs versus BM-MSCs. I detected significant mRNA expression of ion channel genes including KCa1.1, KCa3.1, KCNH1, Kir2.1, SCN9A, CACNA1C and Clcn3 in both human iPSC-MSCs and BM-MSCs; while Kir2.2 and Kir2.3 were only observed in human iPSC-MSCs. Furthermore, I identified five types of currents (BKCa, IKDR, IKir, IKCa and ICl) in iPSC-MSCs, while only four of them (BKCa, IKDR, IKir and IKCa) were observed in BM-MSCs. The rate of cell proliferation was 1.4 fold faster in iPSC-MSCs as compared to BM-MSCs. Interestingly, the proliferation rate of human iPSCMSCs was significantly reduced when inhibiting IKDR with shRNA and hEAG1 channel blockers, 4-AP and astemizole. Though to a lesser extent, the proliferation rate of human BM-MSCs also decreased by IKDR blockage. These results demonstrated that hEAG1 channel plays a crucial role in controlling the proliferation rate of human iPSC-MSCs but to a lesser extent in BM-MSCs. Next, I examined whether forced expression of a transcription factor- myocardin in iPSC-MSC using viral vectors (adenovirus or lentivirus) can further enhance their trans-differentiation to cardiomyocytes and improve their electrophysiological properties for cardiac regeneration. My results on RT-PCR and immunofluorescent staining revealed that myocardin induced the expression of several cardiac and smooth muscle cell markers, including α-MHC, cTnT, GATA4, α-actinin, and cardiac MHC, smooth muscle cell markers MYH11, calponin, and SM α-actin, but not the more mature cardiac markers such as β-MHC and MLC2v in iPSC-MSCs. These findings indicate that forced expression of myocardin in iPSC-MSC resulted in partial trans-differentiation into cardiomyocytes phenotype. Furthermore, I also discovered that myocardin altered the electrophysiological properties of iPSC-MSCs when examined by RT-PCR and patch clamp experiments. Forced expression of myocardin in iPSC-MSC enhanced the expression of Kv4.3, SCN9A and CACNA1C, but reduced that of KCa3.1 and Kir 2.2 in iPSC-MSCs. Moreover, BKCa, IKir, ICl, Ito and INa.TTX were detected in iPSC-MSC with ectopic expression of myocardin; while only BKCa, IKir, ICl, IKDR and IKCa were noted in iPSC-MSC transfected with green florescence protein. Furthermore, as measured by multi-electrode arrays recording plate, the conduction velocity of the neonatal rat ventricular cardiomyocytes cocultured iPSC-MSC monolayer was significantly increased after ectopic expression of myocardin. Taken together, I have demonstrated that hEAG1 channel is important in the regulation of iPSC-MSC proliferation and forced expression of myocardin in iPSC-MSC resulted in their partial transdifferentiation into cardiomyocytes phenotype and improved the electrical conduction during integration with mature cardiomyocytes. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Cell proliferation and differentiation are coordinated by synchronized patterns of gene expression. The regulation of these patterns is achieved, in part, through epigenetic mechanisms that affect the nature of DNA packaging into chromatin. Specifically, post-translational modifications to histone tails impact the structural dynamics of nucleosomes, thereby affecting DNA accessibility to transcriptional complexes. Accumulating evidence suggests that transcriptional regulators facilitate these alterations, resulting in altered local gene transcription. Thus, the structural interpretations of histone modifications are responsible for the establishment and maintenance of discrete programs of gene expression that ultimately correspond with distinct biological outcomes. Most histone lysine methyltransferases catalyze methyl transfer by way of the SET domain, a module encoded within many proteins that regulate diverse processes, including some critical for development and proper progression of the cell cycle. One such group of proteins, the SET and MYND domain (Smyd) family have been demonstrated to be direct regulators of tumorigenesis and essential developmental processes. Presented here is a functional characterization of two members of that family, Smyd2 and Smyd3. Smyd2 is identified as a member of the Smyd family and reported here to possess SET-dependent histone H3, lysine 36-specific methyltransferase activity. Smyd2 specifically associates with the Sin3A histone deacetylase complex, suggesting a link between two independent chromatin modification activities. Finally, over-expression of Smyd2 in fibroblasts is shown to significantly suppress their rate of growth. It is therefore proposed that Smyd2-mediated chromatin modifications regulate specific gene expression, thereby having important implications for normal and neoplastic cell proliferation. Aberrant expression of the histone H3-lysine 4-specific methyltransferase, Smyd3, has been implicated in colorectal, hepatocellular, and breast cell carcinogenesis. Here, Smyd3 is also shown to target histone H4, lysine 20 (H4K20). However, over-expression of Smyd3 in fibroblasts results in global reduction of trimethylation at H4K20 and this is accompanied by a striking increase in cell proliferation. As the methylation of H3K4 and H4K20 are normally associated with conflicting biological functions, I predict that these differential activities of Smyd3 are manifest under spatially and/or temporally distinct conditions, in the presence of different associating complexes, thereby resulting in effects that may be antagonistic of one another. / text

The phenotypic modulation of vascular smooth muscle cells (VSMCs) from a “contractile/quiescent” to an “activated/synthetic” phenotype, with increased proliferative and migratory potential, is critical for the formation of advanced atherosclerotic lesions. Agents that regulate intracellular levels of the cyclic AMP (cAMP) and cyclic GMP (cGMP) have been shown to reduce VSMC migration and proliferation, and to reduce intimal thickening in response to vascular damage. Interestingly, expression of a specific cyclic nucleotide phosphodiesterase, namely PDE1C that is not expressed in contractile VSMCs is induced in activated human VSMCs and this directly impacts human VSMC phenotypic modulation. This study was undertaken to identify potential mechanism(s) by which PDE1C could impact VSMC phenotypic modulation and associated cellular functions. Overall, my data indicate that PDE1C controls store operated calcium (Ca2+) channel (SOCC) activity in activated human VSMCs. Indeed, expression of PDE1C increases store operated Ca2+ entry (SOCE), which in turn activates PDE1C. This linkage between PDE1C and the SOCC complex increases cytosolic [Ca2+] and activates cell proliferation. A potential human VSMC SOCC, the Transient receptor potential channel 1 (TrpC1), was shown to physically associate with PDE1C in HEK293T cells expressing these proteins heterologously, as well as in human aortic Smooth Muscle Cells (HASMCs), which natively express these proteins. In HEK293T cells, I also identified association of adenylyl cyclase 6 (AC6) and of the inositol-trisphosphate receptor (IP3R) iii with TrpC1. Interaction between TrpC1 and PDE1C in HASMCs is decreased upon activation of SOCE, suggesting PDE1C is activated after release from TrpC1-PDE1C complex. From these studies I have established a potential mechanism by which PDE1C signaling impacts SOCE and shown that a TrpC1-PDE1C complex may be important. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2012-03-22 14:34:38.647

No description available.

No description available.

Epithelial-mesenchymal interactions are central to the development of skin and skin appendages in vertebrates. These interactions continue throughout adult life and underpin the cyclic growth and loss of hair in mammals. While the molecular basis of such interactions are being gradually uncovered, at the cellular level many questions remain unanswered. For example, the localisation and role of hair follicle epithelial stem cells remains a subject of debate, as does the function of the dermal sheath component. In embryonic appendage development there is strong evidence for common signalling mechanisms, but the degree to which epithelial-mesenchymal communication diverges in different adult appendages remains as yet undiscovered. I have studied the replicative abilities of germinative epidermal (GE) cells of the rat vibrissa follicle by single and repeated plucking of fibres from individual follicles. In both cases, the cellular events following fibre removal were scrutinised at intervals up to 9 days using histology, and cell proliferation and cell death markers. Follicles that were repeatedly plucked had their growing hairs measured at regular intervals. By analysing cell proliferation patterns I found that the new regenerated epidermal matrix came from residual GE cells left in the follicle base - after both single and repeated depilations. Indeed plucking appeared to cause an initial inhibition of proliferative activity in the follicle upper outer root sheath, the other candidate region for supplying a new matrix. Length measurements of the regenerated hairs demonstrated that the repeatedly plucked follicles produced total cumulative lengths of fibre between 60 and 265% longer than expected, as determined by measuring the original club fibre lengths. In vivo amputation of plucked follicles demonstrated that the residual GE cells have the ability to regenerate a new matrix and new fibre without any contribution from cells from the upper region of the follicle. These studies, along with in vitro observations of prolonged replicative abilities of bulb cells suggest that the GE cells have a proliferative capacity which is beyond that of one cycle. This raises the possibility that GE cells are not transient amplifying cells with limited proliferative potential and strongly suggests that the duration of anagen cannot be attributed to the replicative limitations of the GE cells. The behaviour and interactive abilities of dermal cells isolated from three different skin appendages (rat vibrissa follicle, rat claw unit, and pigeon feather follicle) were characterised by cell culture, immunohistochemistry and dermal-epidermal recombinations. Dermal cells from all the above appendages demonstrated common aggregation properties in culture and all expressed a-smooth muscle actin. When recombined with epidermal cells and implanted onto host rats, dermal sheath cells from the lower part of vibrissa follicles produced a robust skin-like structure with a normal

This thesis examines the motivations, mechanisms and prospects of Turkish Kurdish international migration in relation to the Environment of Insecurity as a set of combined socio-economic and political factors triggered by an ethnic conflict. The analysis focuses on three different, but complementary, levels of analysis. The research comprises first, the analysis of the environment of insecurity in Turkey emphasising its broader socio-economic, legal-political, and demographic aspects; second, the patterns and processes of international migration involving Turkish Kurds investigating the motivations, the mechanisms, and the future migration potentials; third, the role of the expression of ethnicity and of ethnic conflict. A mixed method approach combining qualitative and quantitative methods to address different levels of analysis and different aspects of migration is adopted. The analysis of Turkish Demographic Health Survey data examines the extent to which an environment of insecurity exists for Turkish Kurds. The findings of the Turkish International Migration Survey data outline the patterns of individual migration motives, mechanisms and future intentions. Finally, semi-structured in-depth interviews examine the role of the ethnic conflict and the expression of ethnicity to clarify the relationship between Turkish Kurdish international migration and the ethnic environment of insecurity while also presenting a live account of migration motivations and mechanisms. The research shows that the environment of insecurity is an issue of ethnic conflict and it constitutes the major facilitating factor in Turkish Kurdish international migration resulting in large asylum migration flows. Due to the armed ethnic conflict between the PKK and the Turkish Army in Turkey during the last two decades of the last century, recent migration patterns of Turkish Kurds are dominated by clandestine migration. Along with legal migrations (e.g. economic, family, education), irregular migration appears as a strong trend involving asylum migration and illegal migration. Tightening immigration controls in Europe also prompts this. The conflict situation also serves as an opportunity framework for some who wanted to migrate. While migration is appearing as a liberating event for Turkish Kurdish ethnicity it is found that Kurdish immigrants have not fully exploited the opportunities for exercising their ethnicity. However, for many, migration from Turkey to Germany is an act of escape and so is an expression of ethnicity.

Ulm, Univ., Diss., 2008.

Freiburg i. Br., Univ., Diss., 2008.