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Yes / Articular cartilage is an avascular and flexible connective tissue found in joints. It produces a cushioning effect at the joints and provides low friction to protect the ends of the bones from wear and tear/damage. It has poor repair capacity and any injury can result pain and loss of mobility. Transforming growth factor-beta (TGF-β), a cytokine superfamily, regulates cell function, including differentiation and proliferation. Although the function of the TGF-βs in various cell types has been investigated, their function in cartilage repair is as yet not fully understood. The effect of TGF-β3 in biological regulation of primary chondrocyte was investigated in this work. TGF-β3 provided fibroblastic morphology to chondrocytes and therefore overall reduction in cell proliferation was observed. The length of the cells supplemented with TGF-β3 were larger than the cells without TGF-β3 treatment. This was caused by the fibroblast like cells (dedifferentiated chondrocytes) which occupied larger areas compared to cells without TGF-β3 addition. The healing process of the model wound closure assay of chondrocyte multilayer was slowed down by TGF-β3, and this cytokine negatively affected the strength of chondrocyte adhesion to the cell culture surface.

This study was designed to examine the effect of genistein, a botanical derived primarily from legumes, on pancreatic β-cell proliferation and the related molecular mechanisms. Diabetes mellitus is a major and growing public health problem worldwide. Both in type 1 (T1D) and type 2 diabetes (T2D), the deterioration of glycemic control over time is primarily caused by an inadequate mass and progressive dysfunction of β-cells. Therefore, the search for novel, safe and cost-effective agents that can enhance islet β-cell proliferation, thereby preserving β-cell mass, could be one of the essential strategies to prevent diabetes, given that β-cells have the potential to regenerate by proliferation of pre-existing b-cells in both physiological condition and after onset of diabetes. Genistein has various biological actions. However, studies on whether genistein has an effect on pancreatic β-cell function are very limited. Our laboratory recently found that genistein activates cAMP/protein kinase A (PKA) signaling in both clonal β-cells and mouse islets. Here I present evidence that genistein induced cellular proliferation of clonal rat pancreatic β-cells (INS1) and human islets following 24 h of incubation. This effect was dose-dependent with 5 µM genistein inducing a maximal 41% increase. The effect of genistein on cell proliferation was not dependent on estrogen receptors because this effect was not blocked by the estrogen receptor inhibitor ICI182,780. In addition, the genistein effect on β-cell proliferation was not shared by 17-β-estradiol or a host of structurally related flavonoid compounds, suggesting that this genistein action is structure-specific. Pharmacological or molecular intervention of PKA or MEK1/2, the upstream kinase of p42/44 mitogen activated protein kinases (ERK1/2), completely abolished the genistein-stimulated proliferation of INS1 cells and human islets, suggesting that both molecules are essential for genistein action. Consistent with its effect on cell proliferation, genistein increased intracellular cAMP and subsequently activated PKA in human islets. Genistein also caused a rapid and sustained phosphorylation of ERK1/2 with a maximal increase of 185% at 5 µM genistein. The genistein-induced ERK1/2 activation was completely ablated by inhibition of PKA in INS1 cells and human islets. Furthermore, I found that genistein induced protein expression of cyclin D1, a nuclear target of PKA and ERK1/2 activation and a major cell-cycle regulator essential for ï ¢-cell growth. These findings demonstrated that genistein may be a plant-derived growth factor for pancreatic β-cells involving induction of cyclin D1 via activation of the cAMP/PKA-dependent ERK1/2 signaling pathway, thereby providing a novel role for genistein in the regulation of pancreatic β-cell function. / Master of Science

Poly(α-hydroxy esters) are a class of biocompatible resorbable polyesters including poly(lactic acid) (PLA) and poly(glycolic acid) (PGA) that are FDA-approved for clinical use. Preliminary tissue culture studies have demonstrated that these poly(α-hydroxy esters) support bone tissue development both in vitro and in vivo, but biocompatibility issues still exist. Tissue scaffolds fabricated from these materials by current methods have biocompatibility limitations because they are chemically and topographically inert to cells. The chemical composition of these scaffolds does not influence cell behavior (i.e. proliferation, differentiation) and their surface topography is on a scale length larger than a cell, which is too large to affect cell adhesion or orientation. It is hypothesized that poly(α-hydroxy ester) tissue scaffolds can be made more bioactive by (1) incorporating poly(ethylene glycol) (PEG) into the polymer interface to promote osteoblastic differentiation and (2) controlling topography to direct cell behavior. The novel processing technique of electrospinning allows the fabrication of nanofiber scaffolds with topographical features the size of focal adhesion contacts capable of influencing cell behavior. Thus, the overall objective of this research project is to characterize electrospun PEG-PLA diblock copolymers as substrates for bone tissue engineering. To accomplish this, PEG-PLLA and PEG-PDLLA diblock copolymers were synthesized with target molecular weights of 42,000 g/mol (PEG:2000, PLLA or PDLLA:40,000). Next, these two polymers and commercially available PLLA and PDLLA were electrospun to form scaffolds with fibers of diameters 0.14 to 2.1 μm. Finally, cell culture studies were performed to characterize cell morphology, proliferation, and osteoblastic differentiation. Results indicate electrospun fiber scaffolds limit cell spreading and persist in cell culture for two weeks. Analysis of cells cultured over 14 days revealed that there were no differences in cell density between polymers with and without PEG. Cell density increased with fiber diameter, indicating that fiber diameter affects cell adhesion and proliferation and suggesting that cells may migrate into scaffolds with large diameter fibers. In contrast to cell density, ALP activity, an indicator of osteoblastic differentiation, was unaffected by fiber diameter. / Master of Science

Yes / Cell behavior in terms of adhesion, orientation and guidance, on extracellular matrix (ECM) molecules including collagen, fibronectin and laminin can be examined using micro contact printing (MCP). These cell adhesion proteins can direct cellular adhesion, migration, differentiation and network formation in-vitro. This study investigates the effect of microcontact printed ECM protein, namely fibronectin, on alignment and morphology of HeLa cells cultured in-vitro. Fibronectin was stamped on plain glass cover slips to create patterns of 25μm, 50μm and 100μm width. However, HeLa cells seeded on 50μm induced the best alignment on fibronectin pattern (7.66° ±1.55SD). As a consequence of this, 50μm wide fibronectin pattern was used to see how fibronectin induced cell guidance of HeLa cells was influenced by 100μs and single pulse electric fields (PEF) of 1kV/cm. The results indicates that cells aligned more under pulse electric field exposure (2.33° ±1.52SD) on fibronectin pattern substrate. Thus, PEF usage on biological cells would appear to enhance cell surface attachment and cell guidance. Understanding this further may have applications in enhancing tissue graft generation and potentially wound repair. / Ministry of Higher Education Malaysia and UTHM Tier 1 Research Grant (U865)

According to the National Institute of Neurological Disorders and Stroke, there are more than 600 neurologic disorders that affect approximately 50 million Americans each year. The $91 billion dollars spent by Medicare on Alzheimer's disease and other dementias in 2005 is projected to increase to $189 billion by 2015 [4]. The existence of neural stem cells (NSC's) and neurogenesis makes neural regeneration a viable option. The ethical barriers of using embryonic stem cells, rejection of the transplanted cells, and possible tumor formation, are only a few of the problems that face stem cell transplantation, a widely considered option to repopulate the brain with cells. A noninvasive pharmaceutical approach that can promote neuron regeneration and recovery would be the key to curing many neurodegenerative diseases. The development of MS-818 as a non-invasive enhancer of the proliferation process of NS Cs is revolutionary for the treatment of neurodegenerative diseases. Due to the fact that its mechanism of action remains unknown, the full pharmacological potential of MS- 818 has not been fully exploited [8]. Isolating protein targets of MS-818 is key in identifying the molecular pathways responsible for its mechanism of action. UV-Vis analysis of MS-818 showed absorbance at 275-nm, and this data was used to calculate coupling yield. MS-818 coupled to the NHS-activated sepharose beads of the affinity column with 83% efficiency. Proteins were isolated from human embryonic kidney cells (HEK 293 cells) and applied to the column. Bradford assay confirmed that bound proteins eluted in a concentration dependent manner when an MS-818 gradient was applied to the column. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the eluate revealed two sets of polypeptides migrating at 37-75 kDa and 100-150 kDa. In addition, some trace polypeptides in the sub-35 kDa region could be seen. Although we have not yet identified specific proteins that MS-818 interacts with, we were able to successfully to isolate such proteins.

Thesis (MSc (Physiological Sciences))--University of Stellenbosch, 2006. / Despite a growing interest into the mechanisms of the repeated bout effect, little is known about the consequences of chronic eccentrically biased training and the possible advantageous such training may offer to athletes as well as patients with muscle-debilitating disease. This study investigated the role of satellite cells in the muscle adaptation in response to either downhill or uphill high intensity training (HIT). Welltrained endurance runners were divided into two training groups matched for training volume and 10 km running times (n = 6, uphill training, UP; n = 6, downhill training, DH) and subjects in both groups completed 10 HIT sessions over a period of 4 weeks. Running performance was tested before and after the training intervention by a 10 km road race and peak treadmill speed (PTS) in horizontal and inclined (+5%) laboratory incremental tests to exhaustion. Skeletal muscle biopsies were sampled at baseline, after 2 HIT sessions, and after 4 weeks of HIT. Muscle was analysed immunohistochemically for satellite cell frequency as identified by CD56 and M-cadherin (Mcad) expression. Myogenin protein contents of muscle homogenates were determined by western blotting. Myosin heavy chain (MyHC) isoform proportions and mean fibre crosssectional area was measured. During the HIT intervention, UP exercised at a higher percentage of their HRmax than DH (mean ± SD, 97 ± 1 vs. 92 ± 3 %HRmax, p < 0.005), but at a similar rate of perceived exertion (RPE). DH completed more intervals per session and covered greater distance per session than their UP counterparts. Both training groups increased their training intensity but decreased their training volume during the 4 weeks of HIT. The combined group of 12 athletes improved their PTSgradient (mean ± SD, 16.7 ± 0.8 vs. 17.3 ± 1.0 km/h, p < 0.05). No significant differences between groups were found for PTS, VO2max or 10 km performance. Satellite cell frequency in this cohort of trained runners (48.9 ± 10.3 km/week) at baseline was similar to healthy young males (CD56+ cells/fibre, 0.19 ± 0.10). Satellite cell frequency increased significantly in DH after 4 weeks (Mcad, 123%; CD56, 138%) and non-significantly in UP (Mcad, 45%, CD56, 39%). No significant differences were found after two training sessions or at any time between groups. Mcad and CD56 expression correlated well (r = 0.95, p < 0.0001). Muscle myogenin content increased for both groups (UP: 56%; DH: 60%) after 4 weeks. No notable changes were seen after two training sessions. However, myogenin levels 2 days after session 1 correlated well (r= 0.99, p<0.005) with muscle pain experienced on the same day, as measured by the visual analogue scale. No changes were seen in the MyHC proportions or the fibre cross-sectional area after the training intervention. It was concluded that the training intervention was too short to induce changes in MyHC distribution or fibre area. Is seems likely that satellite cell proliferation was initiated as an early response to DOMS, but the response was maintained for 4 weeks. However, due to the lack of change in fibre morphology and myonuclear number, the role of satellite cell proliferation in fibre type transformation or muscle hypertrophy could not be established. Similarly, various possible roles for increased myogenin protein are offered, but since the origin of myogenin expression (satellite cells vs. myonuclei) was not determined, no definite conclusion regarding the precise function can be made. In conclusion, this study is the first to definitively indicate satellite cell proliferation in well-trained endurance runners in response to a change in training, including specifically downhill HIT. This response was early and sustained. This study asks several questions about the role of satellite cells during muscle adaptation to repetitive downhill training, and lays a foundation for further research into this unexplored field.

There is scarce information on the fate of cardiac progenitor cells (CPC) in the embryonic heart after chamber specification. Furthermore, the role of acetylcholinesterase (AChE) during heart development is unknown, despite record of its presence in the myocardium. Although three molecular variants of AChE (R, H and T) exist due to alternate splicing, temporal and spatial distribution of these splice variants during cardiac ontogeny is not well characterized. We hypothesized that the AChE “R” splice variant (AChE-R) is involved in directing lineage commitment of mouse ventricular CPCs to the conduction cell phenotype. It is possible that AChE may promote the breakdown of ACh and block the effects of ligand-binding via M2 receptors present on the surface of CPCs. Our study has also provided a platform to suggest that AChE may play a role in the molecular mechanisms underlying functional diversification of myocardial cells into conduction system cells during ontogenesis.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase and the limiting factor for the enzyme activity. The expression of TERT and telomerase activity is increased in atherosclerotic plaques. However, the role of TERT dysregulation during atherosclerosis formation remains unknown. The work herein first identified a multi-tiered regulation of TERT expression in smooth muscle cells (SMC) through histone deacetylase (HDAC) inhibition. HDAC inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Furthermore, during vascular remodeling in vivo, TERT protein expression in the neointima is prevented by HDAC inhibition. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition. TERT is highly expressed in replicating SMC of atherosclerotic and neointimal lesions. Using a model of guidewire-induced arterial injury, neointima formation was reduced in TERT-deficient mice. Studies in SMC isolated from TERT-deficient and TERT overexpressing mice with normal telomere length established that TERT is necessary and sufficient for cell proliferation. TERT deficiency did not induce a senescent phenotype but resulted in G1 arrest albeit hyperphosphorylation of the retinoblastoma protein. This proliferative arrest was associated with stable silencing of the E2F1-dependent S-phase gene expression program which could not be reversed by ectopic overexpression of E2F1. Chromatin immunoprecipitation and accessibility assays revealed that TERT was recruited to E2F1 target sites to increase chromatin accessibility for E2F1 by facilitating the acquisition of permissive histone modifications. These data indicate a mitogenic effect of TERT on SMC growth and neointima formation through epigenetic regulation of proliferative gene expression. Furthermore, TERT expression is induced in activated macrophages during experimental and human atherosclerosis formation. To investigate the role for TERT in lesional macrophages and the subsequent effect on atherosclerosis formation, TERT-deficient mice were crossbred with LDL-receptor-deficient (LDLr-/-) mice to generate first generation G1TERT-/-LDLr-/- offsprings, which were then further intercrossed to obtain third generation G3TERT-/-LDLr-/- mice. G1TERT-/-LDLr-/- mice revealed no telomere shortening while severe telomere attrition was evident in G3TERT-/-LDLr-/- mice. When fed an atherogenic diet, G1TERT-/-LDLr-/- and G3TERT-/-LDLr-/- mice were both protected from atherosclerosis formation compared to their wild-type controls, indicating that genetic TERT-deletion prevents atherosclerosis, and formation of the disease is not affected by telomere attrition. Similarly, atherosclerosis development was decreased in chimeric LDLr-/- mice with TERT deletion in hematopoietic stem cells after bone marrow transplantation. TERT deficiency reduced macrophage accumulation in atherosclerotic lesions and altered chemokine expression, including CXC1/2/3, CCL3, CCL5, CCL21, CCR7, IL-6, and IL-1α. In isolated macrophages, gene ontology (GO) enrichment analysis of silenced inflammatory genes indicated that TERT positively regulates signal transducer and activator of transcription (STAT) cascade, which was confirmed by the decreased tyrosine phosphorylation of STAT3 protein resulting from TERT deletion. These findings indicate genetic TERT deficiency decreases atherosclerosis formation by silencing inflammatory chemokine transcription through inactivation of the STAT3 signaling pathway in activated macrophages. In conclusion, the dysregulation of TERT expression within atherosclerotic plaques plays a causative role for vascular remodeling, including injury-induced neointima formation and hypercholesterolemia-induced atherosclerosis, through inducing SMC proliferation and a pro-inflammatory phenotype in infiltrating macrophages. These findings unveil a mechanism of TERT exacerbating the pathological vascular remodeling, which may provide a novel therapeutic target to combating vascular diseases.