Embryonic stem (ES) cells can be maintained indefinitely in culture while retaining the ability to give rise to cellular derivatives from the three germ layers. These unique characteristics hold great promise for regenerative medicine and underscore the importance of understanding the molecular mechanisms behind ES cell maintenance. The embryonic stem cell state is supported by a delicate equilibrium of mechanisms that maintain pluripotency, prevent differentiation, and promote proliferation and survival. We sought to find genes that could contribute to one or more of these processes in human ES cells by using a gain-of-function screen of over 8000 human open reading frames (ORFs). We identify Vestigial-like 4 (Vgll4), a co-transcriptional regulator with no previously known function in ES cells, as a positive regulator for survival of human ES cells. Specifically, Vgll4 protects human ES cells from dissociation stress, and enhances colony formation from single cells. These effects may be attributable in part to the ability of Vgll4 to decrease the activity of initiator and effector caspases. Based on global transcriptional analysis, we hypothesize that Vgll4 enhances survival of hES cells at clonal densities by regulating changes in the cytoskeleton, which may in turn regulate pathways known to result in hES cell death. This dissertation introduces a novel approach for studying hES cell survival in the context of cell dissociation and presents Vgll4 as a novel regulator of this process. We also propose that Vgll4 could have multiple functions in hES cells including possible roles in pluripotency, cell cycle dynamics, Hippo pathway regulation, and \(TGF\beta\) signaling. A direct regulator of survival in human embryonic stem cells could have important implications for facilitating the generation of transgenic cell lines and reporters, thus harnessing the therapeutic application of these cells.

Morphological transitions in Wangiella dermatitidis, a causative agent of human phaeohyphomycosis, influence virulence processes in this polymorphic fungus. My project first involved the cloning and characterizion of the β(1→3) glucan synthase gene WdFKS1, which encodes the enzyme's catalytic subunit, followed by cloning and characterizing the WdRHO1 gene, which encodes its regulatory subunit. To better understand the Rho-type GTPase-mediated regulation of cell polarity and its role in fungal morphological transitions, a homologue of WdRAC1 from a W. dermatitidis was subsequently identified by degenerate PCR and gene walking. Gene deletions of WdFKS1 and WdRHO1 in haploid W. dermatitidis were lethal, whereas the deletion of WdRAC1 was not. RNA interference on WdFKS1 mRNA expression resulted in incomplete septa and damaged cell wall integrity, as well as slow growth rate in W. dermatitidis. Overexpression studies, after site-specific integrations of WdRHO1 and WdRAC1 alleles under control of the glaA promoter into the nonessential WdPKS1 locus, showed the different alleles had different effects on the cell morphological development. For example, whereas overexpression of the wdrho1⁺ allele did not affect the growth rate of W. dermatitidis, the overexpression of wdrho1[superscript G14V], a constitutively active mutation, slowed growth and repressed true filamentous hyphal growth by promoting pseudohyphal growth. While the deletion of WdRAC1 did not affect growth, its loss retarded polarized hyphal growth in a hyphal-inducing minimal medium. Moreover, three new phenotypes of a previously derived WdCDC42 deletion mutant were discovered during this study: in the first, the wdcdc42[Delta] mutant displayed cell lysis when incubated in YPMaltose medium at 37°C; in the second, a dark budding neck abnormality was found after Calcoflour staining; and in the third, the wdcdc42[Delta] mutant displayed no branching during true hyphal growth. Interestingly, the overexpression of wdrac1[superscript G16V] complemented the second and the third phenotypes caused by the WdCDC42 deletion. In addition, the wdcdc42[Delta]/wdrac1[superscript G16V] double mutant unexpectedly displayed an interrupted carotenogenesis pathway. These results support that in W. dermatitidis, Rho-type GTPases play essential roles in growth rate determination and cellular morphogenesis, especially while producing polarized hyphal growth during its many morphological transitions. / text

In vivo, biomechanical stress plays an important role in tissue physiology and pathology, affecting cell and tissue behavior. Even though conventional outflow tissues in the eye are constantly exposed to dynamic changes in intraocular pressure (IOP), the effects of such biomechanical stressors on outflow tissue function have not been analyzed. In particular, changes in IOP with each heartbeat have been measured in human eyes approximating 2.7 mmHg/sec. The purpose of this dissertation is to determine the effect(s) of ocular pulse on conventional outflow tissue regulation and the effect that contractility plays in this mechanical stress-mediated response. The central hypothesis directing this research is that cyclic intraocular pulsations (i.e. ocular pulse) play a significant role in conventional outflow facility.In order to address our hypothesis we studied the effect of biomechanical stressors on conventional outflow physiology using three different strategies: (1) by comparing conventional outflow endothelial cells to blood and lymphatic capillary endothelia, we gained a better understanding of the effects of biomechanical stress on conventional outflow tissue physiology, (2) by modifying the anterior segment perfusion model, we were able to measure the effect of ocular pulse on conventional outflow facility, and (3) by exposing trabecular meshwork cell monolayers to cyclic biomechanical pressure oscillations in the presence of compounds known to affect trabecular meshwork contractility, we were able to analyze the effect of rho-kinase-mediated contractility on the ocular pulse-associated response.Perfused human and porcine anterior segments showed a significant ocular pulse-mediated decrease in outflow facility; in addition, perfused trabecular meshwork monolayers showed an increase in intra-chamber pressure when exposed to cyclic pressure oscillations. This effect was blocked by Y27632 inhibition of rho-kinase-mediated contraction.In conclusion, the work shown in this dissertation demonstrates for the first time that trabecular outflow tissues are capable of responding to a physiologically-relevant cyclic biomechanical stress. This response can be observed as an increase in outflow resistance that translates to lower baselines in outflow facility of anterior segments and lower hydraulic conductivity of trabecular meshwork monolayers. In addition, we concluded that the observed ocular pulse-mediated response of trabecular meshwork cells is regulated by rho-kinase-induced contractility.

Chronic neonatal pulmonary hypertension (PHT) frequently presents with rightventricular (RV) dysfunction. In neonatal rats exposed to chronic hypoxia, RV dysfunction is reversed by sustained rescue treatment with a Rho-kinase (ROCK) inhibitor – the caveat being systemic hypotension. We therefore examined the reversing effects of pulmonary-selective ROCK inhibition. Rat pups were exposed to air or hypoxia from birth for 21 days and received sustained rescue treatment with aerosolized Fasudil (81 mg/ml t.i.d for 15 min) or i.p. Y27632 (15 mg/kg b.i.d) from days 14-21. Inhaled Fasudil normalized pulmonary vascular resistance, and reversed pulmonary vascular remodeling but did not improve RV systolic function. Systemic, but not pulmonary-selective, ROCK inhibition attenuated increased RV ROCK activity. Our findings indicate that RV dysfunction in chronic hypoxic PHT is not merely a result of increased afterload, but rather may be due to increased activity of ROCK in the right ventricle.

Chronic neonatal pulmonary hypertension (PHT) frequently presents with rightventricular (RV) dysfunction. In neonatal rats exposed to chronic hypoxia, RV dysfunction is reversed by sustained rescue treatment with a Rho-kinase (ROCK) inhibitor – the caveat being systemic hypotension. We therefore examined the reversing effects of pulmonary-selective ROCK inhibition. Rat pups were exposed to air or hypoxia from birth for 21 days and received sustained rescue treatment with aerosolized Fasudil (81 mg/ml t.i.d for 15 min) or i.p. Y27632 (15 mg/kg b.i.d) from days 14-21. Inhaled Fasudil normalized pulmonary vascular resistance, and reversed pulmonary vascular remodeling but did not improve RV systolic function. Systemic, but not pulmonary-selective, ROCK inhibition attenuated increased RV ROCK activity. Our findings indicate that RV dysfunction in chronic hypoxic PHT is not merely a result of increased afterload, but rather may be due to increased activity of ROCK in the right ventricle.

No description available.

Statins are widely used to treat hypercholesterolemia. Statins inhibit cholesterol biosynthesis, thereby activating genes involved in cholesterol homeostasis, which are under the control of the Sterol Regulatory Element (SRE). Statins also have cholesterol-independent beneficial cardiovascular effects mediated through the phosphoinositide 3-kinase (PI3-K) / Akt signaling pathway and by inhibition of protein prenylation. Because statins inhibit the synthesis of isoprenoids, they can act by inhibiting the small signaling GTPases Ras and Rho, which require post-translational prenylation to become membrane-anchored and functional. We showed that simvastatin-mediated inhibition of protein prenylation does not appear to play a role in activation of SRE transcriptional activity in HepG2 cells. We also found that when isoprenoids were replenished, basal phospho-Akt decreased, suggesting that inhibition of prenylation by simvastatin mediates Akt phosphorylation. Future studies will be needed to investigate the role that inhibition of protein prenylation plays in the activation of the PI3-K/Akt pathway by simvastatin. / Department of Biology

Access to abstract permanently restricted. / Aldol condensation to synthesize substituted chalcones -- Synthesis and testing of substituted 4,5-dihydropyrazoles -- Biological testing of synthesized 4,5-dihydropyrazoles. / Access to thesis permanently restricted. / Department of Chemistry

Cardiac hypertrophy is a compensatory response to increased work load or stress on the heart, but over time can lead to heart failure and death. The molecular mechanisms underlying this disease are still not completely understood, however the Rho/Rho kinase pathway has been shown to play a role. N-[11C]-methyl-hydroxyfasudil, a PET radiotracer, binds to active Rho kinase and could be a possible tracer for hypertrophy. Hypertrophy was induced in vitro using the β-adrenergic receptor agonist isoproterenol to evaluate optimal Rho kinase activity. Rho kinase activity data was correlated to N-[11C]-methyl-hydroxyfasudil binding. Cardiac hypertrophy was verified with an increase in nuclear size (1.74 fold) and cell size (~2 fold), activation of hypertrophic signalling pathways, and increased Rho kinase activity (1.64 fold). This correlated to a 10.3% increase in N-[11C]-methyl-hydroxyfasudil binding. This data suggests that N-[11C]-methyl-hydroxyfasudil may be useful as a radiotracer for detecting cardiac hypertrophy and merits further in vivo investigation.

Drug development is a long and expensive process. It is often not until potential drug candidates are administered to humans that accurate quantification of their pharmacokinetic characteristics is achieved. The goal of developing quantitative structure-pharmacokinetic relationships (QSPkRs) is to relate the molecular structure of a chemical entity with its pharmacokinetic characteristics. In this thesis artificial neural networks (ANNs) were used to construct in silico predictive QSPkRs for various pharmacokinetic parameters using different drug data sets. Drug pharmacokinetic data for all studies were taken from the literature. Information for model construction was extracted from drug molecular structure. Numerous theoretical descriptors were generated from drug structure ranging from simple constitutional and functional group counts to complex 3D quantum chemical numbers. Subsets of descriptors were selected which best modeled the target pharmacokinetic parameter(s). Using manual selective pruning, QSPkRs for physiological clearances, volumes of distribution, and fraction bound to plasma proteins were developed for a series of beta-adrenoceptor antagonists. All optimum ANN models had training and cross-validation correlations close to unity, while testing was performed with an independent set of compounds. In most cases the ANN models developed performed better than other published ANN models for the same drug data set. The ability of ANNs to develop QSPkRs with multiple target outputs was investigated for a series of cephalosporins. Multilayer perceptron ANN models were constructed for prediction of half life, volume of distribution, clearances (whole body and renal), fraction excreted in the urine, and fraction bound to plasma proteins. The optimum model was well able to differentiate compounds in a qualitative manner while quantitative predictions were mostly in agreement with observed literature values. The ability to make simultaneous predictions of important pharmacokinetic properties of a compound made this a valuable model. A radial-basis function ANN was employed to construct a quantitative structure-bioavailability relationship for a large, structurally diverse series of compounds. The optimum model contained descriptors encoding constitutional through to conformation dependent solubility characteristics. Prediction of bioavailability for the independent testing set were generally close to observed values. Furthermore, the optimum model provided a good qualitative tool for differentiating between drugs with either low or high experimental bioavailability. QSPkR models constructed with ANNs were compared with multilinear regression models. ANN models were shown to be more effective at selecting a suitable subset of descriptors to model a given pharmacokinetic parameter. They also gave more accurate predictions than multilinear regression equations. This thesis presents work which supports the use of ANNs in pharmacokinetic modeling. Successful QSPkRs were constructed using different combinations of theoretically-derived descriptors and model optimisation techniques. The results demonstrate that ANNs provide a valuable modeling tool that may be useful in drug discovery and development.