published_or_final_version / Molecular Biology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Clinical Oncology / Doctoral / Doctor of Philosophy

published_or_final_version / Molecular Biology / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Evaluatiton of the expresion and co-expresion of endoglin and VCAM-1 in the aorta on apoE- deficient mice Diploma thesis Lucie Minaříková Supervisor: Mgr. Jana Rathouská Background: The aim of this work was to monitor the expression and a possible co-expression of endoglin (TGF-β receptor III) and VCAM-1 in mouse aortic endothelium. As an experimental model, we used the mouse strain C57BL/6J, that was genetically modified, and is characteristic by a deficit of apolipoprotein E. Methodes: In the study, we focused on testing the mouse strain C57BL/6J with gene knockout for apolipoprotein E in different stages of the atherosclerotic process. 10 weeks old female mice were divided into three groups and fed diets with different content of cholesterol. One experimental group was fed a standard diet (called "chow type diet") for a period of two months. The other two groups were fed a diet containing 21% fat (called "Western type diet") for a period of two and four months. For the determination of the levels of total cholesterol, a biochemical analysis of blood was performed. Obtained parts of ascending aorta were analyzed by ImmPRESSTM immunohistochemical method with the detection reagent DAB....

Proper functioning of the voltage gated sodium channel, NaV1.5, is essential for maintenance of normal cardiac electrophysiological properties. Changes to the biophysical properties of sodium channels can take many forms and can affect the peak component of current carried during phase zero of the action potential; the “persistent” or “late” current component conducted during the repolarizing phases of the action potential; the availability of the channel as seen by changes in window current; and the kinetics of channel transitions between closed, opened and inactivated states. Mutations in NaV1.5 that alter these parameters of channel function are linked to a number of cardiac diseases including arrhythmias such as atrial fibrillation. In addition, mutations in many of the auxiliary proteins that form part of the sodium channel macromolecular complex have likewise been associated with diseases of the heart. Mutations in regions of the sodium channel responsible for interactions with these auxiliary proteins have also been linked to various dysfunctional cardiac states. Indeed, a large number of disease causing mutations are localized to the C-terminal domain of NaV1.5, a hotspot for interacting proteins. Using a transgenic mouse model, we show that expression of a mutant sodium channel with gain-of-function properties conferring increased persistent current, is sufficient to cause both structural and electrophysiological abnormalities in the heart driving the development of spontaneous and prolonged episodes of atrial fibrillation. The sustained and spontaneous atrial arrhythmias, an unusual if not unique phenotype in mice, enabled explorations of mechanisms of atrial fibrillation using in vivo (telemetry), ex vivo (optical voltage mapping), and in vitro (cellular electrophysiology) techniques. Since persistent sodium current was the driver of the structural and electrophysiological abnormalities leading to atrial fibrillation, we subsequently pursued studies exploring the mechanisms of persistent sodium current. Prior work of heterologously expressed sodium channels identified calmodulin as a regulator of persistent current. Mutation of the calmodulin binding site in the C-terminus of the cardiac sodium channel caused increased persistent current when the channel was expressed heterologously. The role of calmodulin in the regulation of the sodium channel in cardiomyocytes has not been definitively determined. We created transgenic mice expressing human sodium channels harboring a mutation of the calmodulin binding site. Using whole cell patch clamping, we found, in contrast to previously reported findings, that ablation of the calmodulin binding site did not induce increased persistent sodium current. Instead, loss of calmodulin binding stabilized the inactivated state by shifting the V50 for steady-state inactivation in the hyperpolarizing direction. Furthermore, loss of calmodulin binding sped up the transition to the inactivated state demonstrated by a significantly shortened tau of inactivation. In contrast to studies performed in heterologous expression systems, our findings thus suggest that in heart cells, calmodulin binding increases availability, similar to its role in regulating NaV1.4 channels. The studies were then expanded to explore the role of other interacting proteins, fibroblast growth factor (FGF) homologous factors (FHF), in the presence and absence of calmodulin binding. Using whole cell patch clamping, we found that a mutation (H1849R) of the sodium channel causing decreased FHF binding affinity leads to a rightward shift in steady-state inactivation and a slowed rate of inactivation of INa. A third mutant channel, with concurrent decreased FHF and calmodulin binding affinity similarly results in a rightward shift in steady-state inactivation suggesting a dominant effect of the H1849R mutation. Persistent current was not elevated in either of these mutant channels. Importantly, the methodology that we report enables us and other groups to carry out studies of human sodium channels in the native environment of NaV1.5. Our investigation into calmodulin’s role, which yielded conclusions distinct from prior findings in heterologous expression systems, demonstrates the value of this approach.

Thesis (Ph. D.)--University of Chicago, Committee-on-Developmental Biology, 2002. / Includes bibliographical references. Also available on the Internet.

Thesis (M. Sc.)--York University, 1998. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 144-164). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ39236.

Thesis (Ph. D.)--University of Chicago, Dept. of Molecular Genetics and Cell Biology, August 2003. / CD-ROM reproduces dissertation in PDF format; Adobe Acrobat required. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 159-193).