No description available.

No description available.

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....

Cheung Siu Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 86-91). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xiii / List of Figures --- p.xiv / Lists of Abbreviations --- p.xvii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- The plant secretory pathways --- p.2 / Chapter 1.1.1 --- Three different protein sorting pathways to plant vacuoles --- p.3 / Chapter 1.1.2 --- VSD and VSR --- p.6 / Chapter 1.2 --- Prevacuolar compartments --- p.7 / Chapter 1.2.1 --- Lytic PVC --- p.7 / Chapter 1.2.2 --- BP-80 reporter as a lytic PVC marker --- p.8 / Chapter 1.2.3 --- PVC of PSV --- p.9 / Chapter 1.2.4 --- α-TIP CT reporter as a PVC of PSV marker --- p.10 / Chapter 1.3 --- Project objectives --- p.11 / Chapter Chapter 2 --- Development of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Reporters for Golgi and Prevacuolar Compartments / Chapter 2.1 --- Introduction --- p.13 / Chapter 2.2 --- Materials and Methods --- p.15 / Chapter 2.2.1 --- Chemicals --- p.15 / Chapter 2.2.2 --- Oligonucleotides: Primers and Adapters --- p.15 / Chapter 2.2.3 --- Bacterial Strains --- p.17 / Chapter 2.2.4 --- "Preparation of single-reporter constructs (GONST1 -CFP, CFP-BP-80 and CFP-a-TIP CT reporters)" --- p.17 / Chapter 2.2.4.1 --- "Cloning of pGONSTl-CFPK, a Golgi marker" --- p.17 / Chapter 2.2.4.2 --- "Cloning of pCFP-BP-80K, a lytic PVC marker" --- p.20 / Chapter 2.2.4.3 --- "Cloning of pCFP-α-TIP CTK, a putative marker for PVC of PSV" --- p.22 / Chapter 2.2.5 --- "Preparation of double-reporter constructs (CFP-BP-80-GONST1 - YFP, CFP-α-TIP CT-GONST1-YFP, CFP-BP-80-YFP-α-TIP CT and CFP-α-TIP CT-YFP-BP-80 reporters)" --- p.24 / Chapter 2.2.5.1 --- Insertion ofAdapter-XH to pCFP-BP-80K and pCFP-α-TIP CTK --- p.24 / Chapter 2.2.5.2 --- "Cloning of pCFP-BP-80-GONST 1 -YFPK, pCFP-α-TIP CT- GONST 1-YFPK, pCFP-BP-80-YFP-α-TIP CTK and pCFP- α-TIP CT-YFP-BP-80K" --- p.26 / Chapter 2.2.6 --- Agrobacterium electroporation --- p.30 / Chapter 2.2.7 --- Agrobacterium-mediated transformation of tobacco BY-2 cells --- p.30 / Chapter 2.2.8 --- Selection and screening of transformed BY-2 cells --- p.31 / Chapter 2.2.8.1 --- Antibiotic selection --- p.31 / Chapter 2.2.8.2 --- Fluorescence microscopic screening --- p.31 / Chapter 2.2.9 --- Detection of CFP and YFP reporter genes and their expressions --- p.32 / Chapter 2.2.9.1 --- CTAB genomic DNA extraction --- p.32 / Chapter 2.2.9.2 --- PCR test for CFP (and YFP) transgene in genomic DNA --- p.33 / Chapter 2.2.9.3 --- Subcellular fractionation and protein extraction --- p.33 / Chapter 2.2.9.4 --- Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.34 / Chapter 2.2.9.5 --- Confocal microscopic study --- p.35 / Chapter 2.3 --- Results --- p.36 / Chapter 2.3.1 --- Establishment of kanamycin-resistant BY-2 cells expressing CFP (and YFP) reporters --- p.36 / Chapter 2.3.2 --- Fluorescence microscopic screening of transgenic BY-2 cell lines --- p.37 / Chapter 2.3.3 --- CFP (and YFP) reporter was successfully integrated into transgenic BY-2 cell genome --- p.41 / Chapter 2.3.4 --- CFP (and YFP) reporter was expressed in transgenic BY-2 cell lines --- p.44 / Chapter 2.3.5 --- Punctate CFP (and YFP) signals were detected in transgenic BY-2 cell lines expressing single (or double) reporter --- p.48 / Chapter 2.4 --- Discussion --- p.53 / Chapter 2.4.1 --- "Transgenic BY-2 cell lines expressing single reporter marking Golgi, lytic PVC and putative PVC of PSV have been developed" --- p.53 / Chapter 2.4.2 --- "Golgi, lytic PVC and putative PVC of PSV were separate and distinct organelles" --- p.53 / Chapter 2.4.3 --- Transgenic BY-2 cell lines expressing double reporter were not yet suitable for subsequent study --- p.55 / Chapter Chapter 3 --- Characterization of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Reporters for Prevacuolar Compartments / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and Methods --- p.60 / Chapter 3.2.1 --- Confocal immunofluorescence study --- p.60 / Chapter 3.2.2 --- Drug treatment study (for single-reporter transgenic tobacco BY-2 cell line) --- p.62 / Chapter 3.2.2.1 --- Wortmannin treatment --- p.62 / Chapter 3.2.2.1.1 --- Dosage effect --- p.62 / Chapter 3.2.2.1.2 --- Time-course study --- p.62 / Chapter 3.2.2.2 --- Brefeldin A treatment --- p.63 / Chapter 3.2.2.1.1 --- Dosage effect --- p.63 / Chapter 3.2.2.1.2 --- Time-course study --- p.63 / Chapter 3.2.3 --- Drug treatment study (for double-reporter transgenic tobacco BY-2 cell line) --- p.64 / Chapter 3.2.3.1 --- Wortmannin treatment --- p.64 / Chapter 3.2.3.2 --- Brefeldin A treatment --- p.64 / Chapter 3.3 --- Results --- p.65 / Chapter 3.3.1 --- CFP-α-TIP CT reporter-marked compartment was not Golgi apparatus --- p.65 / Chapter 3.3.2 --- Wortmannin induced CFP-α-TIP CT reporter-marked compartment to vacuolate --- p.69 / Chapter 3.3.3 --- BFA induced CFP-α-TIP CT reporter-marked compartment to form aggregates --- p.72 / Chapter 3.3.4 --- Wortmannin and BFA treatment caused lytic PVC to form small vacuole and Golgi to form aggregate respectively in transgenic BY-2 cell lines expressing double-reporter --- p.75 / Chapter 3.4 --- Discussion --- p.77 / Chapter 3.4.1 --- CFP-α-TIP CT reporter-marked compartment was not Golgi apparatus --- p.77 / Chapter 3.4.2 --- CFP-α-TIP CT reporter-marked compartment was not lytic PVC --- p.77 / Chapter 3.4.3 --- Transgenic BY-2 cell lines expressing double reporter could successfully mark two compartments simultaneously in the same cell --- p.78 / Chapter Chapter 4 --- Summary and Future Prospects / Chapter 4.1 --- Summary --- p.80 / Chapter 4.1.1 --- Hypothesis --- p.80 / Chapter 4.1.2 --- Development of transgenic tobacco BY-2 cell lines --- p.81 / Chapter 4.1.3 --- Characterization of α-TIP CT reporter-marked PVC-like compartment --- p.82 / Chapter 4.2 --- Conclusions --- p.84 / Chapter 4.3 --- Future prospects --- p.85 / References --- p.86

Fung Ka Leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 83-93). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- An overview of the secretory pathway in eukaryotic cells --- p.2 / Chapter 1.2 --- The secretory pathway in plants --- p.4 / Chapter 1.2.1 --- Plant cells contain two functionally distinct vacuoles --- p.4 / Chapter 1.2.2 --- Three vesicular pathways to two vacuole --- p.6 / Chapter 1.2.3 --- Transport vesicles in the three vesicular pathways --- p.9 / Chapter 1.2.4 --- Vacuolar sorting determinants (VSDs) --- p.10 / Chapter 1.2.5 --- Vacuolar sorting receptors (VSRs) --- p.12 / Chapter 1.3 --- The PSVs in mature seeds --- p.15 / Chapter 1.3.1 --- Biogenesis of PSV --- p.15 / Chapter 1.3.2 --- The two chimeric integral membrane reporters --- p.16 / Chapter 1.3.3 --- Subcellular localization of the two chimeric integral membrane reporters in PSVs of mature tobacco seeds --- p.17 / Chapter 1.4 --- Project objectives --- p.19 / Chapter Chapter 2 --- Materials and Methods --- p.20 / Chapter 2.1 --- Construction of the YFP-BP-80 and the YFP- a -TIP reporters --- p.21 / Chapter 2.1.1 --- The pYFP-BP-80-K construct --- p.21 / Chapter 2.1.2 --- The pYFP- a -TIP-K construct --- p.22 / Chapter 2.2 --- Construction of GFP-RMR reporter --- p.23 / Chapter 2.2.1 --- Cloning of pGFP-RMR --- p.23 / Chapter 2.2.2 --- Cloning of pGFP-RMR-K --- p.23 / Chapter 2.3 --- Construction of pGONST1-YFP construct --- p.26 / Chapter 2.3.1 --- The pGONSTl-YFP construct --- p.26 / Chapter 2.4 --- Transformation of Agrobacterium by electroporation --- p.27 / Chapter 2.5 --- Tobacco transformation and selection --- p.28 / Chapter 2.5.1 --- Plant materials --- p.28 / Chapter 2.5.2 --- Tobacco transformation --- p.28 / Chapter 2.6 --- Screening of transgenic tobacco plants expressing YFP fusion proteins --- p.30 / Chapter 2.6.1 --- Kanamycin screening --- p.30 / Chapter 2.6.2 --- Extraction of genomic DNA from leaves --- p.30 / Chapter 2.6.3 --- PCR of genomic DNA --- p.31 / Chapter 2.7 --- Southern blot analysis of genomic DNA --- p.32 / Chapter 2.8 --- Western blot analysis of transgenic tobacco plants --- p.33 / Chapter 2.8.1 --- Extraction of total protein from tobacco leaves or seeds --- p.33 / Chapter 2.8.2 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.34 / Chapter 2.9 --- Confocal immunofluorescence studies --- p.35 / Chapter 2.9.1 --- Preparation of sections --- p.35 / Chapter 2.9.2 --- Single labeling --- p.35 / Chapter 2.9.3 --- Double labeling with one polyclonal and one monoclonal antibodies --- p.36 / Chapter 2.9.4 --- Double labeling with two polyclonal antibodies --- p.36 / Chapter 2.9.5 --- Collection of images --- p.37 / Chapter 2.10 --- Chemicals --- p.38 / Chapter 2.11 --- Primers --- p.38 / Chapter 2.12 --- Bacterial strain --- p.38 / Chapter 2.13 --- Antibodies --- p.39 / Chapter 2.14 --- Growing condition of transgenic plants and determining the developmental stage of tobacco flowers --- p.39 / Chapter Chapter 3 --- Results --- p.41 / Chapter 3.1 --- Generation of transgenic tobacco plants --- p.42 / Chapter 3.2 --- PCR screening of transgenic tobacco plants --- p.46 / Chapter 3.3 --- Southern blot analysis --- p.48 / Chapter 3.4 --- Detection of the YFP fusion proteins in transgenic tobacco plants by western blot analysis --- p.50 / Chapter 3.4.1 --- Detection of the YFP fusion proteins in leaves --- p.50 / Chapter 3.4.2 --- Western blot analysis of vegetative tissues --- p.57 / Chapter 3.4.3 --- Western blot analysis of mature seeds --- p.59 / Chapter 3.5 --- Confocal immunofluorescence studies --- p.61 / Chapter 3.5.1 --- Detection of YFP signals in root tip cells --- p.61 / Chapter 3.5.2 --- Detection of YFP signals in developing seeds --- p.65 / Chapter 3.5.3 --- Subcellular localization of the YFP fusion proteins in mature seeds --- p.67 / Chapter Chapter 4 --- Discussion --- p.72 / Chapter Chapter 5 --- Summary and Future Perspectives --- p.77 / Chapter 5.1 --- Summary --- p.78 / Chapter 5.1.1 --- Generation of transgenic tobacco plants expressing the YFP fusion proteins --- p.78 / Chapter 5.1.2 --- Full-length fusion proteins and cleaved soluble YFP were detected in vegetative tissues --- p.79 / Chapter 5.1.3 --- Only cleaved soluble YFP was detected in mature seeds --- p.79 / Chapter 5.1.4 --- The two fusion proteins might localized in different compartments in developing seeds --- p.79 / Chapter 5.1.5 --- Both fusion proteins were localized within the PSVs of mature seeds --- p.80 / Chapter 5.2 --- Future perspectives --- p.81 / References --- p.83

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.

The invention of pronuclear microinjection initiated the field of transgenic research. Over 30 years later microinjection remains the most straight-forward and most commonly used transgene delivery option. In this work we address the current progress of microelectromechanical systems (MEMS) used as transgenic delivery mechanisms. The nanoinjector is a specially designed MEMS device which uses electrostatic charge to manipulate transgene molecules. The process of nanoinjection was designed as an alternative to microinjection which causes less damage to developing embryos, improves embryo survival, birth rates, and overall efficiency of injections. In vivo testing of nanoinjection demonstrates it is both safe and effective. Additionally nanoinjection has the potential to make transgenesis via yeast artificial chromosomes more practical as the nanoinjector may prevent shearing of the YAC molecules. A second nanoinjection protocol termed intracellular electroporetic nanoinjcetion (IEN) was designed to allow for cytoplasmic injections. Cytoplasmic injections are faster and easier than pronuclear injection and do not require the pronuclei to be visible; yet previous attempts to develop cytoplasmic injection have met with limited success. In IEN injections the nanoinjector is used to place transgenic molecules in the cytoplasm. The transgenes are then propelled through the cytoplasm and electroporated into the pronucleus using electrical pulses. Electroporation of whole embryos has not resulted in transgenic animals, but the MEMS device allows localized electroporation to occur within the cytoplasm, giving transgene access to the pronucleus before degradation can occur. In this report we describe the principles which allow for localized electroporation of the pronuclei including: the location of the pronuclei between 21-28 hours post-hCG treatment, modeling data predicting the voltages needed for localized electroporation of pronuclei, and data on the movement of transgenic DNA based on the voltages delivered by IEN. We further report results of an IEN versus microinjection comparative study in which IEN produced transgenic pups with viability, transgene integration, and expression rates statistically comparable to microinjection. The ability to perform injections without visualizing or puncturing the pronuclei will widely benefit transgenic research, and will be particularly advantageous for the production of transgenic animals with embryos exhibiting reduced pronuclear visibility.

No description available.

Thesis (Ph. D.)--University of Chicago, Dept. of Ecology and Evolution, June 1999. / Includes bibliographical references. Also available on the Internet.

Thesis (MSc)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.