The Notch signaling pathway is crucial for mammalian heart development. It controls cell-fate decisions, coordinates patterning processes and regulates proliferation and differentiation. Critical Notch effectors are Hey bHLH transcription factors (TF) that are expressed in atrial (Hey1) and ventricular (Hey2) cardiomyocytes (CM) and in the developing endocardium (Hey1/2/L). The importance of Hey proteins for cardiac development is demonstrated by knockout (KO) mice, which suffer from lethal cardiac defects, such as ventricular septum defects (VSD), valve defects and cardiomyopathy. Despite this clear functional relevance, little is known about Hey downstream targets in the heart and the molecular mechanism by which they are regulated. Here, I use a cell culture system with inducible Hey1, Hey2 or HeyL expression to study Hey target gene regulation in HEK293 cells, in murine embryonic stem cells (ESC) and in ESC derived CM. In HEK293 cells, I could show that genome wide binding sites largely overlap between all three Hey proteins, but HeyL has many additional binding sites that are not bound by Hey1 or Hey2. Shared binding sites are located close to transcription start sites (TSS) where Hey proteins preferentially bind to canonical E boxes, although more loosely defined modes of binding exist. Additional sites only bound by HeyL are more scattered across the genome. The ability of HeyL to bind these sites depends on the C-terminal part of the protein. Although there are genes which are differently regulated by HeyL, it is unclear whether this regulation results from binding of additional sites by HeyL. Additionally, Hey target gene regulation was studied in ESC and differentiated CM, which are more relevant for the observed cardiac phenotypes. ESC derived CM contract in culture and are positive for typical cardiac markers by qRT PCR and staining. According to these markers differentiation is unaffected by prolonged Hey1 or Hey2 overexpression. Regulated genes are largely redundant between Hey1 and Hey2. These are mainly other TF involved in e.g. developmental processes, apoptosis, cell migration and cell cycle. Many target genes are cell type specifically regulated causing a shift in Hey repression of genes involved in cell migration in ESC to repression of genes involved in cell cycle in CM. The number of Hey binding sites is reduced in CM and HEK293 cells compared to ESC, most likely due to more regions of dense chromatin in differentiated cells. Binding sites are enriched at the proximal promoters of down-regulated genes, compared to up-or non-regulated genes. This indicates that up-regulation primarily results from indirect effects, while down-regulation is the direct results of Hey binding to target promoters. The extent of repression generally correlates with the amount of Hey binding and subsequent recruitment of histone deacetylases (Hdac) to target promoters resulting in histone H3 deacetylation. However, in CM the repressive effect of Hey binding on a subset of genes can be annulled, likely due to binding of cardiac specific activators like Srf, Nkx2-5 and Gata4. These factors seem not to interfere with Hey binding in CM, but they recruit histone acetylases such as p300 that may counteract Hey mediated histone H3 deacetylation. Such a scenario explains differential regulation of Hey target genes between ESC and CM resulting in gene and cell-type specific regulation. / Der Notch Signalweg ist essenziell für die Herzentwicklung in Säugetieren. Er kontrolliert Zell-differenzierung, koordiniert Musterbildungsprozesse und reguliert Proliferation und Differenzierung. Kritische Notch Effektoren sind Hey bHLH Transkriptionsfaktoren, welche im Herzen in atrialen (Hey1) und ventrikulären (Hey2) Kardiomyozyten und dem sich entwickelnden Endokardium (Hey1/2/L) exprimiert werden. Die Bedeutung von Hey Proteinen während der Herzentwicklung wird an Hand von verschiedenen KO Mäusen ersichtlich, welche letale Herzdefekte, wie ventrikuläre Septumdefekte, Herzklappendefekte und Kardiomyopathien, entwickeln. Trotz dieser klaren funktionalen Relevanz ist wenig über Hey Zielgene im Herzen und den molekularen Mechanismus bekannt, über den diese reguliert werden. Hier wurde ein Zellkultursystem mit induzierbarer Expression von Hey1, Hey2 oder HeyL verwendet, um Hey Zielgene in HEK293, murinen embryonalen Stammzellen und in differenzierten Kardiomyozyten zu studieren. In HEK293 Zellen konnte ich zeigen, dass die Bindestellen im Genom weitestgehend zwischen allen drei Hey Proteinen überlappen, HeyL jedoch viele zusätzliche Bindestellen aufweist, welche weder von Hey1 noch Hey2 gebunden werden. Gemeinsame Bindestellen befinden sich nahe Transkriptionsstartstellen, präferentiell an kanonische E boxen. Die nur von HeyL gebunden Bindestellen sind mehr über das Genom verteilt. Dabei ist die Fähigkeit von HeyL diese Stellen zu binden vom C-terminalen Teil abhängig. Obwohl es Gene gibt, die unterschiedlich von HeyL reguliert werden, ist es auf Grund der sehr viel größeren Anzahl an HeyL Bindestellen unklar, ob diese Regulation das Resultat von zusätzlicher HeyL Bindung ist. Zusätzlich wurde die Regulation von Hey Zielgenen in embryonalen Stammzellen und differenzierten Kardiomyozyten untersucht, da diese Zellen für die beobachteten kardialen Phänotypen relevanter sind. Differenzierte Kardiomyozyten kontrahieren in Kultur und sind positiv für typische kardiale Marker an Hand von qRT-PCR und Färbungen. Nach diesen Markern ist die Differenzierung durch kontinuierliche Überexpression von Hey1 oder Hey2 unverändert. Die Hey1 und Hey2 regulierten Gene sind weitestgehend redundant. Viele Zielgene sind andere Transkriptionsfaktoren, die zum Beispiel an Entwicklungsprozessen, Apoptose, Zellmigration und dem Zellzyklus beteiligt sind. Diese werden oft Zelltyp spezifisch reguliert, was zur Folge hat, dass in embryonalen Stammzellen auch an der Zellmigration beteiligte Gene reprimiert werden, während es in Kardiomyozyten vor allem Gene sind, die den Zellzyklus betreffen. Die Zahl der Hey Bindestellen ist in Kardiomyozyten und HEK293 Zellen verglichen mit embryonalen Stammzellen reduziert, höchstwahrscheinlich da differenzierte Zellen weniger offenes Chromatin besitzen. Die Bindestellen sind in reprimierten Genen verglichen mit induzierten oder nicht regulierten Genen angereichert. Dies deutet an, dass eine Induktion meist durch indirekte Effekte zu Stande kommt, während eine Repression das direkte Ergebnis der Hey Bindung an Zielpromotoren ist. Die Stärke der Repression korreliert dabei generell mit der Menge an Promoter gebundenem Hey Protein, welches Histon-Deacetylasen rekrutiert und zu einer Reduktion der Histon H3 Acetylierung führt. In Kardiomoyzyten wird der repressive Effekt von Hey für bestimmte Gene unterbunden, wahrscheinlich durch Bindung herzspezifischer Aktivatoren, wie Srf, Nkx2 5 und Gata4. Diese Faktoren scheinen nicht die Bindung von Hey zu beeinflussen, aber sie rekrutieren Acetylasen wie p300, welche Hey vermittelter Histon H3 Deacetylierung entgegenwirken. Dieses Model erklärt die unterschiedliche Regulation von Hey Zielgenen zwischen embryonalen Stammzellen und Kardiomyozyten.

This study examined the differential gene expression patterns between endothelial cells (EC) from 2D monolayer and EC from angiogenic capillary-like network in 3D matrices. Our microarray analysis comparing 3D to 2D EC cultures detected upregulation of 854 protein-coding genes and downregulation of 863 genes. We show that Notch signaling pathway is highly regulated in angiogenesis, induced by change in ECM dimension. Notch target genes Hey1, HeyL, Hes1 and Hes4 transcription factors were upregulated in 3D angiogenic EC, which were confirmed with qRT-PCR. Moreover, we are the first to report enrichment of FoxS1 transcription factor mRNA during angiogenesis in 3D ECM. Next, we asked whether epigenetic mechanisms partly mediate cis-trans response in angiogenesis. Our sodium bisulfite sequencing analyses did not indicate a role for DNA methylation in the expression of key Notch signaling components. However, our pilot studies indicate a potential role for lncRNAs in controlling EC phenotype in angiogenic response.

This study examined the differential gene expression patterns between endothelial cells (EC) from 2D monolayer and EC from angiogenic capillary-like network in 3D matrices. Our microarray analysis comparing 3D to 2D EC cultures detected upregulation of 854 protein-coding genes and downregulation of 863 genes. We show that Notch signaling pathway is highly regulated in angiogenesis, induced by change in ECM dimension. Notch target genes Hey1, HeyL, Hes1 and Hes4 transcription factors were upregulated in 3D angiogenic EC, which were confirmed with qRT-PCR. Moreover, we are the first to report enrichment of FoxS1 transcription factor mRNA during angiogenesis in 3D ECM. Next, we asked whether epigenetic mechanisms partly mediate cis-trans response in angiogenesis. Our sodium bisulfite sequencing analyses did not indicate a role for DNA methylation in the expression of key Notch signaling components. However, our pilot studies indicate a potential role for lncRNAs in controlling EC phenotype in angiogenic response.

This study is based upon the vertical line array (VLA) in South China Sea experiment of the Asian Seas International Acoustic Experiment (ASIAEX), dated from May 3, 2001 to May 16, 2001, in which the ambient noise in shallow water was measured. In this paper, we use the beamforming method to calculate the vertical directionality of ambient noise in shallow water, for discussing the noise source distribution, and environmental effects on vertical directionality. The results show that distant shipping noise was observed near the horizontal angles, and surface noise occurred at high grazing angles. It is also discussed that the wind speed effect on the vertical directionality in this paper. Because of the typhoon Cimaron passed the experimental area during the experiment, which make wind speed changed obviously, so it can be found out the wind effect of ambient noise. By analyze the noise fluctuation, it is concluded that the threshold frequency of wind wave affected ambient noise levels were about 400 Hz. And after analyzing the vertical directionality, it is verified that the threshold was lower to 200 Hz. On the other hand, we also note the phenomena of ¡§noise notch¡¨ appeared at some duration when calculating the vertical directionality. The environmental effects (sound speed profile) on the notch, and the presentation of noise notch at different frequencies were discussed. In the end part of this paper, we determined the time when the notch occurred by using the power difference of vertical directionality, and we expect that the results would be important for similar researches in the future.

Inhibitor of differentiation (Id) family helix-loop-helix proteins regulate the proliferation, survival and differentiation of numerous cell types during development, however their function during retinal development has not been analyzed. Using loss-of-function and overexpression assays in zebrafish, I demonstrate that Id2a levels modulate retinoblast cell cycle kinetics and thereby influence neuron and glia formation in the retina. Id2a-deficient retinas possess increased numbers of cells occupying S phase, at the expense of mitotic cells, and kinetic analyses demonstrate that Id2a is required for S-phase progression and/or the transition from S to M phase. Id2a-dependent defects in retinoblast proliferation lead to microphthalmia and to an absence of nearly all differentiated inner and outer nuclear layer cell types. Overexpression of id2a has the opposite effect on retinoblast cell cycle kinetics: id2a-overexpressing retinoblasts progress from S to M phase more rapidly and they undergo mitosis more frequently, which results in macrophthalmia. Mosaic analyses reveal that Id2a function in facilitating both cell cycle progression and neuronal differentiation in the retina is non-cell-autonomous, suggesting that Id2a functions upstream of the extrinsic pathways that regulate retinogenesis. In an effort to identify which extrinsic pathways function downstream of Id2a, I discovered that Id2a function is necessary and sufficient to limit Notch pathway activity during retinogenesis. Id2a-deficient retinae possess elevated levels of Notch pathway component gene expression, while retinae overexpressing id2a possess reduced expression of Notch pathway component genes. Attenuation of Notch signaling activity by DAPT or by morpholino knockdown of Notch1a is sufficient to rescue both the proliferative and differentiation defects in Id2a-deficient retinae. In addition to regulating Notch pathway activity, through an RNA-Seq and differential gene expression analysis of Id2a-deficient retinae, I identified a number of additional intrinsic and extrinsic regulatory pathway components whose expression is regulated by Id2a. These data highlight the integral role played by Id2a in the gene regulatory network governing the transition from retinoblast proliferation to terminal differentiation during vertebrate retinogenesis. / text

Due to the release of 3.1-10.6 GHz band, UWB systems have a rapidly progressive development. They have been widely employed in short-range communication applications and large-bandwidth handheld devices. As part of the system, the UWB antenna plays an extremely important role. Due to the trend towards integrated printed circuits, co-planar waveguide technology is a feasible solution for designing the UWB antenna. This thesis focuses on designing a UWB co-planar waveguide antenna with a band-stop filter. This band-stop filter offers rejection to unwanted frequencies in the range of the operating band in order to avoid unnecessary interference from other communication applications and improve its own system’s performance. In addition, it can divide the whole wide band into a few sub-bands. This will create more flexibility for practical applications. The professional full-wave field solver software package CST Microwave Studio is used as the analysis tool to obtain the performances of this antenna. It operates from 3.1 GHz to 10.6 GHz with a VSWR < 2 in the pass bands, and a VSWR > 2 in the stop bands. The selected frequencies demonstrate nearly omni-directional characteristics in radiation patterns. Comparing with other published UWB antenna designs, relatively reasonable group delay results are achieved. Measurements on a fabricated prototype validate the design approach. / Graduate / 0537 / wang@uvic.ca

Zugl.: Hohenheim, Universiẗat, Diss., 2008.

Pax7 is a paired box transcription factor expressed by all satellite cells which are critically required for muscle regeneration and growth. The absolute requirements of Pax7 in the maintenance of the satellite cell pool are widely acknowledged. However the mechanisms by which Pax7 executes muscle regeneration or contributes to satellite cell homeostasis remain elusive. We performed cell and molecular analysis of Pax7 null satellite cells to investigate muscle stem cell function. Through genome wide studies, we found that genes involved in cell cell interactions, regulation of migration, control of lipid metabolism and inhibition of myogenic differentiation were significantly perturbed in Pax7 null satellite cells. Analysis of satellite cells in vitro showed that Pax7 null satellite cells undergo precocious myogenic differentiation and have perturbed expression of genes involved in the Notch signaling pathway. We showed that Notch 1 is a novel Pax7 target gene and by using a genetic approach we demonstrated that ectopic expression of the constitutively active intracellular domain of Notch1 (NICD1) in Pax7 null satellite cells is sufficient to maintain the satellite cell pool as well as to restore their proliferation. Instead of differentiating into myogenic cells and in the absence of a myogenic cue, NICD1 Pax7 null satellite cells become a source of ectopic brown fat within muscles and give rise to brown adipocytes both in vivo and in vitro. In conclusion we showed that Notch 1 partially rescues Pax7 deficient satellite cells loss and proliferation. Additionally we provide the first evidence that Notch signalling contributes to satellite cell fate by inhibiting terminal myogenic differentiation and inducing brown adipogenesis.

No description available.

No description available.