Various genes have evolved to protect the cell against stressor-induced damage or death including the heat shock proteins (HSPs). Stressor-induced HSP gene expression involves the activation of heat shock factor (HSF), which binds to the heat shock element (HSE) found in the promoter region of <em>hsp</em> genes. Previously, our laboratory has examined the expression and function of <em>hsp</em> genes in the South African clawed frog, <em>Xenopus laevis</em>. Amphibians are particularly susceptible to adverse environmental conditions, including high temperatures and toxicants. In contrast to the many known inducers of HSF activation in poikilothermic vertebrates, few inhibitors have been either discovered or described in the literature. The present study has compared for the first time the effect of two heat shock response (HSR) inhibitors, quercetin and KNK437, on <em>hsp</em> gene expression in <em>Xenopus</em> A6 cells, demonstrating their efficacy in poikilotherms. Northern blot and densitometric analysis showed that cells treated with either quercetin or KNK437 decreased the heat shock-induced accumulation of <em>hsp70</em>, <em>hsp47</em>, and <em>hsp30</em> mRNAs. Additionally, constitutive levels of <em>hsp47</em> and <em>hsc70</em> mRNAs were reduced. In comparison, neither quercetin nor KNK437 affected the levels of constitutively expressed <em>ef1&alpha;</em> mRNAs under control or heat shock conditions. Western blot and densitometric analysis in this study showed that under heat shock conditions, exposure to quercetin or KNK437 significantly decreased the accumulation of HSP30, and that KNK437 was more effective in doing so than quercetin. In comparison, levels of actin were not significantly affected by either heat shock or exposure to DMSO, quercetin, or KNK437. These findings suggest that one mechanism by which quercetin and KNK437 inhibits the HSR in <em>Xenopus</em> is through the inhibition of HSF activity. <br /><br /> Results of this study also suggest that KNK437 inhibits the acquisition of thermotolerance in poikilotherms, similar to observations in mammalian systems. In the presence of KNK437, cells given a 2 h heat pretreatment at 33ºC followed by a thermal challenge for 1 h at 37ºC, showed numerous ruffled membrane edges and some aggregates of disrupted stress fibers. In comparison, cells directly challenged for 1 h at 37ºC, showed a marked decrease in HSP30, which was located predominantly at the cellular periphery in conjunction with actin aggregates. These cells showed virtually no intact stress fibers spanning cells and no coherent cell-cell connections. A 3-D analysis of cells given a 1 h thermal challenge at 37ºC (after a prior 2 h heat shock at 33ºC) in the absence of KNK437, showed numerous linear actin bundles transversing the entire cell, even extending into areas of cell-cell contact, and abundant HSP30 concentrated in the perinuclear region surrounding an intact nucleus. However, in the presence of KNK437, there was a significant emergence of membrane ruffles indicating global instability of cellular adhesion. This study has demonstrated that KNK437, which is the more specific and efficient HSR inhibitor, will be an important inhibitor to compare with the well-documented quercetin for future investigations.

<p>The means by which cells, tissues, and organisms undergo morphogenesis are variable and highly regulated, and the mechanisms that govern cellular changes in response to signaling cues are poorly understood. This study seeks to address the role of a newly characterized protein in zebrafish in translating signaling cues into physical changes within a cell.</p><p>The <italic>Cordon&ndash;bleu (Cobl)</italic> gene is widely conserved in vertebrates, with developmentally regulated axial and epithelial expression in mouse and chick embryos. <italic>In vitro</italic>, Cobl can bind monomeric actin and nucleate formation of unbranched actin filaments, while in cultured cells it can modulate the actin cytoskeleton. However, an essential role for Cobl <italic>in vivo</italic> has yet to be determined. We have identified the zebrafish <italic>cobl</italic> ortholog and have used zebrafish as a model to assess the requirements for Cobl in embryogenesis. We find that cobl shows enriched expression in ciliated epithelial tissues during zebrafish organogenesis. The utilization of antibodies developed against Cobl shows that the protein is concentrated along the apical domain of ciliated cells, in close proximity to the apical actin cap. </p><p>Reduction of <italic>cobl</italic> by antisense morpholinos reveals an essential role in embryonic morphogenesis and organ development. A requirement for Cobl was shown for the proper function of various and ciliated epithelial organs. Cobl appears to direct the elongation of motile cilia in organs such as Kupffer&rsquo;s vesicle and the pronephros. In Kupffer&rsquo;s vesicle, the reduction in Cobl coincides with a reduction in the amount of apical F-actin. Additionally, Cobl may play a role during gastrulation cell movements and convergence and extension morphogenesis during early embryonic development. Thus, Cobl may represent a molecular activity that couples developmental patterning signals with local intracellular cytoskeletal dynamics to support elongation of motile cilia and tissue morphogenesis.</p> / Dissertation

Bidens pilosa L. is a traditional Chinese herbal medicine of which was considered as a potential COX2 inhibitor and anti-inflammatory agent. The objective of this study is to discriminate the protective effect of aqueous extract of Bidens pilosa L. leaves (BPLAE) against TAA-induced live fibrosis using an animal model. The herb extracts were administrated via intraperitoneal injection once per week (1.25, 2.5 g/kg), and thioacetamide (200 mg/kg) was injected three times per week and the mice were sacrificed at week 4 and week 8, respectively. Immunohistochemistry staining, Hematoxylin-eosin (HE) staining, Sirius red staining were carried out to evaluate the pathological alterations of mouse livers; in addition, Western blotting was performed to measure the differential expression of £\-smooth muscle actin (£\-SMA) between different treatment groups (vehicle, week 4 and week 8). Hepatic hydroxyproline was also detected in order to compare difference in collagen formation of each group. The results showed that Bidens pilosa L. effectively reduced amount of hepatic hydroxyproline and £\-SMA protein in mice with fibrotic liver induced by TAA. Moreover, in histiopathological exam, the BPLAE treated mice demonstrated a lower collagen and £\-SMA expression, which indicated that BPLAE might reduce degree and severity of liver fibrosis in mice. In conclusion, these results suggested that BPLAE potentially against fibrogenesis in TAA- induced mice liver fibrosis. Additionally, we found that BPLAE might involve in the signaling pathway of MAPK (ERK1/ERK2), which reduced the phosporylation level of p44 but not p42. Further studies using cell base assay to confirm the inhibiting role of BPLAE against cell proliferation or migration is warrant.

No description available.

Two phylogenies of Phalaenopsis (Orchidaceae) are presented, one from combined chloroplast DNA data and one from a nuclear actin gene. We used these phylogenies to assess and modify the classification of Phalaenopsis and to examine several morphological characters and geographical distribution patterns. Our results support Christenson’s (2001) treatment of Phalaenopsis as a broadly defined genus that includes the species previously placed in the genera Doritis and Kingidium. Some of Christenson’s subgeneric groups needed to be recircumscribed to reflect a natural classification. We recognized four subgenera and six sections, subgenera Aphyllae, Parishianae (with sections Conspicuum, Delisiosae, Esmeralda, and Parishianae), Phalaenopsis, and Polychilos (with sections Fuscatae and Polychilos). In order to find a set of universally amplifiable, phylogenetically informative, single-copy nuclear regions, we conducted a whole genome comparison of the rice (Oryza sativa) and Arabidopsis thaliana genomes. We constructed a database of both genomes and searched for pairs of sequences using criteria we felt would ensure primers that would reliably amplify using standard PCR protocols. We tested the most promising 142 primer pairs in the lab on eighteen taxa and found four potentially informative markers in Phalaenopsis and one in Helianthus. Our results indicated that it will be difficult to find universal nuclear markers, however our database provides an important tool for finding informative nuclear markers within specific groups. The full set of primer combinations is available online at, “The Conserved Primer Pair Project,” http://aug.csres.utexas.edu:8080/cpp/index.html. We used fourteen Phalaenopsis species and seven horticultural hybrids to create a real dataset with which to test phylogenetic network reconstruction methods. We tested the performance of Neighbor-Net, implemented in SplitsTree, under four different categories of complexity: one hybrid, two independent hybrids (hybrids with no parents in common), three independent hybrids, and two non-independent hybrids (one parent was shared between hybrids). Neighbor-Net was able to predict accurately the parents of hybrids in only about half of the datasets we tested, and there were so many false positives that it was impossible to distinguish the hybrids from the species. We plan to use this dataset to test methods, such as RIATA and RGNet, when they become available. / text

No description available.

published_or_final_version / abstract / toc / Medicine / Master / Master of Philosophy

Da eine Vielzahl von Proteininteraktionen innerhalb zellulärer Organismen an der Grenzfläche zu Membranen stattfindet, ist die Untersuchung dieser Prozesse von gro-ßem wissenschaftlichem Interesse. Ziel dieser Arbeit war es Modellsysteme basierend auf artifiziellen Membranen zu entwickeln, mit deren Hilfe die Untersuchung ausge-wählter Proteininteraktionen ermöglicht werden konnte. Im ersten Abschnitt dieser Arbeit (Kapitel 4-6) wurde ein Biosensorassay basierend auf festköperunterstützten Membranen entwickelt, der die Quantifizierung der Interaktion von C-Polycystin-2 (cPC2) mit seinen Interaktionspartnern C-Polycystin-1 (cPC1) und PIGEA14 mittels der Quarzmikrowaagetechnik ermöglichte. Aufgrund der Tatsache, dass die Affinität von cPC2 zu cPC1 in Anwesenheit von Ca2+ dreifach höher war, wurde eine Ca2+ abhängige Trimerisierung von cPC2 postuliert. Die Unterschiede der ermittelten kinetischen Koeffizienten führten zur Entwicklung eines Bindunsgmodells, welches die dreistufige Adsorption von cPC2 an cPC1 in Abwesenheit bzw. einstufige Adsorption in Anwesenheit von Ca2+ implizierte. Im Falle der Interaktion von cPC2 mit PIGEA14 wurde die Abhänigkeit der cPC2 Bindung von der Pseudophosphorylie-rung des Proteins an Ser812 untersucht. Es wurde festgestellt, dass die Affinität der pseudophosphorylierten Mutante cPC2S812D zu PIGEA14 zweifach niedriger war, als die von cPC2wt. Im zweiten Abschnitt der Arbeit (Kapitel 7 und 8) wurde die spezifische Wechselwir-kung von filamentösem Aktin (F-Aktin) mit festkörperunterstützten und porenüber-spannenden Membranen untersucht. Die kontrollierte Anbindung von F-Aktin in und auf porösen Aluminiumoxidfilmen konnte mit Hilfe verschiedener Funktionalisie-rungsstrategien erzielt werden. Der Einfluss eines F-Aktin Netzwerks auf die Span-nung und viskoelastischen Eigenschaften porenüberspannender Membranen wurde mittels kraftmikroskopischer Studien untersucht. Es wurde nachgewiesen, dass der Einfluss von gebundenem F-Aktin auf die Membranspannung gering war, aber erst durch die F-Aktin Adhäsion viskoelastische Membraneigenschaften induziert wurden.

No description available.

Calcific aortic valve sclerosis is characterized by focal lesions in the valve leaflet. These lesions are rich in myofibroblasts that express α-SMA and cause fibrosis. Lesions tend to occur in regions of the leaflet that are subjected to large bending loads, suggesting a mechanobiological basis for myofibrogenic differentiation and valve pathogenesis. In this thesis, a bioreactor was developed to study the effect of physiological loading on myofibrogenic differentiation of valve interstitial cells. Cyclic loading of native porcine aortic valve leaflets ex vivo resulted in increased α-SMA expression, predominantly in the fibrosa and spongiosa (similar to sclerotic leaflets). Cofilin, an actin-binding protein, was also upregulated by loading, suggesting it plays a role in mechanically-induced myofibrogenesis. Similarly, loading of a tissue engineered aortic valve leaflet model resulted in increased α-SMA transcript and protein expression. These data support an integral role for mechanical stimuli in myofibrogenic differentiation and sclerosis in the aortic valve.