Stress-induced heat shock protein (HSP) gene transcription is controlled primarily by the transcription factor heat shock factor 1 (HSF1). HSF1 activation involves trimerization, heat shock element (HSE)-binding, and transactivation. During prolonged stress or upon removal of stress HSF1 activity attenuates. The mechanism(s) regulating HSF1 activity are unknown. Some reports have suggested that HSF1 may be regulated in some manner by the HSP90 chaperone (Nadeau, K., 'et.al.', 1993; Nair, S., 'et.al.', 1996). Utilizing the 'Xenopus' oocyte model system I tested the hypothesis that the HSP90 chaperone machine, known to function in the folding and maturation of molecules such as steroid receptors, might also participate in HSF1 regulation. Characterization experiments illustrated that the 'Xenopus' oocyte was capable of responding to some but not all forms of stress at the level of HSF1-HSE binding illustrating that certain stress pathways may be absent or inactive in the oocyte. Through transcriptional assaysit was also shown that HSF1-DNA binding and transactivation are regulated by independent mechanisms in the oocyte. HSP90 was shown to interact with and regulate the activity of HSF1 in oocytes. HSP90-HSF1 associations were illustrated ' in vivo' and 'in vitro' by co-immunoprecipitation and gel supershift assays. Immunotargeting HSP90 caused activation of HSF1 under control conditions and delayed deactivation during recovery. These data support a role for HSP90 in the oligomeric changes associated with HSF1 activation/deactivation. Immunotargeting HSP90 also inhibited HSF1 dependent transcription, supporting a role for HSP90 in mediating HSF1 transcriptional activity. HSP90 does not regulate HSF1 alone. Gel supershift analysis showed that p23, HSP90 and FKBP52 exist in a complex with activated HSF1. Furthermore, elevating the levels of various co-chaperones through injection of protein or mRNA had various effects on HSF1 during recovery from stress. Immunotargeting HSP90 or p23 induced HSF1-DNA binding in the absence of stress indicating these proteins may act together to repress HSF1 'in vivo'. Furthermore, injection of HSP90, Hip, Hop, p23, FKBP51, and FKBP52 antibodies significantly delayed HSF1 deactivation supporting a role for these proteins in trimer disassembly. Therefore multiple components of the HSP90 chaperone complex function to regulate HSF1 during its activation and/or deactivation cycle.

Thesis (Ph.D.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Oncology. Title from pdf file main screen (viewed on January 30, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 171-177).

Cilia are evolutionarily conserved microtubule-based organelles projecting from nearly all vertebrate cells, and ciliary defects result in a variety of human disorders known as ciliopathies. Recent studies have shown that several planar cell polarity (PCP) proteins are essential for cilia functions. Here, we focused on Fritz, known as a novel PCP effector protein in Drosophila, in multi-ciliated cells in the epidermis of Xenopus laevis embryos. To investigate the role of Fritz, using confocal and scanning electron microscopy, we discovered that Fritz localizes along the ciliary axonemes and that knockdown of Fritz causes severe reductions in both axoneme length and number. Then, using pull-downs and mass-spectrometry, we identified Chaperonin Containing T-complex polypeptide 1 (CCT) and septin as interacting partners of Fritz. CCT is the key chaperonin interacting with septins, and both have been implicated in ciliogenesis. Using tagged CCT subunit constructs, we found that the tagged CCTα and CCTε co-localize with Fritz along the ciliary axonemes of multi-ciliated cells. Knockdown of Fritz resulted in the accumulation of CCT at the apical cytoplasm of multi-ciliated cells; however, it was confirmed that Fritz does not affect the CCT holoenzyme assembly. Septins, another interacting partner of Fritz, are novel cytoskeletal elements. Using septin antibodies, we found that endogenous septins also localize along the ciliary axonemes and accumulate in the apical cytoplasm of multi-ciliated cells in Fritz morphants. Similar ciliary defects were observed in septin morphants. Our results reveal that Fritz is essential for ciliogenesis, and that CCT and septin interact with Fritz to control ciliogenesis in Xenopus multi-ciliated cells. Additionally, tubulin acetylation is markedly reduced by Fritz knockdown, suggesting that Fritz affects tubulin acetylation.

Xenopus embryos begin development by undergoing a series of extremely rapid cell divisions that occur without growth, gap phases, or cell cycle checkpoints. This cell cycle program, which allows the fertilized egg to rapidly subdivide its contents into many separate cells, is made possible by the extraordinary ability of these embryos to replicate DNA quickly. After a dozen such divisions, the time required to complete S phase and complete the cell cycle increases sharply amidst other embryonic changes during the midblastula transition (MBT). Successful completion of the MBT is essential for viability, but the mechanism responsible for actuating these changes remains unknown. Previous work has shown that the onset of the MBT is dependent upon the embryo reaching a critical nucleus to cytoplasm (N/C) ratio, but it is unclear how this controls cell cycle lengthening. Here, we use Xenopus egg extracts to investigate the mechanism responsible for S phase lengthening at the MBT. As in embryos, high N/C extracts exhibit lengthened S phases, and this is due to both reduced utilization of origins of replication and reduced replication fork progression. Although recent work has suggested that developmental activation of the ATR/Chk1 pathway may provide the stimulus for cell cycle remodeling at the MBT, we find that this pathway is not activated more efficiently at high N/C ratio. Rather, the Chk1 phosphorylation observed at high N/C is simply the aggregated, basal checkpoint activity associated with normal replication in a large number of nuclei. Instead, we provide evidence that the reduced replication rates at high N/C ratio are the result of the depletion of maternal factors by the increased number of nuclei, and these factors are involved in both the initiation of replication and replication fork progression. We provide evidence that protein phosphatase 2A (PP2A) activity is the limiting factor for origin firing in high N/C extracts. Likewise, partial depletion of PP2A is sufficient to prevent the high levels of origin firing observed in low N/C extracts. These results suggest a mechanism by which PP2A levels control the rate of origin firing in Xenopus egg extracts and in Xenopus embryos at the MBT.

Most embryonic development and tissue self-assembly requires the integration of cell movements within multiple cell layers composed of different cell types, which are integrated with the signaling networks in these 3D environments. Although the role of cell mechanics in tissue self-assembly has been demonstrated, little is known about the mechanical responses of 3D multi-layer tissues to chemical cues. To investigate the collective movements within multilayered tissues, I developed a novel microfluidic technique capable of removing desired height or width of tissue from a composite tissue. I call this technique "3D tissue-etching" because it is analogous to techniques used in the microelectromechanics (MEMS) field where complex 3D structures are built by successively removing material from a monolithic solid through subtractive manufacturing. I used a custom-designed microfluidic control system to deliver a range of tissue etching reagents (detergents, chelators, proteases, etc.) to specific regions of multilayered tissues microsurgically isolated from embryos of the African Clawtoed frog, Xenopus laevis. Xenopus embryos and explanted tissues have long been used to elucidate signaling and other cellular processes during development and here provide an ideal model 3D tissue etching. Long exposure to a narrow etchant stream cuts completely through cell-cell layers to expose the substrate. By reducing the exposure time a single layer may be removed. By controlling the width of the etchant and the exposure time a broader swath of the surface layer may be removed. For more refined etching, after removal of a broad swath the resistance circuits can be switched and a second narrow stream can remove only a single narrow band within the swath exposed cells. I developed tissue-etching techniques that allow me to shape complex multi-layered embryonic tissues. The ability to control 3D stimulation and the form of multicellular tissues will provide extend the tools of tissue engineering to synthesize highly complex 3D integrated multicellular biosystems. Integration of tissue etching in my custom microfluidic system provides a "test-bed" where a range of hypotheses concerning the control and regulation of development and cell differentiation can be implemented and tested.

Das Geruchssystem ist in der Lage, mittels sogenannter kombinatorischer Kodierung einen hochdimensionalen Geruchsraum durch eine begrenzte Anzahl von olfaktorischen Rezeptorneuronen (ORN) abzutasten. Hierbei weisen verschiedene ORN-Klassen eine breite und gleichzeitig spezifische Geruchssensitivität auf, durch welche ein geruchsspezifisches Antwortmuster auf Populationen von Mitral-/Tufted Zellen (M/T) des bulbus olfactoris (OB) abgebildet wird. Neueren Untersuchungen zufolge sind diese Antwortmuster nicht notwendigerweise statisch, sondern enthalten Information in ihrer zeitlichen Entwicklung. Im OB von Larven des Krallenfrosches Xenopus laevis wurde herausgefunden, dass sowohl Geruchsidentität als auch -Konzentration besser vorhergesagt wird durch M/T Antwortlatenzmuster als durch durchschnittliche Feuerraten. Diese Arbeit befasst sich mit der Messung von ORN-Aktivität auf verschiedenen raumzeitlichen Skalen. Auf der Ebene von ORN Populationen wurde mit Hilfe von konfokaler Mikroskopie und [Ca2+] -sensitiven Fluoreszenzfarbstoffen untersucht, in wie weit Latenzmuster auftreten. Es wurde gezeigt, dass Latenzmuster im Unterschied zu M/T Zellen eine geringere Vorhersagekraft für die Geruchsstoffkonzentration besitzen als Feuerratenmuster. Außerdem wiesen Ensemble-Feuerraten einen größeren dynamischen Bereich bezüglich der Geruchsstoffkonzentration auf als Latenzen. Durch eine Kombination von schneller (1,25 kHz) [Ca2+] -Bildgebung und whole-cell Patch-Clamp Technik in einzelnen ORNs wurde die zeitliche Entwicklung der dreidimensionalen intrazellulären Ca2+ -Konzentration während eines Depolarisationspulses gemessen. Mit Hilfe von pixelweiser Angleichung eines numerischen Modells wurden Ballungen spannungsabhängiger Ca2+ Kanäle (VGCC) auf der Oberfläche von ORN-Somata lokalisiert. Da der durchschnittliche gemessene VGCC-Kalziumioneneinstrom einen geringen Beitrag im Vergleich zum Ca2+ Generatorstrom darstellt (<80 pA bzw. geschätzt 900 pA), erklärte sich, warum einzelne Aktionspotentiale nicht mittels [Ca2+] Bildgebung gemessen werden konnten. Bezüglich VGCC-Häufung und möglicher Kolokalisation mit Kaliumkanälen hoher Leitfähigkeit (BK) wurde der Effekt von BK Blocker Iberiotoxin auf ORN-Reizantworten untersucht. In einer Untergruppe aller ORNs wurde eine Verringerung der Antwortamplituden nach Anwendung von Iberiotoxin festgestellt. Aus den gezeigten Ergebnissen wurde geschlossen, dass eine wichtige Funktion von Glomeruli im OB die Konversion von Geruchsinformation zwischen Feuerratenkodierung und Latenzkodierung sein müsse.

The sudden appearance of chytridiomycosis, as the cause of amphibian deaths and population declines in several continents suggests that its etiological agent, the amphibian chytrid fungus Batrachochytrium dendrobatidis, was introduced into the affected regions. However, the origin of this virulent pathogen is unknown. Efforts were directed to determine the occurrence of chytridiomycosis in Africa, whether the disease had been introduced into South Africa in recent years and how wild frog populations were affected by infection. A chytridiomycosis survey of 2,300 archived and live specimens involving members of the Pipidae family in sub-Saharan Africa, as well as a number of unrelated frog species in South Africa was conducted by histological diagnosis of skin samples. The epidemiological evidence indicated that chytridiomycosis has been a stable endemic infection in southern Africa for 23 years before any positive specimens were found outside Africa. The occurrence of chytridiomycosis in South Africa can be described as widespread both in terms of geographical distribution and host species and generally infection is not associated with adverse effects at the individual or population level. It was proposed that the amphibian chytrid originated in Africa and that the international trade in the African clawed toad Xenopus laevis that commenced in the mid 1930s was the means of dissemination. A risk assessment of the X. laevis trade demonstrated that chytridiomycosis could spread through this pathway and culminated in the development of a management protocol to reduce the risks of spreading disease through this animate commodity. Initial comparative genetic analysis of B. dendrobatidis strains isolated from South African frogs with a global set of 35 strains, suggests that analysis of a more geographically diverse set of southern African strains is needed before this line of argument can support or reject the "out of Africa" hypothesis. / Thesis (Ph.D. (Zoology))--North-West University, Potchefstroom Campus, 2005.

No description available.

Cadmium is a highly toxic chemical and has been classified by the International Agency for Research on Cancer as a human carcinogen. Cadmium is abundant in the environment, at specific work places, and in food and water. Toxicological responses to cadmium exposure include respiratory diseases, neurological disorders and kidney damage. The present study examined the effects of cadmium on heat shock protein (HSP) accumulation in Xenopus laevis A6 kidney epithelial cells. HSPs are molecular chaperones involved in protein folding and translocation. In response to environmental stress these proteins bind to unfolded protein and inhibit their aggregation. Stress-inducible hsp gene transcription is mediated by the heat shock promoter element (HSE), which interacts with heat shock transcription factor (HSF). In the present study, hsp30 and hsp70 mRNA and protein were induced by heat shock, as determined by northern and western blot analysis. Exposure of A6 cells to cadmium chloride also induced the expression of hsp genes. For example, northern and western blot analysis revealed that exposure of A6 cells to cadmium chloride induced the accumulation of hsp30 and hsp70 mRNA and their respective proteins. Western blot analysis also revealed that A6 cells recovering from a cadmium chloride treatment retained relatively high levels of HSP30 and HSP70 protein accumulation over 24 h after the removal of the stress. Treatments combining a mild heat shock and cadmium chloride resulted in a synergistic increase in hsp30 and hsp70 gene expression at mRNA and protein levels. Further experiments in which two stressors were combined revealed that synergistic effects occurred with varying cadmium concentrations and different temperatures. Immunocytochemistry and confocal microscopy were used to confirm the results attained from western blot analysis. Further, this technique allowed the determination of intracellular localization of HSP30 in A6 cells and the examination of cellular morphology and cytoskeletal structure during cadmium chloride treatments. A 2 h heat shock at 33??C resulted in the accumulation of HSP30 in the cytoplasm, whereas a 2 h heat shock at 35??C resulted in some HSP30 accumulation in the peripheral region of the nucleus. This is in contrast to cells treated with cadmium chloride, where HSP30 accumulation was restricted to the cytoplasm. A 14 h 50 ??M cadmium chloride treatment resulted in the accumulation of HSP30 in approximately 10% of cells. The proportion of cells displaying HSP30 accumulation increased to 80% and 95% in cells treated with 100 ??M and 200 ??M, respectively. HSP30 accumulation frequently occurred in large granular structures. High concentrations of cadmium chloride resulted in cell membrane ruffling at areas of cell-cell contact, as well as actin disorganization. This study characterized the pattern of hsp gene expression, accumulation and localization under various cadmium chloride conditions. These results suggest that hsp30 and hsp70 gene expression can be used as potential biomolecular markers for cadmium exposure.