Current usage of human embryonic stem cells (hES) and induced pluripotent stem cells (iPS) in clinical therapies and personalized medicine are limited as a result of ethical, technical and medical problems that arise from isolation and generation of these cells. Isolation of hES cells faces ethical problems associated with their derivation from human pre-implantation embryos. The most controversial aspect of hES cell isolation targets the generation of autologous hES cell lines which requires the transfer of a somatic-cell nucleus from the patient to an enucleated oocyte. While already established embryonic stem cell lines from IVF embryos can be used in a similar manner, lack of genetic identity can cause therapy rejection from the host, and prevent their use in personalized medicine. Induced pluripotent stem cells on the other hand, are generated from somatic cells that have been reprogrammed in vitro to behave like stem cells. While these cells can potentially be used for personalized medicine without the risk of rejection by the host system, derivation methods prevent their therapeutic use. The most efficient method used to generate iPS cells involves usage of viral particles which can result in viral DNA being integrated in the host cell’s genome and render these cells non-compliant for clinical therapies. Other methods not involving viral particles exist as well, but the reprogramming efficiency is too low and technical problems with generating large enough numbers of cells prevent these methods from being feasible approaches for clinical therapies. Direct reprogramming of a differentiated cell into a developmentally more plastic cell would offer alternatives to applications in regenerative medicine that currently depend on either embryonic stem cells (ES), adult stem cells or iPS cells. We hypothesize that Xenopus laevis egg cytoplasmic extract contains critical factors needed for reprogramming that may allow for non-viral, chemically defined derivation of human induced pluripotent/multipotent cells which can be maintained by addition of exogenous FGF2. In this thesis we investigated a new method for generation of multipotent cells through determining the ability of select fractions of Xenopus laevis egg extract to induce multipotency in already differentiated cells. We were able to identify select fractions from the extract that in combination with exogenously added FGF2 can reprogram and maintain the reprogrammed cells in an undifferentiated state. The findings of this work also determined that Xenopus laevis egg extract mRNA is required for achieving full reprogramming. The body of work presented in this thesis showed the ability of FGF2 isoforms to bind and activate select FGF receptor tyrosine kinases, act as extracellular mitogenic factors to support growth of hES cells in an undifferentiated state as well bind to nuclear DNA and affect expression of endogenous genes. Moreover, we showed that all FGF2 isoforms can induce expression of stem cell specific proteins in human dermal fibroblasts as well as extend lifespan of human dermal fibroblasts in vitro. In this work we identified HECW1, the gene coding for E3 ubiquitin ligase NEDL1, as a novel nuclear target for all FGF2 isoforms and showed that overexpression of recombinant FGF2 isoforms in human dermal fibroblasts can down regulate expression of HECW1 gene.

No description available.

Thesis (M.S. zoology)--Washington State University, December 2007. / Includes bibliographical references (p. 24-26).

De nombreuses kinases participent au bon déroulement de chaque étape du cycle cellulaire. Chez les eucaryotes supérieurs, les kinases Aurora-A et Aurora-B, structuralement très proches, exercent des rôles fondamentaux durant la mitose. Aurora-A est une protéine localisée au niveau des centrosomes, impliquée dans le cycle de division du centrosome et la formation du fuseau mitotique. Aurora-B est une protéine passagère localisée sur les centromères et qui migre, en anaphase, sur le sillon de division et se concentre en cytocinèse sur le corps résiduel. Aurora-B est responsable de la phosphorylation massive, en mitose, du résidu Serine 10 de l'histone H3. Par un système de pseudo-génétique, j'ai ciblé, dans l'extrémité amino-terminale de Aurora-A, le domaine responsable de sa localisation centrosomique. Ces expériences ont montré que les domaines catalytiques de Aurora-A et Aurora-B possèdent tous deux un signal de localisation centromérique. Mais, à l'inverse de Aurora-B, le domaine catalytique de Aurora-A ne se transfert pas des centromères vers le sillon de division en anaphase. Ces travaux montrent également que Aurora-A est capable d'assurer une partie des fonctions mitotiques de Aurora-B. J'ai par ailleurs identifié et cloné la séquence de la protéine passagère TD-60 de Xenopus laevis. J'ai exprimé des domaines protéiques de la protéine xTD60, afin de générer un anticorps spécifique de TD-60 de xénope. Des expériences de co-sédimentation de complexes protéiques d'extraits mitotiques d'œufs de xénope et des expériences d'immunolocalisation cellulaire nous permettent d'envisager pour xTD-60 des fonctions plus larges que celles attribuées aux protéines passagères.

Graduation date: 1994

Vocal communication allows animals to express distress, territoriality, and most important, to attract mates. In the African Clawed frog, Xenopus laevis, vocal communication is unique, because not only do males advertise for mates using elaborate click vocalizations, but also females are able to advertise their reproductive readiness by eliciting a "rapping" call. Sex differences in vocal repertoire match sex differences in vocal circuitry. During development, the vocal circuitry in the male grows increasingly sensitive to circulating androgens. Androgens induce tremendous growth in the cartilage and musculature of the peripheral vocal organ, the larynx. Net addition of synapses and motor fibers soon follow providing communication from the motor nucleus in the hindbrain to the vocal organ. The laryngeal motor nucleus, n. IX-X, accumulates androgens that serve to protect n. IX-X neurons from programmed apoptosis. Females, who have low levels of circulating androgens, experience a profound net loss on n. IX-X neurons during this developmental critical period. Once the frogs reach sexual maturity males possess larger and more numerous n. IX-X neurons than females, as well as sizable sex differences in laryngeal robustness and physiology. These measurable sex differences yield vastly different vocal programs. Androgens continue to maintain a critical role in governing breeding season trophic effects and mediating call production. Because male X. laevis are so susceptible to the effects of androgens, they may also be sensitive to the actions of endocrine disrupting chemical agents. The vocal system of X. laevis and its androgen sensitivity thus provide an ideal model for studying changes imposed to the anatomy and physiology of the system by the brominated flame retardant, PBDE-209, a putative anti-androgen and common pollutant. The present studies investigate how PBDE-209 affects the male vocal system when animals are exposed during the androgen-sensitive critical period of vocal system development and during adulthood when the tissues are utilizing androgens to vocalize. PBDE-209 effectively reduces male n. IX-X number and size at higher concentrations after exposure during the organizational critical period. Similar dose-dependent effects were observed in adult n. IX-X neurons. Moreover, PBDE-209 inhibited male-typical vocalization by reducing the number of calls elicited as well as the average call amplitude. These data strongly suggest that PBDE-209 has cytotoxic effects that alter n. IX-X anatomy and function, and may be mediated through pathways that include blocking the androgens necessary for proper vocal system development.

Gastrulation is a highly complex series of cellular rearrangements that leads to the internalization of the mesoderm and endoderm. The cellular behaviors that underlie morphogenesis are dependent upon changes in cell motility and polarity. Eph receptors belong to a family of receptor tyrosine kinases that are involved in a variety of developmental processes. This study is the first to examine the role EphA4 during Xenopus gastrulation. Morpholino oligonucleotide (MO) mediated knockdown of EphA4 resulted in attenuated mesoderm involution and reduced the expression of the posterior mesoderm marker brachyury (Xbra). Expression of EphA4 in the blastocoel roof was sufficient to promote ectopic Xbra expression. I show that EphA4 can regulate Xbra expression and involution movements by signaling through PAK1. Temporal regulation of Xbra was sufficent to rescue EphA4 induced gastrulation defects. This study has uncovered a novel EphA4/PAK1 pathway which is required for mesoderm involution and Xbra expression during Xenopus gastrulation.

Gastrulation is a highly complex series of cellular rearrangements that leads to the internalization of the mesoderm and endoderm. The cellular behaviors that underlie morphogenesis are dependent upon changes in cell motility and polarity. Eph receptors belong to a family of receptor tyrosine kinases that are involved in a variety of developmental processes. This study is the first to examine the role EphA4 during Xenopus gastrulation. Morpholino oligonucleotide (MO) mediated knockdown of EphA4 resulted in attenuated mesoderm involution and reduced the expression of the posterior mesoderm marker brachyury (Xbra). Expression of EphA4 in the blastocoel roof was sufficient to promote ectopic Xbra expression. I show that EphA4 can regulate Xbra expression and involution movements by signaling through PAK1. Temporal regulation of Xbra was sufficent to rescue EphA4 induced gastrulation defects. This study has uncovered a novel EphA4/PAK1 pathway which is required for mesoderm involution and Xbra expression during Xenopus gastrulation.

The heat shock or stress response is a transient response to stressful stimuli that protects vital cellular proteins from damage and irreversible aggregation. Heat shock proteins (Hsps) are molecular chaperones that bind to unfolded protein and inhibit their aggregation, thereby maintaining their solubility until they can be refolded to their native conformation. Hsp47 is an endoplasmic reticulum (ER)-resident protein that serves as a molecular chaperone during collagen production. Collagen is the major class of insoluble fibrous protein found in the extracellular matrix and in connective tissues. It is the single most abundant protein of the animal kingdom; at least 14 different forms exist, each with distinct structures and binding properties. The various types of collagen all possess protein regions with the distinct triple helical conformation. This complex physical structure requires very organized assembly and HSP47 has been established as an integral component of this process for collagen types I-V. Most of the previous studies examining the expression and function of hsp47 have been conducted with mammalian cultured cells. The present study represented the first investigation of the expression of hsp47 in the poikilothermic vertebrate, <i>Xenopus laevis</i>. Full-length <i>Xenopus</i> hsp47 nucleotide and amino acid sequences were obtained from Genbank and compared with hsp47 from chicken, mouse, rat, human and zebrafish. <i>Xenopus</i> HSP47 protein had an identity of approximately 77% with chicken, 73% with mouse, 72% with rat and human, and 70% with zebrafish. Most of the sequence identity between HSP47 from all investigated organisms occurred centrally in the amino acid sequence and in several carboxyl terminal regions. Three key features were conserved between HSP47 proteins from most species investigated: a hydrophobic leader sequence, two potential glycosylation sites and the ER-retention signal, RDEL. A partial cDNA clone encoding <i>Xenopus</i> hsp47 was obtained from the American Type Culture Collection (ATCC) and used to generate hsp47 antisense riboprobe for the purpose of investigating hsp47 mRNA accumulation in <i>Xenopus</i> A6 kidney epithelial cells and embryos. Northern blot analysis detected hsp47 mRNA constitutively in A6 cells. The expression pattern for hsp47 mRNA was compared with two other <i>Xenopus</i> heat shock proteins that have been previously characterized in our laboratory: hsp70, a cystolic/nuclear hsp and BiP, an ER-resident hsp. The results of hsp47 mRNA accumulation in A6 cells suggested that the expression pattern for <i>Xenopus</i> hsp47 was unique but, with respect to some stressors, resembled that of a cytosolic hsp rather than an ER-resident hsp. HSP47 protein levels were also examined in A6 cells. Heat shock, sodium arsenite and b-aminopropionitrile fumerate treatments enhanced hsp47 accumulation. In some experiments, western blot analysis revealed the presence of two closely sized protein bands. It is possible that minor differences in HSP47 protein size may be due to post-translational modification, namely phosphorylation or glycosylation. The present study also examined the accumulation and spatial pattern of hsp47 mRNA accumulation during <i>Xenopus laevis</i> early development. Hsp47 was constitutively expressed throughout <i>Xenopus</i> early development. Constitutive levels of hsp47 mRNA in unfertilized eggs, fertilized eggs and cleavage stage embryos indicated that these transcripts were maternally inherited. Constitutive hsp47 mRNA accumulation was enhanced in neurula and tailbud embryos compared to earlier stages. This finding may be explained by the shift towards organogenesis during these stages. Whole mount <i>in situ</i> hybridization revealed hsp47 message along the dorsal region of the embryo, in the notochord and somites, as well as in the head region including the eye vesicle. Hsp47 mRNA induction in <i>Xenopus</i> embryos was also examined in response to heat shock. Hsp47 mRNA accumulated in response to heat shock immediately following the midblastula transition (MBT). In tailbud stages, hsp47 mRNA accumulated in the notochord, somites and head region. Northern blot analysis and whole mount <i>in situ</i> hybridization results revealed an expression pattern that coincided well with the development of collagen-rich tissues thereby substantiating the proposed role of HSP47 as a procollagen molecular chaperone.

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 μM 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 μM and 200 μM, 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.