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Au, Yu Ching Eugene. / "December 2011." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 143-158). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese) --- p.iii / Acknowledgments --- p.v / Contents --- p.vi / List of tables --- p.ix / List of figures --- p.xi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.5 / Chapter 2.1 --- Brachyuran systematics --- p.5 / Chapter 2.1.1 --- General brachyuran classification --- p.5 / Chapter 2.1.2 --- Podotremata phylogeny --- p.8 / Chapter 2.1.2.1 --- Dromiacea --- p.8 / Chapter 2.1.2.2 --- Raninoida and Cyclodorippoida --- p.12 / Chapter 2.1.3 --- Relationships of the thoracotreme crabs --- p.16 / Chapter 2.1.4 --- Systematics of Varunidae --- p.19 / Chapter 2.2 --- Use of molecular data in decapod and brachyuran phylogenies --- p.23 / Chapter 2.2.1 --- Use of mitochondrial markers and nuclear ribosomal RNA markers --- p.24 / Chapter 2.2.2 --- Employment of nuclear protein-coding genes --- p.25 / Chapter Chapter 3 --- Molecular Phylogeny of Podotremata sensu Guinot (Decapoda: Brachyura) --- p.35 / Chapter 3.1 --- Introduction --- p.35 / Chapter 3.2 --- Materials and Methods --- p.37 / Chapter 3.2.1 --- Sample collection --- p.37 / Chapter 3.2.2 --- "DNA extraction, amplification and sequencing" --- p.38 / Chapter 3.2.3 --- Data analysis --- p.39 / Chapter 3.3 --- Results --- p.40 / Chapter 3.3.1 --- Dataset of individual markers: Histone 3 --- p.40 / Chapter 3.3.2 --- Dataset of individual markers: Enolase --- p.41 / Chapter 3.3.3 --- Dataset of individual markers: PEPCK --- p.43 / Chapter 3.3.4 --- Dataset of individual markers: 12S rRNA --- p.44 / Chapter 3.3.5 --- Dataset of individual markers: 16S rRNA --- p.45 / Chapter 3.3.6 --- Results from concatenatd dataset --- p.46 / Chapter 3.4 --- Discussion --- p.48 / Chapter 3.4.1 --- Use of markers --- p.48 / Chapter 3.4.2 --- The status of Podotremata sensu Guinot --- p.49 / Chapter 3.4.3 --- Dromiacea --- p.50 / Chapter 3.4.4 --- Dromioidea --- p.50 / Chapter 3.4.5 --- Homoloidea --- p.52 / Chapter 3.4.6 --- Raninoidea --- p.54 / Chapter 3.4.7 --- Cyclodorippoidea --- p.55 / Chapter 3.5 --- Conclusion --- p.56 / Chapter Chapter 4 --- Molecular Phyogeny of Family Varunidae (Decapoda: Brachyura) --- p.86 / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Materials and Methods --- p.87 / Chapter 4.2.1 --- Sample collection --- p.87 / Chapter 4.2.2 --- "DNA extraction, amplification and sequencing" --- p.88 / Chapter 4.2.3 --- Data analysis --- p.90 / Chapter 4.3 --- Results --- p.91 / Chapter 4.3.1 --- Dataset of individual markers: Histone 3 --- p.91 / Chapter 4.3.2 --- Dataset of individual markers: Enolase --- p.91 / Chapter 4.3.3 --- Dataset of individual markers: GAPDH --- p.92 / Chapter 4.3.4 --- Dataset of individual markers: NaK --- p.93 / Chapter 4.3.5 --- Dataset of individual markers: AK --- p.94 / Chapter 4.3.6 --- Dataset of individual markers: 12S rRNA --- p.95 / Chapter 4.3.7 --- Dataset of individual markers: 16S rRNA --- p.96 / Chapter 4.3.8 --- Results of the concatenated dataset --- p.97 / Chapter 4.4 --- Discussion --- p.98 / Chapter 4.4.1 --- On the dataset: Use of markers and taxon coverage --- p.98 / Chapter 4.4.2 --- Position of Varunidae in Thoracotremata --- p.99 / Chapter 4.4.3 --- Intra-varunid relationships --- p.99 / Chapter 4.4.4 --- Geographical and morphological hypotheses of groupings --- p.102 / Chapter 4.5 --- Conclusion --- p.104 / Chapter Chapter 5 --- General conclusion --- p.140 / References --- p.142 / Appendix: Sequences used in the two studies --- p.159

Epidermolysis Bullosa simplex (EBS) is a rare genetic disorder that is typically inherited in an autosomal dominant fashion and affects approximately 1 out of 20 000 individuals. This disease is caused by mutations in either the KRT14, KRT5 or PLEC genes. These genes code for proteins involved in the formation of the cytoskeleton in basal keratinocytes, which form the basal layer of the epidermis. The cytoskeleton provides structural support to the basal keratinocytes and mutations in these genes cause cytoskeletal malformations, making these cells more susceptible to physical stress. This results in the cells undergoing lysis under trivial mechanical stress and causing the epidermis to detach from the dermis, the layer immediately below the epidermis. This leads to the primary symptom of EBS: the formation of blisters. The goal of this project is to recreate EBS in zebrafish using transgenesis and to create stable mutant transgenic line. In the future, high throughput drug screening will be done on mutant zebrafish embryos to find potential drug candidates that can alleviate the symptoms of EBS. To accomplish this, missense and deletion mutations in zebrafish krt5 cDNA using site-directed mutagenesis were performed. It was previously shown that mice models for this disease die shortly after birth and thus no stable mutant lines were able to be created. To ensure embryo survival and avoid a similar fate, mutant krt5 cDNA was expressed in non-essential tissue, such as the embryonic fin fold using a fin epithelial-specific enhancer named epi. These constructs were injected into one-cell stage zebrafish embryos, which were raised and screened for integration of the construct in their germ cells. While results from injected embryos were promising, mutant transgenic zebrafish did not demonstrate any blistering. In an attempt to induce blistering, mutant zebrafish embryos were placed under various environmental stressors known to worsen the symptoms of EBS. This was not successful. Expression of mutant keratin 5 in the basal epidermis of the entire embryo using the 2.3kb upstream region of the zebrafish krt5 gene to drive expression also did not yield any results. More investigations are needed to determine if it will be possible to use the zebrafish to model EBS.

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Graduation date: 1997

Thesis (Ph. D.)--Oklahoma State University, 2006. / Vita. Includes bibliographical references.

The hallmark of chronic myelogenous leukemia (CML) is the Philadelphia chromosome, which arises from the reciprocal translocation of the c-abl proto-onogene on chromosome 9 and the bcr locus on chromosome 22. This translocation results in the expression of a 210 kDa fusion protein (Bcr-Abl) with constitutive tyrosine kinase activity that is responsible for CML pathogenesis. Bcr-Abl activates several signaling proteins important for the proliferation and survival of myeloid progenitors, including the Src family kinases Hck and Lyn, the Stat5 transcription factor and upstream components of the Ras/Erk pathway. Previous work from our laboratory found that kinase-defective Hck blocks Bcr-Abl-induced transformation of DAGM myeloid leukemia cells to cytokine independence, suggesting that activation of the Src kinase family may be essential to oncogenic signaling by Bcr-Abl. Chapter II explores the contribution of Src kinases to Bcr-Abl signaling in vivo, using selective Src family kinase inhibitors. Inhibition of Src family kinases in Ph+ CML cell lines resulted in growth arrest, induction of apoptosis and blocked Stat5 and Erk activaton downstream. These data implicate the Src kinase family in Stat5 and Erk activation downstream of Bcr-Abl, and identify myeloid-specific Src kinases as potential drug targets in CML. In Chapter III, I investigated the biochemical interactions between myeloid Src family members and Bcr-Abl. Hck, Lyn and Fyn each bind the kinase domain, C-terminal tail, and SH3/SH2 region of Bcr-Abl and strongly phosphorylated the Bcr-Abl SH3-SH2 protein in vitro. Seven phosphorylated tyrosine residues were identified and substitution of these residues with phenylalanine in the context of full-length Bcr-Abl blocked transformation of TF-1 myeloid cells to cytokine independence. The position of several of these tyrosines in the crystal structure of c-Abl and transformation defect of the Bcr-Abl mutant suggest that phosphorylation by Src kinases may impact Bcr-Abl autoregulation and downstream oncogenic signaling. Taken together, these data firmly establish an important role for Src family tyrosine kinases in Bcr-Abl-mediated oncogenic signaling and implicate Src kinases as a promising therapeutic target for chronic myelogenous leukemia.

Ionizing radiation (IR) is the primary adjuvant treatment for glioblastoma multiforme (GBM), the most aggressive primary brain tumor in adults. Enhancement of the effects of IR may increase patient survival and quality of life in patients with GBM. The repair of DNA double strand breaks (DSBs) produced by IR proceeds along two pathways, nonhomologous end-joining (NHEJ) and homologous repair (HR). The herpes simplex virus (HSV) immediate-early protein, ICP0, has been shown to induce the degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). DNA-PKcs is the primary component of NHEJ, the major DNA DSB repair pathway in mammalian cells. A replication-defective HSV-1 vector, d106, which solely expresses the immediate-early (IE) protein, ICP0, was used to determine the effect of ICP0 on GBM cell survival and DNA repair after IR treatment. Preinfection of two radioresistant GBM cells lines by d106 resulted in decreased cell survival and proliferation, protein degradation of DNA-PKcs, inhibition of DNA DSB repair, and enhanced apoptosis following IR. Optimal intracerebral delivery of the HSV-1 mutant, d106, was established by convection-enhanced delivery (CED) in a mouse model. Translation of the effects of ICP0 in combination with IR was performed with CED of d106 in a mouse glioma model. CED of d106 in combination with whole-brain irradiation significantly increased animal survival.