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Colorectal cancer (CRC) is the third leading cause of death from cancer in North America. The KRAS gene is mutated in approximately 40-50% of all CRC, and this mutation precludes treatment with promising targeted therapeutics. c-MET is a receptor tyrosine kinase that is overexpressed in ~70% of CRCs, and expression is correlated with disease progression. We hypothesized that high c-MET plus mutant KRAS would result poor survival of CRC patients, by activating unique signalling pathways that may be targeted for therapeutic purposes. To this end, we used phosphoproteomics in a KRAS mutant cell line, and identified proteins phosphorylated on tyrosine in response to HGF stimulation, including a subset of those that contain SRC family kinase consensus motifs. Small molecule inhibitors of either SRC or c-MET reduced tyrosine phosphorylation of both proteins, indicating reciprocal signalling. We chose the c-MET target p190RhoGAP for future study, as it is often ubiquitously bound to p120RasGAP via phosphorylated tyrosine. We found that RasGAP expression is mediated in part by KRAS signalling, and that expression of RasGAP could partly rescue tumourigenicity of a CRC cell line where the mutant KRAS allele has been inactivated, indicating the requirement of both mutant KRAS and RasGAP expression in this model. We then conclude by looking at CRC patient samples to determine the role of KRAS mutation in the progression and survival of CRC. We found that both KRAS and c-MET copy number are correlated to KRAS mutation status, and that c-MET polysomy plus KRAS mutation leads to worse overall survival than KRAS mutation alone. Overall, we identified novel targets of c-MET and KRAS oncogenic signaling, and identify a population which may derive the most benefit from treatments targeting both of these lesions.

Colorectal cancer (CRC) is the third leading cause of death from cancer in North America. The KRAS gene is mutated in approximately 40-50% of all CRC, and this mutation precludes treatment with promising targeted therapeutics. c-MET is a receptor tyrosine kinase that is overexpressed in ~70% of CRCs, and expression is correlated with disease progression. We hypothesized that high c-MET plus mutant KRAS would result poor survival of CRC patients, by activating unique signalling pathways that may be targeted for therapeutic purposes. To this end, we used phosphoproteomics in a KRAS mutant cell line, and identified proteins phosphorylated on tyrosine in response to HGF stimulation, including a subset of those that contain SRC family kinase consensus motifs. Small molecule inhibitors of either SRC or c-MET reduced tyrosine phosphorylation of both proteins, indicating reciprocal signalling. We chose the c-MET target p190RhoGAP for future study, as it is often ubiquitously bound to p120RasGAP via phosphorylated tyrosine. We found that RasGAP expression is mediated in part by KRAS signalling, and that expression of RasGAP could partly rescue tumourigenicity of a CRC cell line where the mutant KRAS allele has been inactivated, indicating the requirement of both mutant KRAS and RasGAP expression in this model. We then conclude by looking at CRC patient samples to determine the role of KRAS mutation in the progression and survival of CRC. We found that both KRAS and c-MET copy number are correlated to KRAS mutation status, and that c-MET polysomy plus KRAS mutation leads to worse overall survival than KRAS mutation alone. Overall, we identified novel targets of c-MET and KRAS oncogenic signaling, and identify a population which may derive the most benefit from treatments targeting both of these lesions.

Programmed cell death (PCD or apoptosis) is an evolutionarily conserved, genetically controlled suicide mechanism for cells, which when deregulated, can lead to developmental defects, cancers and degenerative diseases. In C. elegans, DNA damage induces germ cell death by signaling through cep-1/p53 ultimately leading to the activation of the CED-3/caspase. It has been hypothesized that the major regulatory events controlling cell death occur by cell autonomous mechanisms, that is within the dying cell. In support of this, genetic studies in C. elegans have shown that the core apoptosis pathway genes ced-4/APAF1 and ced-3/caspase are required in cells fated to die. However, it is not known whether the upstream signals that activate apoptosis function in a cell autonomous manner. Here I show that two genes, kri-1, an ortholog of KRIT1/CCM1 that is mutated in the human neurovascular disease cerebral cavernous malformations (CCMs) and daf-2, an insulin-like receptor, are required to activate DNA damage-dependent cell death independently of cep-1/p53. Interestingly, I found that both genes can regulate cell death in a non-autonomous manner, revealing a novel role for non-dying cells in eliciting death in response to DNA damage.

Programmed cell death (PCD or apoptosis) is an evolutionarily conserved, genetically controlled suicide mechanism for cells, which when deregulated, can lead to developmental defects, cancers and degenerative diseases. In C. elegans, DNA damage induces germ cell death by signaling through cep-1/p53 ultimately leading to the activation of the CED-3/caspase. It has been hypothesized that the major regulatory events controlling cell death occur by cell autonomous mechanisms, that is within the dying cell. In support of this, genetic studies in C. elegans have shown that the core apoptosis pathway genes ced-4/APAF1 and ced-3/caspase are required in cells fated to die. However, it is not known whether the upstream signals that activate apoptosis function in a cell autonomous manner. Here I show that two genes, kri-1, an ortholog of KRIT1/CCM1 that is mutated in the human neurovascular disease cerebral cavernous malformations (CCMs) and daf-2, an insulin-like receptor, are required to activate DNA damage-dependent cell death independently of cep-1/p53. Interestingly, I found that both genes can regulate cell death in a non-autonomous manner, revealing a novel role for non-dying cells in eliciting death in response to DNA damage.

Up to 170 million people are infected with Hepatitis C Virus worldwide. Chronic HCV infection is the leading cause of fibrosis, cirrhosis and liver cancer. Treatment options are currently limited to interferon based therapies alone or in conjunction with direct acting antivirals (DAA) such as viral protease inhibitors. While the implementation of DAAs has increased the effective cure rate of HCV infected individuals, treatment is far from being complete or ideal. New DAAs with unique modes of action will be necessary to compliment our current repertoire of anti-HCV therapies. Previously our lab identified three dihydropyridines (DHP) as potent HCV replication inhibitors. We investigated and characterized the anti-HCV properties of nine additional DHP compounds. We also show that DHP compounds inhibit IRES dependent translation in full-length HCV. This inhibition of two separate steps of the viral life cycle may be a unique feature of DHPs making them superior DAAs. Among these DHPs, efonidipine emerged as the most effective HCV replication and translation inhibitor with the least toxicity. Using a real-time evolution strategy, we developed and characterized a mutant virus which was resistant to DHPs and several other drugs which modify intracellular calcium stores. Our results further the understanding of DHP inhibition of HCV providing a solid basis for investigation of more structurally related compounds as potent inhibitors of HCV.

The concentration of ascorbic acid in the plasma was determined on four levels of ascorbic acid intake for seven day periods in 1946-47 and on three levels of ascorbic acid intake for ten day periods in 1947-48. The subjects, eight girls and eight boys, were 16 to 19 year old freshman students at Oregon State College. All of the food eaten by the subjects during the experimental periods was weighed and the quantities were recorded. The reduced ascorbic acid in the foods was determined after each meal by the method of Loeffler and Ponting (1942) and daily fasting plasma ascorbic acid values were determined by the micro-method of Farmer and Abt (1936). The data in this study were analyzed statistically by testing the significance of the differences between the means and by analysis of variance. The recommended allowance of the National Research Council (1945) for these subjects (80 mg for the girls and 100 mg for the boys) did not maintain mean plasma values as high as their respective means during the saturation period when they were receiving 200 mg of crystalline ascorbic acid in addition to the ascorbic acid from their food. On the recommended allowance all the mean plasma values for the girls were above 0.80 mg per cent, ranging from 0.83 to 1.07. The boys values ranged from 0.67 to 0.91 mg per cent; two out of seven values were below 0.80 mg per cent (data for one boy were excluded due to illness). A decrease in ascorbic acid intake to 10 mg less than the recommended allowance of the National Research Council made a statistically significant decrease in the plasma ascorbic acid concentration for only two of the eight girls and for one of the seven boys. The plasma ascorbic acid concentrations of these subjects showed individual variation even when the ascorbic acid intake was considered on the basis of mg of ascorbic acid per kg of body weight. The ten day experimental periods were more desirable than the periods of one week. This was particularly true for the saturation period when some of the subjects had been on diets low in ascorbic acid prior to the study. / Graduation date: 1949

從品牌及顧客觀點看85度C在上海的發展 / 85°C Bakery Café’s successful story has been described as a miracle in Taiwan’s economy. “5 star quality at affordable price” is the slogan firstly revealed to the public in the industry and it is indeed attractive. In merely 5 years since the first store was established in July 2004, 85°C is now the largest coffee chain stores in Taiwan with 420 stores worldwide, covering Taiwan, Australia, China and USA. This case study is to research on its development in Taiwan and the expansion in China, which was considered the biggest opportunity and major source of profits of the company. The concept of “bakery café” is a convergence of coffee shop, bubble tea house and bakery for bread and cake. This business model does help eliminate unnecessary waste of materials and “fast-moving, short life-cycle” finished goods – mainly bread and cake; it minimizes the costs of materials and operations to realize profits. After 2 years in China, 85°C made another successful story. As of the end of 2009, 85°C has nearly 90 stores in China, mostly in Shanghai area. Its first store in China was set up in Shanghai by November 2007; I happened to be relocated in Shanghai since then and had the chance to see how it grew in such a highly competitive market. This study also presents the results of the street surveys performed in October and November 2009, with an attempt to understand the reasons behind the success. Based on the demographic profiles of 68 participants from the streets and my observation, it is clear that “5 star quality at affordable price” and “originated from Taiwan that beat Starbucks” strategy works perfectly fine in Shanghai even though the analysis demonstrated that 85°C and Starbucks are actually in different market segments. However, 85°C does create a niche market of profitability through the convergence of café and bakery.

Jembrana disease is an acute and severe disease of Bali cattle (Bos javanicus) endemic in Indonesia that is caused by a bovine lentivirus designated Jembrana disease virus (JDV). Previous studies have demonstrated that it is possible to induce a protective immunity against the disease by immunisation with a crude whole virus vaccine prepared from the tissues of infected cattle. This vaccine has been demonstrated to ameliorate the clinical signs of disease resulting from exposure to virus infection but a safer vaccine amenable to commercial production techniques is required. JDV, like all lentiviruses, encodes a transcriptional trans-activator Tat protein that is encoded from one or both of two exons of the tat gene. Tat is particularly essential for virus replication and it was hypothesised that the induction of an immune response in cattle against JDV Tat may effect protection against virus infection. Investigations were therefore conducted on JDV Tat to provide basic information on the protein that would enable it to be further investigated as a potential immunogen for incorporation into vaccines for the control of Jembrana disease. Analysis of tat transcripts obtained from tissues of cattle infected with three strains of JDV suggested that, during the acute clinical disease, Tat produced at this stage of the disease process was translated from the first coding exon only. Nucleotide variation in this exon, which would have translated into amino acid variations in the Tat protein, was evident especially between strains from geographically different regions of Indonesia. There was; however, conservation of the essential functional domains of cysteine-rich, core and basic regions, which suggested immunity to a single Tat protein might protect against infection by heterologous strains. Subsequent studies on Tat reported in the thesis therefore concentrated on the protein encoded by tat exon 1 of a single strain of JDV. The exon 1 of tat was cloned into the pGEX vector and recombinant Tat expressed in Escherichia coli. Methods for the purification of the expressed protein were developed. Immunogenicity of the recombinant protein was initially demonstrated by inoculation of the protein into a sheep which developed a high titred specific antibody response. Antibodies induced by this recombinant protein recognised native Tat proteins produced by three JDV strains in Bali cattle and provided a valuable reagent for the subsequent detection of Tat in vitro and in vivo. Aspects of the antibody response to Tat were determined in cattle that had been infected naturally or experimentally with JDV, and compared with the levels of antibody to the immunodominant capsid protein. Tat antibodies were detected in 23 % of 128 Bali cattle from Jembrana disease-endemic areas of Indonesia; in all these cattle, evidence of previous virus infection had been demonstrated by detection of antibody to the JDV capsid protein by Western blot analysis. In cattle experimentally infected with JDV, low levels of serum antibody to Tat were detected by Western blot in the first month post-infection but the levels of antibody then decreased; levels of antibody to the JDV capsid protein increased over the 6-month observation period following infection. The detection of Tatantibody soon after the acute clinical disease suggested that this protein is secreted extracellularly during JDV infection in cattle. In contrast to the antibody response to Tat in JDV-infected cattle, an apparently greater antibody response to Tat was induced by injection of recombinant Tat in Bali cattle. The strong antibody response resulting from inoculation of the recombinant Tat and low levels of Tat antibody in animals that had been naturally or experimentally infected with virus suggested there might be a conformational difference in the recombinant and native Tat protein and that the native protein was a poor immunogen, or that the levels of Tat in infected cattle were too low to induce a strong antibody response. As an alternative means of inducing an immune response to JDV Tat, perhaps one associated with a greater cell-mediated rather than an antibody response, a candidate tat DNA vaccine was produced by insertion of tat exon 1 into a DNA vaccine vector. Transfection of this naked DNA plasmid into mammalian cells induced the expression of a functional Tat protein which maintained antigenicity. The results suggested this construct merits further animal studies attempting to induce a protective immune response against Jembrana disease in cattle. A method of assaying the trans-acting function of Tat was also developed which will have application for quality control procedures for large-scale production of tat DNA vaccine.

Thesis (Ph. D.)--Bob Jones University, 1988. / Includes bibliographical references (leaves 430-459).