A digoxigenin-labeled nonradioactive detection system was used to screen a zebrafish cDNA library for p53-like and ras-like genes. One clone was isolated and identified as an incomplete p53-like gene. The insert size of this clone is 1777 bp, which encodes part of evolutionarily conserved region II and all of regions III, IV, and V. A magnetically enriched whole zebrafish cDNA library was constructed to enhance possible recovery of ras-like genes in zebrafish. One clone, termed Zras-Bl, carried an insert of 2592 bp with an open reading frame encoding a 188 amino acid residue ras p21 protein. Based on total protein sequence, this expressed zebrafish ras p21 is most closely related to human N-ras (91% homology), with lesser homology to Ha-ras (84%) and Ki-ras (85%). Preliminary partial sequence data obtained by genomic and reverase transcriptasepolymerase chain reaction (RT-PCR) screening indicate the presence of at least one additional expressed ras gene in zebrafish. The tumorigenicity and Ki-ras mutational properties of dietary 7,12-dimethylbenz[a]anthracene (DMBA) and dibenzo[a,l]pyrene (DBP) were compared in rainbow trout. Both chemicals elicited predominantly 12(1)G->A and 12(2)G->T mutations in trout liver tumors. Two {12(1)G->T and 12(2)G->T} and one {12(1)G->A and 12(2)G->T} double mutation were also observed in DBP livers tumors, but not in DMBA liver tumors. Some stomach tumors from both chemicals exhibited so much DNA degradation that routine PCR amplification was not possible. Among sixteen DMBA stomach tumors with intact DNA, no Ki-ras mutations were found. Of sixteen DBP stomach tumors examined, one had 12(1)G->A and two had 13(1)G->C mutations. The observed G->T transversions are compatible with apurinic mutagenesis driven by unstable DNA adducts arising from one-electron oxidation, but this is not true for the major G->A transitions or G->C transversions and rare double mutations found in this study. The low sensitivity of direct sequencing may limit the frequency of ras mutant detection in this study. / Graduation date: 1996

Graduation date: 1992

Zebrafish (Brachydanio rerio) are small, freshwater teleost fishes in the family Cyprinidae, the true minnows. They are native to the tropical latitudes of India, but have become widespread through their use as aquarium fish and as models for several branches of biological research. Their ease of rearing, short generation time, year-around egg laying potential, brief developmental period, and embryo transparency have made them especially desirable as models for developmental biology, genetics, and neurobiology. Because of their popularity, they were also the first small aquarium fish to be used as test organisms for carcinogenesis in the early 1960's. For reasons that have never been stated, their use as a model for carcinogenesis research did not continue. Due to the number of positive characteristics that this species has, the goal of this research effort was to systematically evaluate the potential of zebrafish for use as an environmental monitor, to evaluate the toxicology and carcinogenesis of surface and/or ground waters. The overall project was multidisciplinary in nature, but the focus of this thesis research was on the whole animal, dose-response to a number of well-known carcinogens, administered by multiple exposure routes, and the pathological description of the resulting lesions. Exposure to N-nitrosodiethylamine (DEN) and N-nitrosodimethylamine (DMN) in the diet was ineffective, but static water bath exposure of fry and embryos to these nitrosamines resulted in neoplasms, primarily in the liver. Embryo exposure to DEN resulted in a low response of neoplasms in several other organs as well. Dietary exposure of zebrafish to aflatoxin B₁ resulted in few hepatic neoplasms, revealing a marked resistance to this carcinogen. Dietary exposure to methylazoxymethanol acetate (MAM-Ac) produced mostly liver tumors, as did both fry and embryo water bath exposures. Each water bath exposure also produced neoplasms at other tissue and organ sites, but the embryo stage produced the greatest variety. These results demonstrate a relative resistance to neoplastic development compared to the well-known rainbow trout model. But in one comparative trial, zebrafish were similar to Japanese medaka in their response to dietary MAM-Ac. The major limitation of this species, that will prevent its use as a model for environmental monitoring, however, is its narrow range of temperature tolerance. Temperatures below 15°C produce marked sluggishness, and below 10-12°C cause anesthesia and death. Therefore, this research indicates that this species is not as versatile as some other small fish species for laboratory and especially field monitoring of environmental carcinogenic hazards. / Graduation date: 1997

Objectives: To establish a mouse orthotopic bladder cancer model with consistent tumor-take rate. This orthotopic model was subsequently used to evaluate small animal imaging techniques and investigate new therapeutic agents for bladder cancer treatment. / Materials and Methods: Different orthotopic implantation techniques have been tested. MBT-2 cells and syngeneic C3H/He mice were used in all experiments. Chemical bladder pre-treatment with different agents (saline, hydrochloric acid, trypsin and poly-L-lysine) and different concentration of instilled tumor cells (1 x 10⁶ or 2 x 10⁶) were investigated. In the second part of the experiment, trans-abdominal micro-ultrasound imaging (MUI) technique was investigated and validated. Bladder tumor growths were monitored with longitudinal measurement. Mice were killed at every MUI session. Bladder tumor volumes were measured and correlated with gross stereomicroscopy. Using the optimized orthotopic bladder cancer model, targeted contrast enhanced micro-ultrasound imaging has been investigated. VEGFR2 targeted contrast agent was prepared and injected intravenously before imaging sessions. The intra-tumoral perfusion, VEGFR2 expression and blood volume in real time were quantified. Contrast enhanced MUI was performed on Days 14 and 21. The feasibility of targeted contrast enhanced micro-ultrasound imaging was confirmed. After the establishment of orthotopic model and in vivo molecular imaging techniques, this robust platform was used for investigating new treatment agent in localized bladder cancer. Tumor-bearing mice were randomized into control and sunitinibtreated (40 mg/kg) groups. Tumor volume, intra-tumoral perfusion, and in vivo VEGFR2 expression were measured using a targeted contrast-enhanced micro-ultrasound imaging system. The effects of sunitinib malate on angiogenesis and cellular proliferation were measured by CD31 and Ki-67 immunohistochemistry. The clinical outcomes including total bladder weight, tumor stage, and survival were evaluated. / Results: A consistent tumor take-rate of over 90% was achieved by using poly-L-lysine pretreatment with 2 x 10⁶ MBT-2 cells in all of the experiments. MUI identified all tumors that were present on final histology. Measurements of tumor size by MUI and gross microscopy had a high correlation coefficient (r = 0.97). Measurements of intra-tumoral perfusion and in vivo VEGFR2 expression were also proved to be feasible. After the technical refinement and modification, complete measurements could be performed in all mice (n = 10) at 2 consecutive imaging sessions. No adverse effects occurred due to anesthesia or the ultrasound contrast agent. This is the first report of applying targeted contrast enhanced MUI in orthotopic bladder cancer model. Finally, sunitinib was found to have significant tumor growth inhibition in both in vitro and in vivo experiments. In the orthotopic model, tumors in sunitinib-treated mice had reduced tumor volume and stage, lower proliferation index and micro-vessel density. Sunitinib prolonged survival in tumor-bearing mice as compared to control group. / Conclusions: The development of reliable orthotopic animal models assists in the discovery of novel therapeutic agents. The establishment in the methods of implantation with improved tumor-take rate and the advances in imaging technology form the important foundation of basic research in bladder cancer. Trans-abdominal MUI is proven to be a valuable tool for translational studies involving orthotopic mouse bladder cancer models. Furthermore, the first report of the application of targeted contrast enhanced MUI in deep-seated tumor in bladder has been published. It enables investigators to monitor tumor angiogenesis and vascular changes after treatment. It will be useful for direct, noninvasive, in vivo evaluation of anti-angiogenesis therapeutic agents. The preclinical study has demonstrated the activities of a new class of targeted therapy against localized bladder cancer in an orthotopic mouse model. Sunitinib inhibits tumor growth and thus decreases the tumor burden and prolongs survival compared with placebo. These results provide a rationale for future clinical trials using VEGFR-targeted treatments of localized bladder cancer in the neo-adjuvant and adjuvant settings. / Chan, Shu Yin Eddie. / Thesis (M.D) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 189-212).

Lymphomas and leukemias are the most common cancer in children and young adults and in utero exposure to carcinogens may contribute to the etiology of these cancers. A polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DBP), was administered to pregnant mice (15 mg/Kg b.w., gavage) on gestation day 17. Significant mortalities in young offspring were observed due to T-cell lymphoma. Lung and liver tumors also were observed in survivors at 10 months of age. To assess the role of the Aryl Hydrocarbon Receptor (AHR), we utilized crosses of B6129SF1/J (responsive) mice with strain 129S1/SvImJ (non-responsive). Offspring born to AHR non-responsive mothers had greater susceptibility to lymphoma, irrespective of offspring genotype. Responsive offspring displayed increased mortality if the mother was responsive. Lung adenomas showed Ki-ras mutations and exhibited a 50% decrease and a 35-fold increase in expression of Rb and p19/ARF mRNA, respectively. To examine the risk/benefit of maternal dietary phytochemical treatment against transplacental cancer, 2000 ppm indole-3-carbinol (I3C) was given to pregnant mice through diet from gestation day 9 till weaning. I3C significantly lowered mortality caused by lymphomas regardless of the maternal genotype, and also reduced lung tumor multiplicity in offspring born to AHR [superscript b-l/d] dams. Distribution of I3C in most maternal and fetal tissues was quantified following a single gavage of [¹⁴C]-I3C to the pregnant mice. DBP-DNA adducts were observed in both maternal and fetal tissues by ³³P postlabeling and HPLC analysis and were modulated by I3C and AHR genotype. I3C also modulated phase I and phase II enzyme protein expression in dams and gene expression in newborn thymus. I3C chemoprotection may involve modification of the bioavailability of DBP to the fetus and/or modulation of gene expression in the fetus as well. This is the first demonstration that transplacental exposure to an environmental PAH can induce a highly aggressive lymphoma in mice. These results raise the possibility that PAH exposures to pregnant women could contribute to similar cancers in children and young adults and, that the addition of chemoprotective agents to the maternal diet may reduce cancer risk among offspring. / Graduation date: 2006

Ionizing radiation (IR), along with being an important diagnostic and treatment modality, is a potent tumor-causing agent, and the risk of secondary radiation treatment-related cancers is a growing clinical problem. Now some studies propose to link secondary radiation-induced cancers to an enigmatic phenomenon of bystander effects, whereby the exposed cells send signal damage and distress to their naïve neighbors and result in genome destabilization and carcinogenesis. Yet, no data existed on the bystander effects in an organ other than an exposed one. With this in mind, we focused on the analysis of existence and mechanisms of radiation-induced bystander effects in vivo. We have found that bystander effects occur in vivo in distant skin and spleen following half-body or cranial irradiation. These bystander effects resulted in elevated DNA damage, profound dysregulation of epigenetic machinery, and pronounced alterations in apoptosis, proliferation and gene expression. Bystander effects also exhibited persistency and sex specificity. The results obtained while using the animal model systems can potentially be extrapolated to different animals and humans. / xiii, 208 leaves : ill. ; 29 cm.

Protein kinase A (PKA) is an important enzyme inside the body; it is responsible for phosphorylation of gene regulatory elements and thus regulation of gene expression inside the nucleus. Malfunction of PKA affects transcriptional and translational levels of cell signaling ligands, leading to abnormal activity of various signaling pathways. PKA holoenzyme is composed of two regulatory and two catalytic subunits; four main regulatory subunit isoforms (R1α, R1β, R2α and R2β) and four main catalytic subunit isoforms (Cα, Cβ, Cγ and Prkx) of PKA have been identified. Mutations in these subunits lead to altered total PKA activities and PKAT-I to PKAT-II ratios, leading to diseases both in human and mice. These diseases include Carney Complex (CNC), fibrous dysplasia (FD) and Cushing syndrome. We studied the effect of PKA subunit mutations on intracellular PKA activities, PKAT-I to PKAT-II ratios, and bone and adrenal gland phenotypes in transgenic mouse models. Firstly, we generated whole-body transgenic mice single or double heterozygous for PKA regulatory subunits. Tail vertebral bone lesions including osteosarcomas, osteochondromas and osteochondrosarcomas were found in these mice and we found that mutations in different PKA subunits affect bone lesion formation, new bone generation, and bone organization and mineralization in mouse tail vertebrae. Elevated Cβ subunit expression in Parkar1a+/-Prkar2a+/- and Prkar1a+/-Prkar2b+/-double heterozygous mice leads to a less severe vertebral bone lesion phenotype, an increased osteogenic activity and a better bone regeneration activity. We then studied mice with tissue specific knock out of Prkar1a, the gene coding for type I regulatory subunit, specifically in adrenal cortex (AdKO). AdKO mice developed pituitary-independent Cushing syndrome with increased PKA activity. They also demonstrated increased plasma corticosterone levels resistant to dexamethasone suppression. Dietary treatment of both mice with bone lesions and mice with adrenal lesions with COX2 inhibitor Celecoxib led to partial rescue of phenotypes; this is due to inhibition of the positive feedback loop between PKA signaling and inflamasome pathway at COX2 induction level by Celecoxib. / 蛋白激酶A（PKA）是人體中重要的蛋白酶， 它通過燐酸化基因調控元件來實現對細胞核內基因表達的調節。PKA異常影響細胞內信號傳遞因子的基因轉錄和蛋白翻譯水平，從而導致各細胞信號通路的異常活動。PKA全酶由兩個調節亞基和兩個催化亞基組成，目前已經發現的有四個調節亞基 (R1α, R1β, R2α 和R2β) 以及四個催化亞基(Cα, Cβ, Cγ和Prkx)。發生在這些亞基中的基因突變會改變總的PKA活動水平，PKA-I 和PKA-II的比例，在人類和實驗鼠中引起疾病。這些疾病包括卡尼綜合症 （CNC），骨纖維性發育不良（FD）和庫欣綜合症。我們在轉基因鼠模型中研究PKA亞基突變對細胞中PKA總活性， PKA-I和PKA-II比例的影響，以及由此帶來的骨和腎上腺表型的改變和病變。我們首先製造了有一個或兩個PKA亞基雜合性缺失的全身轉基因鼠。在這些轉基因鼠中，我們發現了包括骨肉瘤，骨軟骨瘤和骨軟骨肉瘤在內的尾椎骨病變。研究發現在不同PKA亞基中的基因變異對實驗鼠尾椎骨病變的發生，新骨的形成和骨的結構和纖維化均有影響。在Prkar1a+/-Prkar2a+/-和Prkar1a+/-Prkar2b+/-實驗鼠中我們發現了較高的Cβ催化亞基表達，這兩個基因型因此具有更輕度的骨病變和更強的骨再生能力。我們繼續研究了在腎上腺中敲除了標記PKA 第一調節亞基的Prkar1a基因的實驗鼠 （AdKO）。AdKO實驗鼠中產生了與垂體無關的庫欣綜合症，並伴隨PKA活性的增加。它們還表現出耐地塞米松抑制的血漿皮質酮水平增加。對骨病變或腎上腺病變的實驗鼠通過飲食進行COX2抑制劑塞來昔布的治療可以部分緩解病變表型。這是由對PKA和炎性體的正反饋機制在COX2誘導步驟的抑制造成的。 / Liu, Sisi. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 115-130). / Abstracts also in Chinese. / Title from PDF title page (viewed on 09, September, 2016). / Detailed summary in vernacular field only.

Approximately 45% of men will develop cancer during their lifetime; some of which will be of reproductive age (Canadian Cancer Society, 2008). Current advances in treatment regimens such as radiotherapy have significantly lowered cancer-related mortality rates; however, one major quality-of-life issue in cancer survivors is the ability to produce healthy offspring. Exposure to ionizing radiation (IR) leads to genomic instability in the germline, and further to transgeneration genome instability in unexposed offspring of preconceptionally exposed parents. The results presented in this thesis define, in part, the molecular consequences of direct and indirect irradiation for the male germline. Direct exposure results in a significant accumulation of DNA damage, altered levels of global DNA methylation and microRNAome dysregulation of testis tissue. Localized cranial irradiation results in a significant accumulation of unrepaired DNA lesions and loss of global DNA methylation in the rodent (rat) germline. Biological consequences of the changes observed are discussed. / xii, 121 leaves : ill. ; 29 cm.