published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

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The preimplantation mouse embryo undergoes many metabolic changes as development proceeds. One major change is the switch from a pyruvate based metabolism, to a glucose based metabolism. The phosphofructokinase enzyme is the regulatory enzyme of glycolysis and is thought to be a major contributor in controlling the block to glycolysis in early preimplantation mouse embryos. This study was undertaken to construct a system that would allow detection of RNA for the highly glycolytically active subunit (muscletype) of the phosphofructokinase (PFK) enzyme. A muscle specific mutant PFK plasmid was generated to provide mutant internal control RNA. Using this internal control, initial reverse transcriptionpolymerase chain reaction data collected from early embryo stages suggest that the muscle type PFK subunit RNA is not expressed in the preimplantation mouse at the 1-cell or blastocyst stages. This result suggests that PFK activity detected at the later morula and blastocyst stages must be from either a different PFK subunit or a novel embryonic form of PFK. / Department of Biology

The ability to modulate behavior and physiology when encountering novel social contexts is essential for the survival and fitness of socially living species. During social interactions, individuals must assess their current social environment and integrate this information with their own internal state and past social experiences to facilitate appropriate social behavior. This process leads to adaptive modulation of behavior and physiology. The behavioral dynamicswithin social dominance hierarchies are an exemplar of how individuals adaptively modulate theirsocial behaviors and physiology. However, much remains unknown about the behavioral, physiological, and neurobiological underpinnings of plasticity among individuals living in social hierarchies within complex social environments. This dissertation is composed of several studies aimed at investigating the behavioral and physiological plasticity and associated neurobiological characteristics of group-living mice as they form stable and consistent social relationships with unfamiliar social partners to achieve a social hierarchy. In Chapter 2, I analyze how the patterns of both aggressive and non-aggressive social behaviors change as unfamiliar male mice establish social relationships in dyads, providing new statistical methods to define the resolution of a dominance relationship. In Chapter 3, I use an ethologically relevant experimental paradigm to investigate social hierarchies in large groups and explore how mice change their urination pattern (scent-marking) and metabolic investment in major urinary proteins as they acquire dominance status. In Chapter 4, I demonstrate the association between individual social ranks and foraging dynamics of mice living in social hierarchies. Collectively, the results of these studies suggest thatmaintaining high social status, particularly alpha status, can be energetically costly. Investment byalpha males in reproduction and territorial defense may come at the cost of resources available topromote long-term health, particularly responses to immune challenges. To explore thishypothesized trade-off, inChapter 5, I test the hypothesis that individuals of different socialstatus vary significantly in immune system functioning. I demonstrate that dominant males are primed to utilize adaptive immunity while subordinate males invest more in innate immunity. In Chapter 6, I explore the neurobiological characteristics of social dominance, with a particular focus on the association between the oxytocin and vasopressin neuropeptide systems and social status. I idenfity several brain regions, such as nucleus accumbens and lateral preoptic area, inwhich alpha, subdominant and subordinate mice show significant differences in the levels ofoxytocin receptors and vasopressin 1a receptors. To better understand how the brain responds to social cues, in Chapter 7, I identify brain regions in dominant and subordinate mice that respond to cues regarding social status and familiarity. I demonstrate that brain regions in the social decision-making network respond distinctively depending on the social cue types sensory information and the internal state. Finally, in Chapter 8, I explore brain transcriptomic profiles associated with behavioral differences among alpha, subdominant, and subordinate male mice. Overall, this dissertation contributes significantly to our understanding of how an individual’s social context leads to plastic and adaptive changes in the brain, behavior and physiology.

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Dilantin (diphenylhydantoin or DPH) has been given to epileptic mothers to control seizures during pregnancy. Previous research has demonstrated that exposure of human embryos to Dilantin in vivo results in an increased probability of abnormal development and early fetal loss. Preliminary results with cultured 1-cell and 2-cell mouse embryos demonstrated that Dilantin causes mouse embryonic cleavage events to slow during preimplantation development (Chatot et al., unpublished). Dilantin may be responsible for this by inhibiting the rate of DNA synthesis during cleavage or by affecting the expression of proteins that control cell cycle progression. The standard expression pattern of these cell cycle regulatory proteins (cyclins) has not previously been determined in the mouse preimplantation embryo model. In this study, immunolabellingtechniques have been used to determine the expression pattern of cyclins D, A, and B 1 in the first three cell cycles of preimplantation mouse embryo development.This study reveals a unique expression pattern of cyclins D, A, and B1 in the first three cell cycles of preimplantation embryo development. Examination of the beginning of the first cell cycle, or G1, indicated a moderate expression of cyclin B1 and A but no cyclin D expression. During DNA synthesis (S-phase) all cyclin expression was virtually nonexistent. Toward the end of the cell cycle at G2/M, cyclin D expression appeared to be at moderate levels while cyclins A and B 1 exhibited minimal degrees of expression.In G 1 of the second cell cycle, cyclins D and A were minimally to moderately expressed and cyclin B 1 expression was minimal. At S-phase, cyclin D expression dropped to minimal levels whereas cyclins A and B 1 were at minimal to moderate levels of expression. At G2/M of the second cell cycle, cyclin B1 was expressed at minimal to moderate levels and cyclins A and D were both expressed at minimal levels.The third cell cycle began at G 1 with cyclin B 1 being expressed at moderate levels followed by minimal to moderate levels of cyclin D expression and minimal expression for cyclin A. Cyclin D expression increased to moderate levels at S-phase and cyclin A exhibited minimal to moderate levels of expression. Cyclin B 1 was observed at moderate levels of expression at S-phase of the third cell cycle. G2 of the third cell cycle included a drop to minimal levels of expression of cyclin D, while cyclin A expression remained at minimal to moderate levels and cyclin B remained at moderate levels of expression.The cyclin expression pattern for the first three cell cycles in preimplantation mouse embryos is unique compared to known cyclin expression patterns in other species. Cyclin D is expressed in G1 and is known to be necessary for advancement to S-phase in human glioblastoma cell lines (Xiong et al., 1991). Cyclin A is active at S-phase through Win human fibroblasts and xenopus oocytes (Giordino et al., 1991; Minshul et al., 1990). Cyclin B is present at G2 through mitosis in human fibroblasts and xenopus oocytes (Pines and Hunter, 1990; Minshul et al, 1990). / Department of Biology

The anticonvulsant drug, Dilantin, in many cases must be taken by epileptic mothers to control seizures during pregnancy, but unfortunately, it has been characterized as a human teratogen. It has also been demonstrated that many of the teratogenic effects of Dilantin occur during postimplantation, but some studies implicate a detrimental role for Dilantin during the preimplantation stages of development. Some of the postimplantation effects include congenital malformations and the potential'loss of the fetus. Our lab has proposed that in preimplantation mouse embryos the drug may be altering the timing of expression of cell cycle regulatory proteins and therefore, we have begun to examine the expression of these proteins. Thus, it was the goal of this study to characterize and localize various cell cycle proteins at specific time points in normal in vivo preimplantation mouse embryos, as this will provide important baseline information for studies on how anticonvulsant drugs may alter cell cycle regulation in embryos.Western blotting has confirmed the presence of cyclin BI in G1 of the first cell cycle. Both cyclin E and CDK2 were not detected in GI or G2/M of the first cell cycle or GI of the second cell cycle.From the immunogold TEM experiments, the density of cyclin B1 staining was observed to be the highest at G1 of the first cell cycle and declined at S and G2/M. Cyclin B 1 was detected in all regions of the embryo including the microvilli, cortical cytoplasm, interior cytoplasm, and was observed to be associated with vesicles and some filaments. The gold particles at GI, S, and G2/N4 of the first cell cycle and G1 of the second cell cycle appear to be associated with filamentous and membraneous structures and not free in the cytoplasmic spaces. Cyclin B 1 expression was more concentrated around vesicles at G1 of the first cell cycle and in general, was more concentrated around vesicles than in microvilli and cortical cytoplasm, interior cytoplasm, or around filaments at each cell cycle stage tested. / Department of Biology

Li Mei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 182-202). / Abstracts in English and Chinese. / Abstract --- p.i / 中文摘要 --- p.iii / Acknowledgements --- p.v / List of Abbreviations --- p.vi / CONTENTS --- p.viii / Chapter Chapter 1 --- General introduction --- p.1 / Chapter 1.1 --- Alkaline phosphatase --- p.1 / Chapter 1.1.1 --- Distribution --- p.1 / Chapter 1.1.2 --- Molecular characteristics of alkaline phosphatase --- p.4 / Chapter 1.1.3 --- Properties of alkaline phosphatase --- p.8 / Chapter 1.1.4 --- Role of alkaline phosphatase --- p.10 / Chapter 1.2 --- Mouse embryonic brain development --- p.18 / Chapter 1.2.1 --- General developing process --- p.18 / Chapter 1.2.2 --- The crainal nerve nuclei in the embryonic mouse brainstem --- p.20 / Chapter 1.2.3 --- The process of neurogenesis in central nerve system --- p.22 / Chapter 1.3 --- Alkaline phosphatase expressed in developing neural tube --- p.26 / Chapter 1.4 --- Summary --- p.30 / Chapter 1.5 --- Objectives of study --- p.31 / Chapter Chapter 2 --- AP expression pattern in embryonic mouse brainstem --- p.33 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.1.1 --- AP expressed in developing neural tube --- p.33 / Chapter 2.1.2 --- Methods for alkaline phosphatase detection --- p.35 / Chapter 2.2 --- Materials and methods --- p.39 / Chapter 2.2.1 --- Animal and procedure --- p.39 / Chapter 2.2.2 --- Preparation of tissue sections and histochemistry --- p.39 / Chapter 2.2.3 --- Electron microscopy study of AP location --- p.41 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Histochemical demonstration of AP --- p.42 / Chapter 2.3.2 --- Stage-specificity and tissue-specificity of AP activity in the neural tube --- p.43 / Chapter 2.3.3 --- Cytochemical localization of AP activity --- p.46 / Chapter 2.3.4 --- Sencitivity to pH of the histochemical staining for AP --- p.46 / Chapter 2.3.5 --- Inactivation of AP activity --- p.47 / Chapter Chapter 3 --- Quantitative studies of AP activity in embryonic mouse brainstem --- p.48 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.1.1 --- Basic knowledge about enzyme kinetic study --- p.48 / Chapter 3.1.2 --- Enzyme assay for alkaline phosphatase --- p.50 / Chapter 3.2 --- Materials and methods --- p.52 / Chapter 3.2.1 --- Animals and sample preparation --- p.52 / Chapter 3.2.2 --- Measurement of AP activities --- p.53 / Chapter 3.2.3 --- Data analysis --- p.54 / Chapter 3.3 --- Results --- p.54 / Chapter 3.3.1 --- "Determination of reaction duration, initial velocity and Km of AP activity" --- p.54 / Chapter 3.3.2 --- Comparision of AP activity in the brainstem and cortex and at different stages --- p.55 / Chapter 3.3.3 --- Effects of physical and chemical factors on AP activity --- p.55 / Chapter Chapter 4 --- Electrophoresis study of AP activity --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Materials and methods --- p.60 / Chapter 4.2.1 --- AP extraction --- p.60 / Chapter 4.2.2 --- Polyacrylamide gel electrophoresis (PAGE) --- p.61 / Chapter 4.2.3 --- Detection of AP activity --- p.61 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- Demonstration of AP activity on the gels --- p.62 / Chapter 4.3.2 --- Comparison of AP from the brain at different stages --- p.62 / Chapter 4.3.3 --- "Comparison of AP in the embryonic brainstem with those in the adult mouse placenta, kidney, liver and intestine" --- p.63 / Chapter 4.3.4 --- Effect of heating and chemical factors on AP activity in the embryonic brainstem --- p.63 / Chapter Chapter 5 --- Study of the cell types expressing AP activity --- p.65 / Chapter 5.1 --- Introduction --- p.65 / Chapter 5.2 --- Materials and methods --- p.67 / Chapter 5.2.1 --- Materials --- p.67 / Chapter 5.2.2 --- Immunostaining of AP in the embryonic brainstem --- p.68 / Chapter 5.2.3 --- Double staining for AP and cells markers --- p.70 / Chapter 5.3 --- Results --- p.70 / Chapter 5.3.1 --- Effectiveness of anti-TNAP antibody on the embryonic mouse brain --- p.70 / Chapter 5.3.2 --- Expression pattern of different neural cell markers at E13.5 --- p.71 / Chapter 5.3.3 --- Co-localization of AP and specific cell markers in E13.5 brain --- p.72 / Chapter Chapter 6 --- Discussion --- p.74 / Chapter 6.1 --- Stage-dependence and tissue-specificity of AP expression in the developing mouse brainstem --- p.75 / Chapter 6.2 --- Possible molecular nature of AP expressed in the developing mouse brainstem --- p.80 / Chapter 6.3 --- The possible cell types that express the enzyme activity --- p.83 / "Figures, Tables, Graphs and Legends" --- p.87 / Appendices --- p.165 / Appendix A: Sources of materials --- p.165 / Appendix B: The process of sample for staining --- p.167 / Appendix C: Protocol of histochemical staining for AP --- p.170 / Appendix D: Protocol of electron microscopy study for AP activity --- p.172 / Appendix E: Protocol of enzyme assay for AP activity --- p.174 / Appendix F: Protocol of immunostaining (ABC method) --- p.175 / Appendix G: Protocol of double staining with fluorescent detection --- p.177 / Appendix H: Protocol of electrophoresis analysis for AP --- p.179 / References --- p.182

The importance of metabolism in tumor initiation and progression is becoming increasingly clear. Metabolic changes induced by oncogenic drivers of cancer contribute to tumor growth and are attractive targets for cancer treatment. Phosphatase and Tensin homolog deleted from chromosome ten (PTEN) is one of the most commonly mutated tumor suppressors in cancer and operates in multiple roles, rendering it a hub for understanding cancer biology and for developing targeted therapy. PTEN’s canonical function is its ability to antagonize the phosphoinositide 3-kinase (PI3K) pathway by dephosphorylating the lipid second messenger phosphatidylinositol (3,4,5) tri-phosphate (PIP3). This thesis focuses on the effects of PTEN loss on cellular metabolism, and the therapeutic vulnerability that stems from metabolic alterations. First, we discovered that loss of Pten in mouse embryonic fibroblasts (MEFs) increases cellular proliferation and the number of replication forks per cell, launching our investigation into metabolic pathways that may be altered to support increased growth. Indeed, we found that Pten-/- cells exhibited a dependence on glutamine for their faster rate of growth, and that glutamine was channeled into the de novo synthesis of pyrimidines. The next chapter examined dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme for pyrimidine ring synthesis in the de novo pyrimidine synthesis pathway. We found that PTEN-deficient primary cells and cancer cell lines were more sensitive to inhibition of DHODH than PTEN WT cells were, and that the growth inhibition could be rescued by metabolites downstream of DHODH. Furthermore, we found that xenografted human triple negative breast cancer tumors in mice could be diminished by treatment with leflunomide, a DHODH inhibitor. In the following chapter, we aimed to identify the mechanisms leading to cell death in PTEN mutant cells upon DHODH inhibition. We found that inherent defects in checkpoint regulation in PTEN-deficient cells were exacerbated by the stress of obstructed de novo pyrimidine synthesis, leading to a buildup of DNA damage at replication forks and ultimately chromosomal breaks. This was instigated by AKT-mediated phosphorylation of TOPBP1 that caused inadequate ATR activation, as well as AKT-mediated phosphorylation and inactivation of CHK1. In sum, the findings of this thesis indicate that enhanced glutamine flux to de novo pyrimidine synthesis in PTEN mutant cells generates vulnerability to DHODH inhibition. The integration of altered glutamine regulation with PTEN’s effect on replication, DNA damage, and the checkpoint response manifests as synthetic lethality upon DHODH inhibition in cells with PTEN inactivation. Inhibition of DHODH could thus be a promising therapy for patients with PTEN mutant cancers.

This project was the first study of the genetics and embryo-pathology of exencephaly in a partially inbred mouse stock, SELH/Bc. Exencephaly was found in 17% of SELH fetuses. Analysis of day 8-9 gestation embryos indicated that SELH embryos were collectively normal in general development, but delayed in neural tube closure relative to overall or general development compared to two normal strains of mice, ICR/Be and SWV/Bc. Exencephaly was observed to be caused by a failure of fusion of the cranial neural folds in the mesencephalon region in SELH. All SELH embryos appeared to be abnormal in their pattern of cranial neural tube closure. They fail to make initial contact at the prosencephalon/mesencephalon junction region of the cranial neural folds (the first fusion in the cranial neural folds in normal embryos). SELH embryos, fused their anterior neural folds via an alternate (possibly passive) mechanism compared to normal strains of mice (SWV/Bc, and ICR/Be), by fusing the folds in a "zipper-like" fashion from the rostral base of the prosencephalon. This closure of the neural tube in genetically liable embryos by an abnormal sequence of events suggests a new model for anterior neural tube closure failure. Liability to exencephaly appeared to be fixed in the SELH stock. Of the 53 SELH males tested, all produced exencephaly. SELH animals were found to be heterogeneous in the frequency of exencephaly they produced, indicating that there are still genes segregating in the stock which affect the ability of embryos to complete anterior neural tube closure. Exencephaly in SELH does not appear to be caused by an autosomal dominant, sex-linked dominant or recessive, or simple autosomal recessive single gene, although F2, BCl, and BC2 exencephaly frequencies (after an outcross to ICR/Be) suggest that only a small number of genes are involved. A marked excess of female exencephalics was observed in SELH, F2, BCl, and BC2 fetuses. / Medicine, Faculty of / Medical Genetics, Department of / Graduate