Despite the extraordinary diversity of behavior across the animal kingdom, the genes and molecules that contribute to such natural diversity are largely unknown. In this thesis, I leverage the dramatic divergence in behavior between two closely related species of deer mice (genus Peromyscus) to investigate the genetic, cellular, and neuroendocrine basis of behavior. In chapter 2, I show that the monogamous oldfield mouse (Peromyscus polionotus subgriseus) has evolved a novel cell type in the adrenal gland that expresses the enzyme AKR1C18, which converts progesterone into 20α-hydroxyprogesterone (20α-OHP). I then demonstrate that 20α-OHP is more abundant in oldfield mice than in the closely-related promiscuous prairie deer mouse (P. maniculatus bairdii) and that it increases monogamous-typical parental behaviors when administered to both monogamous fathers. Using quantitative trait locus mapping in a cross between these species, I discover interspecific genetic variation that drives expression of the glycoprotein tenascin N and ultimately contributes to gain of adrenal AKR1C18 expression in oldfield mice. In chapter 3, I investigate the genetic architecture underlying the striking difference in exploratory behavior between prairie deer mice and oldfield mice. Through congenic fine-mapping, I identify a 15-Mb locus that strongly contributes to species differences in exploratory behavior. I then investigate the potential contributions of one of the 18 genes in the locus, Olfm4, which harbors cis-regulatory variants that drives its expression in the oldfield hypothalamus. Taken together, my research advances our understanding of the genetic and molecular causes that drive rapid behavioral divergence between species.

No description available.

Yuen, Yee Lok. / On t.p. "alpha" appears as the Greek letter. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 165-177). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vii / Table of Contents --- p.viii / List of Abbreviations --- p.xiii / List of Figures --- p.xv / List of Tables --- p.xx / Chapter Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Peroxisome proliferator-activated receptor (PPAR) --- p.1 / Chapter 1.1.1 --- PPARα isoforms --- p.1 / Chapter 1.2 --- PPARα ligands --- p.2 / Chapter 1.3 --- Biological roles of PPARα --- p.3 / Chapter 1.3.1 --- Lipid metabolism --- p.3 / Chapter 1.3.2 --- Bile acid metabolism --- p.4 / Chapter 1.3.3 --- Biotransformation --- p.6 / Chapter 1.4 --- Roles of PPARα in hepatocarcinogenesis --- p.7 / Chapter 1.4.1 --- Cell proliferation and apoptosis --- p.7 / Chapter 1.4.2 --- Oxidative stress --- p.8 / Chapter 1.5 --- Discovery of novel PPARα target genes --- p.9 / Chapter 1.5.1 --- Identification of a novel PPARα-regulated gene L5#55 by fluorescent differential mRNA display (FDD) analysis --- p.9 / Chapter 1.6 --- Sulfotransferase (SULT) --- p.15 / Chapter 1.7 --- Objective of the present study --- p.16 / Chapter Chapter 2 --- Molecular cloning and characterization of mouse liver sulfotransferase-like (mL-STL) gene --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and methods --- p.17 / Chapter 2.2.1 --- Animals --- p.17 / Chapter 2.2.2 --- Treatments --- p.18 / Chapter 2.2.3 --- Total RNA extraction --- p.18 / Chapter 2.2.3.1 --- Materials --- p.18 / Chapter 2.2.3.2 --- Methods --- p.19 / Chapter 2.2.4 --- Rapid amplification of cDNA ends (RACE) --- p.19 / Chapter 2.2.4.1 --- Materials --- p.19 / Chapter 2.2.4.2 --- Methods --- p.20 / Chapter 2.2.4.2.1 --- Primer design --- p.20 / Chapter 2.2.4.2.2 --- Rapid amplification of 5'- and 3'-cDNA ends --- p.20 / Chapter 2.2.5 --- Cloning of the 5'- and 3' RACE products --- p.25 / Chapter 2.2.5.1 --- Materials --- p.25 / Chapter 2.2.5.2 --- Methods --- p.25 / Chapter 2.2.6 --- Northern blot analysis --- p.28 / Chapter 2.2.6.1 --- Materials --- p.28 / Chapter 2.2.6.2 --- Methods --- p.28 / Chapter 2.2.6.2.1 --- Formaldehyde-agarose gel electrophoresis and blotting of RNA --- p.31 / Chapter 2.2.6.2.2 --- PCR DIG-labeling --- p.31 / Chapter 2.2.6.2.3 --- Hybridization and signal detection --- p.32 / Chapter 2.2.7 --- Reverse transcription (RT)-PCR --- p.34 / Chapter 2.2.7.1 --- Materials --- p.34 / Chapter 2.2.7.2 --- Methods --- p.34 / Chapter 2.3 --- Results and discussion --- p.37 / Chapter 2.3.1 --- Cloning of the full-length mL-STL cDNA --- p.37 / Chapter 2.3.2 --- In silico analysis of the mL-STL cDNAs --- p.50 / Chapter 2.3.3 --- Genomic organization of the mL-STL gene --- p.61 / Chapter 2.3.4 --- Tissue distribution of mL-STL mRNA transcript --- p.68 / Chapter 2.3.5 --- "PPARα-dependent regulation of mL-STL mRNA expression by fasting and Wy-14,643 treatment" --- p.74 / Chapter Chapter 3 --- Identification of the native mL-STL protein in mouse liver --- p.86 / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.2 --- Materials and methods --- p.87 / Chapter 3.2.1 --- Animal and treatments --- p.87 / Chapter 3.2.2 --- Cloning of the mL-STL cDNA into a modified pRSET (mpRSET) expression vector --- p.88 / Chapter 3.2.2.1 --- Materials --- p.88 / Chapter 3.2.2.2 --- Methods --- p.88 / Chapter 3.2.2.2.1 --- Amplification of mL-STL cDNA fragments --- p.88 / Chapter 3.2.2.2.2 --- Preparation of mpRSET expression vector --- p.92 / Chapter 3.2.2.2.3 --- "Ligation, transformation, and screening of recombinants" --- p.92 / Chapter 3.2.3 --- Over-expression of the mL-STL recombinant proteins in E coli strains --- p.94 / Chapter 3.2.3.1 --- Materials --- p.94 / Chapter 3.2.3.2 --- Methods --- p.94 / Chapter 3.2.4 --- Mass spectrometry analysis of the mL-STL recombinant proteins --- p.95 / Chapter 3.2.4.1 --- Materials --- p.96 / Chapter 3.2.4.2 --- Methods --- p.96 / Chapter 3.2.4.2.1 --- Trypsin digestion and peptide extraction --- p.96 / Chapter 3.2.4.2.2 --- Matrix-assisted laser desorption/ionization time-of- flight (MALDI-TOF) mass spectrometry --- p.97 / Chapter 3.2.5 --- Purification of the mL-STL recombinant proteins --- p.98 / Chapter 3.2.5.1 --- Materials --- p.98 / Chapter 3.2.5.2 --- Methods --- p.98 / Chapter 3.2.5.2.1 --- Semi-purification of the mL-STL recombinant proteins by preparative SDS-PAGE --- p.98 / Chapter 3.2.5.2.2 --- Purification of mL-STL recombinant proteins by column chromatography --- p.99 / Chapter 3.2.6 --- Rabbit immunization using purified mL-STL recombinant proteins --- p.101 / Chapter 3.2.7 --- Subcellular fractionation of mouse liver by ultracentrifugation --- p.101 / Chapter 3.2.7.1 --- Materials --- p.101 / Chapter 3.2.7.2 --- Methods --- p.102 / Chapter 3.2.8 --- Western blot analysis of the native mL-STL protein --- p.104 / Chapter 3.2.8.1 --- Materials --- p.104 / Chapter 3.2.8.2 --- Methods --- p.104 / Chapter 3.2.8.2.1 --- SDS-PAGE and electro-blotting of proteins --- p.104 / Chapter 3.2.8.2.2 --- Immunostaining and signal detection --- p.105 / Chapter 3.3 --- Results and discussion --- p.106 / Chapter 3.3.1 --- Cloning of the mL-STLl and mL-STL2 cDNAs into a modified pRSET (mpRSET) vector --- p.106 / Chapter 3.3.2 --- IPTG induction of the mpRSET-mL-STL protein expression --- p.106 / Chapter 3.3.3 --- Confirmation of mL-STL recombinant proteins by mass spectrometry --- p.118 / Chapter 3.3.4 --- Purification of mL-STL recombinant proteins for rabbit immunization and polyclonal antisera production --- p.130 / Chapter 3.3.5 --- Antigenicity of mL-STL antisera --- p.134 / Chapter 3.3.6 --- Identification of mL-STL native protein and its induction pattern in mouse liver --- p.139 / Chapter 3.3.7 --- "Time-course of fasting and Wy-14,643 treatment on the mL- STLl native protein expression" --- p.147 / Chapter Chapter 4 --- Overall discussion --- p.153 / Future study --- p.163 / References --- p.165 / "Appendix A. Alignment of nucleotide sequences of mouse chromosome 7，Riken2810007J24, mL-STLl, and mL-STL2 cDNA sequences" --- p.178 / Appendix Bl. Theoretical tryptic peptide masses of mpRSET- mL-STLl protein --- p.217 / Appendix B2. Raw data from mass spectrometry analysis of mpRSET-mL-STLl protein --- p.218 / Appendix C1. Residue molecular mass of amino acids --- p.219 / Appendix C2. Di-peptide table --- p.220 / Appendix D1. Theoretical tryptic peptide masses of mpRSET- mL-STL2 protein --- p.221 / Appendix D2. Raw data from mass spectrometry analysis of mpRSET-mL-STL2 protein --- p.222

Xu, Jian. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 238-255). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016).

Not available / text

published_or_final_version / Surgery / Master / Master of Philosophy

In order to maintain genetic stability in eukaryotes, tight regulation of the relative sizes of deoxyribonucleoside triphosphate (dNTP) levels inside the cell is essential for optimal fidelity of DNA replication. Ribonucleotide reductase (RNR) is the enzyme responsible for proportional production of DNA precursors. Studies on regulation of this enzyme, the focus of this thesis, are important because mutations affecting RNR control mechanisms result in dNTP pool imbalance, thus promoting mutagenesis. By using mouse RNR as a model for mammalian forms of the enzyme, three major factors--allosteric effectors, rNDP substrate concentrations, and hypoxic conditions--that influence the substrate specificity of RNR have been investigated. Allosteric regulation has been studied by the four-substrate assay, which permits simultaneous monitoring of the four reactions catalyzed by this enzyme in one reaction mixture. Individual dNTPs affect the four activities differentially in a concentration-dependent manner with discrete effects of dTTP and dGTP on reduction of ADP and GDP, respectively. Ribonucleoside diphosphate (rNDP) substrate concentrations are equally important, as their variations lead to different product ratios. Results from nucleotide binding assays indicate that rNDPs directly influence binding of dNTP effectors at the specificity site, one of the two classes of allosteric sites, whereas ADP has an indirect effect, displacing other substrates at the catalytic site and consequently removing effects of those substrates upon dNTP binding. Hence, this is the first evidence of a two-way communication between the catalytic site and the specificity site. Oxygen limitation also plays an important role in controlling the enzyme specificity. Reactivation of the enzyme at different oxygen tensions, after treatment of the enzyme with hydroxyurea (HU) followed by removal of HU, reveals a distinct sensitivity of GDP reductase to low 0��� levels. Although the basis for specific inhibition of GDP reduction remains to be determined, some possibilities have been ruled out. This research proves that in addition to allosteric regulation by nucleoside triphosphates, mouse RNR is also controlled by other factors. Since these components can simultaneously exert their effects upon enzyme specificity, complex regulatory patterns of RNR to provide a proportional supply of the DNA building blocks in vivo are suggested. / Graduation date: 2003

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

During the growth phase of oogenesis, mammalian oocytes increase several hundred-fold in volume. Although it is known that ovarian granulosa cells send growth promoting signals, neither these external signals nor the transduction pathways that become activated in the oocyte are known. Therefore, the presence and the activity of candidate signaling pathways in growing murine oocytes were investigated. By immunoblotting, the MAP kinases, ERK1 and ERK2, as well as their activating kinase MEK, were detected in oocytes at all stages of growth. However, using a phospho-specific anti-ERK antibody, no immunoreactive species were detectable in isolated granulosa cells or oocytes at any stage of growth, except metaphase II. Phosphorylated ERK was also present, although in smaller quantities, in oocyte-granulosa cell complexes at the later stages of growth. Furthermore, when ovarian sections were stained with an anti-ERK antibody, the protein was found to be highly concentrated in the cytoplasm of oocytes at all stages of growth, with lower levels in the nucleus. Another member of the MAP kinase family, Jun kinase (JNK), was investigated. By immunoblotting, JNK was detected in growing oocytes. Experiments using an anti-JNK antibody on ovary sections revealed the protein to be uniformly distributed in non-growing and growing oocytes with no evidence of preferential nuclear localization. These results imply that an interaction between the oocyte and the granulosa cells may be required to generate phosphorylated ERK. They also imply that growth signals probably are not relayed through ERK, but do not exclude a role for Jun kinase in mediating oocyte growth.

Childhood epileptic encephalopathies (EE) are severe neurodevelopmental diseases that manifest in early development. EE is characterized by abnormal electroencephalographic (EEG) activity, intractable seizures comprising of various seizure types, as well as cognitive, behavioral and neurological defects. Developmental and epileptic encephalopathies (DEEs) are a subclass of EEs where the progressive and permanent cognitive and neurophysiological deterioration is not caused by seizure activity alone, but is caused by the same underlying etiology. Recent advances in whole exome sequencing revealed an important role for synaptic dysregulation in DEE and identified multiple new causative variants in synaptic genes. Indeed, mutations in various genes associated with neuronal functions like synaptic transmission and recycling, including transporters, neurotransmitter receptors, and ion channels, have all been identified as causative of DEE. In total, pathogenic DEE-causing variants in over eighty-five genes have been identified and more are likely to follow as next-generation sequencing becomes widely available. DEEs comprise a large group of genetically and phenotypically heterogenous diseases that have been difficult to treat. While in many cases the etiology is unknown, de novo heterozygous missense mutations have often been identified as the underlying cause of DEE. Existing pharmacological interventions by way of antiepileptic drugs leave approximately seventy-percent of DEE patients with intractable seizures. Moreover, these pharmacological treatments do not address the cognitive impairments and associated comorbidities caused by the underlying pathophysiological mechanism. In fact, treatment with antiepileptic drugs may actually worsen cognitive comorbidities due to side effects. Additionally, there are no pharmacological treatments for these cognitive comorbidities other than mood stabilizers and antipsychotics. Therefore, alternative approaches to treatments that address the underlying genetic etiology are necessary. Indeed, the recent utilization of gene therapeutic approaches in other genetic disease models such as spinal muscular atrophy (SMA) has spurred the investigation of gene therapies to treat DEEs. Here, we executed a molecular, behavioral and functional characterization of three preclinical mouse models of DEE involved in synaptic function (Dnm1) and ion channel function (Kcnq3). The human orthologs of the Dnm1 and Kcnq3 genes cause some of the most severe DEE syndromes. Understanding the pathophysiological mechanisms by which mutations in these genes cause disease, is important in identifying and assessing future gene therapeutic interventions. Patients with heterozygous DNM1 pathogenic mutations present with early onset seizures, severe intellectual disability, developmental delay, lack of speech and ambulation, and hypotonia. For the DNM1 dominant-negative model of DEE, we first characterized the Dnm1Ftfl mouse which phenocopies the key disease-defining phenotypes and comorbidities observed in DNM1 patients. Further, we modelled a gene therapy approach in Dnm1Ftfl mice using an RNA interference-based, virally delivered treatment construct. Dnm1Ftfl homozygous mice showed early onset lethality, seizures, growth deficits, hypotonia, and severe ataxia. Molecular analysis of Dnm1Ftfl homozygous mice showed gliosis, cellular degeneration, increased neuronal activation and aberrant metabolic activity, all indicative of recurrent seizure activity. Importantly, our gene therapy treatment significantly rescued all the severe phenotypes associated with DEE, including seizures, early-onset lethality, growth deficits, and aberrant neuronal phenotypes. Thus, our gene therapy approach provided a proof-of-principle for the efficacy of gene silencing to treat DEEs caused by dominant-negative mutations. Second, a DNM1 human variant modelled in mice was generated and characterized. The Dnm1G359A mutation, unlike the Dnm1Ftfl mouse-specific mutation has been identified in patients suffering from DNM1 DEE. Thus, this model allows for a more clinically relevant assessment of the impact of a human DNM1 mutation in mice. In the long run, this model will help validate gene therapeutic approaches that may be clinically relevant to DNM1 DEE patients. The Dnm1G359A mutation, like the Dnm1Ftfl mutation, led to early onset seizures, growth deficits, and lethality, establishing the Dnm1G359A mouse model as a viable model to study DNM1 DEE. In the gain-of-function KCNQ3 model of DEE, Kcnq3R231H mice were characterized molecularly and behaviorally. Patients with KCNQ3 mutations show electrical status epilepticus during sleep (ESES), as well as cognitive and behavioral impairments. The Kcnq3R231H variant led to severe spike-wave discharge phenotype on EEG, decreased maximal seizure threshold, and anxiety-like behavior. Additionally, Kcnq3R231H led to increased localization of Kcnq3 protein at neuronal membranes, suggesting a role for membrane aggregation on disease phenotypes. Altogether, these findings show the viability of preclinical models of both dominant-negative and gain-of-function mutations in replicating key disease-defining phenotypes associated with severe DEEs. Additionally, the results presented here establish a proof-of-principle demonstration that gene silencing can rescue severe phenotypes caused by dominant-negative mutations in DEE. Future studies on both dominant-negative and gain-of-function models should enable an in-depth understanding of mechanistic implications for each mutation, and lead to gene therapeutic strategies to mitigate the debilitating phenotypes of these DEEs.