Law, Sau Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 111-117). / Abstract also in Chinese. / Thesis Committee --- p.i / Acknowledgements --- p.ii / Contents --- p.iii / Abstract --- p.vii / 論文摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xiii / List of Tables --- p.xxiii / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1.1 --- Embryonic Stem (ES) Cells / Chapter 1.1.1 --- Characteristics of ES Cells l / Chapter 1.1.2 --- Therapeutic Potential of ES Cells --- p.3 / Chapter 1.1.3 --- Myocardial Infarction and ES cells-derived Cardiomyocytes --- p.4 / Chapter 1.1.4 --- Current Hurdles of Using ES cells-derived Cardiomyocytes for Research and Therapeutic Purposes --- p.6 / Chapter 1.2 --- Transcription Factors for Cardiac Development / Chapter 1.2.1 --- GATA-binding Protein 4 (GATA-4) --- p.8 / Chapter 1.2.2 --- Myocyte Enhancer Factor 2C (MEF2C) --- p.10 / Chapter 1.2.3 --- "NK2 Transcription Factor Related, Locus 5 (Nkx2.5)" --- p.11 / Chapter 1.2.4 --- Heart and Neural Crest Derivatives Expressed 1 /2 (HANDI/2) --- p.11 / Chapter 1.2.5 --- T-box Protein 5 (Tbx5) --- p.13 / Chapter 1.2.6 --- Serum Response Factor (SRF) --- p.14 / Chapter 1.2.7 --- Specificity Protein 1 (Spl) --- p.15 / Chapter 1.2.8 --- Activator Protein 1 (AP-1) --- p.16 / Chapter 1.3 --- Reactive Oxygen Species (ROS) / Chapter 1.3.1 --- Cellular Production of ROS --- p.18 / Chapter 1.3.2 --- Maintenance of Redox balance --- p.18 / Chapter 1.3.3 --- Redox Signaling --- p.19 / Chapter 1.4 --- Nitric Oxide (NO) and NO Signaling --- p.20 / Chapter 1.5 --- Aims of the Study --- p.22 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- Mouse Embryonic Fibroblast (MEF) Culture / Chapter 2.1.1 --- Derivation of MEF --- p.23 / Chapter 2.1.2 --- Maintenance of MEF Culture --- p.24 / Chapter 2.1.3 --- Irradiation of MEF --- p.25 / Chapter 2.2 --- Mouse ES Cell Culture / Chapter 2.2.1 --- Maintenance of Undifferentiated Mouse ES Cell Culture --- p.26 / Chapter 2.2.2 --- Differentiation of Mouse ES Cells --- p.26 / Chapter 2.2.3 --- Exogenous addition of hydrogen peroxide (H2O2) and NO --- p.27 / Chapter 2.3 --- ROS Localization Study / Chapter 2.3.1 --- Frozen Sectioning --- p.28 / Chapter 2.3.2 --- Confocal microscopy for ROS detection --- p.28 / Chapter 2.4 --- Intracellular ROS Measurement / Chapter 2.4.1 --- "Chemistry of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA)" --- p.29 / Chapter 2.4.2 --- Flow Cytometry for ROS Measurement --- p.29 / Chapter 2.5 --- Gene Expression Study / Chapter 2.5.1 --- Primer Design --- p.30 / Chapter 2.5.2 --- RNA Extraction --- p.31 / Chapter 2.5.3 --- DNase Treatment --- p.32 / Chapter 2.5.4 --- Reverse Transcription --- p.32 / Chapter 2.5.5 --- Quantitative Real Time PCR --- p.33 / Chapter 2.5.6 --- Quantification of mRNA Expression --- p.34 / Chapter 2.6 --- Protein Expression Study / Chapter 2.6.1 --- Total Protein Extraction --- p.34 / Chapter 2.6.2 --- Nuclear and Cytosolic Protein Extraction --- p.35 / Chapter 2.6.3 --- Measurement of Protein Concentration --- p.36 / Chapter 2.6.4 --- De-sumoylation Assay --- p.36 / Chapter 2.6.5 --- De-phosphorylation Assay --- p.37 / Chapter 2.6.6 --- De-glycosylation Assay --- p.38 / Chapter 2.6.7 --- Western Blot --- p.39 / Chapter 2.7 --- Statistical Analysis --- p.41 / Chapter CHAPTER THREE --- RESULTS / Chapter 3.1 --- Study of Endogenous ROS / Chapter 3.1.1 --- Level and Distribution of Endogenous ROS --- p.47 / Chapter 3.1.2 --- Quantification of intracellular ROS --- p.48 / Chapter 3.2 --- Effect of Exogenous Addition of Nitric Oxide (NO) on Cardiac Differentiation / Chapter 3.2.1 --- Beating Profile of NO-treated Embryoid Bodies (EBs) --- p.50 / Chapter 3.3 --- Effect of Exogenous Addition of H2O2 on Cardiac Differentiation / Chapter 3.3.1 --- Beating Profile of H2O2-treated EBs --- p.51 / Chapter 3.3.2 --- mRNA Expression of Cardiac Structural Genes --- p.52 / Chapter 3.3.3 --- Protein Expression of Cardiac Structural Genes --- p.54 / Chapter 3.3.4 --- mRNA Expression of Cardiac Transcription Factors --- p.58 / Chapter 3.3.5 --- Protein Expression of Cardiac Transcription Factors --- p.67 / Chapter 3.3.6 --- Post-translational Modifications of Cardiac Transcription Factors --- p.74 / Chapter 3.3.7 --- Translocation of Cardiac Transcription Factors --- p.89 / Chapter CHAPTER FOUR --- DISCUSSION / Chapter 4.1 --- Changes in the Level of Endogenous ROS During Cardiac Differentiation of Mouse ES Cells --- p.96 / Chapter 4.2 --- H2O2 and NO Have Opposite Effects Towards Cardiac Differentiation --- p.97 / Chapter 4.3 --- Exogenous Addition of H2O2 Advances Differentiation of Mouse ES Cells into Cardiac Lineage --- p.99 / Chapter 4.4 --- Possible Role of H2O2 in Mediating Cardiac Differentiation of Mouse ES Cells --- p.103 / Chapter 4.5 --- Future Directions --- p.108 / Conclusions --- p.110 / References --- p.111

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in the embryonic neural development and adult neurogenesis. But the effects of 5-HT on stem cells are not fully known. In this study, the effects and underlying signal pathways of 5- HT on proliferation and neural differentiation of mouse embryonic stem (ES) cells, neural progenitor (NP) cell line C 17.2 and embryonic neural stem (NS) cells were explored. Molecular analysis, immunostaining and western blotting revealed that NP/NB cells expressed the rate-limiting enzyme tryptophan hydroxylase (TPH) and produced endogenous 5-HT. While mouse ES cells showed no expression of TPH. Quantitative PCR demonstrated that ES cells and NPINS cells expressed majority of 5-HT receptor sUbtypes. In serum free propagation culture, WST1, BrdU incorporation and neural colony forming cell assay demonstrated that 5-HT enhanced proliferation of ES cells and NPINS cells in a dose-dependent manner. Tryptophan hydroxylase (TPH) inhibitor para-chlorophenylalanine (PCPA) which can inhibit biosynthesis of endogenous 5-HT decreased viability of mouse NP/NS cells. Mouse ES cells derived embryoid bodies (EB) and NS/NP cells were subjected to neural induction in serum-free medium with and without 5-HT or PCPA. On day 8 of EB cultures, immunofluorescence staining displayed a less percentage of SSEA-1+ cells derived from cultures supplemented with 5-HT. Nestin positivity are comparable. Quantitative PCR analysis suggested that supplement of 5-HT in EB culture inhibit neural differentiation of ES cells and induce mesodermal commitment. On day 21 of ES cells neural induction, compared to cultures without 5-HT treatment, a significantly less number of ß-tubulin III+ neurons, GEAP+ astrocytes and GaIC+ oligodendrocytes were noted in 5-HT -supplemented cultures. For NS/NP cells, the inhibitory effects of 5-HT on neuronal and oligodendrocytic commitment were also observed. And the application of PCPA exerted a promoting effect on neural differentiation of NS cells. Manipulating 5-HT level can affect the expression level of key genes which involved in 5-HT metabolism. ES and NS/NP cells treated with 5-HT showed decreased production of endogenous reactive oxygen species (ROS). 5-HT demonstrated a significant anti-apoptotic effect on NP cells and this antiapoptotic effect may be mediated by up-regulated expression of anti-apoptotic gene Bel- 2. Whole genome cDNA microarray analysis and quantitative RT-PCR revealed that notch signal pathway was involved in mediating the biological effects of 5-HT. Western blotting further confirmed that 5-HT treatment up-regulated the protein level of NICD and notch downstream effectors Hes-l and Hes-5. Finally, the therapeutic effects of ES cell-derived neural cells were testified in a mouse model of global ischemia. Two weeks post-transplantation, BrdU labeled ES cell-derived neural cells survived and migrated throughout brain parenchyma. A majority of transplanted cells remained nestin positive. The cognitive functions of cell transplanted groups showed significant recovery compared with untransplanted arms, but no significant difference was observed between transplanted groups treated with and without 5-HT. Taken together, data of this study indicated 5-HT play an important role in neural development and ES cell-derived neural cells might be applicable in the treatment of stroke. / Li, Jin. / "November 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 195-241). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstracts in English. / ACKNOWLEDGEMENTS --- p.i / LIST OF PUBLICATIONS --- p.ii / ABSTRACT --- p.iii / ABSTRACT [in Chinese] --- p.v / TABLE OF CONTENT --- p.vi / LISTS OF FLOWCHARTS --- p.xii / LISTS OF FIGURES --- p.xiii / LIST OF TABLES --- p.xvi / LIST OF EQUIPMENTS --- p.xvii / LIST OF ABBREVATIONS --- p.xvii / Chapter Chapter1 --- Introduction --- p.1 / Chapter 1.1 --- Central nervous system disorder --- p.1 / Chapter 1.1.1 --- Stroke --- p.1 / Chapter 1.1.2 --- Spinal cord injuries --- p.4 / Chapter 1.1.3 --- Parkinson's disease --- p.6 / Chapter 1.1.4 --- Amyotrophic Lateral Sclerosis --- p.8 / Chapter 1.2 --- Stem cell therapy --- p.10 / Chapter 1.2.1 --- General considerations in stem cell therapy --- p.11 / Chapter 1.2.2 --- Stem cell therapy for stroke --- p.11 / Chapter 1.2.3 --- Stem cell therapy for spinal cord injury --- p.15 / Chapter 1.2.4 --- Stem cell therapy for Parkinson's disease --- p.16 / Chapter 1.2.5 --- Stem cell therapy for ALS --- p.18 / Chapter 1.3 --- Stem cells --- p.20 / Chapter 1.3.1 --- Embryonic stem cells --- p.21 / Chapter 1.3.1.1 --- Derivation and characterization --- p.21 / Chapter 1.3.1.2 --- Biology of ES cells --- p.21 / Chapter 1.3.1.2.1 --- Pluripotency of ES cells --- p.21 / Chapter 1.3.1.2.2 --- Differentiation of ES cells to multiple lineages --- p.24 / Chapter 1.3.1.2.2.1 --- Ectodermal differentiation --- p.25 / Chapter 1.3.1.2.2.2 --- Mesodermal differentiation --- p.27 / Chapter 1.3.1.2.2.3 --- Endodermal differentiation --- p.28 / Chapter 1.3.2 --- Neural stem cells --- p.30 / Chapter 1.3.2.1 --- Derivation and characterization --- p.30 / Chapter 1.3.2.2 --- Biology of NS cells --- p.32 / Chapter 1.3.3 --- Induced pluripotent stem cells --- p.34 / Chapter 1.3.4 --- Mesenchymal stem cells --- p.35 / Chapter 1.4 --- Serotonin (5-HT) --- p.36 / Chapter 1.4.1 --- Distribution --- p.37 / Chapter 1.4.2 --- Metabolism --- p.37 / Chapter 1.4.3 --- Biological effects of 5-HT --- p.38 / Chapter 1.4.4 --- Serotonin receptor subtypes and receptor signal transduction pathways --- p.40 / Chapter Chapter2 --- Aim --- p.43 / Chapter 2.1 --- Hypothesis and study objectives --- p.43 / Chapter Chapter3 --- Materials and Methods --- p.49 / Chapter 3.1 --- Chemicals and Reagents --- p.49 / Chapter 3.1.1 --- Cell culture --- p.49 / Chapter 3.1.2 --- Serotonin, serotonin receptor subtypes specific agonists/antagonists and drugs that regulate serotonin metabolism --- p.51 / Chapter 3.1.3 --- Cell proliferation assay --- p.52 / Chapter 3.1.4 --- Cell apoptosis assay --- p.52 / Chapter 3.1.5 --- Immunohistochemistry and staining --- p.52 / Chapter 3.1.6 --- Western blotting --- p.55 / Chapter 3.1.7 --- Molecular biology --- p.56 / Chapter 3.1.8 --- Whole genome cDNA micro array --- p.58 / Chapter 3.1.9 --- MAO activity assay --- p.58 / Chapter 3.1.10 --- Endogenous ROS production assay --- p.58 / Chapter 3.2 --- Consumable --- p.58 / Chapter 3.3 --- Cells --- p.60 / Chapter 3.3.1 --- Feeder cell --- p.60 / Chapter 3.3.1.1 --- Mouse embryonic fibroblasts --- p.60 / Chapter 3.3.2 --- ES cells --- p.61 / Chapter 3.3.2.1 --- ES cell D3 --- p.61 / Chapter 3.3.2.2 --- ES cell-E14TG2a --- p.61 / Chapter 3.3.3 --- NS cells --- p.61 / Chapter 3.3.3.1 --- Neural progenitor cells line C172 --- p.61 / Chapter 3.3.3.2 --- Mouse embryonic neural stem cells --- p.61 / Chapter 3.4 --- In-house prepared solutions --- p.62 / Chapter 3.4.1 --- Stock solution ofInsulin, Transferrin, Selentine (ITS) Supplement --- p.63 / Chapter 3.4.2 --- Gelatin solution 01% --- p.62 / Chapter 3.4.3 --- Paraformaldehyde solution 4% (PFA) --- p.62 / Chapter 3.4.4 --- Tritox X-lOO solution 03% --- p.63 / Chapter 3.4.5 --- Popidium iodide solution 1 ug/ml (PI) --- p.63 / Chapter 3.4.6 --- Poly-L-ornithine solution --- p.63 / Chapter 3.4.7 --- Laminin solution --- p.64 / Chapter 3.4.7 --- MEF Maintenance medium --- p.64 / Chapter 3.4.9 --- Cryopreservation Media for MEF and C172 (2X) --- p.64 / Chapter 3.4.10 --- Cryopreservation Media for mouse ES cell (2X) --- p.65 / Chapter 3.4.11 --- Cryopreservation Media for mouse NS cell (2X) --- p.65 / Chapter 3.4.12 --- Serum based maintenance medium for C172 --- p.65 / Chapter 3.4.13 --- Serum free maintenance medium for C172 --- p.66 / Chapter 3.4.14 --- Serum-based propagation medium for ES cells --- p.66 / Chapter 3.4.15 --- Serum-free propagation medium forES cells --- p.67 / Chapter 3.4.16 --- Serum-free induction medium for ES cells --- p.67 / Chapter 3.4.16.1 --- Serum-free induction medium I --- p.67 / Chapter 3.4.16.2 --- Serum-free induction medium II --- p.68 / Chapter 3.4.16.3 --- Serum-free induction medium III --- p.68 / Chapter 3.4.17 --- Tris-HCl (1 M), pH 74 --- p.68 / Chapter 3.4.18 --- Tris-HCl (1 M), pH 87 --- p.69 / Chapter 3.4.19 --- Tris-HCI (1 M), pH 69 --- p.69 / Chapter 3.4.20 --- APS 10% (wt/vol) --- p.69 / Chapter 3.4.21 --- Protease inhibitor (10X) --- p.70 / Chapter 3.4.22 --- RIPA --- p.70 / Chapter 3.4.23 --- Resolving buffer (8X) --- p.70 / Chapter 3.4.24 --- Stacking buffer (4X) --- p.71 / Chapter 3.4.25 --- Protein running buffer (lOX) --- p.71 / Chapter 3.4.26 --- Transfer buffer (10X) --- p.72 / Chapter 3.4.27 --- Transfer buffer (IX) --- p.72 / Chapter 3.4.28 --- Blocking buffer (lOX) --- p.72 / Chapter 3.4.29 --- TBS (10X) --- p.73 / Chapter 3.4.30 --- TBS-T (IX) --- p.73 / Chapter 3.4.31 --- Stacking gel --- p.73 / Chapter 3.4.32 --- Resolving gel --- p.74 / Chapter 3.5 --- Methods --- p.75 / Chapter 3.5.1 --- Cell culture --- p.75 / Chapter 3.5.1.1 --- Preparation of acid washed cover slips --- p.75 / Chapter 3.5.1.2 --- Preparation of gelatinized culture wares --- p.75 / Chapter 3.5.1.3 --- Poly-L-omithine and laminin coating --- p.76 / Chapter 3.5.1.4 --- Thawing cryopreserved cells --- p.76 / Chapter 3.5.1.5 --- Passage of culture --- p.77 / Chapter 3.5.1.5 --- 6 Cell count --- p.78 / Chapter 3.5.1.7 --- Cytospin --- p.78 / Chapter 3.5.1.8 --- Trypan blue dye exclusion test --- p.78 / Chapter 3.5.1.9 --- Cryopreservation --- p.79 / Chapter 3.5.1.10 --- Derivation and culture of mouse embryonic fibroblasts (MEF) --- p.79 / Chapter 3.5.1.11 --- Propagation of ES cells in serum-based/free medium --- p.81 / Chapter 3.5.1.12 --- Neural differentiation ofES cells --- p.83 / Chapter 3.5.1.13 --- Propagation ofNP cell C172 in serum-based or serum-free medium --- p.84 / Chapter 3.5.1.14 --- Neural differentiation ofC172 --- p.85 / Chapter 3.5.1.15 --- Derivation and propagation of embryonic NS cells --- p.85 / Chapter 3.5.1.13 --- Neural differentiation of embryonic NS cells --- p.86 / Chapter 3.5.1.17 --- BrdU labeling of the ES cells derived products --- p.87 / Chapter 3.5.2 --- Cell proliferation assay --- p.87 / Chapter 3.5.2.1 --- Cell morphology --- p.87 / Chapter 3.5.2.2 --- WST-1 assay --- p.88 / Chapter 3.5.2.3 --- BrdU incorporation assay --- p.88 / Chapter 3.5.2.4 --- NCFC assay --- p.89 / Chapter 3.5.3 --- Conventional and quantitative RT-PCR --- p.89 / Chapter 3.5.3.1 --- RNA extraction --- p.89 / Chapter 3.5.3.2 --- RNA quantitation --- p.90 / Chapter 3.5.3.3 --- Reverse Transcription ofthe First Strand complementary DNA --- p.90 / Chapter 3.5.3.4 --- Polymerase chain reaction --- p.91 / Chapter 3.5.3.5 --- RNA Integrity Check --- p.91 / Chapter 3.5.3.6 --- Electrophoresis and visualization of gene products --- p.91 / Chapter 3.5.3.7 --- Real-time quantitative PCR --- p.92 / Chapter 3.5.4 --- Microarray --- p.94 / Chapter 3.5.5 --- Immunofluoresent staining --- p.94 / Chapter 3.5.6 --- Western blot --- p.95 / Chapter 3.5.6.1 --- Harvesting samples --- p.95 / Chapter 3.5.6.2 --- Protein extraction --- p.96 / Chapter 3.5.6.3 --- Protein quantification --- p.96 / Chapter 3.5.6.4 --- SDS-PAGE --- p.97 / Chapter 3.5.6.5 --- Wet transfer of protein to PVDF membrane --- p.97 / Chapter 3.5.6.6 --- Blocking the membrane --- p.97 / Chapter 3.5.6.7 --- Immunoblotting --- p.97 / Chapter 3.5.6.8 --- Signal detection --- p.98 / Chapter 3.5.7 --- Cell apoptosis assay --- p.98 / Chapter 3.5.7.1 --- ANNEXINV-FITC apoptosis detection --- p.98 / Chapter 3.5.7.2 --- TUNEL --- p.99 / Chapter 3.5.8 --- Endogenous ROS assay --- p.100 / Chapter 3.5.9 --- In vivo studies --- p.101 / Chapter 3.5.9.1 --- Induction of cerebral ischemia in mice --- p.101 / Chapter 3.5.9.2 --- Transplantation --- p.101 / Chapter 3.5.9.3 --- Assessment of learning ability and memory --- p.102 / Chapter 3.5.10 --- Histological analysis --- p.103 / Chapter 3.5.10.1 --- Animal sacrifice for brain harvest --- p.103 / Chapter 3.5.10.2 --- Cryosectioning --- p.103 / Chapter 3.5.10.3 --- Haematoxylin and eosin staining --- p.104 / Chapter 3.6 --- Data analysis --- p.104 / Chapter Chapter4 --- Results --- p.113 / Chapter 4.1 --- Expression profile of 5-HT receptors and metablism of endogenous 5-HT --- p.113 / Chapter 4.1.1 --- Expression profiles of 5-HT receptors in stem cells --- p.113 / Chapter 4.1.2 --- Biosynthesis of endogenous 5-HT --- p.115 / Chapter 4.2 --- Effects of 5-HT on proliferation of mouse ES cells and NS cells --- p.115 / Chapter 4.2.1 --- Effects of 5-HT on proliferation ofES cells --- p.115 / Chapter 4.2.2 --- Effects of 5-HT on proliferation ofNP and NS cells --- p.117 / Chapter 4.3 --- Effects of 5-HT on differentiation of mouse ES cells and NS cells --- p.119 / Chapter 4.3.1 --- Neural differentiation ofES cells --- p.119 / Chapter 4.3.2 --- Effects of 5-HT on differentiation ofES cells --- p.119 / Chapter 4.3.3 --- Neural differentiation ofNP and NS cells --- p.120 / Chapter 4.3.4 --- Effects of 5-HT on differentiation ofNP and NS cells --- p.121 / Chapter 4.4 --- 5-HT metabolism in mouse ES cells and NS cells --- p.122 / Chapter 4.4.1 --- Expression of key 5-HT metablic genes in stem cells --- p.122 / Chapter 4.4.2 --- Detection ofROS generation in mouse NS cells --- p.123 / Chapter 4.4.3 --- Effects of 5-HT on expression level of MAO-A, MAO-B and SERT --- p.123 / Chapter 4.5 --- Anti-apoptotic effect of 5-HT on NP and NS cells in neural induction --- p.127 / Chapter 4.6 --- Potential signaling pathways mediated by 5-HT --- p.130 / Chapter 4.7 --- Therapeutic effects of 5-HT treated mouse ES cell-derived cells in a stoke model --- p.130 / Chapter 4.7.1 --- Induction of global ischemia by transient BCCAO --- p.130 / Chapter 4.7.1.1 --- HE staining of post ischemic brain --- p.131 / Chapter 4.7.1.2 --- TUNEL analysis of cell apoptosis at post ischemia day 3 --- p.132 / Chapter 4.7.2 --- Cell labelling --- p.132 / Chapter 4.7.3 --- Cognition monitoring post transplantation --- p.133 / Chapter 4.7.4 --- Survival, migration and differentiation of transplanted neural cells --- p.135 / Chapter Chapter5 --- Discussion --- p.180 / Chapter Chapter6 --- Conclusions --- p.192 / References --- p.195

Professional research project (Au.D.)--University of South Florida, 2003. / Title from PDF of title page. / Document formatted into pages; contains 20 pages. / Includes bibliographical references. / Text (Electronic thesis) in PDF format. / ABSTRACT: The purpose of this study was to determine the effects of an atypical pattern of early acoustic stimulation on auditory development. Previous human research suggests that the acoustic environment of pre-term human infants in the Neonatal Intensive Care Unit (NICU) negatively affects some aspects of auditory development. Animal research suggests that premature auditory stimulation interrupts auditory development. Because mice are born before their auditory systems are developed, they make an excellent model for research on fetal and postnatal plasticity of the auditory system. The premature auditory state of newborn mice is similar to that of the NICU pre-term infant, albeit, natural for mice C57 mouse pups were exposed to an augmented acoustic environment (AAE) of a nightly 12-hour regiment of 70 dB SPL noise burst, beginning before age 12 days (onset of hearing) and lasting for one month. / ABSTRACT: The prepulse inhibition (PPI) of mice exposed to the AAE was compared to that of non-exposed mice to observe short-term and long-term effects. Results showed that the prepulse inhibition of the AAE exposed mice did not differ significantly from that of the non-exposed mice. However, it is possible that the measurement used, PPI, may not have been appropriate or that the AAE may not have been an appropriate simulation of the NICU environment. / System requirements: World Wide Web browser and PDF reader. / Mode of access: World Wide Web.

Chemotherapy persists as one of the mainstays of breast cancer treatment, particularly for triple-negative breast cancer which currently has no targeted treatment methods. While chemotherapy is beneficial for killing the malignant tumor cells, it leads to the release of damage-associated molecular patterns into the tumor microenvironment. Damage-associated molecular patterns are a contributing factor to cancer-related inflammation which can potentiate metastatic spread through several mechanisms such as the development of tumor microenvironments at metastastic sites. These damage-associated molecular patterns include nucleic acids, nucleic acid-associated lipids and vesicles, cytokines, and proteins such as high mobility group protein B1. Polyamidoamine (PAMAM) is a biodegradable, water-soluble dendrimer polymer with the ability to possess different charges and sizes depending on its terminal branches and degree of branching (i.e. generation number), respectively. Amine-terminated PAMAM-NH2 is positively charged and can bind to circulating DNA and RNA. Since most DAMP molecules are negatively charged, I hypothesized that a polycation such as PAMAM-NH2 would be an efficient nanomaterial to remove pathogenic NA DAMPs generated by chemotherapy. Building on this dendrimer, we synthesized modified cationic PAMAM-generation 3 derivatives with an aim to balance toxicity with NA-binding affinity and capacity to encapsulate chemodrugs. Our results found that these soluble and nanoparticle PAMAM materials can bind to both cell-free DNA and RNA released as a result of treating triple-negative breast cancer cells with chemotherapy drugs such as doxorubicin and paclitaxel. These PAMAM-G3 materials are termed as nucleic acid binding polymers and nucleic-acid binding polymeric nanoparticles.My thesis dissertation explores the anti-metastatic effects of nucleic-acid binding polymeric nanoparticles delivering the chemotherapy drug paclitaxel using in-vitro and in-vivo models. Two murine metastatic breast cancer models served as the basis for assessing the effects of conventional paclitaxel delivery compared to paclitaxel delivery from within PAMAM nucleic-acid binding polymeric nanoparticles with respect to primary tumor growth, extent of lung metastasis, and the systemic inflammatory response reflected in murine serum. Compared to treatment with unencapsulated paclitaxel, delivery of paclitaxel within the PAMAM nucleic-acid binding polymeric nanoparticles resulted in significantly decreased serum cell-free DNA levels, decreased inflammatory cytokines, and a lower degree of lung metastasis in the mice. The decrease in the degree of lung metastasis in mice receiving paclitaxel within the PAMAM nanoparticles was confirmed by assessing the photon flux signal of 4T1-luciferase breast cancer cells invading the murine lungs in both in-vivo and ex-vivo imaging and by using a machine learning method to quantify the degree of metastasis in H&E- stained sections of the lungs. The ability to mitigate the phenomenon of chemotherapy-induced cancer metastasis while effectively delivering the chemotherapy to the tumor microenvironment could help improve the outcomes of patients being treated with chemotherapy. This work developed a therapeutic cationic PAMAM nanocarrier-based strategy to inhibit paclitaxel-induced metastasis by scavenging cell-free nucleic acids and mitigating cell-free nucleic acid-induced inflammation.

Indiana University-Purdue University Indianapolis (IUPUI) / The present study sought to identify the effects of chronic lithium administration on ethanol intakes in high alcohol-preferring (HAP) mice. Lithium is a well-established treatment for bipolar disorder and has demonstrated efficacy in reducing impulsivity, an endophenotype of the disease. Impulsivity is also a prominent trait of alcoholism. HAP mice display a preference for consuming substantial amounts of ethanol and exhibit abnormally high levels of impulsivity. Previous work has determined that chronic lithium exposure in HAP mice reduces their levels of impulsivity. The present study analyzed fluctuations in established intake patterns after lithium exposure and how pre-exposure to lithium would affect ethanol intake acquisition. The results showed an increase in ethanol intake and no change in preference for ethanol over water in lithium treated mice. There was an increase in overall total fluid consumption in these mice, likely resulting from polydipsic effects. There also appeared to be a potentiated lithium toxicity effect found in those mice pre-exposed to lithium. The conclusion was that lithium therapy does not decrease ethanol consumption in HAP mice.

Indiana University-Purdue University Indianapolis (IUPUI) / Background: Group II metabotropic glutamate receptors (mGluR2/3) are predominately presynaptically located Gi/o coupled receptors that are highly expressed in the cortex, nucleus accumbens, amygdala, and hippocampus. Previous studies suggest that group II mGluRs are involved in regulating ethanol (EtOH) consumption and seeking following extinction (Backstrom and Hyytia, 2005; Kufahl, et al., 2011). The sipper tube model, which allows for procedural separation of seeking and consumption, was used to further clarify the role of mGluR2/3 in EtOH-seeking and consumption. The non-selective group II mGluR agonist LY379268 (LY37) and selective mGluR2 positive allosteric modulator (PAM) BINA were used to determine the relative contribution of mGlu2 and mGlu3 receptors on EtOH seeking and consumption. Following characterization of the agonist and PAM on EtOH reinforcement, a microinjection study was performed examining the effect of blockade of nucleus accumbens core mGluR2/3 on systemic agonist induced suppression of EtOH-seeking. Methods: For the systemic agonist/PAM experiments, separate groups of male Wistar rats [n=8-9 group; LY37 (0-2.0 mg/kg) and BINA (0-20 mg/kg)] were trained to complete a response requirement (RR) of 10 lever presses that resulted in access to 10% EtOH or 2% sucrose (in separate groups) for a 20-minute drinking period. For consummatory testing, animals received weekly drug injections with a RR1. The RR was then increased over sessions to a RR20. For appetitive testing, animals received weekly drug injections followed by a non-reinforced extinction session. To determine effects of blockade of NAc core mGluR2/3 receptors on agonist-induced suppression of EtOH-seeking, a separate group of male Wistar rats (n=15) was trained to complete a RR10 for access to 10% EtOH. Animals were surgically implanted with bilateral guide cannulae terminating 1mm above the NAc core. Following recovery, animals received four sets of microinjections in a balanced design (systemic vehicle + core vehicle, systemic LY37 + core vehicle, systemic LY37 + core LY34, and systemic vehicle + core LY34). A final non-balanced microinjection of LY37 was then performed. Results and Conclusions: Systemic administration of the mGluR2/3 agonist LY37 significantly reduced EtOH- and sucrose- seeking with no systematic effect on locomotion. Systemic administration of the selective mGluR2 PAM BINA had no significant effect on either seeking or consumption. These findings suggest that modulation of glutamatergic neurotransmission by a systemic mGluR2/3 agonist, but not allosteric modulation of mGluR2, significantly reduces reinforcer seeking. Intra- accumbens core administration of LY37 significantly reduced EtOH-seeking, suggesting a role of NAc core mGluR2/3 modulation in EtOH-seeking during maintenance drinking. Systemic administration of LY37 was also found to significantly reduce sucrose consumption and body weight 24-hours following systemic administration, meriting further examination of the role of mGluR2/3 receptors on feeding behavior.

Indiana University-Purdue University Indianapolis (IUPUI) Indiana University School of Medicine / The islet β cell is central to the maintenance of glucose homeostasis as the β cell is solely responsible for the synthesis of Insulin. Therefore, better understanding of the molecular mechanisms governing β cell function is crucial to designing therapies for diabetes. Pdx1, the master transcription factor of the β cell, is required for the synthesis of proteins that maintain optimal β cell function such as Insulin and glucose transporter type 2. Previous studies showed that Pdx1 interacts with the lysine methyltransferase Set7/9, relaxing chromatin and increasing transcription. Because Set7/9 also methylates non-histone proteins, I hypothesized that Set7/9-mediated methylation of Pdx1 increases its transcriptional activity. I showed that recombinant and cellular Pdx1 protein is methylated at two lysine residues, Lys123 and Lys131. Lys131 is involved in Set7/9 mediated augmented transactivation of Pdx1 target genes. Furthermore, β cell-specific Set7/9 knockout mice displayed glucose intolerance and impaired insulin secretion, accompanied by a reduction in the expression of Pdx1 target genes. Our results indicate a previously unappreciated role for Set7/9 in the maintenance of Pdx1 activity and β cell function. β cell function is regulated on both the transcriptional and translational levels. β cell function is central to the development of type 1 diabetes, a disease wherein the β cell is destroyed by immune cells. Although the immune system is considered the primary instigator of the disease, recent studies suggest that defective β cells may initiate the autoimmune response. I tested the hypothesis that improving β cell function would reduce immune infiltration of the islet in the NOD mouse, a mouse model of spontaneous type 1 diabetes. Prediabetic NOD mice treated with pioglitazone, a drug that improves β cell function, displayed an improvement in β cell function, a reduction in β cell death, accompanied by reductions in β cell autoimmunity, indicating that β cell dysfunction assists in the development of type 1 diabetes. Therefore, understanding the molecular mechanisms involved in β cell function is essential for the development of therapies for diabetes.

Indiana University-Purdue University Indianapolis (IUPUI)

Indiana University-Purdue University Indianapolis (IUPUI) / The immune system protects the body from foreign organisms. T cells and B cells are integral components of the ability of the immune system to generate focused immune responses. The development of specialized subsets of T helper cells is governed by transcription factors. Previous work demonstrated a requirement for the transcription factor PU.1 in the development of IL-9-secreting Th9 cells. Work in this dissertation demonstrates that the Th9 subset is not stable in vitro, and that PU.1 expression decreases during long-term culture. To examine a role for PU.1 in Th9-independent immunity we examined a model of multiple sclerosis termed experimental autoimmune encephalomyelitis (EAE). Mice that lack PU.1 expression in T cells (Sfpi1lck-/- mice) demonstrated more severe disease with attenuated recovery compared to control mice, and this was accompanied by an increase of T cells in the central nervous system. We also observed that following multiple routes of immunization Sfpi1lck-/- mice had increased numbers of T follicular helper (Tfh) cells and increased germinal center responses. This correlated with increased expression of the cytokine IL-21 and the surface protein CD40L in T cells that lacked PU.1 expression and resulted in increased numbers of germinal center B cells and antigen-specific antibody titers compared to control mice. The increased germinal center B cells and antibody titers were attenuated with blocking CD40L antibody but not with neutralizing IL-21 antibody. These results suggest that PU.1 limits the expression of CD40L on Tfh cells to regulate the humoral immune response. Together, the data in this dissertation demonstrate Th9-independent functions of PU.1. Moreover, this work shows that transcription factors promoting the development of one subset of T helper cells can simultaneously have negative effects on distinct T cell lineages.

Indiana University-Purdue University Indianapolis (IUPUI) / CXXC finger protein 1 (Cfp1), a component of the Set1 histone methyltransferase complex, is a critical epigenetic regulator of both histone and cytosine methylation. Murine embryos lacking Cfp1 are unable to gastrulate and Cfp1-null embryonic stem (ES) cells fail to undergo cellular differentiation in vitro. However, expression of wild type Cfp1 in Cfp1-null ES cells rescues differentiation capacity, suggesting that dynamic epigenetic changes occurring during lineage specification require Cfp1. The domain structure of Cfp1 consists of a DNA binding CXXC domain and an N-terminal plant homeodomain (PHD). PHDs are frequently observed in chromatin remodeling proteins, functioning as reader modules for histone marks. However, the histone binding properties and underlying functional significance of Cfp1 PHD are largely unknown. My research revealed that Cfp1 PHD directly and specifically binds to histone H3K4me1/me2/me3 marks. A point mutation that abolishes binding to methylated H3K4 (W49A) does not affect rescue of cellular differentiation, but, point mutations that abolish both methylated H3K4 (W49A) and DNA (C169A) binding result in defective in vitro differentiation, indicating that PHD and CXXC exhibit redundant functions. The mammalian liver has the unique ability to regenerate following injury. Previous studies indicated that Cfp1 is essential for hematopoiesis in zebrafish and mice. I hypothesized that Cfp1 additionally plays a role in liver development and regeneration. To understand the importance of Cfp1 in liver development and regeneration, I generated a mouse line lacking Cfp1 specifically in the liver (Cfp1fl/fl Alb-Cre+). Around 40% of these mice display a wasting phenotype and die within a year. Livers of these mice have altered global H3K4me3 levels and often exhibit regenerative nodules. Most importantly, livers of these mice display an impaired regenerative response following partial hepatectomy. Collectively, these findings establish Cfp1 as an epigenetic regulator essential for ES cell function and liver homeostasis and regeneration.