Adult neural stem cells are specialized astrocytes that generate neurons in restricted regions of the mammalian brain. The largest neurogenic region is the ventricular-subventricular zone, which lines the lateral ventricles and generates olfactory bulb neurons. Stem cell astrocytes give rise to new neurons in both homeostatic and regenerative conditions, suggesting that they can potentially be harnessed for regenerating the brain after injury, stroke, or neurodegenerative disease. Previous work has shown that stem cell astrocytes exist in both quiescent and activated states, but due to a lack of markers, it was not feasible to purify them. Using a novel fluorescence activated cell sorting (FACS) strategy that allows quiescent neural stem cells (qNSCs) and activated neural stem cells (aNSCs) to be purified for the first time, we performed transcriptome profiling to illuminate the molecular pathways active in each population. This analysis revealed that qNSCs are enriched in signaling pathways, especially G-protein coupled receptors, as well as for adhesion molecules, which facilitate interactions with the niche. qNSCs and aNSCs utilize different metabolic pathways. qNSCs are enriched for lipid and glycolytic metabolism, while aNSCs are enriched for DNA, RNA, and protein metabolism. Many receptors and ligands are reciprocally distributed between qNSCs and aNSCs, suggesting that they may regulate each other. Finally, comparison of the transcriptomes of qNSCs and aNSCs with their counterparts in other organs revealed that pathways underlying stem cell quiescence are shared across diverse tissues. A key step in recruiting adult neural stem cells for brain repair is to define the molecular pathways regulating their switch from a quiescent to an activated state. MicroRNAs are small non-coding RNAs that simultaneously target hundreds of mRNAs for degradation and translational repression. MicroRNAs have been implicated in stem cell self-renewal and differentiation. However, their role in adult neural stem cell activation is unknown. We performed miRNA profiling of FACS-purified quiescent and activated adult neural stem cells to define their miRNA signatures. Bioinformatic analysis identified the miR-17~92 cluster as highly upregulated in activated (actively dividing) stem cells in comparison to their quiescent counterparts. Conditional deletion of the miR-17~92 cluster in FACS purified neural stem cells in vitro reduced adult neural stem cell activation, proliferation, and self-renewal. In addition, miR-17~92 deletion led to a selective decrease in neuronal differentiation. Using an in vivo conditional deletion model, we showed that loss of miR-17~92 led to an increase in the proportion of GFAP+ cells and decrease in MCM2+ cells, as well as decreased neurogenesis. Finally, I identify Sphingosine 1 phosphate receptor 1 (S1pr1) as a computationally predicted target of the miR-17~92 cluster. S1pr1 is highly enriched in quiescent neural stem cells. Treatment of quiescent neural stem cells with S1P, the ligand for S1PR1, reduced their activation and proliferation. In vivo deletion of miR-17~92 lead to an increase in S1PR1+ cells, even among MCM2+ cells. Together, these data reveal that the miR-17~92 cluster is a key regulator of adult neural stem cell activation from the quiescent state and subsequent proliferation.

<p> While it is clear that thermodynamics plays a nontrivial role in biological processes, exactly how this affects the macroscopic structuring of living systems is not fully understood. Thus, the objective of this dissertation was to investigate how thermodynamic variables such as exergy, entropy, and information are involved in biological processes such as cellular metabolism, ecological succession, and evolution. To this end, I have used a combination of mathematical modelling, <i>in silico</i> simulation, and both laboratory- and field-based experimentation. </p><p> To begin the dissertation, I review the basic tenets of biological thermodynamics and synthesize them with modern fluctuation theory, information theory, and finite time thermodynamics. In this review, I develop hypotheses concerning how entropy production rate changes across various time scales and exergy inputs. To begin testing these hypotheses I utilized a stochastic, agent-based, mathematical model of ecological evolution, The Tangled Nature Model. This model allows one to observe the dynamics of entropy production over time scales that would not be possible in real biological systems (i.e., 10⁶ generations). The results of the model&rsquo;s simulations demonstrate that the ecological communities generated by the model&rsquo;s dynamics have increasing entropies, and that this leads to emergent order, organization, and complexity over time. To continue to examine the role of thermodynamics in biological processes I investigated the bioenergetics of marine microbes associated with benthic substrates on coral reefs. By utilizing both mesocosm and <i> in situ</i> experiments I have shown that these microbes change their power output, oxygen uptake, and community structure depending upon their available exergy. </p><p> Overall, the data presented herein demonstrates that ecological structuring and evolutionary change are, at least in part, determined by underlying thermodynamic mechanisms. Recognizing how physical processes affect biological dynamics allows for a more holistic understanding of biology at all scales from biochemistry, to ecological succession, and even long-term evolutionary change.</p><p>

<p> The major barrier to curing HIV-1 infection is latency. HIV-1 latent cells are those in which the viral genome has been integrated into the host cell genome but the virus does not produce the primary infectious agents, viral RNA and proteins. Latency can occur when the virus directly infects long-lived memory CD4+ T cells or infects active CD4+ T cells that have the potential to become memory T cells. The virus persists inside those cells as long as they are alive. This dormancy provides a reservoir of HIV-1 virus in memory T cells, which can cause infection relapse whenever antiretroviral therapy (ART) is discontinued. The situation is further complicated by the fact that multiple reservoirs of HIV-1 virus can be established at early stages of infection. </p><p> The major reservoir lies in the CD4+ T cells present in blood, lymph nodes and the spleen. Unfortunately, ART fails to target hidden HIV-1 virus that persists in resting T-cells. Furthermore, life-long ART use increases the chances that mutant virus will develop which will be resistant to continued therapy. Therefore, various studies have explored mechanisms to eradicate the latent HIV-1 reservoir. One proposed strategy to target this reservoir is known as &ldquo;shock and kill&rdquo;. The proposed shock and kill strategy initially &ldquo;shocks&rdquo; the HIV-1 virus out of latency with latency reversing agents (LRAs). The reactivated virus can then be controlled by ART and cytotoxic CD8+ T cells (CTLs), which kill the infected cells. Despite the great findings regarding reactivating HIV-1 latency <i>in vitro</i> and <i>ex vivo</i>, tested LRAs proved unsuccessful in reactivating HIV-1 virus in clinical trials. </p><p> Different factors can contribute to establishment of HIV-1 latency and different reservoirs in different immune cells and tissues are established early after HIV-1 infection. Therefore, synergy between multiple LRAs should be sought and studied for successful reactivation of the latent viral pool. </p><p> Runt Related Transcription Factor 1 (RUNX1) is a key transcription factor that is important during T cell development and has been shown to recruit different transcription factors in a context dependent manner. It has been shown to be involved in repressing various genes and it also interacts with chromatin modifiers that can alter the landscape of the chromatin and modify its compaction. Our lab has shown that there is a putative RUNX1 binding site on HIV-1 long terminal repeats (LTR) and the transfection of RUNX1 can suppress HIV-1 transcription. In addition, our lab has shown that the benzodiazepine, RO5-3335, which pharmacologically inhibits RUNX1, synergizes with vorinostat (SAHA), an HDAC inhibitor to reactivate latent HIV-1. </p><p> Using DNA cloning, an HIV-1 virus with a mutated RUNX1 binding site was constructed. Then, replication, infectivity and fitness of the mutated virus were examined and compared to a control virus using ELISA, RT, PCR, and TA cloning techniques. We have found that this mutated virus replicates faster and has more fitness and infectivity than the control virus with an intact RUNX1 binding site. Our results show that inhibition of RUNX1 binding to HIV-1 3&rsquo; long terminal repeat (LTR) positively affects viral replication and infectivity. This suggests that RUNX1 host transcription factor suppresses HIV-1 replication through its transcriptional repressor function and it possibly contributes to establishment of latency. </p><p> We screened clinically prescribed benzodiazepines (BDZs) to identify reactivators for latent HIV-1 virus. Using flow cytometry, we have found most of these BDZs synergized with SAHA in reactivation of latent HIV-1. Unlike the other BDZs tested, alprazolam was able to reactivate HIV-1 even when not in combination with SAHA. The effect of alprazolam on RUNX1 responsive genes was further investigated using qPCR. Alprazolam was found to affect RUNX1 responsive genes similarly to RO5-3335, a known RUNX1 inhibitor. The effect of alprazolam on IFN&gamma; and TNF&alpha; that are produced from cytotoxic T cells (CTLs) was also examined. Alprazolam enhanced CTL function that was shown in the literature to be attenuated by SAHA. Thus, alprazolam successfully reverses HIV-1 latency and decreases the side effects of SAHA on CTL function when used in combination. </p><p>

Chu Wai-ling. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 106-114). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese) --- p.iii / Acknowledgments --- p.v / Contents --- p.vi / List of Tables --- p.ix / List of Figures --- p.x / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.8 / Chapter 2.1 --- Introduction to phylogenetic biology --- p.8 / Chapter 2.1.1 --- Definition of phylogenetics --- p.8 / Chapter 2.1.2 --- Why employ molecular genetic markers in phylogenetics? --- p.8 / Chapter 2.2 --- DNA analysis and the contributions to phylogenetics --- p.10 / Chapter 2.2.1 --- Historical development of DNA analysis in phylogenetics --- p.10 / Chapter 2.2.2 --- Nuclear ribosomal DNA (rDNA) --- p.12 / Chapter 2.2.3 --- Animal mitochondrial DNA (mt DNA) --- p.14 / Chapter 2.3 --- Molecular phylogeny of crustaceans --- p.16 / Chapter 2.3.1 --- Phylogenetic studies of crustaceans using nuclear ribosomal DNA --- p.16 / Chapter 2.3.2 --- Phylogenetic studies of crustaceans using mitochondrial DNA --- p.17 / Chapter 2.4 --- Taxonomy of the genus Heterocarpus --- p.22 / Chapter Chapter 3 --- Materials and Methods --- p.36 / Chapter 3.1 --- Collection and storage of specimens --- p.36 / Chapter 3.2 --- DNA extraction --- p.36 / Chapter 3.3 --- Amplification of mitochondrial genes --- p.38 / Chapter 3.3.1 --- PCR profile --- p.39 / Chapter 3.3.1.1 --- 16SrRNA gene --- p.39 / Chapter 3.3.1.2 --- COI gene --- p.42 / Chapter 3.3.1.2.1 --- Amplification of COI gene segments using primers LCD1490/HCO2198 --- p.42 / Chapter 3.3.1.2.2 --- Amplification of COI gene segments using primers COIf/COIa and COIp3/COIa --- p.43 / Chapter 3.4 --- DNA sequencing --- p.44 / Chapter 3.4.1 --- Purification of extension products --- p.45 / Chapter 3.4.2 --- Electrophoresis and data collection --- p.46 / Chapter 3.5 --- Data analysis --- p.47 / Chapter Chapter 4 --- Results --- p.50 / Chapter 4.1 --- PCR products of 16S rRNA and COI genes --- p.50 / Chapter 4.2 --- Genetic variability in Heterocarpus based on partial DNA sequence of 16S rRNA gene --- p.52 / Chapter 4.3 --- Genetic variability in Heterocarpus based on COI gene --- p.61 / Chapter 4.3.1 --- Genetic variability in Heterocarpus based on partial DNA sequence of COI gene --- p.61 / Chapter 4.3.2 --- Genetic variability in Heterocarpus based on amino acid sequence of COI --- p.69 / Chapter 4.4 --- Phylogenetic analysis --- p.75 / Chapter 4.4.1 --- Phylogenetic analysis based on 16S rDNA sequence --- p.75 / Chapter 4.4.2 --- Phylogenetic analysis based on DNA sequence of COI gene --- p.80 / Chapter 4.4.3 --- Phylogenetic analysis based on amino acid sequence of COI --- p.84 / Chapter 4.5 --- Kishino-Hasegawa and Shimodaira-Hasegawa tests --- p.86 / Chapter Chapter 5 --- Discussion --- p.90 / Chapter 5.1 --- Examination on the validity of the four Heterocarpus complexes --- p.90 / Chapter 5.2 --- Phylogenetic relationship of Heterocarpus species within each complex --- p.91 / Chapter 5.2.1 --- Phylogenetic relationship of Heterocarpus species within H.gibbosus complex --- p.92 / Chapter 5.2.2 --- Phylogenetic relationship of Heterocarpus species within H.woodmasoni complex --- p.94 / Chapter 5.2.3 --- Phylogenetic relationship of Heterocarpus species within H. ensifer and H. sibogae complexes --- p.96 / Chapter 5.3 --- Phylogenetic relationship among Heterocarpus complexes --- p.98 / Chapter 5.4 --- "Comparisons of phylogenetic resolving power of 16S rRNA, COI and 28S rRNA genes" --- p.100 / Chapter Chapter 6 --- Conclusions --- p.104 / Literature Cited --- p.106

Cheng Tat Cheung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 106-114). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of content --- p.iii / Abbreviations --- p.iv / List of figures --- p.v / List of tables --- p.vi / Chapter Chapter One --- Introduction / Chapter 1.1 --- Ribozyme --- p.1 / Chapter 1.1.1 --- RNA world hypothesis --- p.2 / Chapter 1.1.2 --- Hammerhead ribozyme --- p.3 / Chapter 1.1.3 --- Applications of hammerhead ribozymes --- p.4 / Chapter 1.1.4 --- Allosteric ribozyme --- p.4 / Chapter 1.1.5 --- Ribozyme screening system --- p.5 / Chapter 1.2 --- Other RNA as gene silencing agents --- p.7 / Chapter 1.2.1 --- RNAi --- p.7 / Chapter 1.2.2 --- Antisense RNA --- p.10 / Chapter 1.3 --- Project Overview --- p.11 / Chapter 1.3.1 --- Construction of anti-GFP ribozymes and their in vitro and in vivo studies --- p.11 / Chapter 1.3.2 --- Construction of anti-Elfin ribozyme and its application on gene silencing study --- p.11 / Chapter Chapter Two --- Materials and Methods / Chapter 2.1 --- Cloning techniques --- p.13 / Chapter 2.1.1 --- Polymerase Chain Reaction (PCR) --- p.13 / Chapter 2.1.2 --- Restriction digestion of DNA --- p.13 / Chapter 2.1.3 --- Ligation of DNA fragments --- p.14 / Chapter 2.1.4 --- Preparation of competent cells --- p.15 / Chapter 2.1.5 --- Transformation of competent cells --- p.16 / Chapter 2.1.6 --- Gel extraction --- p.16 / Chapter 2.1.7 --- Plasmid preparation --- p.17 / Chapter 2.1.7.1 --- Mini scale plasmid preparation --- p.17 / Chapter 2.1.7.2 --- Medium scale plasmid preparation --- p.19 / Chapter 2.1.8 --- DNA agarose gel electrophoresis --- p.20 / Chapter 2.1.9 --- Buffer and reagents --- p.21 / Chapter 2.2 --- In vitro cleavage of target RNA by ribozymes --- p.22 / Chapter 2.2.1 --- In vitro transcription of target RNA and ribozymes --- p.22 / Chapter 2.2.2 --- Purification of transcription products --- p.22 / Chapter 2.2.3 --- Ribozymatic cleavage reaction --- p.24 / Chapter 2.2.4 --- Preparation of RNA size marker templates --- p.24 / Chapter 2.2.5 --- Urea-acrylamide gel electrophoresis --- p.25 / Chapter 2.2.6 --- Autoradiography --- p.26 / Chapter 2.2.7 --- Buffer and reagents --- p.26 / Chapter 2.3 --- Detection of cellular RNA expression RT-PCR detection --- p.28 / Chapter 2.3.1 --- RNA extraction --- p.28 / Chapter 2.3.2 --- DNase I digestion --- p.29 / Chapter 2.3.3 --- Reverse transcription --- p.29 / Chapter 2.4 --- Mammalian cell culture techniques --- p.31 / Chapter 2.4.1 --- Transfection into mammalian cells --- p.31 / Chapter 2.4.2 --- Counting the number of cells --- p.32 / Chapter 2.4.2 --- Buffer and reagents --- p.32 / Chapter Chapter Three --- Construction of anti-GFP ribozymes and their in vitro and in vivo studies / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.1.1 --- Objectives --- p.34 / Chapter 3.1.2 --- Why anti-GFP ribozymes? --- p.34 / Chapter 3.2 --- Construction of anti-GFP ribozymes that are active in vitro --- p.36 / Chapter 3.2.1 --- Design of the anti-GFP ribozymes --- p.36 / Chapter 3.2.2 --- Construction of DNA templates for in vitro transcription --- p.40 / Chapter 3.2.3 --- GFP RNA was successfully cleaved by ribozymes --- p.45 / Chapter 3.3 --- In vivo activities of anti-GFP ribozymes --- p.48 / Chapter 3.3.1 --- Design of systems that detected anti-GFP ribozyme activities in --- p.48 / Chapter 3.4 --- The first trial - system α --- p.50 / Chapter 3.4.1 --- Cloning of ribozymes into pACYC184 --- p.51 / Chapter 3.4.2 --- IPTG interfered with GFP expression --- p.54 / Chapter 3.5 --- The second trial - system β --- p.57 / Chapter 3.5.1 --- Insertion of new multiple cloning sites into pET3d --- p.58 / Chapter 3.5.2 --- Cloning of GFP into pET-neu --- p.60 / Chapter 3.5.3 --- GFP expression was interfered by the additional T7 promoter --- p.62 / Chapter 3.6 --- The third trial - system δ --- p.65 / Chapter 3.6.1 --- Insertion of a new cloning site into pET-neu --- p.66 / Chapter 3.6.2 --- Cloning of GFP and ribozymes into pET-fn --- p.68 / Chapter 3.6.3 --- Ribozymes sequence upstream of GFP interfered with GFP expression / Chapter 3.7 --- The fourth trial - system co --- p.73 / Chapter 3.7.1 --- Insertion of new cloning sites into pET-neu --- p.74 / Chapter 3.7.2 --- Cloning of GFP and ribozymes into pET-nr --- p.76 / Chapter 3.7.3 --- Anti-GFP ribozymes did not turn off green fluorescence of GFP --- p.79 / Chapter 3.7.4 --- No in vivo cleavage of GFP was detected --- p.81 / Chapter 3.8 --- Summary --- p.83 / Chapter Chapter Four --- Construction of an anti-Elfin ribozyme and its application on gene silencing study / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.1.1 --- Objectives --- p.84 / Chapter 4.1.2 --- Elfin --- p.84 / Chapter 4.1.3 --- Experimental plan --- p.85 / Chapter 4.2 --- In vitro cleavage of Elfin RNA by ribozyme --- p.86 / Chapter 4.2.1 --- Design of anti-Elfin ribozyme --- p.86 / Chapter 4.2.2 --- Preparation of DNA template for in vitro transcription --- p.88 / Chapter 4.2.3 --- Successful in vitro cleavage of Elfin RNA by ribozyme --- p.88 / Chapter 4.3 --- In vivo gene silencing studies of RNA tools --- p.90 / Chapter 4.3.1 --- Design of antisense RNA --- p.90 / Chapter 4.3.2 --- Design of siRNA --- p.92 / Chapter 4.3.3 --- Cloning of RNA tools into pSilencer --- p.94 / Chapter 4.3.4 --- Cloning of a neomycin resistance gene into pSilencer-R --- p.94 / Chapter 4.3.5 --- Elfin RNA was not down-regulated --- p.97 / Chapter 4.4 --- Summary --- p.100 / Chapter Chapter 5 --- Discussions --- p.101 / Reference / Appendix

Lo Sze Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 108-115). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract (in English) --- p.vi / Abstract (in Chinese) --- p.viii / Table of content --- p.x / List of tables --- p.xv / List of figures --- p.xvi / List of abbreviations --- p.xix / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The secretory pathway --- p.2 / Chapter 1.1.1 --- Endoplasmic reticulum --- p.2 / Chapter 1.1.2 --- Golgi complex --- p.3 / Chapter 1.1.3 --- Vacuoles --- p.3 / Chapter 1.1.4 --- Prevacuolar compartment --- p.4 / Chapter 1.2 --- The secretory pathway in plant cells --- p.5 / Chapter 1.2.1 --- The secretory pathway in yeast and mammalian cells --- p.7 / Chapter 1.2.2 --- The lytic pathway in plant cells --- p.8 / Chapter 1.2.3 --- The protein storage vacuole pathway in plant cells --- p.10 / Chapter 1.3 --- Dynamic studies between organelles --- p.12 / Chapter 1.4 --- Objectives of this thesis research --- p.13 / Chapter Chapter 2 --- Development of Transgenic Cell Lines Expressing PVC and Golgi Markers --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.1.1 --- Putative PVC marker --- p.16 / Chapter 2.1.2 --- Golgi marker --- p.17 / Chapter 2.1.3 --- Dynamic studies --- p.18 / Chapter 2.1.4 --- Cell culture study --- p.18 / Chapter 2.2 --- Materials and Methods --- p.21 / Chapter 2.2.1 --- Plant material --- p.21 / Chapter 2.2.2 --- Construction of fusion reporters --- p.22 / Chapter 2.2.2.1 --- Cloning materials --- p.22 / Chapter 2.2.2.2 --- Vector preparation --- p.22 / Chapter 2.2.2.3 --- Cloning of pGFP-BP-80K and pGFP-BP-80H --- p.24 / Chapter 2.2.2.4 --- Cloning of pGFP-α-TIPH --- p.28 / Chapter 2.2.3 --- Transformation of tobacco BY-2 cells --- p.30 / Chapter 2.2.3.1 --- Agrobacterium transformation --- p.30 / Chapter 2.2.3.2 --- BY-2 cell transformation --- p.30 / Chapter 2.2.4 --- Screening of transgenic BY-2 cells --- p.31 / Chapter 2.2.4.1 --- Killing curve study --- p.31 / Chapter 2.2.4.2 --- Antibiotic selection --- p.32 / Chapter 2.2.4.3 --- Fluorescence microscopy screening (For single-construct cell lines) --- p.33 / Chapter 2.2.4.4 --- Confocal laser scanning microscopy (CLSM) screening (For double-construct cell lines) --- p.33 / Chapter 2.2.5 --- Detection of fluorescent protein expression --- p.35 / Chapter 2.2.5.1 --- Confocal imaging --- p.35 / Chapter 2.2.5.2 --- Protein extraction and subcellular fractionation --- p.36 / Chapter 2.2.5.3 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.36 / Chapter 2.2.5.4 --- Western blot analysis --- p.37 / Chapter 2.2.5.5 --- Cell culture study --- p.37 / Chapter 2.3 --- Results --- p.39 / Chapter 2.3.1 --- Hygromycin concentration at 50 mg/L was optimal for selection --- p.39 / Chapter 2.3.2 --- Lower transformation efficiency for double-construct cell lines --- p.40 / Chapter 2.3.3 --- Screening of transgenic cell lines --- p.41 / Chapter 2.3.4 --- Both pGFP-BP-80K and pGFP- a -TIPH expressed as punctate signals in single-construct cell lines --- p.45 / Chapter 2.3.5 --- Weak punctate or diffuse signals were detected from PVC markers in double-construct cell lines --- p.47 / Chapter 2.3.6 --- GFP reporters were successfully transformed into BY-2 cells --- p.51 / Chapter 2.3.7 --- Profiles of fluorescent signals in transgenic cells during cell culture --- p.53 / Chapter 2.4 --- Discussion --- p.59 / Chapter 2.4.1 --- Abnormal cell growth might be due to high selection pressure --- p.59 / Chapter 2.4.2 --- Double-construct cell lines developed were not yet suitable for further study --- p.60 / Chapter 2.4.3 --- Single-construct cell lines expressing putative PVC markers were developed --- p.62 / Chapter 2.4.4 --- 2- to 3-day-old cells were more suitable for subsequent studies --- p.63 / Chapter Chapter 3 --- Characterization of Transgenic Tobacco BY-2 Cell Expressing Reporters for Distinct Prevacuolar Compartments --- p.66 / Chapter 3.1 --- Introduction --- p.67 / Chapter 3.1.1 --- Wortmannin --- p.69 / Chapter 3.1.2 --- Brefeldin A --- p.70 / Chapter 3.1.3 --- FM4-64 --- p.71 / Chapter 3.2 --- Materials and Methods --- p.73 / Chapter 3.2.1 --- Plant material --- p.73 / Chapter 3.2.2 --- Confocal immunofluorescence studies --- p.73 / Chapter 3.2.3 --- Drug treatment studies --- p.74 / Chapter 3.2.3.1 --- Wortmannin treatment --- p.74 / Chapter 3.2.3.2 --- BFA treatment --- p.75 / Chapter 3.2.4 --- FM4-64 uptake study --- p.76 / Chapter 3.3 --- Results --- p.78 / Chapter 3.3.1 --- Organelles marked by GFP- a -TIP CT reporters did not localize at Golgi compartment --- p.78 / Chapter 3.3.2 --- Wortmannin induced GFP- a -TIP marked organelles to vacuolated --- p.81 / Chapter 3.3.3 --- GFP- a -TIP CT reporters partially colocalized with VSRin wortmannin-treated cells --- p.83 / Chapter 3.3.4 --- BFA induced GFP- a -TIP marked organelles to form BFA- induced compartments --- p.88 / Chapter 3.3.5 --- GFP-α -TIP CT reporter colocalized with internalized FM4-64 --- p.91 / Chapter 3.4 --- Discussion --- p.94 / Chapter 3.4.1 --- GFP- α -TIP CT reporter was a putative PVC marker --- p.94 / Chapter 3.4.2 --- GFP- a -TIP marked organelles behaved differently from lytic PVCs --- p.95 / Chapter 3.4.3 --- GFP- a -TIP marked organelles were not lytic PVCs --- p.96 / Chapter 3.4.4 --- FM4-64 uptake study reveals a new PVC marker --- p.98 / Chapter Chapter 4 --- Summary and Future Prospects --- p.100 / Chapter 4.1 --- Summary --- p.101 / Chapter 4.1.1 --- Hypothesis --- p.101 / Chapter 4.1.2 --- Development of transgenic cell lines --- p.102 / Chapter 4.1.3 --- Characterization of organelles marked by GFP- a -TIP CT reporter --- p.103 / Chapter 4.2 --- Future prospects --- p.106 / Reference --- p.108

The Rbfox proteins are a family of splicing regulators in post-mitotic neurons, predicted to be required for control of hundreds of alternative exons in neuronal development. However, their contribution to the cellular processes in developing and adult nervous system remains unclear and few candidate target exons were experimentally confirmed due to functional redundancy of the three Rbfox proteins. In this thesis, I combined CRISPR/Cas9 genome engineering with in vitro differentiation of embryonic stem cells into spinal motor neurons to unravel the Rbfox regulatory network and to study the functional importance of Rbfox-dependent splicing regulation for neuronal maturation. Global analysis revealed that neurons lacking Rbfox proteins exhibit developmentally immature splicing profile but little change in the gene expression profile. Integrative modeling based on splicing changes in Rbfox triple knockout (Rbfox tKO) neurons and HITS-CLIP Rbfox binding mapping identified 547 cassette exons directly regulated by Rbfox proteins in maturing neurons. Strikingly, many transcripts encoding structural and functional components of axon initial segment (AIS), nodes of Ranver (NoR) and synapses undergo Rbfox-dependent regulation. I focused on the AIS whose assembly, which occurs during the early stages of neuronal maturation, is poorly understood. I found that the AIS of Rbfox tKO neurons is perturbed and contains disorganized ankyrin G, as revealed by super-resolution microscopy. This is in part due to an aberrant splicing of ankyrin G, resulting in destabilization of its interaction with βII- and βIV-spectrin. Thus, Rbfox factors play a crucial role in regulating a neurodevelopmental splicing program underlying structural and functional maturation of post-mitotic neurons. These data highlight the importance of alternative splicing in neurodevelopment and provide a novel link between alternative splicing regulation and AIS establishment.

<p>It is widely known that orexin A and B peptides as well as their receptors are expressed in the hypothalamus and distributed throughout the central nervous system, but there have been few studies regarding its presences in other parts of the body. There is now evidence that orexin (ORX) and its receptors (ORXR1/2) are present in the avian liver; however, their regulation under different environmental conditions is still unknown. In the current study, we sought to determine the effects of heat and oxidative stress using hydrogen peroxide (H2O2) and 4-hydroxynonenal (4-HNE) on the hepatic expression of ORX and ORXR1/2 in the avian species. Overall, heat stress significantly down regulated the expression of ORX, and ORXR1/2 mRNA and pro1tein in quail liver and LMH cells. LMH cells treated with H2O2 had decreased ORX protein and increased ORX mRNA levels (P < 0.05). There was a biphasic effect of 4-HNE on the expression of ORX and ORXR1/2 in LMH cells. There was a significant upregulation at low doses (10 and 20 ?M) and significant down-regulation at a high dose (30?M) of 4-HNE. In light of the current data, the hepatic expression of orexin could serve as a molecular signature in the heat and oxidative stress response.

abstract: The RASopathies are a collection of developmental diseases caused by germline mutations in components of the RAS/MAPK signaling pathway and is one of the world’s most common set of genetic diseases. A majority of these mutations result in an upregulation of RAS/MAPK signaling and cause a variety of both physical and neurological symptoms. Neurodevelopmental symptoms of the RASopathies include cognitive and motor delays, learning and intellectual disabilities, and various behavioral problems. Recent noninvasive imaging studies have detected widespread abnormalities within white matter tracts in the brains of RASopathy patients. These abnormalities are believed to be indicative of underlying connectivity deficits and a possible source of the behavioral and cognitive deficits. To evaluate these long-range connectivity and behavioral issues in a cell-autonomous manner, MEK1 loss- and gain-of-function (LoF and GoF) mutations were induced solely in the cortical glutamatergic neurons using a Nex:Cre mouse model. Layer autonomous effects of the cortex were also tested in the GoF mouse using a layer 5 specific Rbp4:Cre mouse. Immunohistochemical analysis showed that activated ERK1/2 (P-ERK1/2) was expressed in high levels in the axonal compartments and reduced levels in the soma when compared to control mice. Axonal tract tracing using a lipophilic dye and an adeno-associated viral (AAV) tract tracing vector, identified significant corticospinal tract (CST) elongation deficits in the LoF and GoF Nex:Cre mouse and in the GoF Rbp4:Cre mouse. AAV tract tracing was further used to identify significant deficits in axonal innervation of the contralateral cortex, the dorsal striatum, and the hind brain of the Nex:Cre GoF mouse and the contralateral cortex and dorsal striatum of the Rbp4:Cre mouse. Behavioral testing of the Nex:Cre GoF mouse indicated deficits in motor learning acquisition while the Rbp4:Cre GoF mouse showed no failure to acquire motor skills as tested. Analysis of the expression levels of the immediate early gene ARC in Nex:Cre and Rbp4:Cre mice showed a specific reduction in a cell- and layer-autonomous manner. These findings suggest that hyperactivation of the RAS/MAPK pathway in cortical glutamatergic neurons, induces changes to the expression patterns of P-ERK1/2, disrupts axonal elongation and innervation patterns, and disrupts motor learning abilities. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2018

No description available.