The PHB-d protein family is an evolutionarily ancient family of integral membrane proteins with members in all taxa that are involved in a wide variety of biological process but share several common molecular properties: oligomerization, detergent resistant membrane (DRM) association, and regulation of other proteins. To better understand the biological roles of the PHB-d gene family and provide a starting point for analysis of their individual functions, I determined the expression patterns of all of the uncharacterized PHB-d genes in the nematode C. elegans. All eight of the proteins similar to mammalian stomatin are detectably expressed in neurons. The specificity of expression varies from 34 neuron types that express sto-4 to only one that expresses sto- 3. STO-1 protein is localized to amphid sensory cilia and is needed for optimal chemotaxis to diacetyl. This phenotype is likely to affect chemotaxis mediated by the AWA sensory neurons. Involvement of PHB-d proteins in ! chemosensation may be conserved in mammals. Two murine PHB-d genes are highly expressed in olfactory sensory neurons, but no olfactory phenotype has been described in mice lacking either gene. C. elegans senses gentle body touch via a mechanosensory channel complex that contains two Degenerin/Epithelial sodium channel (DEG/ENaC) subunits and two prohibitin homology domain (PHB-d) proteins. One of the PHB-d proteins (UNC-24) has a C-terminal sterol carrier protein 2 (SCP-2) domain and only a subtle mechanosensory abnormal (Mec) phenotype. The other PHB-d (MEC-2) is essential for channel function. I show here that unc-24 is needed for proper localization of MEC-2 to the channel complex, but that this function is unlikely to underlie the unc-24 phenotype. This result supports the hypothesis that PHB-d/SCP-2 proteins have a conserved role in localizing other PHB-d proteins and demonstrates that UNC-24 must have a second, unknown, role in mechanosensation. MEC-2 is embedded in the plasma membrane via a non-spanning hydrophobic hook. Mutation of a conserved proline (P134S) in the hydrophobic hook leads to complete touch insensitivity. Mutation of the equivalent proline in human stomatin converts the hook to a transmembrane domain and expels the PHB-d and C-terminus of the protein. We show here that the P134S mutation has the same effect on MEC-2 topology in HEK293T cells. This result has implications for MEC-2 function because the P134S mutation has previously been shown to affect some but not all MEC-2 biochemical properties. The conserved proline is found in all C. elegans and human stomatins and its mutation in human podocin leads to kidney failure.

TDP43 is an RNA and DNA binding protein that has been shown to play an integral role in disease mechanisms that underlie ALS. In fact, a common feature of the vast majority of ALS cases is the presence of TDP-43 aggregates in postmortem tissue from the brain and spinal cord. This finding has spurred research to understand the physiological roles of TDP-43 in the absence of disease, and how these roles are affected by disease. Current TDP-43 mouse models fail to faithfully and reproducibly recapitulate key aspects of ALS, possibly due to the transgenic approaches used. To address these concerns, we generated a targeted, conditional mouse model, and embryonic stem cell lines expressing either human WT or M337V mutant TDP-43 at equivalent levels. We show that expression of mutant hTDP-43 in mice with a mixed genetic background leads to selective motor neuron loss, muscle weakness and premature death. However this disease phenotype is not observed with the same TDP43 mutation in a pure Bl6 background. We next sought to identify alterations in the biochemistry of the mutant protein that may underlie its toxicity, such as its interactions with RNA and protein. By creating a library of RNAs bound by TDP-43 in the mouse spinal cord, we found that the M337V mutation does not compromise the ability of TDP43 to bind to target mRNA transcripts, although the mutation does lead to changes in expression of genes known to be involved in inflammation. In addition, we identified 22 proteins that bind to TDP43 in an RNA-dependent manner, and found that the M337V mutation does not alter these interactions. This work establishes novel mouse and cellular models that provide insights into the functions of normal and ALS-causing mutant TDP43 protein.

We investigated the role of Notch signaling in the vascular microenvironment, with particular attention paid to the vascular consequences of Notch signaling disruption in myeloid cells. We adapted an established in vitro model of angiogenesis to recreate interactions between endothelial sprouts and vascular support cells, including macrophages and vascular pericytes. We found that inflammatory polarization of macrophages increased their ability to foster angiogenesis, and that intact Notch signaling was essential to this phenomenon. We also demonstrated a role for Notch/Jagged1 signaling in the interaction between vascular pericytes and endothelial sprouts, the disruption of which limits the growth and maturation of vessel networks. We have also investigated the role of myeloid Notch signaling in vivo, using a number of developmental and pathological models of angiogenesis. We found that Notch inhibition leads to decreased myeloid cell recruitment to a broad variety of functionally distinct angiogenic sites. Importantly, we observed that myeloid Notch disruption has vascular consequences in both physiological and pathological angiogenesis. Myeloid Notch- inhibited mice exhibit decreased vascular complexity in the deep retinal plexus during development. Additionally, these mice show significantly increased vascular tuft formation in the setting of oxygen-induced retinopathy, suggestive of a heretofore- undescribed role for myeloid Notch signaling in the pathogenesis of this significant human disease. This body of work increases our understanding of the role of Notch signaling both in the dynamics of myeloid cells and in their specific contribution to angiogenesis in multiple disparate contexts. It also contributes to our understanding of a number of key models of human disease, and may prove useful in the development of novel therapies to treat those diseases. Further, we are confident that our new experimental methodology will allow continued fruitful reductive study of the complex intercellular interactions within the vascular microenvironment.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological tumor resulting from the malignant transformation of immature T-cell progenitors. Originally associated with a dismal prognosis, the outcome of T-ALL patients has improved remarkably over the last two decades as a result of the introduction of intensified chemotherapy protocols. However, these treatments are associated with significant acute and long-term toxicities, and the treatment of patients presenting with primary resistant disease or those relapsing after a transient response remains challenging. Oncogenic activation of NOTCH1 signaling plays a central role in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), with mutations on this signaling pathway affecting more than 60% of patients at diagnosis. However the transcriptional regulatory circuitries driving T-cell transformation downstream of NOTCH1 remain incompletely understood. Here we identify HES1, a transcriptional repressor controlled by NOTCH1 as a critical mediator of NOTCH1 induced leukemogenesis strictly required for tumor cell survival. Mechanistically, we demonstrate that HES1 inhibits leukemia cell death by repressing BBC3, the gene encoding the PUMA BH3-only proapototic factor. Finally, we identify perhexiline, a small molecule inhibitor of mitochondrial carnitine palmitoyltransferase-1, as a HES1-signature antagonist drug with robust antileukemic activity against NOTCH1 induced leukemias in vitro and in vivo.

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