Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 106-116.

Exposure to external stimuli promotes a variety of cellular responses including changes in morphology, gene expression and cell division status. These responses are promoted by signaling pathways composed of modules that are conserved from lower to higher eukaryotes. In Saccharomyces cerevisiae response to the external stimuli provided by mating pheromone is governed by the pheromone response pathway. This pathway is composed of a G protein coupled receptor/heterotrimeric G protein (Gαβγ) module and a MAP kinase cascade. Activation of this pathway allows the heterotrimeric G protein βγ dimer (Gβγ) to recruit polarity proteins to promote changes in cell morphology and to activate signaling through the MAP kinase cascade. Here we investigate the regulation of these pheromone-induced responses. We first examine how an asymmetric polarization response is generated. Normally, a gradient of pheromone serves as a spatial cue for formation of a polarized mating projection, but cells can still polarize when pheromone is present uniformly. Here we show that an intact receptor/Gαβγ module is required for polarization in response to both a gradient and uniform concentration of pheromone. Further investigation into regulation of Gβγ by Gα revealed that the two interaction interfaces between Gα and Gβ have qualitatively different roles. Our results suggest that one interface controls signaling whereas the other governs coupling to the receptor. Overall our results indicate that communication between the receptor and Gαβγ is required for proper polarization. We then examine how G1 CDKs regulate MAP kinase signaling. Response to pheromone is restricted to the G1 stage of the cell cycle. Once cells commit to a round of division they become refractory to mating pheromone until that round of division is complete. One contributor to this specificity involves inhibition of signaling through the MAP kinase cascade by G1 CDKs, but it was not known how this occurs. Here, we show that the MAP kinase cascade scaffold Ste5 is the target of this inhibition. Cln/CDKs inhibit signaling by phosphorylating sites surrounding a small membrane-binding domain in Ste5, thereby disrupting the membrane localization of Ste5. Furthermore, we found that disrupting this regulation allows cells to arrest at an aberrant non-G1 position. Our findings define a mechanism and a physiological benefit for restricting pheromone-induced signaling to G1. This thesis describes findings related to generation of an asymmetric polarization response, heterotrimeric G protein function, and coordination of differentiation signaling with cell division status. Lessons learned here might be applicable to the regulation of polarization and differentiation responses in other systems as the signaling modules are conserved.

In mammalian cells, homeostasis and fate during development relies on the proper transport of membrane-bound cargoes to their designated cellular locations. The hetero-tetrameric adaptor protein complexes (APs) are required for sorting and concentration of cargo at donor membranes, a crucial step during targeted transport. AP2, which functions at the plasma membrane during clathrin-mediated endocytosis, is well characterized. In contrast, AP1 a clathrin adaptor mediating the delivery of lysosomal hydrolases via mannose 6-phosphate receptors (MPRs) and AP3 an adaptor ensuring the proper targeting of lysosomal membrane protein are difficult to study by classic cell biology tools. To gain new insights on these APs, our lab has previously designed an in vitro system. Reconstituted liposomes were modified with small peptides mimicking the cytosolic domains of bona fide cargoes for AP1 and AP3 respectively and thereby enabling the selective recruitment of these APs and the identification of the interacting protein network. In the study at hand we utilize above-described liposomes to generate supported lipid bilayers and Giant Unilamellar Vesicles (GUVs), large-scale membrane systems suited for analysis by fluorescence microscopy. By using cytosol containing fluorescently-tagged subunits, we visualized clathrin coats on artificial membranes under near physiological conditions for the first time. Moreover, we demonstrated clathrin-independent recruitment of AP3 coats on respective GUVs. Presence of active ARF1 was sufficient for the selective assembly of AP1-dependent clathrin coats and AP3 coats on GUVs. By using dye-conjugated ARF1, we show that ARF1 colocalized with AP3 coats on GUVs and that increased association of ARF1 with GUVs coincided with AP1-dependent clathrin coats. Our previous study identified members of the septin family together with AP3 coats on liposomes. Here we show on GUVs, that active ARF1 stimulated the assembly of septin7 filaments, which may constrain the size and mobility of AP3 coats on the surface. Subsequent cell biology studies in HeLa cells linked septins to actin fibers on which they may control mobility of AP3-coated endosomes and thus their maturation. An actin nucleation complex, based on CYFIP1 was identified together with AP1 on liposomes before. Here we show on GUVs, that CYFIP1 is recruited on the surface surrounding clathrin coats. Upon supply of ATP, sustained actin polymerization generated a thick shell of actin on the GUV surface. The force generated by actin assembly lead to formation of long tubular protrusions, which projected from the GUV surface and were decorated with clathrin coats. Thereby the GUV model illustrated a possible mechanism for tubular carriers formation. The importance of CYFIP1-reliant actin polymerization for the generation of MPR-positive tubules at the trans-Golgi network (TGN) of HeLa cells was subsequently demonstrated in our lab. The notion that tubulation of artificial membranes could be triggered by actin polymerization allowed us to perform a comparative mass spectrometry screen. By comparing the abundance of proteins on liposomes under conditions promoting or inhibiting actin polymerization, candidates possibly involved in stabilization, elongation or fission of membrane tubules could be identified. Among the proteins enriched under conditions promoting tubulation, we identified type I phosphatidylinositol-4-phosphate 5-kinases. Their presence suggested an involvement of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in tubule formation. By cell biology studies in HeLa we show, that down regulation of these enzymes altered the dynamics of fluorescently-tagged MPRs, illustrating the importance of locally confined PI(4,5)P2 synthesis during formation of coated carriers at the TGN. Bin–Amphiphysin–Rvs (BAR) domains are known to sense membrane curvature and induce membrane tubulation. Among various BAR domain proteins, Arfaptin2 was enriched under conditions allowing tubulation of liposomes. By microscopy studies on HeLa cells we show, that Arfaptin2 as well as its close paralog Arfaptin1 were present on AP1-coated MPR tubules emerging from the TGN. We further show, that tubule fission occurred at regions were Arfaptin1 is concentrated and that simultaneous down regulation of both Arfaptins lead to increased number and length of MPR tubules. Since fission of coated transport intermediates at the TGN is poorly understood, our findings contribute a valuable component towards a model describing the entire biogenesis of coated post-Golgi carriers. In conclusion, combining artificial membrane systems and cell biology studies allowed us to propose new models for formation as wall as for fission of AP1-coated transport intermediates at the TGN. Further we gained new insights on AP3 coats and the possible involvement of septin filaments in AP3-dependent endosomal maturation.

In cancer, aberrant activation of developmental signaling pathways such as the Hippo Pathway has been shown to drive proliferation and invasion of cancer cells. Therefore, understanding the normal function of the Hippo Pathway during embryonic development can provide critical insight into how aberrant activity contributes to tumorigenesis. This dissertation explores the role of the Hippo Pathway members YAP and TAZ in gastrointestinal (GI) development and tumorigenesis. I use mouse genetics to systematically dissect the roles of YAP/TAZ in the endoderm-derived gastrointestinal epithelia and mesoderm-derived gastrointestinal mesenchyme during mammalian development. In the GI epithelium, I demonstrate that YAP/TAZ are dispensable for development and homeostasis. However, YAP/TAZ are required for Wnt pathway-driven tumorigenesis. I find that YAP/TAZ are direct transcriptional targets of Wnt/TCF4 signaling. In the GI mesenchyme, I describe a previously unknown requirement for YAP/TAZ activity during mammalian GI development. YAP/TAZ are involved in normal GI mesenchymal differentiation and function as transcriptional co-repressors in a progenitor cell population. In this way, YAP/TAZ act as molecular gatekeepers prior to Hedgehog-mediated differentiation into smooth muscle cells. This work unveils a previously unknown requirement for Hippo pathway signaling in the mammalian GI tract and a novel mechanism wherein YAP/TAZ function as transcriptional co-repressors to maintain a mesenchymal progenitor cell population.

Adaptive immunity requires T cell responses to foreign pathogens to be counterbalanced with the need to limit collateral destruction of the host’s own tissues. Further, the presence of a substantial pool of lymphocytes capable of recognizing selfantigen in the periphery poses a threat to the maintenance of peripheral tolerance and prevention of autoimmunity. Regulatory T cells (Treg) that can suppress potentially self-reactive T cells are critical regulators of peripheral tolerance as well as initiation of immune responses. Treg cells employ several context-dependent mechanisms to establish regulation. In this thesis, we describe two distinct pathways of regulation used by Treg cells involving negative costimulation by CTLA-4 and immunomodulation by the morphogen, TGFβ. CTLA-4 is a co-inhibitory receptor on T cells essential for maintaining T cell homeostasis and tolerance to self. CTLA-4 expression is induced in conventional T cells following activation, whereas it is constitutively expressed in regulatory FOXP3+CD4+ regulatory T cells. Mice lacking CTLA-4 develop an early onset, fatal breakdown in T cell tolerance. Whether this autoimmune disease occurs because of the loss of CTLA-4 function in regulatory T cells, conventional T cells, or both, is not known. We present evidence here that in addition to a critical CTLA-4 function in regulatory T cells, CTLA-4 in conventional T cells is also necessary for controlling the consequences of abnormal T cell activation. CTLA-4 expression in activated conventional T cells only in vivois unable to compensate for the impaired function of CTLA-4-less regulatory T cells that results in systemic lymphoproliferation, but it can prevent the aberrantly activated T cells from infiltrating and fatally damaging non-lymphoid tissues. These results demonstrate that CTLA-4 has a dual function in maintaining T cell homeostasis: CTLA-4 in regulatory T cells inhibits inappropriate naïve T cell activation and CTLA-4 in conventional T cells can prevent the harmful accumulation of inappropriately activated pathogenic T cells in vital organs. In addition, we have identified Disabled-2 (Dab2), a TGFβ signaling intermediate, as a FOXP3 target gene that is expressed exclusively in Treg cells and is critical for in vitro and in vivo regulation by Treg cells. During T cell development, DAB2 is also expressed in a Foxp3-independent manner in thymic precursor cells, and acts as a sensor of TGFβ signals that is required for programming normal TGFβ responsiveness in T cell progenies. Naïve CD4+ T cells that differentiate from Dab2-deficient precursors favor Th17 cell generation at the expense of FOXP3+ Treg cells as a result of altered sensitivity to TGFβ. Importantly, retinoic acid can restore TGFβ signaling capacity of naïve CD4+ T cells generated from Dab2-deficient precursors, emphasizing the cooperative nature of retinoic acid and TGFβ signaling pathways in promoting Treg cell development and maintenance.

No description available.

No description available.

An emerging problem in organisations is that there exist a large number of tools storing data that communicate with each other too often, throughout the process of an application or product development. However, no means of communication without the intervention of a central entity (usually a server) or storing the schema at a central repository exist. Accessing data among tools and linking them is tough and resource intensive. As part of the thesis, we develop a software (also referred to as ‘adaptor’ in the thesis), which, when implemented in the lifecycle management systems, integrates data seamlessly. This will eliminate the need of storing database schemas at a central repository and make the process of accessing data within tools less resource intensive. The adaptor acts as a wrapper to the tools and allows them to directly communicate with each other and exchange data. When using the developed adaptor for communicating data between various tools, the data in relational databases is first converted into RDF format and is then sent or received. Hence, RDF forms the crucial underlying concept on which the software will be based. The Resource description framework (RDF) provides the functionality of data integration irrespective of underlying schemas by treating data as resource and representing it as URIs. The model of RDF is a data model that is used for exchange and communication of data on the Internet and can be used in solving other real world problems like tool integration and automation of communication in relational databases. However, developing this adaptor for every tool requires understanding the individual schemas and structure of each of the tools’ database. This again requires a lot of effort for the developer of the adaptor. So, the main aim of the thesis will be to automate the development of such adaptors. With this automation, the need for anyone to manually assess the database and then develop the adaptor specific to the database is eliminated. Such adaptors and concepts can be used to implement similar solutions in other organisations faced with similar problems. In the end, the output of the thesis is an approachwhich automates the process of generating these adaptors. / Resource Description Framework (RDF) ger funktionaliteten av dataintegration, oberoende av underliggande scheman genom att behandla uppgifter som resurs och representerar det som URI. Modellen för Resource Description Framework är en datamodell som används för utbyte och kommunikation av uppgifter om Internet och kan användas för att lösa andra verkliga problem som integrationsverktyg och automatisering av kommunikation i relationsdatabaser. Ett växande problem i organisationer är att det finns ett stort antal verktyg som lagrar data och som kommunicerar med varandra alltför ofta, under hela processen för ett program eller produktutveckling. Men inga kommunikationsmedel utan ingripande av en central enhet (oftast en server) finns. Åtkomst av data mellan verktyg och länkningar mellan dem är resurskrävande. Som en del av avhandlingen utvecklar vi en programvara (även hänvisad till som "adapter" i avhandlingen), som integrerar data utan större problem. Detta kommer att eliminera behovet av att lagra databasscheman på en central lagringsplats och göra processen för att hämta data inom verktyg mindre resurskrävande. Detta kommer att ske efter beslut om en särskild strategi för att uppnå kommunikation mellan olika verktyg som kan vara en sammanslagning av många relevanta begrepp, genom studier av nya och kommande metoder som kan hjälpa i nämnda scenarier. Med den utvecklade programvaran konverteras först datat i relationsdatabaserna till RDF form och skickas och tas sedan emot i RDF format. Således utgör RDF det viktiga underliggande konceptet för programvaran. Det främsta målet med avhandlingen är att automatisera utvecklingen av ett sådant verktyg (adapter). Med denna automatisering elimineras behovet att av någon manuellt behöver utvärdera databasen och sedan utveckla adaptern enligt databasen. Ett sådant verktyg kan användas för att implementera liknande lösningar i andra organisationer som har liknande problem. Således är resultatet av avhandlingen en algoritm eller ett tillvägagångssätt för att automatisera processen av att skapa adaptern.

Hao, Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 98-115). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.iii / 摘要 --- p.v / List of Abbreviations --- p.vii / List of Figures --- p.x / List of Tables --- p.xi / List of Primers --- p.xii / Publications arising from this study --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Alzheimer's disease --- p.1 / Chapter 1.2 --- APP and its functions --- p.4 / Chapter 1.2.1 --- APP processing --- p.7 / Chapter 1.3 --- APPc-interacting proteins --- p.10 / Chapter 1.3.1 --- FE65 --- p.10 / Chapter 1.3.2 --- Xllα and Xl1β --- p.12 / Chapter 1.3.3 --- JIP-1 --- p.13 / Chapter 1.3.4 --- Dabl and Dab2 --- p.15 / Chapter 1.3.5 --- SNX17 --- p.15 / Chapter 1.3.6 --- Numb --- p.15 / Chapter 1.3.7 --- AIDA-1 --- p.16 / Chapter 1.4 --- Objectives of the project --- p.18 / Chapter 1.4.1 --- Engulfment adaptor protein 1 (GULP1) --- p.19 / Chapter 1.4.2 --- Specific aims of my study --- p.20 / Chapter Chapter 2 --- General Methodology --- p.22 / Chapter 2.1 --- Bacterial culture --- p.22 / Chapter 2.2 --- Mini-preparation/Midi-preparation of plasmid DNA --- p.22 / Chapter 2.3 --- Spectrophotometric analysis of DNA --- p.22 / Chapter 2.4 --- Agarose gel electrophoresis for DNA --- p.23 / Chapter 2.5 --- Preparation of competent E. coli --- p.23 / Chapter 2.6 --- Transformation of competent E. coli --- p.24 / Chapter 2.7 --- Molecular cloning --- p.24 / Chapter 2.7.1 --- Preparation of the cloning vector and insert --- p.25 / Chapter 2.7.2 --- Isolation of DNA from agarose gel --- p.25 / Chapter 2.7.3 --- DNA ligation and transformation --- p.25 / Chapter 2.7.4 --- Rapid screening for ligated plasmid --- p.26 / Chapter 2.8 --- Site-directed mutagenesis --- p.26 / Chapter 2.9 --- Cell culture and transfection --- p.27 / Chapter 2.10 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) --- p.28 / Chapter 2.11 --- Western blotting --- p.29 / Chapter Chapter 3 --- Investigation of the GULP1-APP interaction and the effect of GULP1 on APP processing --- p.31 / Chapter 3.1 --- Introduction --- p.31 / Chapter 3.2 --- Materials and methods --- p.34 / Chapter 3.2.1 --- DNA constructs --- p.34 / Chapter 3.2.2 --- Antibodies --- p.34 / Chapter 3.2.3 --- GST pull-down assays --- p.35 / Chapter 3.2.4 --- Rat tissues preparation --- p.36 / Chapter 3.2.5 --- Immunostaining --- p.36 / Chapter 3.2.6 --- "siRNA knockdown of GULPl in CHO, HEK293 and SHSY5Y cells" --- p.37 / Chapter 3.2.7 --- Luciferase assays --- p.37 / Chapter 3.2.9 --- Tricine-SDS/PAGE analysis for APP CTFs --- p.38 / Chapter 3.2.9 --- Aβ enzyme-linked immunosorbent assay (ELISA) --- p.39 / Chapter 3.2.10 --- Statistical analysis --- p.40 / Chapter 3.3 --- Results --- p.40 / Chapter 3.3.1 --- GULP1 F145V mutant abandons the GULP1-APP interaction --- p.40 / Chapter 3.3.2 --- GULP1 and APP colocalize in neurons --- p.45 / Chapter 3.3.3 --- "siRNA mediated knockdown of GULPl in CHO, HEK293 and SHSY5Y cells" --- p.48 / Chapter 3.3.4 --- GULP1 enhances the cleavage of APP in APP-GAL4 cleavage system --- p.49 / Chapter 3.3.5 --- GULP1 alters APP processing by increasing the secretion of APP CTFs --- p.52 / Chapter 3.3.6 --- GULP1 stimulates Aβ secretion --- p.55 / Chapter 3.4 --- Discussion --- p.57 / Chapter Chapter 4 --- Identification and characterization of GULPl phosphorylation sites --- p.60 / Chapter 4.1 --- Introduction --- p.60 / Chapter 4.2 --- Materials and Methods --- p.60 / Chapter 4.2.1 --- DNA constructs --- p.61 / Chapter 4.2.2 --- Antibodies --- p.61 / Chapter 4.2.3 --- Expression and purification of GST fusion proteins --- p.61 / Chapter 4.2.4 --- In vitro phosphorylation of GULP1 by cdk5/p35 --- p.62 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- GULP1 Ser223 can be phosphorylated by cdk5/p35 in vivo --- p.62 / Chapter 4.3.2 --- The phosphorylation ofGULPl Thr35 completely abolished the GULP1-APP interaction --- p.67 / Chapter 4.4 --- Discussion --- p.70 / Chapter Chapter 5 --- Crystallization of the PTB domains of GULPl and GULP1t35d…… --- p.72 / Chapter 5.1 --- Introduction --- p.72 / Chapter 5.2 --- Materials and Methods --- p.72 / Chapter 5.2.1 --- DNA constructs --- p.72 / Chapter 5.2.2 --- Small-scale protein expression and purification --- p.73 / Chapter 5.2.3 --- Large-scale protein expression and purification --- p.73 / Chapter 5.2.4 --- Dynamic light scattering measurement --- p.76 / Chapter 5.2.5 --- Crystallization screening GULP1-PTB --- p.76 / Chapter 5.2.6 --- Optimization of GULP1-PTB crystals by grid screen --- p.76 / Chapter 5.2.7 --- Optimization of GULPl -PTB crystals by additive screen and detergent screen --- p.79 / Chapter 5.3 --- Results --- p.79 / Chapter 5.3.1 --- Large-scale expression and purification of GULP 1-PTB --- p.79 / Chapter 5.3.2 --- Small-scale expression and purification of GULP1T35d-PTB --- p.86 / Chapter 5.3.3 --- Crystallization screening and optimization --- p.88 / Chapter 5.4 --- Discussion --- p.91 / Chapter Chapter 6 --- Conclusion and future perspective --- p.94 / Chapter 6.1 --- Conclusion --- p.94 / Chapter 6.2 --- Future perspective --- p.95 / References --- p.98

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 117-147). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;