Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, March 2000. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: The regulation of LRP in the CNS. Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (M.S.)--Missouri State University, 2008. / "August 2008." Includes bibliographical references (leaves 70-75). Also available online.

Thesis (M.S.)--Missouri State University, 2009. / "May 2009." Includes bibliographical references (leaves 38-42). Also available online.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 146-165).

Assembly of branched actin filaments produces dynamic structures required during membrane associated processes including cell motility and endocytosis. The Actin Related Protein 2/3 (Arp2/3) complex is the only known regulator capable of nucleating actin branches. To specify the sub cellular localization and timing of actin assembly the complex is tightly regulated. Canonical activation of the Arp2/3 complex by Wiskott-Aldrich Syndrome proteins (WASP), requires preformed actin filaments, ensuring the complex nucleates new actin filaments off the sides of preformed filaments. WASP proteins can therefore propagate branch formation but cannot initiate a Y-branch without performed filaments. A key question, then, is what is the source of preformed filaments that seed branched actin network formation in cells? It is unclear how activation of Arp2/3 by multiple regulators is balanced to specify actin filament architectures that are productive in vivo. In this dissertation, we identified WISH/DIP1/SPIN90 (WDS) family proteins as activators of the Arp2/3 complex that do not require preformed filaments, and evaluated whether WDS proteins seed branching nucleation. In chapter II, we dissected the biochemical properties of WDS proteins and found they activate the Arp2/3 complex using a non-WASP like mechanism. Importantly, we discovered WDS-mediated Arp2/3 activation produces linear, unbranched filaments, and this activity is conversed from yeast to mammals. These observations highlight that WDS proteins have the biochemical capacity to seed actin branches. In chapter III, we observed WDS-generated linear filaments can seed WASP-mediated branching directly using single molecule microscopy with fluorescently labeled Dip1. We find that WDS-mediated nucleation co-opts features of branching nucleation. In chapter IV, we investigated how WDS activity is balanced with WASP. We discovered WDS proteins use a single turnover mechanism to activate Arp2/3 and this is conserved during endocytosis. In contrast, WASP-mediated activation is multi-turnover, highlighting a crucial difference between WDS proteins and WASP. Our observations explain how Arp2/3 may limit linear filament production to initiate networks and favor branches during network propagation. Finally, we use fission yeast to show that increasing Dip1 is sufficient to cause defects in actin assembly and the timing of actin patches at sites of endocytosis.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-04-27T17:39:55Z No. of bitstreams: 1 José Geraldo Bomfim Lima Estudo do efeito....pdf: 7893051 bytes, checksum: a043a24c2d08a6c835942fd381ead5a4 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-04-27T17:40:27Z (GMT) No. of bitstreams: 1 José Geraldo Bomfim Lima Estudo do efeito....pdf: 7893051 bytes, checksum: a043a24c2d08a6c835942fd381ead5a4 (MD5) / Made available in DSpace on 2016-04-27T17:40:27Z (GMT). No. of bitstreams: 1 José Geraldo Bomfim Lima Estudo do efeito....pdf: 7893051 bytes, checksum: a043a24c2d08a6c835942fd381ead5a4 (MD5) Previous issue date: 2015 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / INTRODUÇÃO: A influência da autofagia em processos celulares que participam da homeostase celular, como a endocitose e a adesão celular, até o momento, foi pouco estudada. A endocitose consiste na internalização de material extracelular, quando as vesículas endocíticas são menores que 500nm é chamada de endocitose em microescala e quando as vesículas formadas são maiores que essa medida trata-se de endocitose em macroescala. Foi demonstrado que a conexão da via endocítica com a via autofágica é fundamental para a degradação de material citosólico e, subsequente, produção de energia e disponibilização de substrato para o metabolismo celular. Estudos controversos da literatura mostraram que a autofagia pode favorecer ou não interferir com a endocitose em macroescala. Além disso, alguns trabalhos demonstraram que o processo autofágico foi capaz de reduzir a reciclagem de integrinas para a membrana plasmática por alterar a endocitose em microescala envolvida na internalização desse tipo de proteína, reduzindo a capacidade de adesão e, consequentemente, a migração celular. Assim, em conjunto, esses achados evidenciam que a autofagia pode interagir e interferir com eventos celulares dependentes da participação da membrana plasmática como a endocitose e a adesão celular. OBJETIVO: No presente estudo, hipotetizamos que a prévia indução de autofagia em macrófagos é capaz de reduzir a endocitose em micro e macroescala, além de reduzir a capacidade de adesão celular. Desta forma, o objetivo desse estudo foi determinar o efeito da indução de autofagia, in vitro, sobre a endocitose e a adesão de macrófagos murino. MATERIAL E MÉTODOS: Macrófagos foram induzidos à autofagia por privação de nutrientes (starvation) ou pelo tratamento com um indutor farmacológico, a rapamicina, seguida da exposição a macromoléculas ou grandes partículas de diferentes naturezas. Além disso, após indução de autofagia, macrófagos em suspensão foram incubados em superfícies como o vidro ou uma matriz de colágeno e fibronectina para avaliação da capacidade de adesão. Os percentuais de endocitose em microescala, em macroescala e de adesão foram estimados. RESULTADOS: Mostramos que a indução de autofagia promoveu redução da capacidade fagocítica em cerca de 60% no percentual de macrófagos que internalizam grandes partículas, como levedo, sendo um mecanismo precoce e reversível. Ao passo que a indução de autofagia por privação de aminoácidos ou farmacológica não interferiu na endocitose em microescala. A indução de autofagia não alterou a endocitose de transferrina (endocitose mediada por receptores) e endocitose de BSA (endocitose de fase fluida). Em contraste, a indução de autofagia promoveu redução em aproximadamente 70% da quantidade de macrófagos que aderem a matriz de colágeno e fibronectina. Uma possível explicação para a redução da endocitose em macroescala pode estar relacionada à autofagia diminuir a disponibilidade de grandes extensões de membrana necessárias à internalização de partículas maiores que 500nm. Alternativamente, a indução de autofagia pode estar levando a célula a uma indisponibilidade de receptores na membrana plasmática que justificaria a redução da capacidade fagocítica e de adesão do 11 macrófago murino. CONCLUSÕES: A indução de autofagia diminui a capacidade fagocítica e a capacidade de adesão do macrófago murino. / INTRODUCTION: The influence of autophagy on cellular processes that participate in cellular homeostasis, such as endocytosis and cell adhesion has been poorly evaluated. Endocytosis consists in the internalization of extracellular material and includes microscale endocytosis, when endocytic vesicles are smaller than 500nm, and macroscale endocytosis, when the formed vesicles are larger than this measure. It has been shown that the connection between the endocytic and the autophagic pathways is essential for degradation of cytosolic material and, subsequently, power generation and provision of substrate for cellular metabolism. Controversial studies showed that autophagy can improve or do not interfere with macroscale endocytosis. Furthermore, some studies demonstrated that the autophagic process reduced integrin recycling to the plasma membrane through the modulation of microscale endocytosis involved in the internalization of this protein, reducing cell adhesion and migration. Taken together, these findings show that autophagy can interact and interfere with cellular events that depend on plasma membrane participation, such as endocytosis and cell adhesion. OBJECTIVES: In the present study, we hypothesized that prior autophagy induction in macrophage reduces micro and macroscale endocytosis, as well as cell adhesion. Thus, the aim of this study was to determine the effect of autophagy induction, in vitro, on endocytosis and adhesion of murine macrophages. MATERIAL AND METHODS: Autophagy by nutrient deprivation (starvation) or by treatment with an inducer drug, rapamycin, was induced in macrophages, followed by exposure to macromolecules or large particles of different natures. Furthermore, after autophagic induction, macrophages were plated on different surfaces like glass or collagen-fibronectin matrix to evaluate cell adhesiveness. After that, the percentage of endocytosis in micro and macroscale and adhesion were determined. RESULTS: We showed that autophagy induction decreases phagocytic ability to 60% in macrophages that internalized large particles like yeast. This is a reversible mechanism that occurs at early stages after autophagy induction. On the other hand, autophagy by amino acid deprivation or pharmacological induction does not interfere with the microscale endocytosis. The autophagy induction doesn’t alter transferrin endocytosis (receptor-mediated endocytosis) and BSA endocytosis (fluid-phase endocytosis). By contrast, the autophagy induction leads to a reduction of approximately 70% in macrophages adhesion on a collagen-fibronectin matrix. The reduction of macroscale endocytosis may be related to the decreased availability of large areas of membrane required for internalization of particles larger than 500nm caused by autophagy. Alternatively, autophagy induction may be leading to receptors unavailability in plasm membrane, which would explain the reduction of the phagocytic and adhesion ability. CONCLUSIONS: autophagy induction reduces

Eukaryotic cells are characterised by membranes with varied and dynamic compositions and shapes. Consequently, membrane-binding proteins are tuned to recognise and modify these membrane states to perform their functions. To study the curvature sensitivity of proteins, I have developed a single-particle assay using NanoSight technology that tracks the Brownian motion of particles to measure their size. I optimised this system to track fluorescently labelled lipid-binding domains bound to liposomes of different sizes moving freely in solution. The comparison of the size distribution of the total liposomes with the fluorescently labelled population allowed me to determine their curvature preferences. To validate the method I tested proteins from the Bin/Amphiphysin/Rvs (BAR) superfamily, which are inherently curved and have known curvature preferences. My method was capable of recapitulating the behaviour of BAR domains with different curvature preferences. I then expanded the range of targets and showed that this assay is also capable of detecting curvature preferences for a variety of other lipid-binding domain families. As such, I identified AKT PH domain as a new curvature-sensing domain. Finally, using the ENTH domain of Epsin1 that causes vesicle budding, I demonstrated that this method can also be used to study membrane remodelling. Trafficking involves generation and sensing of membrane curvature combined with recognition of specific cargo. Endophilin consists of a curvature-sensitive BAR domain followed by an SH3 (Src-homology 3) domain and has recently been identified in a clathrin-independent endocytosis pathway, FEME (fast endophilin-mediated endocytosis), involved in the uptake of cell surface receptors. Endophilin recognises ligands via its SH3 domain, binding G-protein coupled receptors (GPCRs) directly in their intracellular loop 3 and receptor tyrosine kinase (RTKs) via adaptor proteins. However, a specific recognition motif has not been identified yet. Here, using a combination of biophysical approaches and NMR spectroscopy, I characterised the Endophilin binding motif of ALIX (ALG-2-interacting protein X) adaptor protein and of the GPCR $\alpha$2A adrenergic receptor. Comparison of SH3-peptide models resulted in a putative Endophilin recognition site.

Endosomes are now recognized as important sites for regulating signal transduction. Here we show that the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIβ) regulates both endocytic kinetics and receptor signaling in breast cancer cells. PI4KIIIβ generates phosphatidylinositol 4-phosphate from phosphatidylinositol and is highly expressed in a subset of breast cancers. However, the molecular mechanism by which PI4KIIIβ promotes breast cancer is unclear. We demonstrate that ectopic PI4KIIIβ expression increases the rates of both endocytic internalization and recycling. Furthermore, PI4KIIIβ deletion reduces endocytic kinetics. Regulation of endocytic function by PI4KIIIβ is independent of its kinase activity but requires interaction with the Rab11a GTPase. Additionally, we find that PI4KIIIβ activates IGF-IRβ signaling, dependent on endosome function. Finally, we observe that PI4KIIIβ deletion decreases the growth rate of mammary tumours in mice. Our work suggests a novel regulatory role for PI4KIIIβ in endosome function and plasma membrane receptor signaling, providing a mechanism by which increased PI4KIIIβ expression could promote breast cancer oncogenesis.