背景和目的：胰腺導管腺癌（簡稱胰腺癌）是世界範圍內惡性程度最高的癌癥之一，目前它的5 年生存率不到5%。大部分的病人在診斷初期就已經發展到了局部浸潤或遠處轉移的階段，因此失去了根治性手術切除的机会。輔助性化療對於胰腺癌病人來說是一個首選的治療方案，但是目前只有一小部分病人對化療藥物有良好的反應，而臨床化療失敗常與腫瘤細胞對化療藥物產生耐藥有關。吉西他濱是目前臨床上常用的一線抗癌藥物，但是它的耐藥現象在胰腺癌病人中廣泛存在，也是阻礙其臨床應用的主要原因之一。盡管已經有很多研究致力於揭示吉西他濱在胰腺癌細胞中的耐藥機理，目前臨床上仍然沒有有效的方法應對吉西他濱耐藥。我們的研究主要是為了探討一些以前沒有报道過的參與吉西他濱耐藥機理的基因，借此揭示胰腺癌細胞的吉西他濱耐藥的深層機制，為臨床上的治療提供理論依據。 / 實驗方法：我們實驗室之前在胰腺癌細胞株Capan2 中用全基因組RNAi篩選的方法確定LLGL1 作為抑癌基因能增強吉西他濱在胰腺癌細胞中的細胞毒性。我們隨後用體外細胞毒性分析實驗和皮下腫瘤動物模型來驗證LLGL1 是否能增強吉西他濱的細胞毒性，用蘇木素-伊紅染色和原味末端轉移酶標記技術分析抑制LLGL1 的表達是否會影響吉西他濱誘導的細胞雕亡反應。我們還應用微陣列分析技術進一步探尋LLGL1 的下遊靶蛋白，用實時定量PCR(qRT-PCR) 、蛋白印跡法（western blotting）、熒光素酶檢測等技術來進一步證實LLGL1 與下遊靶蛋白的關系，用免疫組織化學方法探究LLGL1 下遊靶蛋白在胰腺癌組織中的表達情況，以及該蛋白與LLGL1 的表達相關性，還應用染色體免疫共沈澱的方法探討轉錄因子Sp1(pThr453) 和RNA 聚合酶 II 在LLGL1 下遊靶蛋白的啟動子上的富集情況。 / 實驗結果：LLGL1 能增強吉西他濱在胰腺癌中的細胞毒性，抑制該基因的表達能誘導胰腺癌細胞對吉西他濱的耐藥，而上調該基因的表達則會增強胰腺癌細胞對吉西他濱的細胞毒性反應。OSMR 是LLGL1 的下遊靶蛋白, 其在胰腺癌組織中的表達與LLGL1 呈負性相關，抑制OSMR 的表達可以逆轉由LLGL1表達下調引起的吉西他濱耐藥現象。OSMR 表達上調可以增強腫瘤幹細胞標記物CD44 和CD24 的表達。另外，在胰腺癌細胞中，抑制LLGL1 的表達能激活ERK2/Sp1 信號通路，導致磷酸化Sp1(pThr453)的表達升高。OSMR 啟動子既沒有TATA 元件也沒有INR 元件，但是有Sp1 结合元件可供Sp1 結合。磷酸化Sp1(pThr453)可以結合到OSMR 啟動子的Sp1 结合元件上，從而促使RNA 轉錄酶II 結合到該啟動子上，啟動OSMR 基因的轉錄。 / 結論：我們的研究發現：1，LLGL1 能增強吉西他濱在胰腺癌中的細胞毒性，抑制該基因在胰腺癌細胞中的表達能上調OSMR 的表達，並誘導吉西他濱耐藥；2，OSMR 的表達在胰腺癌組織中與LLGL1 呈負性相關；3，下調LLGL1的表達能激活ERK2/Sp1 信號通路，進一步導致磷酸化Sp1(pThr453)和RNA 轉錄酶II 在OSMR 啟動子上的聚集，最終促使OSMR 的高表達，而下調LLGL1的表達能抑制該調節通路，從而抑制OSMR 的轉錄。 / Background & Aims: Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant cancers worldwide. Its 5-year survival rate is less than 5%, because most patients have already developed to the advanced stage of local invasion or distant metastasis once diagnosed, and missed the chances of curable surgical resection. Adjuvant chemotherapy is an alternative therapeutic strategy against PDAC. Yet, only very small proportion of patients could benefit from chemotherapy due to the innate and easily-acquired chemo-resistance in PDAC cells, especially to the first-line chemotherapeutic drug, gemcitabine. Many studies have been conducted to exploring the mechanisms underlying gemcitabine resistance in PDAC cells, but gemcitabine resistance is still the major obstacle impeding PDAC patients benefits from chemotherapy. Our studies aimed to investigate novel genes involved in gemcitabine response and to explore the undefined mechanisms generating gemcitabine resistance in PDAC cells. / Methods: Our colleagues previously performed genome-wide RNAi screening in gemcitabine-sensitive Capan2 cells. Lethal giant larvae homolog 1 (LLGL1) was identified as a potential gemcitabine-sensitizing gene which was then validated by our subsequent in-vitro drug cytotoxicity assay in LLGL1-inhibited Capan2 and SW1990 cells and in vivo subcutaneous xenograft mouse model. Hematoxylin & Eosin staining and terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling were applied for the assessment of apoptotic effects induced by gemcitabine in subcutaneous xenografts. We did gene expression microarray analysis to explore the potential downstream targets of LLGL1. Western blotting, qRT-PCR, and luciferase assay were applied to validate the downstream target of LLGL1 that were figured out by microarray analysis. We also did immunohistochemical staining to investigate the expression levels and correlationship of LLGL1 and its downstream target in PDAC specimens. Chromatin immunoprecipitation was performed to explore the enrichment of the transcriptional factor Sp1(pThr453) and RNA polymerase II (Pol II) at the promoter of the downstream targets of LLGL1. / Results: LLGL1 was identified as a gemcitabine-sensitizing gene, whose inhibition remarkably reduced gemcitabine response in gemcitabine-sensitive Capan2 and SW1990 cells, and ectopic expression induced gemcitabine response in gemcitabine-resistant PANC1 cells. Oncostatin M receptor (OSMR) was identified as a downstream target of LLGL1, whose expression was negatively correlated with LLGL1, and knockdown of OSMR significantly reversed gemcitabine resistance induced by LLGL1 inhibition in Capan2 and SW1990 cells. Additionally, activation of OSMR signaling was associated with the elevated expression of cancer stem cell markers, CD44 and CD24, both of which had already been identified to contribute to gemcitabine resistance in PDAC cells. Moreover, OSMR up-regulation induced by LLGL1 inhibition in SW1990 cells depended on the activation of ERK2/Sp1 signaling and subsequent accumulation of Sp1(pThr453) and Pol II at the TATA-less, INR-less but Sp1-binding-site-rich promoter of OSMR, while ectopic expression of LLGL1 in PANC1 cells inactivated ERK2/Sp1 signaling and subsequently reduced the enrichment of Sp1(pThr453) and Pol II at OSMR promoter. / CONCLUSIONS: Our studies revealed the novel tumor suppressive role of LLGL1 as a gemcitabine-sensitizing gene in PDAC cells. Loss of LLGL1 resulted in the activation of ERK2/Sp1 signaling and up-regulation of OSMR expression, and ultimately desensitized gemcitabine response in PDAC cells. More importantly, ectopic expression of LLGL1 disrupted such regulatory axis and improved gemcitabine response. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhu, Yinxin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 154-183). / Abstracts also in Chinese.

A revascularização cardíaca utilizando a ponte de safena é um procedimento bastante utilizado para restabelecer o fluxo coronariano. Apesar do sucesso deste procedimento, a patência deste enxerto pode chegar a menos de 50% em 10 anos. Atribui-se parte deste insucesso a variações no processo adaptativo à nova condição hemodinâmica, onde o shear stress e o estiramento aumentados podem estar interferindo na função endotelial e vascular. Este processo envolve a participação de diversas proteínas e o estudo de como elas participam conjuntamente é uma importante abordagem para entender as alterações fisiológicas e patológicas que ocorrem no enxerto vascular. Neste trabalho, tecnologias proteômicas, gel 2-D e ICAT, foram utilizadas para identificar as proteínas que são modificadas nas fases precoces da arterialização do enxerto venoso. Foi utilizado um sistema ex vivo de perfusão controlada, desenvolvido em nosso laboratório, onde a veia safena humana foi cultivada tanto em regime hemodinâmico venoso (5 mL/min) e arterial (50 mL/min, 80 mmHg) por 24 horas. Dentre as proteínas identificadas, a maioria apresenta funções estruturais como, por exemplo, -actina de músculo liso, CRP1, colágeno VI, tropomiosina, miosina, desmina e vimentina. Para avaliação funcional foram selecionadas a -SMA e a CRP. A -SMA mostrou-se diminuída nas fases mais precoces da arterialização venosa, com quase desaparecimento após 3 dias da cirurgia, seguido de um aumento nos períodos subseqüentes. A CRP3 mostrou-se com expressão predominantemente arterial tanto em amostra humana como de rato. A arterialização de segmentos venosos induziu a expressão da CRP3, sendo dependente do aumento do estiramento (stretch) nas células musculares lisas e não do aumento do shear stress na superfície endotelial. Coletivamente, neste trabalho caracterizamos duas proteínas que foram modificadas durante o processo de arterialização e/ou adaptação da veia à condição hemodinâmica arterial. As proteínas identificadas contribuirão para o melhor entendimento do processo de arterialização venosa e poderão ser testadas como novos alvos terapêuticos para melhorar a patência destes enxertos / Coronary artery bypass surgery by saphenous vein graft is still widely used to revascularization of ischemic heart. Despite the success of this procedure, about 50% occlude after 5-10 years. The vein graft is subjected to increased tensile stress and the adaptive vein response to the arterial hemodynamic condition may predispose to bypass occlusion. Several proteins are modulated during arterialization, the understanding of the molecular changes of this process may be useful to new therapeutics approaches development attempting to increase vein graft patency. In this work, proteomics plataform, gel 2-D and ICAT, were used to identify the proteins that are modified in the early stages of vein graft rterialization. Human saphenous vein were cultured in an ex vivo flow through system in both venous (5 ml / min) and arterial (50 ml / min, 80 mm Hg) hemodymanic conditions for 24 hours. The identified proteins were related to cell structural function, such as -SMA, CRP1, collagen VI, tropomyosin, myosin, desmin and vimentin. To functional characterization, -SMA and CRP were selected. In rat vein arterialization model, - SMA showed to be decreased during the early stages of arterialization and almost disappeared after 3 days of surgery. Later on, -SMA-positive cells increase reaching similar expression levels of normal jugular vein. The expressiom of CRP3 showed to be predominantly to arterial beds both in human and rat. When vein segment were submitted to arterial hemodynamic condition, it was observed a significant induction of CRP3 expression. Interestingly, the increase of CRP3 is dependent of stretch stimulus in smooth muscle cells while shear stress did not modify its expression in endothelial cells. Collectively, we successfully identified proteins differentially expressed during the vein arterialization by using proteomic technique. -SMA and CRP3 were modified in vein segments exposed to arterial hemodynamic condition and efficiently discriminate smooth muscle cell phenotype. The identified proteins will contribute to the better understanding of the venous arterialization process and may be tested as new therapeutic targets for improving the patency these grafts

Spinal muscular atrophy (SMA) is the leading genetic cause of death of young children. It is an autosomal recessive disease caused by the mutation and/or the deletion within the ubiquitously expressed survival motor neuron 1 (SMN1) gene. SMA pathology is characterized by spinal cord motor neuron degeneration, neuromuscular junction (NMJ) defects and muscular atrophy. Upon disease onset, SMA patients progressively become paralyzed and in the most severe cases, they die due to respiratory complications. Over the years, it has become clear that SMN is a multi-functional protein with important roles in small nuclear ribonucleoprotein (snRNP) assembly, RNA metabolism, axonal outgrowth and pathfinding, mRNA transport as well as in the functional development of NMJs, skeletal muscle and cardiac muscle. However, it remains unclear which of these functions, and the respective perturbed molecular pathways, dictate SMA pathogenesis. Here, we have established Smn-depleted PC12 cells and an intermediate SMA mouse model to characterize a role for Smn in the regulation of actin cytoskeleton dynamics. We find that Smn depletion results in the increased expression of profilin IIa and active RhoA (RhoA-GTP) as well as the decreased expression of plastin 3 and Cdc42. Importantly, the inhibition of rho-kinase (ROCK), a direct downstream regulator of RhoA, significantly increased the lifespan of SMA mice and shows beneficial potential as a therapeutic strategy for SMA. In an addition, we have uncovered a muscle- and motor neuron-independent role for SMN in the regulation of pancreatic development and glucose metabolism in SMA mice and type 1 SMA patients. This finding highlights the importance of combining a glucose tolerance assessment of SMA patients with their existing clinical care management. Thus, our work has uncovered two novel and equally important roles for the SMN protein, both of which contribute significantly to SMA pathogenesis.

Schaut man durch ein Mikroskop auf eine biologische Zelle mit angefärbten Zytoskelett, so erblickt man lange, mehr oder minder gerade Objekte. Mit ziemlicher Sicherheit gehören diese zu einer von drei Arten von Zytoskelettfilamenten -- Aktin- oder Mikrofilamente, Intermediärfilamente und Mikrotubuli. Schon seit mehreren Jahrzehnten versucht man die mechanischen Eigenschaften lebender Zellen nicht nur zu beschreiben, sondern ihr Verhalten von zwei tieferen Ebenen ausgehend zu verstehen: Inwiefern beschreiben die Eigenschaften von Filamentnetzwerken und -gelen die Zellmechanik und, noch tiefgreifender, wie bestimmen eigentlich die einzelnen Filamente die Netzwerkmechanik. Das Verständnis der Mechanik homogener und isotroper, verhedderter als auch quervernetzter Gele ist dabei erstaunlich detailreich, ohne jedoch vollständig dem jüngeren Verständnis von Zellen als glassartige Systeme zu entsprechen. In den letzten Jahren sind daher anisotrope Strukturen mehr und mehr in den Fokus gerückt, die die Bandbreite möglichen mechanischen Verhaltens enorm bereichern. Die vorliegende Arbeit beschäftigt sich mit solch einem hochgradig anisotropen System -- nämlich Aktinbündeln -- unter drei Gesichtspunkten. Mit Hilfe von aktiven Biegedeformationen wird ein funktionales Modul, das eine differentielle Antwort auf verschiedenen Zeitskalen liefert, identifiziert. Es handelt sich um Aktinfilamente, die durch transiente Quervernetzer gebündelt werden. Während sich das System nach kurz anhaltenden Deformation völlig elastisch verhält, sorgt eine Restrukturierung der Quervernetzer während langanhaltender Deformationen für eine plastische Verformung des Bündels. In einem weiteren Aspekt widmet sich die Arbeit der frequenz- und längenabhängigen Biegesteifigkeit. Die Methode des Bündel-Wigglings, das Induzieren von \"Seilwellen\", wird dabei genutzt, um aus der Wellenform die Biegesteifigkeit zu berechnen. Bündel von Aktinbündeln zeigen dabei ein Verhalten, das vom klassischen Worm-like-chain-Modell abweicht und stattdessen durch das Worm-like-bundle-Modell beschrieben werden kann. Der letzte Aspekt dieser Arbeit untersucht den Musterbildungsprozess bei der Entstehung von Aktinbündeln. Gänzlich unerwartet entstehen quasi-isotrope Strukturen mit langreichweitiger Ordnung, wenn der Bündelungsprozess erst nach der Polymerisation von Filamenten frei von zusätzlichen mechanischen Einwirkungen einsetzt. Da dieser Zustand nicht von der klassischen Flüssigkristalltheorie vorhergesagt wird, soll eine Simulation eine Hypothese zum Entstehungsmechanismus testen. Die Annahme einer lateralen Kondensation von Filamenten zu Bündeln reicht demnach aus, um die beobachteten Strukturen zu erzeugen. Diese Arbeit leistet somit einen Beitrag zum Verständnis hochgradig anisotroper Strukturen und deren Überstrukturen, wie sie auch in lebendigen Zellen reichlich vorhanden sind. / Being the most basic unit of living organisms, the cell is a complex entity comprising thousands of different proteins. Yet only very few of which are considered to play a leading part in the cell’s mechanical integrity. The biopolymers actin, intermediate filaments and microtubules constitute the so-called cytoskeleton – a highly dynamic, constantly restructuring scaffold endowing the cell not only with integrity to sustain mechanical perturbations but also with the ability to rapidly reorganize or even drive directed motion. Actin has been regarded to be the protagonist and tremendous efforts have been made to understand passive actin networks using concepts from polymer rheology and statistical mechanics. In bottom-up approaches isotropic, homogeneous actin-gels are well-characterized with rheological methods that measure elastic and viscous properties on different time scales. Cells, however, are not exclusively isotropic networks of any of the mentioned filaments. Rather, actin alone can already be organized into heterogeneous and highly anisotropic structures like bundles. These heterogeneous structures have only come into focus recently with theoretical work addressing bundle networks. and, in the case of the worm-like bundle theory, individual bundles. This work aims at characterizing bundles and bundle-crosslinker systems mechanically in two complementary approaches – in the time as well as in the frequency domain. In addition, it illuminates a bundle formation mechanism that leads to bundle networks displaying higher ordering.

Spinal muscular atrophy (SMA) is the leading genetic cause of death of young children. It is an autosomal recessive disease caused by the mutation and/or the deletion within the ubiquitously expressed survival motor neuron 1 (SMN1) gene. SMA pathology is characterized by spinal cord motor neuron degeneration, neuromuscular junction (NMJ) defects and muscular atrophy. Upon disease onset, SMA patients progressively become paralyzed and in the most severe cases, they die due to respiratory complications. Over the years, it has become clear that SMN is a multi-functional protein with important roles in small nuclear ribonucleoprotein (snRNP) assembly, RNA metabolism, axonal outgrowth and pathfinding, mRNA transport as well as in the functional development of NMJs, skeletal muscle and cardiac muscle. However, it remains unclear which of these functions, and the respective perturbed molecular pathways, dictate SMA pathogenesis. Here, we have established Smn-depleted PC12 cells and an intermediate SMA mouse model to characterize a role for Smn in the regulation of actin cytoskeleton dynamics. We find that Smn depletion results in the increased expression of profilin IIa and active RhoA (RhoA-GTP) as well as the decreased expression of plastin 3 and Cdc42. Importantly, the inhibition of rho-kinase (ROCK), a direct downstream regulator of RhoA, significantly increased the lifespan of SMA mice and shows beneficial potential as a therapeutic strategy for SMA. In an addition, we have uncovered a muscle- and motor neuron-independent role for SMN in the regulation of pancreatic development and glucose metabolism in SMA mice and type 1 SMA patients. This finding highlights the importance of combining a glucose tolerance assessment of SMA patients with their existing clinical care management. Thus, our work has uncovered two novel and equally important roles for the SMN protein, both of which contribute significantly to SMA pathogenesis.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Trypanosoma cruzi é o agente causador da doença de Chagas, uma das doenças tropicais mais negligenciadas. Estima-se que cerca de 11 milhões de pessoas no mundo estão infectadas por T. cruzi e cerca de 6 a 7 milhões de pessoas estão em risco por encontrarem-se em áreas endêmicas. Durante o processo de invasão moléculas do parasita e do hospedeiro interagem permitindo a transdução de sinal e a expressão de genes de modulação em resposta à invasão. A diversidade de proteínas e vias acionadas para reparar a lesão pela ruptura da membrana plasmática nos interessou e dessa forma o presente estudo desenvolveu uma nova forma de detecção e quantificação por reação em cadeia da polimerase em tempo real (qPCR) da carga parasitária de T. cruzi e a quantificou os níveis transcricionais relativos (RT-qPCR) da Disferlina, Esfingomielina esferase ácida (ASM), Fator de Transcrição EB (TFEB), Galectinas 1 e 3 e Anexina A2. Neste estudo, foi demonstrado que a quantificação por PCR em tempo real utilizando os iniciadores P21fw e P21rv foi específico e sensível para a detecção de T. cruzi in vivo e in vitro, bem como os níveis transcricionais de genes relacionados a organização do citoesqueleto e reparo de membrana plasmática são moduladas em resposta ao dano gerado pelo parasita. / Trypanosoma cruzi is causative agent of Chagas disease, one of most neglected tropical diseases. Estimated that about 11 million people worldwide are infected by T. cruzi and about 6 to 7 million people are at risk in endemic areas. During the process of invasion of host and parasite interact enabling signal transduction and gene expression modulation in response to invasion. The diversity of activated proteins and pathways to repair the damage by disruption of the plasma membrane interest to us and thus present study developed a new form of detection and quantitation by polymerase chain reaction in real time (qPCR) of parasitic load T. cruzi and quantified transcriptional levels relative (RT-qPCR) of dysferlin, Sphingomyelin acid esferase (ASM), transcription factor EB (TFEB) Galectins 1 and 3 and Annexin A2. This study demonstrated that quantification by real time PCR using primers P21fw and P21rv was specific and sensitive for detection of T. cruzi in vivo and in vitro, as well as transcriptional levels of genes related to cytoskeletal organization and repair plasma membrane are modulated in response to damage generated by parasite. / Tese (Doutorado)

Synapse is the fundamental unit of synaptic transmission. Learning, memory and neurodegenerative diseases of the brain are attributed to the maintenance and alteration in synaptic connections. The efficiency for synaptic transmission depends on how well the post synapse receives the signals from the presynapse; this in turn depends on the receptors present in the post synaptic density (PSD). PSD is present in the post synapse right opposite to the neurotransmitter release site in presynapse (active zone) is an indispensable part of the synapse. The PSD is comprised of receptors and scaffold proteins, which is ultimately supported by the actin cytoskeleton of the dendritic spines. Cytoskeletal dynamics is shown to influence the structural plasticity of spine and also PSD, but how it regulates the dynamicity of the synaptic transmission is not completely understood. Here we studied the influence of actin depolymerisation on sub synaptic organization of an excitatory synapse. In order to study the organization of the synapse at molecular resolution, the conventional microscopy cannot be employed due to the limit of diffraction. Super resolution microscopy circumvents this diffraction limitation. In this study we have used custom built fluorescence microscope with Total Internal Reflection Fluorescence (TIRF) modality to observe the nanometre sized structures inside spines of mouse hippocampal primary neurons. The setup was integrated with Metamorph imaging software for both operating the microscope and imaging acquisition purpose with a separate appropriate laser system. This setup was successful in achieving the lateral resolution of ~30nm and axial resolution of ~51nm. Over all we were able to observe the loss of spines and significant reduction in area of nanometer sized protein clusters in postsynaptic density with in the spines of latrunculin A treated mouse hippocampal primary neurons compared to the native neurons. Along with the morphological alterations in neurons we also observed the changes in nanoscale organization of few key molecules in the postsynaptic density.

Le métabolisme des phosphoinositides constitue un processus crucial parmi les systèmes permettant la terminaison de la transmission intracellulaire d’un stimulus extracellulaire. En effet, l’une de principales voies de signalisation intracellulaire engendrée en réponse à la liaison d’hormones ou de facteurs de croissance sur leurs récepteurs spécifiques fait intervenir la phosphorylation du PtdIns(4,5)P2 en PtdIns(3,4,5)P3 par une PI 3-kinase. Les enzymes responsables du métabolisme de ce second messager sont donc essentielles à la fonction normale de la cellule. L’ADNc de la 5-phosphatase SHIP2 a été cloné dans notre laboratoire. Cette enzyme présente au sein de sa structure primaire, en plus d’un domaine catalytique 5-phosphatase, un grand nombre de motifs permettant des interactions spécifiques avec d’autres protéines :un domaine SH2 N-terminal, des séquences riches en prolines, un motif NPXY et un domaine SAM. <p>SHIP2 joue un rôle de régulateur négatif dans la voie PKB et MAPK in vitro dans plusieurs modèles cellulaires. De plus, l’affinité de SHIP2 pour le cytosquelette a été mise en évidence notamment dans les plaquettes sanguines.<p> Notre travail de thèse s’intègre dans le cadre de l’étude biochimique de SHIP2 et plus particulièrement dans la recherche de ses partenaires protéiques. La Vinexine, protéine du cytosquelette impliquée dans l’adhérence cellulaire, avait été identifiée comme une protéine interagissant avec SHIP2 par la technique du double hybride. Nous avons confirmé cette association par des expériences d’immunoprécipitation en système transfecté (cellules COS-7) ainsi qu’en système natif (cellules HeLa et MEF). Nous avons montré que cette interaction n’était ni modulée par une stimulation à l’EGF (dans des cellules COS-7) ni par le sérum (dans des cellules MEF). Nous avons démontré une colocalisation de SHIP2 avec la Vinexine &61537; à la périphérie des cellules COS-7 stimulées à l’EGF. Par la suite, nous nous sommes intéressés au rôle potentiel de cette interaction dans l’adhérence cellulaire. Nous avons montré que la surexpression de SHIP2 ou de la Vinexine &61537; augmente l’adhérence des cellules COS-7 sur un substrat de collagène I. L’adhérence à ce même substrat est diminuée dans les cellules MEF issues des souris déficientes en SHIP2 comparées à des cellules contrôles SHIP2 +/+. De plus, il apparaît que la partie carboxy-terminale de SHIP2 ainsi que son activité catalytique sont importantes pour l’adhérence des cellules au substrat.<p>Ces résultats suggèrent un rôle important de SHIP2 dans l’adhérence cellulaire et l’organisation du cytosquelette d’actine faisant intervenir un mécanisme probable de déphosphorylation du PtdInsP3.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Angiogenesis, the process of new vessel formation, appears to be a central mechanism that underlies the development of haemangiomas. Recently, intralesional bleomycin injection was used to treat paediatric haemangiomas with very good results. The purpose of this study was to determine whether there was significant systemic circulatory spill-over of bleomycin in haemangioma patients treated with intralesional bleomycin to determine safety of use. Furthermore, in order to elucidate bleomycin’s mechanism of action in inducing haemangioma regression, this study aimed at determining the effects of bleomycin on aspects of angiogenesis, namely, endothelial cell migration, growth and apoptosis, and comparing these effects with those of drugs previously reported to inhibit various aspects of the angiogenic process (mitomycin C, 2-methoxyestradiol, taxol, vincristine, vinblastine, colchicine, nocodazole and cytochalasin D). Lastly, the effects of bleomycin, mitomycin C, 2-methoxyestradiol, taxol, vincristine, vinblastine, colchicine, nocodazole and cytochalasin D were studied in an animal haemangioma model. A rapid and highly sensitive high performance liquid chromatographic (HPLC) method was developed. Blood samples were collected from four haemangioma patients before and after (over a 24 hour period) intralesional bleomycin (IB) therapy. As a control, blood samples were also collected at identical time intervals from four patients undergoing intravenous (IV) bleomycin chemotherapy for various malignant tumours. The HPLC method was used to quantitate bleomycin fractions in patient samples. The mean bleomycin concentration detected in plasma samples obtained from IB treated patients was 0.00 ìg/ml for both bleomycin A<Sub>2 and B2 over the 24-hour period following therapy. Plasma bleomycin A2 and B2 levels of 360.79 and 158.85 ìg/ml respectively were detected in samples obtained from cancer patients treated with bleomycin IV. These findings indicate that the low levels detected may translate to a significantly lesser risk of pulmonary fibrosis following IBI. The effect of drugs on endothelial cell migration was analyzed by wounding a confluent monolayer of cells and determining the number of cells that had migrated from the wound edge. Endothelial cell growth was determined in cells treated with various drug concentrations while apoptosis was examined using hematoxylin and eosin staining, DNA fragmentation assay and acridine orange staining. The effect of test drugs on in vitro angiogenesis was determined on endothelial cells induced to form capillary-like tubes in collagen gel. Test drugs were then evaluated for antitumour activity in an animal haemangioma model. Data demonstrated that test drugs inhibited endothelial cell migration, with the exception of mitomycin C. All test drugs induced a reduction in the percentage of viable endothelial cell in a dose-dependant manner, and also induced endothelial cell apoptosis. The drugs inhibited angiogenesis in vitro and inhibited tumour development in vivo with varying potency. In general, results from this study indicated that there was negligible systemic spill-over of bleomycin following IB administration in patients with haemangiomas, suggesting a much lesser risk of developing bleomycin-induced pulmonary fibrosis. This study also showed that test drugs inhibited angiogenesis in vitro and haemangioma development in vivo in a mouse model. Taken together, these observations demonstrate that bleomycin may inhibit haemangioma growth by inhibiting angiogenesis. In addition, mitomycin C, 2-methoxyestradiol, taxol, vincristine, vinblastine, colchicine, nocodazole and cytochalasin D may have potential in the treatment of haemangiomas of infancy, and should be investigated further in a murine haemangioma model to determine effective dose schedules. / Thesis (PhD)--University of Pretoria, 2009. / Physiology / unrestricted

Spinal muscular atrophy (SMA) is the leading genetic cause of death of young children. It is an autosomal recessive disease caused by the mutation and/or the deletion within the ubiquitously expressed survival motor neuron 1 (SMN1) gene. SMA pathology is characterized by spinal cord motor neuron degeneration, neuromuscular junction (NMJ) defects and muscular atrophy. Upon disease onset, SMA patients progressively become paralyzed and in the most severe cases, they die due to respiratory complications. Over the years, it has become clear that SMN is a multi-functional protein with important roles in small nuclear ribonucleoprotein (snRNP) assembly, RNA metabolism, axonal outgrowth and pathfinding, mRNA transport as well as in the functional development of NMJs, skeletal muscle and cardiac muscle. However, it remains unclear which of these functions, and the respective perturbed molecular pathways, dictate SMA pathogenesis. Here, we have established Smn-depleted PC12 cells and an intermediate SMA mouse model to characterize a role for Smn in the regulation of actin cytoskeleton dynamics. We find that Smn depletion results in the increased expression of profilin IIa and active RhoA (RhoA-GTP) as well as the decreased expression of plastin 3 and Cdc42. Importantly, the inhibition of rho-kinase (ROCK), a direct downstream regulator of RhoA, significantly increased the lifespan of SMA mice and shows beneficial potential as a therapeutic strategy for SMA. In an addition, we have uncovered a muscle- and motor neuron-independent role for SMN in the regulation of pancreatic development and glucose metabolism in SMA mice and type 1 SMA patients. This finding highlights the importance of combining a glucose tolerance assessment of SMA patients with their existing clinical care management. Thus, our work has uncovered two novel and equally important roles for the SMN protein, both of which contribute significantly to SMA pathogenesis.