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1
Vilímek, Hynek. "Převod PPTX do HTML". Master's thesis, Vysoké učení technické v Brně. Fakulta informačních technologií, 2016. http://www.nusl.cz/ntk/nusl-255407.
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PowerPoint is an excellent tool for creating presentations and people are accustomed to using it. Its only handicap is that it is not installed everywhere and it exists in numerous versions. But there is an application that is installed almost everywhere and that application is the web browser. This work aims to create the PowerPoint presentation viewer for the web browser. With the internet as the environment, it may have a wide range of applications from the content sharing point of view. The solution is a web application that allows to upload the PowerPoint file and then the application displays the content of the file. The application also offers functionality such as navigation between slides and full-screen mode. The rendered slides in the web browser are very similar to the slides in PowerPoint. It does not support advanced features, but it supports displaying text, pictures, video and audio. Further, it supports basic styling options such as colours, margins, position and line height.
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Anzi, C. "Pattern di espressione del gene Ppi1 e analisi di mutanti ppi1 e ppi 2 in Arabidopsis thaliana". Doctoral thesis, Università degli Studi di Milano, 2008. http://hdl.handle.net/2434/61194.
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The proton pump interactor (Ppi) gene family of Arabidopsis thaliana: expression pattern of Ppi1 and characterisation of knockout mutants for Ppi1 and 2. Plant plasma membrane H+-ATPases (PM H+-ATPase) are essential for establishing a proton electrochemical gradient across the cell plasma membrane. Their regulation is poorly understood, except for the role of 14-3-3 proteins, which relieve autoinhibition from the C-terminal domain. A novel protein interacting with this domain was recently identified in Arabidopsis and named PPI1 (Proton Pump Interactor 1). PPI1 stimulates PM H+-ATPase activity in vitro. Here, we analyse the expression pattern of Ppi1 using b-glucuronidase as a reporter. Expression is strong in root and shoot vascular systems, particularly in meristematic and sink tissues, as well as in pollen, stigmas and siliques, but not in developing embryos. Removal of the first intron decreased GUS expression 45-fold. We also analysed the transcription of Ppi2, another gene in the family, and demonstrated that Ppi2 is expressed in seedlings, cultured cells and flowers. Insertional mutants for both Ppi1 and Ppi2 were isolated. Two different mutants of Ppi1 showed aberrant mRNAs and lacked any detectable protein and are therefore true knockouts. At the plant level, neither of the single mutants nor the double ppi1ppi2 mutant showed an altered phenotype in standard growth conditions under acid load or salt stress.
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Soler, David C. "The PP1 gamma isoforms restore spermatogenesis but not fertility in PP1 gamma null mice". [Kent, Ohio] : Kent State University, 2009. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=kent1259087463.
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Thesis (Ph.D.)--Kent State University, 2009.Title from PDF t.p. (viewed May 17, 2010). Advisor: Srinivasan Vijayaraghavan. Keywords: sperm; spermatogenesis; PP1gamma2; PP1gamma1; mice; transgene. Includes bibliographical references (p. 102-123).
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Gregório, Luís Korrodi Mineiro Marques. "Characterization of PPP1 interacting proteins in male reproduction". Doctoral thesis, Universidade de Aveiro, 2012. http://hdl.handle.net/10773/7826.
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Doutoramento em BioquímicaA fosforilação reversível de proteínas é um importante mecanismo de controlo em eucariotas. A fosfoproteína fosfatase 1 (PPP1) é uma fosfatase de serina/treonina envolvida em vários processos celulares. Existem três isoformas da subunidade catalítica (α/CA, δ/β/CB e γ/CC) com pequenas diferenças nos terminais amino e carboxílico. O gene PPP1CC sofre ainda splicing alternativo para produzir duas isoformas, a PPP1CC1 ubíqua e a PPP1CC2 enriquecida em testículo e específica de esperma. A localização e especificidade de substratos da PPP1 está dependente da formação de complexos oligoméricos com proteínas que interagem com a PPP1 (PIPs). O objetivo principal desta tese foi estudar novas PIPs, específicas de testículo e esperma, a fim de melhor caracterizar o papel desta fosfatase e dos respetivos complexos na reprodução em mamíferos. Com este fim, estudou-se a presença, localização e possíveis funções de uma PIP previamente conhecida, PPP1R2, e de duas novas PIPs, PPP1R2P3 e Tctex1d4. PPP1R2 e PPP1R2P3 estão presentes em esperma humano colocalizando com a PPP1CC2, na cabeça e na cauda. A hipótese é que as holoenzimas localizadas na cabeça terão um papel na reação acrossómica, enquanto que as holoenzimas presentes no axonema são relevantes para o controlo da motilidade flagelar. De seguida foram estudados os pseudogenes da PPP1R2, em termos de história evolutiva e de possíveis funções. Na espécie humana, a PPP1R2 tem 10 pseudogenes, 7 deles específicos de primatas. Estudos de bioinformática e dados de expressão mostram que os PPP1R2P1/P3/P9 são os pseudogenes com maior probabilidade de serem transcritos e traduzidos. Também identificámos o PPP1R2P9 em esperma humano e mostrámos que alguns pseudogenes poderão estar associados a estados fisiopatológicos. Isto indica que o processo de evolução poderá estar ligado á formação de novos genes ou ao controlo do mRNA da PPP1R2. A sobre-expressão da PPP1R2 ou PPP1R2P3 em testículo de ratinho também foi realizada, para caracterizar os mecanismos envolvidas na função dos complexos PPP1R2/PPP1R2P3-PPP1CC2 na espermatogénese e fisiologia dos espermatozoides. A dineína de cadeia leve, Tctex1d4, foi encontrada como interagindo com a PPP1C e como estando presente em testículo de ratinho e em esperma humano. Demonstrámos que a Tctex1d4 e a PPP1 colocalizam no centro organizador de microtúbulos e nos microtúbulos e que o motivo de ligação à PPP1 presente na Tctex1d4 parece ser importante para manter a PPP1 no centro organizador de microtúbulos e/ou para disromper ou atrasar o seu movimento ao longo dos microtúbulos emergentes. Estes resultados abrem novos caminhos para os possíveis papéis do complexo Tctex1d4-PPP1 na dinâmica dos microtúbulos, motilidade do esperma, reação acrossómica e na regulação da barreira hemato-testicular, provavelmente, através da via de sinalização do TGFß. A análise do motivo de ligação à PPP1 mostra que este é altamente conservado entre os mamíferos, com exceção das Pikas, sugerindo que esta perda aconteceu antes da radiação das Pikas, há 6-20 milhões de anos atrás. Através de um rastreio por mutações demonstrámos que a capacidade da Tctex1d4 se ligar à PPP1 é mantida nas Pikas, embora o motivo de ligação à PPP1 esteja disrompido. Este estudo abre portas para novas descobertas na área da reprodução mostrando o papel da PPP1CC2 na espermatogénese e fisiologia do esperma.
Reversible phosphorylation of proteins is an important intracellular control mechanism in eukaryotes. Phosphoprotein Phosphatase 1 (PPP1) is a major serine/threonine protein phosphatase involved in a wide range of cellular processes. Three closely related catalytic subunit isoforms (/CA, δ//CB and /CC) exist with only minor differences at their N- and C-terminus. PPP1CC gene can also undergo tissue-specific processing to yield a ubiquitously expressed PPP1CC1 and the testis-enriched and sperm-specific PPP1CC2 isoforms. PPP1C exists in the cell as an oligomeric complex binding to a spectrum of PPP1 interacting proteins (PIPs), which modulate both its intracellular localization and substrate specificity. The main goal of this thesis was to study novel PIPs in testis and sperm, in order to further characterize the role of PPP1CC2 and the respective complexes in mammalian reproduction. To this end we addressed the presence, localization and putative roles of a previously known PIP, PPP1R2, in testis and sperm, and two novel PPP1CC2 testis/sperm specific PIPs, PPP1R2P3 and Tctex1d4. PPP1R2/PPP1R2P3 were shown to be present in human sperm co-localizing with PPP1CC2, in the head and tail. It was shown that PPP1R2P3 is a heat stable inhibitor of PPP1CC that cannot be phosphorylated by GSK3. We hypothesize that the holoenzymes localized in the head may have a role in the acrosome reaction while the axoneme bound holoenzymes are relevant for the control of flagellar motility. To further address the PPP1R2 significance, its pseudogenes were described in terms of evolutionary history and putative functions. In human specie, PPP1R2 has ten pseudogenes most of them primate-specific. Besides PPP1R2P3, bioinformatic studies and expression data show that PPP1R2P1, PPP1R2P2 and PPP1R2P9 are the pseudogenes with more probability of being transcribed and eventually translated. Moreover, we identified PPP1R2P9 in human sperm and showed that several pseudogenes appear to be associated with physiological and pathological states. This indicates that evolution processes might be in part related with the formation of new genes or in the control of the parental PPP1R2 message. Overexpression of human PPP1R2 or PPP1R2P3 in mouse testis was also pursued to provide the molecular tools to initiate the characterization of the mechanisms behind PPP1R2/PPP1CC2 and PPP1R2P3/PPP1CC2 role in spermatogenesis and sperm physiology. Dynein light chain, Tctex1d4, was found to bind to PPP1C and to be present in mouse testis and human sperm. Tctex1d4-PPP1CC complex was shown to co-localize in the microtubule organizing centre and in microtubules. Moreover, the Tctex1d4 PPP1 binding motif seems to be important to retain PPP1CC in the microtubule organizing centre, and also to disrupt or delay its movement along microtubules. These results open new avenues to the possible roles of Tctex1d4-PPP1 complex in microtubule dynamics, sperm motility, acrosome reaction and in regulation of the blood testis barrier possibly via TGFß signaling. Moreover, PPP1 binding motif is highly conserved among mammals, except in Pikas, suggesting that this event happened before the Pikas radiation, 6-20 Million years ago. Mutational screening shows that the ability of Tctex1d4 to bind to PPP1 is maintained in Pikas, although the PPP1 binding motif is disrupted. This work opens doors to new discoveries in male reproduction and unravels the roles of PPP1CC2 and its PIPs in spermatogenesis and sperm physiology.
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Sousa, Luís Miguel dos Santos. "TCTEX1D4 and PPP1: TGFß pathway and prostate cancer". Master's thesis, Universidade de Aveiro, 2013. http://hdl.handle.net/10773/11571.
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Mestrado em Biomedicina MolecularT-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) é uma cadeia leve de dineina que foi identificada como sendo uma proteína que interage com a PPP1. As funções específicas da TCTEX1D4 ainda permanecem desconhecidas mas a identificação dos seus interactores pode elucidar sobre as suas funções em sistemas biológicos. No interactoma da TCTEX1D4 merece particular destaque a presença de diversas proteínas associadas à via de sinalização do TGFβ e cuja desregulação se encontra associada ao cancro da próstata. Desta forma, foi objectivo deste trabalho avaliar a existência de TCTEX1D4 e do complexo TCTEX1D4-PPP1 em células de cancro da próstata, procurar desvendar o papel da TCTEX1D4 na via de sinalização do TGFβ e identificar eventuais alterações associadas à malginidade no cancro da próstata.
T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) is a dynein light chain that has been identified as interacting with PPP1. Whilst specific functions of TCTEX1D4 remain unclear, the identification of its interactors may help elucidating its biological function. Concerning to TCTEX1D4 interactome, the presence of several proteins of the TGFβ signaling pathway which deregulation is associated with prostate cancer is of particular interest. Thereof, it was purpose of this work to assess of existence of TCTEX1D4 and the TCTEX1D4-PPP1 complex in prostate cancer cells, clarify its role within the TGFβ signaling pathway and identify possible alterations during prostate cancer carcinogenesis.
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Chaudry, Tanya N. "Characterisation of PP71 homologues encoded by mammalian cytomegaloviruses". Thesis, Connect to e-thesis, 2008. http://theses.gla.ac.uk/377/.
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Thesis (Ph.D.) - University of Glasgow, 2008.Ph.D. thesis submitted to the Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.
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Zhao, Yiqiang. "Functional Analysis of Human Cytomegalovirus (HCMV) US3 and pp71". Ohio University / OhioLINK, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou995293805.
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Prechova, M. "Functional analysis of PP1 regulator Phactr1". Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10046874/.
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The Phosphatase and actin regulator (Phactr) protein family has been identified as a group of four proteins (Phactr1,2,3,4), interacting with Protein Phosphatase 1 (PP1) and G-actin. G-actin binding to Phactr1 RPEL motifs has been shown to sterically inhibit Phactr1 interaction with PP1 and its nuclear import. Members of the Phactr family are highly expressed in the nervous system and have been implicated in the regulation of the actin cytoskeleton, cell migration and angiogenesis. The molecular mechanism of their signalling is however not well understood. Here I show that Phactr1 functions as a novel regulatory subunit of PP1. Phactr1 contains several binding motifs typical of PP1 regulatory subunits, including an RVxF motif, located in the C-terminal part of Phactr1, partially overlapping with the G-actin binding RPEL motif. Phactr1 binding leaves the PP1 active site accessible for substrates, blocks one of the substrate-binding grooves and creates a highly basic pocket along the PP1 hydrophobic substrate binding groove, thereby defining Phactr1-PP1 specific protein substrates for dephosphorylation. A phosphoproteomic analysis revealed that activation of Phactr1-PP1 complex formation leads to the dephosphorylation of several cytoskeletal proteins (such as IRSp53 and Afadin), leading to rearrangements of the actin cytoskeleton. Induction of Phactr1-PP1 interaction leads to the formation of aberrant actomyosin structures in fibroblasts. The converse phenotype could be induced by non-PP1-binding Phactr1 mutant expression, leading to the formation of long cytoplasmic extensions. In neurons, Phactr1 was enriched in dendritic spines and activation of Phactr1-PP1 complex formation led to changes in dendritic spine morphology. Moreover, I show that Phactr1 L519R mutation, that has been implicated in epilepsy, decreases Phactr1 affinity to G-actin, and thus indirectly activates Phactr1 interaction with PP1. All this suggests that Phactr1 targets PP1 to dephosphorylate specific cytoskeletal proteins, leading to changes in the actin cytoskeleton, which in neurons modulates dendritic spine morphology, leading to changes in neuronal signalling.
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Broad, William. "Elucidating the function of the suppressor of ppi1 locus 2". Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:81cb5fb1-e735-453c-9c4b-332a5aa16b27.
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Fox, Helen. "PP1 localisation and function during fungal morphogenesis". Thesis, University of East Anglia, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327586.
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Molero, Merinero Cristina. "Molecular characterization of the Ppz1 phosphatase and its regulatory subunit Hal3". Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/454816.
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La proteína Ser / Thr fosfatasa Ppz1 de S. cerevisiae es un componente importante en el mantenimiento de la homeostasis de cationes monovalentes (K+ y Na+) y, como tal, incide en múltiples procesos celulares, tales como la progresión del ciclo celular y el mantenimiento de la integridad celular. La homeostasis de cationes es un factor clave para la supervivencia de cualquier célula, particularmente para los microorganismos que, como las levaduras, deben hacer frente a los cambios ambientales. Aunque Ppz1 es un enzima relacionado con la ubicua fosfatasa tipo 1 (PP1c), las enzimas tipo Ppz sólo se encuentran en hongos, incluyendo patógenos. Se han identificado dos subunidades reguladoras de S. cerevisiae Ppz1, Hal3 y Vhs3, que se unen a la mitad C-terminal catalítica de Ppz1 e inhiben fuertemente su actividad fosfatasa. La modulación de PP1c por sus muchas subunidades reguladoras ha sido ampliamente caracterizada, interesantemente, Hal3 no parece estructuralmente similar a los inhibidores de PP1c conocidos, con la excepción de la presencia de una secuencia relacionada con RVxF, que se encuentra en la mayoría de los reguladores de PP1c. Este motivo interactúa con un surco hidrofóbico de la fosfatasa, cuya estructura también se conserva en Ppz1. Sin embargo, Hal3 no se une o inhibe in vitro la PP1c de levadura y este motivo parece no ser relevante para su interacción con Ppz1. Por lo tanto, Hal3 parece ser bastante específico hacia Ppz1, lo que sugiere que el mecanismo de regulación de Ppz1 por Hal3 podría diferir de los encontrados para PP1c subunidades reguladoras.
Un objetivo principal de este trabajo fue obtener una visión de los mecanismos específicos de regulación de Ppz1 por Hal3. Con este fin, dos enfoques experimentales se llevaron a cabo. En primer lugar, se diseñó un cribado genético para identificar las mutaciones de Ppz1 que liberarían Ppz1 de la regulación de Hal3. Se generó una biblioteca de las versiones de Ppz1 que portaban mutación aleatoria en su dominio catalítico C-terminal y se llevó a cabo un cribado funcional en el fondo slt2. Este cribado produjo 36 variantes, de las cuales 9 llevaron simple y 4 doble cambios de aminoácidos. La caracterización in vivo e in vitro de estas variantes demostró que todas ellas afectaron la capacidad inhibitoria de ser inhibida por, pero no para interactuar con Hal3. La asignación de los residuos mutados en un modelo de Ppz1 localizó estos residuos en una región equivalente a la reconocida en PP1c por el Inhibitor-2. Segundamente, desarrollamos estrategias exitosas para coexpresar en E. coli y purificar el complejo de proteínas formado por Ppz1 (mitad C-terminal) y Hal3. Estas preparaciones permitieron la generación de diversos cristales. Sin embargo, a pesar de los muchos esfuerzos para mejorar los cristales obtenidos, no fueron de suficiente calidad para obtener conjuntos de datos de alta resolución como para resolver la estructura tridimensional del complejo.
Hace años se demostró que Hal3 es una proteína multifuncional, porque además de su papel en la regulación de Ppz1, Hal3 contribuye (junto con Cab3 y Vhs3) a la generación de un enzima PPCDC heterotrimérico atípico. Este enzima esencial y conservado existe como una flavoproteína homotrimérica en la mayoría de las especies eucarióticas, y requiere en el centro activo la presencia de residuos His y Cys específicos, así como un Asn conservado. La búsqueda de genomas fúngicos para la presencia de enzimas putativo PPCDC reveló que Schizosaccharomyces pombe contiene un único ORF (SPAC15E1.04, llamado aquí SpHal3)) que podría codificar un Hal3 homólogo. Demostramos que este gen tiene una estructura inusual, siendo el resultado de un evento de fusión génica, que contiene un segmento de tipo Hal3 necesario para la actividad PPCDC (similar a ScHal3) en la región N-terminal y la secuencia que codifica la enzima timidilato sintasa en el extremo C-terminal. Nuestros resultados mostraron que SPAC15E1.04 se expresa como un único polipéptido y que la mitad N-terminal de SpHal3 es suficiente para determinar la actividad de PPCDC, si la mitad C-terminal es capaz de proporcionar la función TS en S. cerevisiae.The S. cerevisiae Ser/Thr protein phosphatase Ppz1 is an important component in the maintenance of monovalent cation (K+ and Na+) homeostasis and, as such, impinges in multiple cellular processes, such as cell cycle progression, cell integrity maintenance. Cation homeostasis is a key factor for the survival of any single cell, particularly for microorganisms that, like yeasts, must confront environmental changes. Although Ppz1 is an enzyme related to the ubiquitous type-1 phosphatase (PP1c), Ppz-like enzymes are only found in fungi, including pathogenic ones. Two regulatory subunits of S. cerevisiae Ppz1 have been identified, Hal3 and Vhs3, which bind to the catalytic C-terminal half of Ppz1 and strongly inhibit its phosphatase activity. Modulation of PP1c by its many regulatory subunits has been extensively characterized, interestingly, Hal3 does not appear structurally similar to known PP1c inhibitors, with the exception of the presence of an RVxF related sequence, which is found in most PP1c regulators. This motif interacts with a hydrophobic groove of the phosphatase, whose structure is also conserved in Ppz1. However, Hal3 does not bind or inhibit in vitro yeast PP1c and this motif seems to not be relevant for its interaction with Ppz1. Therefore, Hal3 seems to be rather specific towards Ppz1, suggesting that the mechanism of Ppz1 regulation by Hal3 might differ from those found for PP1c regulatory subunits. A major goal of this work was to gain insight into the specific regulatory mechanisms of Ppz1 by Hal3. To this end, two experimental approaches were undertaken. Firstly, a genetic screen was devised to identify Ppz1 mutations that would release Ppz1 from Hal3 regulation. A library of Ppz1 versions carrying random mutation in its C-terminal catalytic domain was generated and a functional screen in the slt2 background carried out. This screen yielded 36 variants, of which 9 carried single and 4 double amino acid changes. In vivo and in vitro characterization of these variants demonstrated that all of them affected the inhibitory capacity to be inhibited by, but not to interact with Hal3. Mapping the mutated residues in a Ppz1 model localized these residues in one of the region equivalent to that recognized on PP1c by Inhibitor-2. Secondly, we developed successful strategies to co-express in E. coli and to purify the protein complex formed by Ppz1 (C-terminal half) and Hal3. These preparations allowed the generation of diverse crystals. However, despite the many efforts to improve the obtained crystals, they were not of enough quality to obtain a high resolution data sets as to resolve the three-dimensional structure of the complex. It was demonstrated years ago that Hal3 is a moonlighting protein, because in addition to its role in Ppz1 regulation, Hal3 contributes (together with Cab3 and Vhs3) to the generation of an atypical heterotrimeric PPCDC enzyme This essential and conserved enzyme exists as a homotrimeric flavoprotein in most eukaryotic species, and requires in the active site the presence of specific His and Cys residues, as well as a conserved Asn. The search of fungal genomes for the presence of putative PPCDC enzymes revealed that Schizosaccharomyces pombe contains a unique ORF (SPAC15E1.04, named here SpHal3)) that could encode a Hal3 homolog. We demonstrate that this gene has an unusual structure, being the result of a gene fusion event, containing a Hal3-like segment necessary for PPCDC activity (similar to ScHal3) in the N-terminal region and the sequence that encodes the thymidylate synthase enzyme in the C-terminus. Our results showed that SPAC15E1.04 is expressed as a single polypeptide and that the SpHal3 N-terminal half is sufficient to decide PPCDC activity, were the C-terminal half is able to provide TS function in S. cerevisiae. Our results confirmed the multifunctional nature of this fusion gene and demonstrated that it is an essential gene.
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Tryggvason, Thorir. "Analysis of the PPTP and IPSec protocols in Virtual Private Networks". Thesis, University of Skövde, Department of Computer Science, 2000. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-415.
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Today increasing numbers of individuals are working away from the ordinary workplace while still requiring access to the server located at the workplace. New technology is meeting this demand allowing for safe and secure transmission of the data over the Internet. The aim of this project is to analyse two protocols that are used within the Virtual Private Network (VPN) structure today, with the focus on installation, transmission speed on both Local Area Networks (LAN) and via telephone line and security aspects of the protocols.
The results show that it is quite complicated to setup a VPN network and to get operational. The results also show that there are security compromises within the VPN structure that indicate that if proper precaution is not taken it may give a false sense of security, where the user believes that it is a secure communication when in reality it is not.
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Candon, Heather L. "Polyphosphate kinase 1 (PPK1) is a pathogenesis determinant in Campylobacter jejuni". Thesis, University of British Columbia, 2007. http://hdl.handle.net/2429/31547.
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Campylobacter jejuni (C. jejuni) is the leading cause of bacterial gastroenteritis in the developed world. Despite the prevalence of C. jejuni as a human pathogen, relatively little is known about it's precise pathogenesis mechanisms, particularly in comparison to other well-studied enteric pathogens like E. coli and Salmonella spp. Altered expression of phosphate genes in a C.jejuni stringent response mutant, together with known correlations between the stringent response, polyphosphate (poly P), and virulence in other pathogens, led us to investigate the role of poly P in C. jejuni physiology and pathogenesis. All sequenced C. jejuni strains harbour a conserved putative polyphosphate kinase (PPK1) predicted to be principally responsible for poly P synthesis. We generated a targeted ppkl deletion mutant (Δppk1) in C. jejuni strain 81-176 and found that this mutant, as well as the ΔspoT stringent response mutant, exhibited low levels of poly P at all growth stages. In contrast, wild-type C. jejuni poly P levels increased significantly as the bacteria transitioned from log to stationary phase. Phenotypic analyses revealed that the Δppk1 mutant was defective for survival during osmotic shock and low-nutrient stress. However, certain phenotypes associated with ppk1 deletion in other bacteria (i.e., motility, oxidative stress) were unaffected in the C. jejuni mutant, which also displayed a surprising increase in biofilm formation. The C. jejuni Δppk1 mutant was also defective for the virulence-associated phenotype of intra-epithelial cell survival in a tissue culture infection model and exhibited a striking defect in dose-dependent chick colonization. These results indicate that poly P utilization and accumulation contribute significantly to C. jejuni pathogenesis and affect its ability to adapt to specific stresses and stringencies. Furthermore, our study demonstrates that poly P likely plays both similar and unique roles in C. jejuni compared to other bacteria, and that poly P metabolism is linked with stringent response mechanisms in C. jejuni.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Nicholson, Iain Peter. "Characterisation of two homologues of the human cytomegalovirus transactivating protein pp71". Thesis, University of Glasgow, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398661.
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Fernandes, Emanuel Ferreira. "The role of Synphilin-1A/PPP1 complex in Lewy bodies formation". Master's thesis, Universidade de Aveiro, 2013. http://hdl.handle.net/10773/12488.
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Mestrado em Biomedicina MolecularOne of the major Parkinson’s disease hallmarks is the development of cytoplasmic inclusions, termed Lewy bodies, mainly within surviving neurons in the brainstem of affected patients. Many proteins have been identified in the Lewy bodies, but their formation mechanism remains unclear. Among the proteins already identified in the Lewy bodies are synphilin-1, a α-synucleininteracting protein, and synphilin-1A, a synphilin-1 splice variant. Synphilin-1 and synphilin-1A have been considered key elements in Parkinson’s disease as their overexpression in human embryonic kidney 293 cells, with or without α-synuclein, leads to the formation of Lewy body-like cytoplasmic inclusions. Therefore, efforts have been made to clarify the regulatory mechanisms behind synphilin-1 and synphilin-1A aggregation as a means to uncover new aspects of Lewy bodies formation. Although kinases able to phosphorylate synphilin-1 have been described, there are no specific data concerning the phosphatases responsible for its dephosphorylation. This gap was filled with the identification of synphilin-1A as a novel phosphoprotein phosphatase 1-interacting protein in human brain, through yeast two hybrid. Hence, in the present work, the physiological role of synphilin-1A/phosphoprotein phosphatase 1 complex is studied, being demonstrated the ability of synphilin-1A to specifically target phosphoprotein phosphatase 1 to inclusion bodies, using immunofluorescence experiments. Moreover, the consequences of disrupting this interaction are explored using a synphilin-1A mutant unable to interact with phosphoprotein phosphatase 1, revealing an enhancement of synphilin-1A aggregative properties. Also, the ability of wild type synphilin-1A and the mutant form to produce aggresomes upon overexpression and without proteasome inhibition is addressed but the results are unclear, does not allowing the classification of the inclusions documented in this work as aggresomes. All together, these results suggest that synphilin-1A is able to affect phosphoprotein phosphatase 1 targeting within cells, being inclusion bodies formation dependent and, most specifically, controlled by phosphoprotein phosphatase 1 activity. It is postulated that decreased phosphoprotein phosphatase 1 recruitment to inclusion bodies produces hyperphosphorylated states that favor protein aggregation.
Uma das características principais da doença de Parkinson é o aparecimento de inclusões citoplasmáticas, chamadas corpos de Lewy, maioritariamente nos neurónios dopaminérgicos remanescentes, no tronco cerebral dos pacientes afetados. Várias proteínas têm sido identificadas nos corpos de Lewy, mas o seu mecanismo de formação permanece por clarificar. Entre as várias proteínas já identificadas encontram-se a sinfilina-1, uma proteína interactora da α-sinucleina, e a sinfilina-1A, uma variante da sinfilina-1. Ambas têm sido consideradas elementos chave na doença de Parkinson, já que a sua sobreexpressão em células embrionárias 293 de rim humano, com ou sem a α-sinucleina, conduz à formação de inclusões citoplasmáticas parecidas com corpos de Lewy. Posto isto, têm sido envidados esforços no sentido de clarificar os mecanismos reguladores da agregação da sinfilina-1 e da sinfilina- 1A, como forma de revelar novos aspetos da formação dos corpos de Lewy. Embora tenham sido descritas cinases capazes de fosforilar a sinfilina-1, não há informações concretas sobre as fosfatases responsáveis pela sua desfosforilação. Este vazio começou a ser preenchido com a identificação da sinfilina-1A como uma nova proteína interactora da fosfoproteína fosfatase 1 em cérebro humano, através do sistema dois híbrido de levedura. Deste modo, no presente trabalho, procede-se ao estudo da função fisiológica do complexo sinfilina-1A/fosfoproteína fosfatase 1, demonstrando-se a capacidade da sinfilina-1A de recrutar de forma específica a fosfoproteína fosfatase 1 para corpos de inclusão, com recurso a imunofluorescência. Adicionalmente, as consequências do bloqueio desta interação são exploradas utilizando um mutante da sinfilina-1A incapaz de interagir com a fosfoproteína fosfatase 1, revelando um aumento das propriedades agregativas da sinfilina-1A. Finalmente, também é avaliada a capacidade de a sinfilina-1A selvagem e mutada produzirem agressomas, quando sobreexpressas e sem inibição do proteassoma, mas os resultados não são claros e não permitem a classificação das inclusões documentadas no presente trabalho como agressomas. Em conjunto, estes resultados sugerem que a sinfilina-1A tem a capacidade de afetar o endereçamento da fosfoproteína fosfatase 1 nas células, sendo a formação de corpos de inclusão dependente e, mais concretamente, controlada pela atividade da fosfoproteína fosfatase 1. Postulase que um menor endereçamento da fosfoproteína fosfatase 1 para os corpos de inclusão conduza a estados hiperfosforilados que favorecem a agregação proteica.
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Felgueiras, Juliana Carina Cardoso. "The role of TCTEX1D4/PPP1 complex in cell proliferation and migration". Master's thesis, Universidade de Aveiro, 2013. http://hdl.handle.net/10773/12507.
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Mestrado em Biomedicina MolecularProstate cancer is one of the most prevalent and incident cancer in men, being a disquieting cause of men’s death worldwide. The widespread implementation of prostate-specific antigen (PSA) screening and the improvements on prostate cancer therapy have been leading to a markedly decrease in mortality. However, PSA use is controversial since it has been leading to the diagnosis and treatment of several prostate cancer cases that would not otherwise cause symptoms or threaten man’s life. Hence, the establishment of a panel of biomarkers and the discovery of new therapeutical targets for prostate cancer is a pressing need. Changes in several cell signaling pathways have been associated with de onset, development, and progression of prostate cancer. TGFβ signaling counterbalanced the mitogenic effects of androgens, thus being one of the most prominent pathways involved in controlling prostate growth. Mediators of TGFβ signaling pathway are strictly regulated by several mechanisms, among which reversible phosphorylation. This process involves a fine equilibrium between the action of protein kinases and phosphatases. PPP1 is a major serine/threonine phosphatase that regulates numerous cellular events, including TGFβ signaling. The majority of its functions are accomplished by association with regulatory subunits, known as PPP1-interacting proteins (PIPs). Recently, a novel PIP was found: TCTEX1D4, a dynein light chain protein that was already established as an interactor of TGFβ receptors. Its functions, nevertheless, remain poorly understood. This work identified TCTEX1D4 as an anti-proliferative agent in human prostate cells. Also, TCTEX1D4 activity seems to be regulated through binding to PPP1, as the disruption of TCTEX1D4/PPP1 complex resulted in enhancement of cell proliferation inhibition. Alternatively or in addition, TCTEX1D4 may indirectly regulate PPP1 function by modulating its subcellular localization. This complex doesn´t appear to play a major role in cell migration. Given the results, TCTEX1D4/PPP1 complex may constitute a potential target for the development of new prostate cancer therapies.
O cancro da próstata é um dos cancros mais prevalentes e incidentes em homens, sendo uma preocupante causa de morte a nível mundial. A ampla utilização do antigénio específico da próstata (PSA) no seu rastreio e as melhorias na sua terapêutica têm permitido uma diminuição marcada da mortalidade. No entanto, o uso do PSA é controverso uma vez que se verifica um diagnóstico excessivo e o tratamento de vários casos que, eventualmente, não representariam qualquer ameaça para a vida do individuo. Assim, o estabelecimento de novos biomarcadores e a descoberta de novos alvos terapêuticos para o cancro da próstata constitui um importante desafio na atualidade. Alterações em várias vias de sinalização têm sido relacionadas com o desenvolvimento e a progressão do cancro da próstata. A via do TGFβ é uma das vias mais proeminentes no controlo do crescimento da próstata, uma vez que contrabalança os efeitos proliferativos dos androgénios. Um dos principais mecanismos de regulação da via do TGFβ é a fosforilação reversível, executada de modo complementar por proteínas cinases e fosfatases. Uma das fosfatases envolvidas na regulação da via do TGFβ é a PPP1. A PPP1 é uma fosfatase reguladora de inúmeros eventos celulares. A maioria das suas funções envolve a sua ligação a subunidades reguladoras conhecidas como PIPs. Recentemente, foi descrita uma nova PIP, a TCTEX1D4, que já havia sido referida como interactor dos recetores da via do TGFβ. No entanto, pouco se sabe acerca da sua função. Este trabalho permitiu identificar a TCTEX1D4 como agente inibidor da proliferação em células humanas de próstata. A sua atividade parece ser modulada através da ligação à PPP1, uma vez que a disrupção do complexo TCTEX1D4/PPP1 conduziu a uma maior inibição da proliferação celular. Por outro lado, a TCTEX1D4 pode regular indiretamente a atividade da PPP1 ao modular a sua localização no interior da célula. Este complexo não parece atuar ao nível da migração celular. Tendo em conta os presentes resultados, o complexo TCTEX1D4/PPP1 parece constituir um potencial alvo para o desenvolvimento de novas terapias para o cancro da próstata.
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Voll, Ane-Idun. "Samhandling mellom skole og PPT : En kvalitativ og kvantitativ studie fra et småskalaprosjekt i regi av Faglig Løft for PPT". Thesis, Norges teknisk-naturvitenskapelige universitet, Pedagogisk institutt, 2012. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-17568.
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Faglig Løft for PPT er et 4-årig modellforsøk som avsluttes i 2012. Etablering av prosjektet begrunnes i et behov for kompetanseheving hos PPT. Som en del av det større prosjektet Faglig løft for PPT er det etablert småskalaprosjekt i to kommuner, Samhandlingsprosjektet. Hensikten med å opprette et slikt småskalaprosjekt er å prøve ut ulike samhandlingsstrukturer, bidra til kompetansebygging og prøve ut ulike roller. I den ene kommunen, som er fokus for denne studien, er det opprettet arbeidsgrupper på hver av de seks grunnskolene i kommunen. Bakgrunnen for at kommunen deltar i Samhandlingsprosjektet er et ønske om å se nærmere på og kvalitetssikre den spesialpedagogiske praksisen. Denne kvalitative og kvantitative studien har sett på Samhandlingsprosjektet og arbeidsgruppen ut i fra lærernes opplevelse og erfaring. Hensikten med studien er å undersøke om Samhandlingsprosjektet har hatt effekt for lærerne i arbeidsgruppens opplevelse av samhandling mellom skole og PPT. Funnene fra studien kan med noe forbehold tyde på at Samhandlingsprosjektet har hatt effekt for arbeidsgruppens opplevelse av samhandling mellom skole og PPT. Studien tar utgangspunkt i to forskningsspørsmål, der det første ønsket å undersøke hvilke faktorer lærere som har deltatt i Samhandlingsprosjektet opplever som viktige for samhandling mellom skole og PPT. Funnene fra studien kan tyde på at lærere som har deltatt i Samhandlingsprosjektet opplever avklaring av rolleforventinger, PPTs tilgjengelighet og tilstedeværelse, kvaliteten på hjelpen som tilbys av PPT og handlingskompetanse på egen skole som viktige faktorer for samhandling. Det andre forskningsspørsmålet hadde til hensikt å undersøke om lærere som har vært med i Samhandlingsprosjektet og lærere som ikke har deltatt, har ulik opplevelse av samhandling mellom skole og PPT. Funn fra studien viser at det på enkelte områder kan se ut som det er ulik opplevelse av samhandling mellom skole og PPT, mellom lærere som har deltatt i samhandlingsprosjektet og lærere som ikke har deltatt.
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Fardilha, Margarida Sâncio da Cruz. "Characterization of the PP1 interactome from human testis". Doctoral thesis, Universidade de Aveiro, 2003. http://hdl.handle.net/10773/4490.
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Doutoramento em BiologiaProtein phosphorylation is a major regulatory mechanism notably of signal transduction cascades in eukaryotic cells. Protein phosphorylation is catalysed by protein kinases and can be reversed by the action of protein phosphatases. Although phosphatases were discovered more than sixty years ago, their importance as central players in multiple cellular mechanisms was only recently recognized. PP1, a Serine/Threonine specific phosphatase, is involved in important cellular mechanisms such as the cell cycle, muscle contraction and apoptosis, among others. Its role in such diverse cellular processes depends on its interactions with targeting/regulatory subunits. To date, more than 50 regulatory subunits have been identified that bind the catalytic subunit of PP1 determining its function in a specific cellular location. Several isoforms of PP1 are known, termed PP1 , PP1 and PP1 . The gamma isoform undergoes alternative splicing to yield a ubiquitously expressed PP1 1 and a testis-specific PP1 2 isoform. Incubation of non-motile immature sperm with phosphatase inhibitors induces sperm motility, and PP1 2 was implicated in this process. This led us to search for PP1 2-specific interactors in human sperm that could be targeted for infertility therapeutics or in male contraception. To achieve this goal the Yeast Two Hybrid system was used to screen a human testis library for new PP1 binding proteins using both PP1 1 (YTH1) and PP1 2 (YTH2) as baits. We recovered 120 positive clones in YTH1 and 155 positive clones in YTH2. Among these were clones encoding “bona fide” PP1 interactors such as Nek2 and NIPP1, and also previously uncharacterized proteins. We undertook a more detailed study of a novel gene encoding a novel protein that we termed SEARP-T. This protein of 93KDa is expressed mainly in testis and fluorescence immunocytochemistry was used to determine its intra sperm localization. Both PP1 2 and SEARP-T proteins are present in the tail and in the equatorial segment of the head.These results provide new insights into PP1 function in human testis and sperm motility, and indicates that the Yeast Two Hybrid System provides a mean to understand the roles PP1 plays in diverse cellular regulatory events.
A fosforilação de proteínas é um dos principais mecanismos reguladores de cascatas de transdução de sinais em eucariotas. A fosforilação é catalizada por proteínas cinases e é revertida pela acção de proteínas fosfatases. Embora as proteínas fosfatases tenham sido descobertas à mais de sessenta anos, a sua importância central em múltiplos mecanismos celulares só muito recentemente foi reconhecida. PP1, uma fosfatase específica para serina/treonina, está envolvida em importantes mecanismos celulares, como o ciclo celular, a contracção muscular e a apoptose, entre outros. O seu papel em tão variados processos celulares depende das suas interacções com subunidades reguladoras. Até à data, foram descritas mais de 50 subunidades reguladoras que se ligam à subunidade catalítica da PP1, sendo determinantes para a sua função num local específico da célula. Das três isoformas da PP1 conhecidas, PP1 , PP1 e PP1 , a isoforma gama sofre splicing alternativo, originando a PP1 1, ubíqua e, a PP1 2 específica de testículo. Incubação de espermatozóides imaturos com inibidores de fosfatases induz a sua motilidade, sendo a PP1 2 a fosfatase envolvida. Este facto levou-nos a procurar proteínas de testículo humano capazes de interagir especificamente com a PP1 2 que possam ser alvos terapêuticos no tratamento da infertilidade, ou na contracepção masculina. Para atingir este objectivo utilizou-se o sistema Dois Híbrido de Levedura no rastreio de uma biblioteca de testículo humano, na busca de novas proteínas que se ligam à PP1 usando como isco a PP1 1 (no YTH1) ou a PP1 2 (no YTH2). Obtivemos 120 clones positivos no YTH1 e 155 no YTH2. Entre eles encontravam-se clones que codificam reguladores da PP1 previamente descritos, como a Nek2 e a NIPP1, assim como novas proteínas. Efectuámos um estudo detalhado de um novo gene, codificando uma nova proteína, que denominamos SEARP-T. Esta proteína de 93kDa é expressa maioritariamente em testículo e, a sua localização intracelular foi determinada por imunocitoquímica de fluorescência. Ambas as proteínas, PP1 2 e SEARP-T, estão presentes na cauda e no segmento equatorial da cabeça do espermatozóide. Estes resultados clarificam as funções da PP1 no testículo humano e na motilidade do esperma e, indicam que o sistema Dois Híbrido de Levedura é um bom método para compreender o papel da PP1 em múltiplos eventos de regulação celular.
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Wright, Thomas Matthew. "Properties of nonwoven assemblies containing mechanically processed and photo-aged PPTA fibres". Thesis, University of Leeds, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613623.
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Degradation of poly-para phenylene terephthalamide (PPTA) fibres during the manufacture of carded and hydroentangled fabric is investigated. During PPTA fibre processing operations such as fibre opening and carding, the material is subjected to applied mechanical forces resulting from interactions with machine surfaces and inter-fibre friction. Additionally, post-industrial and post-consumer sources of PPTA fibre are exposed to UV irradiation, which also has potential to influence fibre properties. The degree to which such agencies of degradation are encountered during fabric manufacture can influence fibre morphology and ultimate nonwoven fabric properties is not fully understood. This question is relevant both to the processing of virgin PPTA fibre and to PPTA fibre recyclates. Substantial morphological changes in PPTA fibres resulting from carding included kink banding, skin peeling and microfibrillation, the magnitude of which depended upon card feed rate and the number of passes through the system. Additionally, it was found that PPTA fibre degradation as a result of both UV irradiation and mechanical processing could initially increase rather than decrease fabric burst strength partly as a result of extensive fibril entanglement. A modified burst testing system that obviated fibre• bridging effects was developed to assist in the evaluation of PPTA hydroentangled fabrics. The magnitude of UV degradation in PPTA fibre webs was characterised using the b• colour coordinate (yellowness) in CIElab. There is an interaction between the irradiation of PPTA fibres and subsequent mechanical processes (carding and hydroentangling) wherein the fibres respond differently to non-irradiated fibres.
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Juhem, Aurélie. "Identification d’un inhibiteur sélectif PP1 aux propriétés anti-tumorales". Université Joseph Fourier (Grenoble ; 1971-2015), 2008. http://www.theses.fr/2008GRE10170.
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La chimiothérapie reste une des thérapies les plus utilisées pour lutter contre le cancer. Bien qu'il existe de nombreuses molécules anti-cancéreuses, il demeure un besoin constant de nouvelles drogues thérapeutiques plus efficaces et moins toxiques. Parmi l'ensemble des médicaments disponibles pour traiter les patients, « les poisons microtubulaires », qui agissent sur la tubuline et/ou les microtubules (MTs), sont largement utilisés puisqu'ils ciblent préférentiellement les cellules qui se divisent, en provoquant un arrêt en mitose et la mort cellulaire. Cependant, l'usage de ces drogues anti-tubuline est limité par la neurotoxicité périphérique qu'elles induisent et les phénomènes de chimiorésistance que les patients développent. Dans ce contexte, il devient évident de rechercher des molécules anti-mitotiques agissant sur de nouvelles cibles moléculaires. Par le biais d'un criblage phénotypique, nous avons identifié un dérivé d'isoquinoline, la molécule D5, qui perturbe la division cellulaire sans cibler directement la tubuline ou les MTs. La caractérisation de son activité in vitro sur les cellules HeLa, a montré, une perturbation de la cytocinèse par l'induction de protrusions au niveau des cellules mitotiques, entraînant la mort cellulaire. D5 déstabilise le réseau microtubulaire, un autre phénomène qui induit aussi un arrêt mitotique et la mort cellulaire. Des tests biochimiques et l'utilisation de siRNA, nous ont permis d'identifier la PP1 comme cible de D5. En parallèle, l'effet anti-prolifératif de D5 a été évalué sur un panel d'une dizaine de lignées cancéreuses humaines, et D5 a présenté une efficacité particulière pour les lignées de cellules cancéreuses suivantes : HTB-177, HeLa, 786-0, RT-112, U87, MCF7 et A549. Les études préliminaires de toxicité sur des souris nude ont montré que la drogue est tolérée in vivo, et ont permis de démarrer des tests pilotes sur des modèles de xénogreffes avec la lignée de carcinomes pulmonaires humains HTB 177. Ces premiers essais démontrent que la molécule est efficace per os chez la souris, provoquant une diminution de la taille des tumeurs par un phénomène de nécrose. L'ensemble de ces résultats met en évidence le potentiel de D5 en tant que candidat médicament et fournit la base pour son entrée dans des essais pré-cliniques et cliniquesChemotherapy and/or radiotherapy remain the cornerstones of the anti-cancer therapy. Although dozens of cancer drugs have been approved for clinical use, there is a continuous need for more selective, more efficient and less toxic therapeutics. Drugs perturbing the microtubule cytoskeleton via binding to tubulin dimers or polymers are widely used in cancer treatment because they arrest in mitosis and kill fast-dividing tumor cells. However, these anti-tubulin drugs have their limitations, especially in terms of chemoresistance and peripheral neurotoxicity, strongly supporting the search for anti-mitotic drugs with new molecular targets. Using phenotypical screening, we identified an isoquinoline derivative, D5, which perturbs cell division without targeting microtubules or tubulin. We characterized the activity of the drug in vitro to show that in tumor cells,D5 prevents normal cytokinesis by inducing ectopic furrows, followed by cell death. The drug also indirectly destabilizes the microtubule cytoskeleton, another phenomenon leading to cell arrest and death in mitosis. Using biochemical assays and RNAi techniques, we identified the protein target of D5. Using a panel of ten diverse human cancer cell lines, we found that HTB-177, HeLa, 786-0, RT-112, U87, MCF7 and A549 cells were the most sensitive to D5. Pilot toxicity studies on nude mice showed that D5 was well tolerated in vivo. Pilot in vivo studies in nude mice xenografted with HTB- l 77 cells showed that D5 therapy induced necrosis in all tumors. These results with other on-going experiments on D5 will provide the basis for its entering regulatory pre-clinical and clinical trials central to anti-cancer drug development
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Ning, Shunbin y Ling Wang. "Identification of PP1 as the First Phosphatase for IRF7". Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etsu-works/6527.
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Esteves, Sara Luísa de Castro. "PP1 interactomes as a means of characterizing protein functions". Doctoral thesis, Universidade de Aveiro, 2012. http://hdl.handle.net/10773/10998.
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Doutoramento em BioquímicaA maioria das funções celulares, incluindo expressão de genes, crescimento e proliferação celulares, metabolismo, morfologia, motilidade, comunicação intercelular e apoptose, é regulada por interações proteína-proteína (IPP). A célula responde a uma variedade de estímulos, como tal a expressão de proteínas é um processo dinâmico e os complexos formados são constituídos transitoriamente mudando de acordo com o seu ciclo funcional, adicionalmente, muitas proteínas são expressas de uma forma dependente do tipo de célula. Em qualquer instante a célula pode conter cerca de centenas de milhares de IPPs binárias, e encontrar os companheiros de interação de uma proteína é um meio de inferir a sua função. Alterações em redes de IPP podem também fornecer informações acerca de mecanismos de doença. O método de identificação binário mais frequentemente usado é o sistema Dois Hibrido de Levedura, adaptado para rastreio em larga escala. Esta metodologia foi aqui usada para identificar os interactomas específicos de isoforma da Proteína Fosfatase 1 (PP1), em cérebro humano. A PP1 é uma proteína fosfatase de Ser/Thr envolvida numa grande variedade de vias e eventos celulares. É uma proteína conservada codificada por três genes, que originam as isoformas α, β, e γ, com a última a originar γ1 e γ2 por splicing alternativo. As diferentes isoformas da PP1 são reguladas pelos companheiros de interação – proteínas que interagem com a PP1 (PIPs). A natureza modular dos complexos da PP1, bem como a sua associação combinacional, gera um largo reportório de complexos reguladores e papéis em circuitos de sinalização celular. Os interactomas da PP1 específicos de isofoma, em cérebro, foram aqui descritos, com um total de 263 interações identificadas e integradas com os dados recolhidos de várias bases de dados de IPPs. Adicionalmente, duas PIPs foram selecionadas para uma caracterização mais aprofundada da interação: Taperina e Sinfilina-1A. A Taperina é uma proteína ainda pouco descrita, descoberta recentemente como sendo uma PIP. A sua interação com as diferentes isoformas da PP1 e localização celulares foram analisadas. Foi descoberto que a Taperina é clivada e que está presente no citoplasma, membrana e núcleo e que aumenta os níveis de PP1, em células HeLa. Na membrana ela co-localiza com a PP1 e a actina e uma forma mutada da Taperina, no motivo de ligação à PP1, está enriquecida no núcleo, juntamente com a actina. Mais, foi descoberto que a Taperina é expressa em testículo e localiza-se na região acrossómica da cabeça do espermatozoide, uma estrutura onde a PP1 e a actina estão também presentes. A Sinfilina-1A, uma isoforma da Sinfilina-1, é uma proteína com tendência para agregar e tóxica, envolvida na doença de Parkinson. Foi mostrado que a Sinfilina-1A liga às isoformas da PP1, por co-transformação em levedura, e que mutação do seu motivo de ligação à PP1 diminuiu significativamente a interação, num ensaio de overlay. Quando sobre-expressa em células Cos-7, a Sinfilina-1A formou corpos de inclusão onde a PP1 estava presente, no entanto a forma mutada da Sinfilina-1A também foi capaz de agregar, indicando que a formação de inclusões não foi dependente de ligação à PP1. Este trabalho dá uma nova perspetiva dos interactomas da PP1, incluindo a identificação de dezenas de companheiros de ligação específicos de isoforma, e enfatiza a importância das PIPs, não apenas na compreensão das funções celulares da PP1 mas também, como alvos de intervenção terapêutica.
Most of the crucial functions in the cell, including gene expression, cell growth and proliferation, metabolism, morphology, motility, intercellular communication and apoptosis, are regulated by protein-protein interactions (PPIs). Cells respond to a variety of stimuli, thus protein expression is a dynamic process and the complexes formed are transiently assembled and change during their functional cycle; additionally, many proteins are expressed in a cell type-dependent manner. At any time, cell may contain about hundreds of thousands of binary PPIs, and finding interaction partners of a certain protein it’s a mean of discovering its function. Changes in PPIs networks may also provide information about disease mechanisms. The most frequently used binary identification method is the Yeast Two Hybrid system, adapted to high-throughput screening. This approach was here used in order to identify the Protein Phosphatase 1 (PP1) isoform specific interactomes, in the human brain. PP1 is a Ser/Thr protein phosphatase involved in a large variety of cellular pathways and events. It is a conserved protein codified by three genes giving rise to the α, β and γ isoforms, with the last originating γ1 and γ2 by alternative splicing. PP1 isoforms are regulated by the binding partners – PP1 interacting proteins (PIPs). The modular nature of the PP1 complexes, as well as their combinational assembly, generates a large repertoire of regulatory complexes and roles in signaling circuits. The human brain isoform specific interactomes of PP1 were here described, with a total of 263 interactions identified and integrated with the data collected from several PPIs databases. Also, two PIPs were selected for further characterization of the interaction: Taperin and Synphilin-1A. Taperin is a poorly described protein, recently found to be a PIP. Its interaction with PP1 different isoforms and localization in the cell was analyzed. Taperin was found to be cleaved and to be present in the cytoplasm, membrane and nucleus and to increase the levels of PP1, in HeLa cells. In the membrane it co-localizes with PP1 and actin and a mutant form of Taperin, in the PP1 binding motif, is enrich in the nucleus together with actin. Moreover, Taperin was found to be expressed in testis and to localize in the acrossome region of the sperm head, a structure where PP1 and actin are also present. Synphilin-1A, an isoform of Synphilin-1, is an aggregation prone and toxic protein involved in Parkinson`s Disease. Synphilin-1A was shown to bind PP1 isoforms, by yeast co-transformation, and mutation of its PP1 binding motif decrease significantly the interaction in an overlay assay. When overexpressed in Cos-7 cells, synphilin-1A formed inclusion bodies where PP1 was present, but the mutated form of Synphilin-1A was also able to aggregate indicating that inclusions formation was not dependent on PP1 binding. This work gives a new perspective of PP1 interactomes, including the identification of dozens of isoform specific binding partners, and emphasizes the importance of PIPs, not only in the understanding of PP1 physiological functions but also, as targets for therapeutic interventions.
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Alves, Helder Luís da Costa. "The impact of Aβ on the Neurabin/PP1 complex". Master's thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/15017.
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Mestrado em Biomedicina MolecularOur brain is a complex structure constituted by neurons capable of communicating through synapses, which generally occur between the axon terminal and the dendritic spine of two different neurons. These dendritic spines are dynamic, which allow for the rapid adaptation to the different stimuli they receive. PP1 is a phosphatase protein which catalyzes the majority of dephosphorylation reactions that occur in our body. It is involved in several different functions, from glycogen metabolism to synaptic regulation. Neurabins are two structurally and functionally similar proteins, highly concentrated in dendritic spines, where they interact with several proteins – PP1 included –, and target them to receptors, the cytoskeleton and to other cellular compartments. Thus, neurabins regulate neuronal morphology and synaptic transmission, and hence, synaptic plasticity. Alzheimer’s disease is the most common neurodegenerative disease and it is characterized by the deposition of Aβ and the presence of neurofibrilliary tangles, by the loss of synapses and neuronal death, and by the gradual loss of memory and other cognitive functions. APP is an integral membrane protein which, in Alzheimer’s disease, is abnormally cleaved via the amyloidogenic pathway, thus resulting in the overproduction of a toxic peptide, Aβ, believed to be the major culprit of the changes observed in this disease. The main aim of this thesis was to study the effects of Aβ on neurabins expression and to evaluate its effects on the neurabin/PP1 complex. The results here reported showed a slight decrease in both neurabins expression levels when Aβ was added to the cells, possibly due to the morphological changes and synaptic dysfunction this peptide induces. It was also here reported that Aβ interferes with the neurabin-1/PP1 complex. This may be related to the direct effect of Aβ on neurabin-1 or due to the imbalance of phosphatases and kinases seen when Aβ is added, which could result in a decrease of neurabin-1’s affinity for PP1. The same effect was not seen with the neurabin-2/PP1 complex, possibly because they are differently regulated by several kinases. The immunocytochemistry study here performed did not show any changes between the co-localization of neurabin-1 and PP1, and allowed for assessment of cellular distribution of neurabin-1 and neurabin-2 in SH-SY5Y cells. The experimental procedures here performed allowed us to conclude that Aβ interferes with the expression of both neurabins and with the interaction between neurabin-1 and PP1. However, additional studies need to be conducted in order to understand the physiological relevance of this complex.
O nosso cérebro é uma estrutura complexa constituída por neurónios capazes de comunicar entre si através de sinapses, que geralmente ocorrem entre o terminal axonal e a espinha dendrítica de dois neurónios. Estas espinhas dendríticas são dinâmicas, permitindo assim que se adaptem aos estímulos que recebem, quer estimulantes quer inibitórios. A PP1 é uma proteína fosfatase que catalisa grande parte das reações de desfosforilação que ocorrem no nosso corpo, encontrando-se envolvida, por isso, em diversas funções, desde o metabolismo do glicogénio até à regulação sináptica. As neurabinas são duas proteínas estrutural e funcionalmente similares, muito concentradas nas espinhas dendríticas, onde interagem com diversas proteínas, incluindo a PP1, e as direcionam ou para os recetores que aqui se encontram, ou para o citoesqueleto de actina ou para outras regiões do neurónio. Assim, as neurabinas são responsáveis pela regulação da morfologia neuronal, pela transmissão sináptica e, por conseguinte, pela plasticidade sináptica. A doença de Alzheimer é a doença neurodegenerativa mais comum e é caraterizada por depósitos de Aβ e presença de tranças neurofibrilares, pela destruição de sinapses e morte neuronal, e pela perda gradual da memória e de outras funções cognitivas. A PPA é uma proteína transmembranar que, na doença de Alzheimer, é processada anormalmente pela via amiloidogénica, resultando na sobreprodução de Aβ, um peptídeo tóxico, que se crê ser o principal causador das alterações caraterísticas da doença de Alzheimer. O principal objetivo desta tese de mestrado foi avaliar o efeito do Aβ na expressão das neurabinas e na interação destas com a PP1. Os resultados aqui reportados demonstram uma ligeira diminuição de ambas as neurabinas quando o Aβ se encontra presente nas células, provavelmente pelas alterações a nível morfológico e destruição de sinapses que ocorre. Também foi aqui reportado que o Aβ interfere com o complexo neurabina-1/PP1, talvez pelo efeito direto do Aβ na neurabina-1 ou pela desregulação de fosfatases e cinases existente quando o Aβ se encontra presente no meio, levando a uma diminuição da afinidade entre a neurabina-1 e a PP1. O mesmo efeito não se verificou no complexo neurabina-2/PP1, talvez por serem reguladas de forma diferente por diferentes cinases. O estudo imunocitoquímico não demonstrou alterações a nível da co-localização entre a neurabina-1 e a PP1, e permitiu a caraterização da distribuição celular de ambas as neurabinas em células SH-SY5Y. As experiências laboratoriais realizadas nesta tese permitiram concluir que o Aβ interfere com a expressão das neurabinas e com o complexo neurabina-1/PP1. No entanto, terão que ser realizados estudos adicionais para compreender a relevância fisiológica do complexo neurabina-1/PP1.
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Holley, Aaron K. "Investigations of supramolecular and catalytic properties of PP1 dendrimers". Huntington, WV : [Marshall University Libraries], 2003. http://www.marshall.edu/etd/descript.asp?ref=334.
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Brusnický, Pavel. "Útoky na bezdrátovou síť". Master's thesis, Vysoké učení technické v Brně. Fakulta elektrotechniky a komunikačních technologií, 2010. http://www.nusl.cz/ntk/nusl-218580.
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Objective of this thesis is to point out to almost everywhere present flaw in realization of second level network security access to WiFi networks with using traffic tunneling over DNS protocol. Realization has been accomplished by existing utilities OzymanDNS, DNS2TCP, NSTX, Iodine, Heyoka. Measurements were done on realistic traffic on the network. The effort was to show outline of these implementations. Transfer speeds in some implementations can be marked as applicable thanks to high speeds, which are on the same level as broadband internet. Functionality was tested on WiFi network, where was also compromised PPTP VPN tunnel, its function was to provide security of the communication on wireless network due to absence of first level security mechanisms such as WPA, WPA 2 and so on, with the help of Asleap, which comes out of Cisco LEAP attack. At the end of the work are suggested possible countermeasures for securing network by the topology change of the network infrastructure or by implementing IDS.
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Calafi, Pascual Carlos Alberto. "1 La proteína fosfatasa Ppz1 de levadura: estudios estructurales y funcionales en sobreexpresión". Doctoral thesis, TDX (Tesis Doctorals en Xarxa), 2020. http://hdl.handle.net/10803/670425.
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Els enzims Ppz són proteïnes fosfatases que es troben només en fongs i es caracteritzen per un domini catalític C-terminal molt conservat, semblant al de les fosfatases PP1c, i una regió N-terminal poc conservada. En Saccharomyces cerevisiae, les fosfatases Ppz estan codificades en els gens paràlegs PPZ1 i PPZ2. Ppz1 és la proteïna més tòxica en sobreexpressió en llevat, alterant la proliferació cel·lular de manera dependent de la seva activitat fosfatasa, tot i que els mecanismes darrer d’aquesta toxicitat encara no han estat establerts. En aquest estudi intentem conèixer aquests mecanismes. Hem identificat alguns gens que codifiquen proteïnes ribosòmiques, així com factors implicats en la biogènesi de ribosomes com a supressors en multicòpia de l’efecte tòxic de Ppz1. Aquesta fosfatasa s’uneix als ribosomes que comencen a traduir, i l’excés de Ppz1 produeix una disminució en el contingut de polisomes. L’absència de la quinasa Gcn2 (regulador negatiu de l’inici de la traducció) parcialment suprimeix el defecte de creixement d’una soca que sobreexpressa Ppz1. Per tot això, proposem que part dels efectes de la sobreexpressió de Ppz1 es deuen a l’alteració de la síntesi de proteïnes.
Malgrat el seu domini catalític conservat, descrivim que Ppz2 no és tòxica quan se sobreexpressa en les mateixes condicions que Ppz1, tot i que els nivells de Ppz2 són més baixos. Sorprenentment, una versió híbrida composada pel segment N-terminal de Ppz1 i el domini catalític de Ppz2 va presentar la mateixa toxicitat que Ppz1, tot i mostrar nivells de proteïna comparables a Ppz2. Per tant, creiem que la extensió N-terminal és important per a la toxicitat de Ppz1.
També vam analitzar el grau de toxicitat de dos versions de Ppz1: G2A (no miristilable) i R451L (catalíticament inactiva). El canvi G2A va atenuar lleument la toxicitat de Ppz1, mentre que la versió R451L va eliminar gran part de la toxicitat. És conegut que Ppz1 és capaç d’inhibir l’entrada de K+ via Trk1. L’addició de K+ va millorar el creixement de les cèl·lules que portaven les versions tant de la Ppz1 nativa com de la versió G2A. Vam analitzar també el creixement d’alguns mutants de la via de resposta a l’estrès osmòtic (HOG) quan se sobreexpressaven aquestes versions de Ppz1. La deleció de HOG1 (que codifica la MAPK central de la via HOG) va reduir de manera notable la toxicitat de les 2 versions. L’absència de Nha1, un antiportador Na+,K+/H+, va produir una reducció molt dràstica de la toxicitat de la versió G2A. Per últim, hipotetitzem un model on la sobreexpressió de Ppz1 podria alterar el funcionament dels transportadors Nha1 y Trk1, explicant el comportament de la versió G2A de Ppz1.Los enzimas Ppz son proteínas fosfatasas que se encuentran solo en hongos y se caracterizan por un dominio catalítico C-terminal muy conservado, parecido al de las fosfatasas PP1c, y una región N-terminal poco conservada. En Saccharomyces cerevisiae, las fosfatasas Ppz están codificadas en los genes parálogos PPZ1 y PPZ2. Ppz1 es la proteína más tóxica en sobreexpresión en levadura, alterando la proliferación celular de manera dependiente de su actividad fosfatasa, aunque los mecanismos que recaen sobre esta toxicidad aún no han sido descubiertos. En este estudio intentamos conocer estos mecanismos. Hemos identificado varios genes que codifican proteínas ribosómicas, así como factores implicados en la biogénesis de ribosomas como supresores en multicopia del efecto tóxico de Ppz1. Esta fosfatasa se une a los ribosomas que empiezan a traducir, y el exceso de Ppz1 produce una caída en la cantidad de polisomas. La ausencia de la quinasa Gcn2 (regulador negativo del inicio de traducción) parcialmente suprime el defecto de crecimiento de una cepa que sobreexpresa Ppz1. Por todo esto, proponemos que parte de los efectos de la sobreexpresión de Ppz1 se deben a la alteración de la síntesis de proteínas. A pesar de su dominio catalítico conservado, describimos que Ppz2 no es tóxica cuando se sobreexpresa en las mismas condiciones que Ppz1, aunque los niveles de Ppz2 son menores. Sorprendentemente, una versión híbrida compuesta por el segmento N-terminal de Ppz1 y el dominio catalítico de Ppz2 presentó la misma toxicidad que Ppz1, incluso mostrando niveles de expresión comparables a los de Ppz2. Por tanto, creemos que la extensión N-terminal es importante para la toxicidad de Ppz1. También analizamos el nivel de toxicidad de dos versiones de Ppz1: G2A (no miristilable) y R451L (catalíticamente inactiva). El cambio G2A atenuó solo levemente la toxicidad de Ppz1, mientras que la versión R451L eliminó gran parte de la toxicidad. Es sabido que Ppz1 es capaz de inhibir la entrada de K+ vía Trk1. La adición de K+ logró mejorar el crecimiento de las células que portaban las versiones tanto de la Ppz1 nativa como de la versión G2A. Analizamos también el crecimiento de varios mutantes de la vía de respuesta a estrés osmótico (HOG) al sobreexpresar estas versiones de Ppz1. La deleción de HOG1 (que codifica la MAPK central de la vía HOG) redujo visiblemente la toxicidad de las 2 versiones. La ausencia de Nha1, un antiportador Na+,K+/H+, produjo una reducción muy drástica de la toxicidad de la versión G2A. Por último, hipotetizamos un modelo en el que la sobreexpresión de Ppz1 podría alterar el funcionamiento de los transportadores Nha1 y Trk1, explicando el comportamiento de la versión G2A de Ppz1.
The Ppz enzymes are Ser/Thr protein phosphatases present only in fungi that are characterized by a highly conserved C-terminal catalytic region, related to PP1c phosphatases, and a more divergent N-terminal extension. In Saccharomyces cerevisiae, Ppz phosphatases are encoded by two paralog genes, PPZ1 and PPZ2. Ppz1 is the most toxic protein when overexpressed in budding yeast, halting cell proliferation, and this effect requires its phosphatase activity. However, the reasons for such toxicity have not been elucidated. In this study, we tried to unveil the mechanisms behind the toxicity of Ppz1. We have identified several genes encoding ribosomal proteins and ribosome assembly factors as mild high-copy suppressors of the toxic Ppz1 effect. This phosphatase binds to ribosomes engaged in translation, and the Ppz1 excess leads to a decrease in the polysome content. The absence of the Gcn2 kinase (a translation initiation negative regulator) partially suppresses the growth defect of a Ppz1 overexpressing strain, consistently with an impact in the initiation process. We propose that the deleterious effects of Ppz1 overexpression are in part due to alteration in normal protein synthesis. Despite its conserved catalytic domain, we report that Ppz2 was not toxic when overexpressed in the same conditions that Ppz1, albeit Ppz2 levels were somewhat lower. Remarkably, a hybrid protein composed of the N-terminal extension of Ppz1 and the catalytic domain of Ppz2 was as toxic as Ppz1 even if its expression level was comparable to that of Ppz2. Thus, the N-terminal extension of Ppz1 plays a key role in defining Ppz1 toxicity. The toxic effect of two Ppz1 versions was also analyzed. The G2A mutation generates a non-myristoylable Ppz1 and the R451L version is catalytically inactive. The G2A change slightly attenuated the toxicity of Ppz1, while the R451L mutation strongly reduced the growth defect associated with the excess of Ppz1. Ppz1 is capable to inhibit K+ uptake via the Trk1 transporter. The addition of external potassium ameliorated the growth of cells carrying the native Ppz1 and G2A version. We also analyzed the growth of mutants related to the HOG pathway containing these Ppz1 versions. The deletion of HOG1, which encodes the central MAPK of the HOG pathway, notably reduced the toxicity associated with the 2 Ppz1 versions. The absence of Nha1, a Na+,K+/H+ antiporter, led to a strong reduction of the toxic effect of the G2A variant. Finally, we hypothesize a model in which overexpression of Ppz1 might alter the function of both Nha1 and Trk1 transporters, thus explaining the behavior of the G2A version of Ppz1.
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Khuong, Thi thu huong. "Investigation of the regulation of photosynthesis at the molecular level for improvement of plant growth and productivity under limiting light conditions". Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM4001.
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La lumière est indispensable à la survie des plantes via le processus photosynthétique, pourtant les plantes doivent s'adapter à différentes conditions environnementales où la quantité et la qualité de la lumière peuvent être non optimales pour la photosynthèse. Cela peut provoquer des dégâts photo-induits par formation d'espèces réactives de l'oxygène (ROS), qui sont dangereux pour la plante. Pour limiter la formation des ROS, les plantes mettent en place une régulation importante qui est la dissipation thermique de l'énergie absorbée en excès, appelé Non photochemical quenching (NPQ). Il est connu que la protéine PsbS joue le rôle clé de senseur du pH bas du lumen thylacoïdal, qui est le signal initial pour activer le NPQ. Dans le contexte de cette thèse, on propose d'étudier l'hypothèse que l'absence de la protéine PsbS (diminué NPQ) pourrait augmenter la croissance et la productivité des plantes en conditions contrôlées de faible lumière par l'éminilation de la protéine PsbS chez Arabidopsis thaliana et chez la tomate. Les résultats obtenus indiquent qu'en lumière faible les plantes mutantes montrent une augmentation du rendement de photosystème II conduisant une croissance et un nombre de fleurs significativement augmentés par rapport aux plantes sauvages.De plus, une autre régulation de la photosynthèse, nommée « transitions d'état », est importante pour optimiser la photosynthèse en réponse aux variations de la quantité et de la qualité de la lumière, grâce à la migration réversible des antennes collectrices d'énergie LHCII phosphorylées du PSII au PSI, c'est aussi étudié dans ma thèseLight is indispensable for plant survival, but plants have to cope with different environmental situations where light quantity and quality can be not optimal for photosynthesis. This can cause photodamage due to the formation of harmful reactive oxygen species (ROS). To limit ROS formation, plants developed a mechanism important as the dissipation of excess absorbed energy as heat and is called Non Photochemical Quenching (NPQ). The PsbS protein plays the key role of sensor of the low lumenal pH, the signal to activate NPQ. In this thesis, we proposed and investigated the hypothesis that PsbS absence (NPQ decrease) would improve growth under controlled low light upon elimination of the PsbS in Arabidopsis and tomato plants. Results showed that the increase of photosystem II yield in mutant plants leaded to a significant improvement of growth and flower number in mutants as compared with wild type plants under low light, suggesting that this mutation could be useful to improve plant performances in controlled conditions where light is strongly limiting. In addition, another photosynthetic regulation, called “state transitions”, which is important to optimize photosynthesis under variable light for intensity and quality thank to reversible migration of phosphorylated light harvesting complexes LHCII from PSII to PSI also investigated in my thesis
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Spencer, Eleanor Mary. "Transcriptional regulation of preprotachykinin-A (PPT-A) in the hippocampus". Thesis, University of Liverpool, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433413.
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Schneider, Georg [Verfasser]. "300 ppt Measurement of the Proton g-Factor / Georg Schneider". Mainz : Universitätsbibliothek Mainz, 2018. http://d-nb.info/1162145307/34.
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Figueiredo, João Daniel Amaral. "The effect of anticancer drugs prodiginines in PP1 in melanoma". Master's thesis, Universidade de Aveiro, 2011. http://hdl.handle.net/10773/6861.
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Mestrado em Biomedicina MolecularUm dos principais mecanismos reguladores da função celular é a fosforilação de proteínas. É de focar que a fosforilação anormal de proteínas-chave pode estar associada a várias patologias, incluindo o cancro. Embora já existam muitos estudos sobre cinases no cancro, o conhecimento sobre as fosfatases que antagonizam a acção das cinases é muito menos. A PP1, uma das principais proteínas fosfatase de serina/treonina expressa em todas as células eucarióticas, está envolvida em vários processos celulares incluindo apoptosis e ciclo celular. Na realidade, diversos estudos demonstram que a PP1 regula variadas proteínas que são elementos-chave no processo de tumorigenesis. A AKT, uma cinase serina/treonina que se encontra desregulada em vários tipos de cancro, é um factor crucial na progressão e sobrevivência de melanoma. Prodigiosina, um membro da família de metabolitos secundários tripirrolicos pigmentados de vermelho, as prodigininas, demonstra propriedades anticancerigenas em vários tipos de cancro. Na verdade alguns estudos verificaram que a AKT é desfosforilada pela prodigiosina embora ainda seja desconhecido o mecanismo pelo qual tal acontece. Dada a importância da AKT na progressão e sobrevivência do melanoma e a capacidade da PP1 em desfosforilar a AKT é possível que a PP1 esteja envolvida em tal mecanismo. Os resultados preliminares demonstraram que a PP1 liga-se a um membro da família das prodigininas provando a interacção entre estas moléculas. Por outro lado, ensaios em linhas celulares de melanoma usando tratamentos com prodigiosina e cantaridina, um inibidor da PP1, demonstraram que a prodigiosina afecta isoformas da PP1 diferencialmente. Estes resultados sugerem que a prodigiosina actua em duas vias de sinalização distintas em melanoma, a via da AKT e a da MAPK, uma vez que alteração nos níveis de PP1α, uma das isoformas da PP1, se correlaciona com a variação dos níveis de fosforilação da AKT e as mudanças nos níveis da PP1γ com a variação dos níveis de fosforilação da MAPK. Com estes resultados propomos um modelo de como a prodigiosina desfosforila a AKT e como este processo contribui para a indução da morte celular em células de melanoma. Esperamos que este modelo ajude na compreensão do mecanismo de acção da prodigiosina bem como no reconhecimento das fosfatases como novos alvos terapêuticos no tratamento de cancro.
Protein phosphorylation is a major regulatory mechanism for cell function. It is noteworthy that several pathologies, including cancer to be associated with abnormal phosphorylation of key proteins. Although many studies have addressed the kinases that are misregulated in cancer, much less is known about the phosphatases that counteract their actions. PP1, a major serine/threonine protein phosphatase that is ubiquitously expressed in all eukaryotic cells, is involved in many cellular processes including apoptosis and cell cycle. In fact, several studies demonstrate that PP1 regulates several proteins that are key elements in the tumorogenesis process. AKT, a serine/threonine kinase that is disregulated in several types of cancer is a crucial factor in melanoma progression and survival. Prodigiosin, a family member of the natural red pigmented tripyrrolic secondary metabolites, prodiginines, show anticancer properties in numerous types of cancer. In fact, some prodigiosin studies demonstrate that AKT is dephosphorylated by prodigiosin by an unknown mechanism. Given the importance of AKT in melanoma progression and survival and the capacity of PP1 to dephosphorylate AKT it is possible that PP1 is involved in this mechanism. Our preliminary results showed that PP1 binds to one member of prodiginine family proving the interaction between these molecules. On the other hand, experiments with melanoma cell lines, using prodigiosin and cantharidin, a PP1 inhibitor, treatments, demonstrate that prodigiosin affect differently PP1 isoforms. These results suggest that prodigiosin acts in a different way in two altered pathways in melanoma, AKT and MAPK, since the alterations in PP1α levels, one of PP1 isoforms, are correlated with the conversion in AKT dephosphorylation and the variations in PP1γ levels with the changes in MAPK dephosphorylation. Given these results we propose a model of how prodigiosin dephosphorylates AKT and how this process contributes to induce cell death in melanoma cells. We expect that this model helps to understand prodigiosin action mechanism as well as acknowledge phosphatases as a therapy target in cancer treatment.
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Silva, Joana Vieira da. "Electron microscopy study of PP1 and SARP2 in human testis". Master's thesis, Universidade de Aveiro, 2011. http://hdl.handle.net/10773/7698.
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Mestrado em Biomedicina MolecularA proteína fosfatase-1 (PP1) é uma fosfatase específica para a desfosforilação de resíduos de serina e treonina, cujas funções celulares dependem da formação de complexos com proteínas que interagem com a PP1 (PIPs). Dada a importância da PP1γ2, isoforma específica de testículo/espermatozóides, na espermatogénese e na função dos espermatozóides, é fundamental identificar as funções das PIPs específicas destes tecidos, tais como a SARP2 (Several Ankyrin Repeat Protein 2). A PP1 alcança a sua especificidade, não só pela diversidade de proteínas com as quais interage, mas também através de diferentes subunidades catalíticas. No entanto, a maioria dos estudos não aborda directamente a importância das diferentes isoformas da PP1. Neste trabalho relatamos, pela primeira vez, os padrões de expressão de PP1γ2, PP1α e SARP2 no testículo humano, colhido in vivo, por microscopia electrónica. Os nossos resultados demostram que a PP1γ2 é a isoforma mais abundante nos estágios finais de maturação das células germinativas masculinas, enquanto que a PP1α está presente no núcleo dos espermatídeos e durante a formação do acrossoma. A SARP2 é altamente abundante no testículo no qual é expressa apenas na fase pós-meiótica, mais especificamente na fase média/tardia dos espermatídeos haplóides elongados. Dado este padrão de localização de SARP2, propomos que esta possa desempenhar um papel importante na diferenciação e/ou função dos espermatozóides. Em conclusão, a SARP2 e a PP1γ2, proteínas abundantes em testículo e espermatozóides, apresentam a mesma localização no testículo humano. O complexo PP1γ2/SARP2 revela especificidade a nível tecidular (testículo), de estadío de espermatogénese (espermiogénese), a nível celular (espermatídeos elongados) e da localização sub-celular (núcleo), o que torna o complexo um novo alvo para a contracepção masculina ou tratamento de infertilidade masculina.
Protein Phosphatase 1 (PP1) is a major eukaryotic serine/threonine specific phosphatase whose cellular functions depend on the complexes it forms with PP1 Interacting Proteins (PIPs). Given the importance of the testis/sperm-enriched variant, PP1γ2, in spermatogenesis and sperm function it is imperative to identify the roles of the testis/sperm specific PIPs, such as SARP2 (several ankyrin repeat protein 2). PP1 achieves its specificity towards the substrates not only by the diversity of the binding partners but also through different catalytic subunits. Most studies have not directly addressed the significance of the different isoforms. Here, we report, for the first time, the expression patterns of PP1α, PP1γ2 and SARP2 in human testis by immunoelectron microscopy, using tissue previously collected in vivo. Our results show that PP1γ2 is the most abundant isoform in the late stages of germ cell maturation, whereas for the most part, PP1α is found in spermatids nucleus and during the acrosome formation. SARP2 exhibits a tissue, cell type, and spermatogenic stage specificity. SARP2 is more abundant in testis, and within the testis it is expressed only in post-meiotic haploid spermatids, mainly in mid/late stages of elongated spermatids. Given this unique expression pattern of SARP2, we propose that it plays a significant role in sperm differentiation and/or function. In conclusion, SARP2 shows the same localization pattern in human testis as PP1γ2, the PP1 testis/sperm-specific isoform. PP1γ2/SARP2 complex is tissue (testis), spermatogenic stage (spermiogenesis), cell type (elongated spermatids) and subcellular compartment (nucleus) specific. Thus making this complex a putative novel target for male contraception or male infertility treatment.
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Ferreira, Mónica Alexandra dos Santos. "I-2L, a novel putative PP1 inhibitor in human sperm". Master's thesis, Universidade de Aveiro, 2010. http://hdl.handle.net/10773/8796.
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Mestrado em Biologia Molecular e CelularA proteína fosfatase-1 (PP1) é uma proteína fosfatase ubíqua e específica para a desfosforilação de resíduos de serina e treonina, que participa na regulação de diversos processos celulares. Em mamíferos, a actividade da PP1 está relacionada com a motilidade dos espermatozóides durante o percurso no tracto epididimal. Esta holoenzima é composta por uma subunidade catalítica, denominada PP1c, e por proteínas que interagem com a PP1 (PIPs) e a dirigem para a proximidade de substratos, determinando a sua actividade. A PP1 é codificada por três genes distintos: PP1α, PP1β e PP1γ. As isoformas PP1γ1 e PP1γ2 são originadas por splicing alternativo do gene PP1γ. PP1γ1 é uma isoforma ubíqua e a PP1γ2 é expressa maioritariamente no testículo e espermatozóides. O inhibidor-2 (I-2) de mamífero é uma PIP ancestral que se liga à PP1c, resultando na inibição da actividade da PP1. A reactivação deste complexo é desencadeada pela desfosforilação do I-2 no resíduo de Thr73 pela GSK-3. Atendendo às diversas funções que têm sido atribuídas à PP1, com base nas suas subunidades reguladoras, é crucial identificar novas PIPs. Assim, com o objectivo de identificar novas PIPs em testículo humano, procedeu-se ao rastreio de uma biblioteca de cDNA de testículo humano pelo método de dois híbrido de levedura, usando a PP1γ1 e PP1γ2 como isco. Foi assim obtido um clone muito semelhante ao I-2, denominado I-2L. Após um estudo comparativo por análise bioinformática, a principal diferença detectada foi a substituição de um resíduo de Thr73 por um de Pro73, no I-2L, conduzindo à ausência do local de fosforilação pela GSK-3, o que resulta num novo mecanismo de regulação para o complexo PP1γ2-I-2L. A presença de I-2/I-2L em espermatozóides humanos foi confirmada pelos métodos de Western blot e imunocitoquímica. As proteínas I-2/I-2L e a PP1γ2 co-localizam na peça intermédia e principal do flagelo dos espermatozóides, em concordância com o papel do complexo PP1γ2-(I-2/I-2L) na motilidade dos espermatozóides. A semelhança entre o I-2 e I-2L, em termos da sequência de aminoácidos (95%), origina um novo desafio na diferenciação destas duas proteínas. Consequentemente, foi analisado o fingerprint proteolítico do I-2 e I-2L para posterior análise por espectrometria de massa, sendo esta uma abordagem eficaz para provar a existência do I-2L in vivo. Estudos estão presentemente a decorrer no sentido de validar, por espectrometria de massa, a sequência de aminoácidos do I-2L imunoprecipitado de espermatozóides humanos.
Protein phosphatase-1 (PP1) is a ubiquitously expressed serine/threonine protein phosphatase that controls several cellular processes. In mammalian sperm, changes in PP1 activity correlates with sperm motility development during the transit through the epididymis. The PP1 holoenzyme structure is composed of a conserved subunit termed PP1c and PP1-interacting proteins (PIPs) which direct the holoenzyme to the proximity of its substrates, determining their activity. The catalytic subunit of PP1 is encoded by three different genes termed PP1α, PP1β and PP1γ. PP1γ1 and PP1γ2 are alternatively spliced isoforms of the PP1γ gene. PP1γ1 is ubiquitously expressed whereas PP1γ2 is mainly expressed is testis and sperm. Mammalian inhibitor-2 (I-2) is an ancient PIP that binds to PP1c, resulting in the inhibition of PP1 activity. The reactivation of this complex is triggered by phosphorylation of I-2 at Thr73 residue by GSK-3. Given all the roles that have been proposed to PP1 based on its binding subunits it seemed very important to identify novel PIPs. Thus, in order to search for novel PP1 regulator in human testis yeast two-hybrid screens were performed using PP1γ1 and PP1γ2 as baits to screen a human testis cDNA library. A clone was obtained very similar to I-2, termed I-2 Like (I-2L). After bioinformatics analysis comparison, the main difference detected was Thr73/Pro73 substitution which abolishes the GSK-3 phosphorylation site. This introduces a new regulatory mechanism for the PP1γ2-I-2L complex. The presence of I-2/I-2L in human sperm was confirmed by immunoblot and immunocytochemistry analysis. I-2/I-2L and PP1γ2 co-localize endogenously in the middle- and principal- piece of spermatozoa flagellum consistent with the role of PP1γ2-(I-2/I-2L) complex in sperm motility. The similarities between I-2/I-2L in terms of amino acid sequences (95%) introduced a challenge to differentiate I-2 and I-2L proteins. Hence, the proteolytic fingerprint of I-2 and I-2L was analyzed for further mass spectrometry (MS) analysis, and concluded that this was a fine approach to prove the existence of I-2L in vivo. Studies are now in progress in order to validate, via MS analysis, the amino acid sequence of I-2L immunoprecipitated from human sperm.
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Chakrabarti, Rumela. "Role for PP1 [gamma] 2 in spermatogenesis and sperm morphogenesis". [Kent, Ohio] : Kent State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=kent1176430377.
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Thesis (Ph.D.)--Kent State University, 2007.Title from PDF t.p. (viewed Mar. 12, 2009). Advisor: Srinivasan Vijayaraghavan. Keywords: sperm, testes, spermatogenesis, protein phosphatase, knock out, spermatid. Includes bibliographical references (p. 115-129).
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Muñoz, Muñoz Ivan. "Caracterització molecular de subunitats reguladores de la fosfatasa Ppz1 en el llevat saccharomyces cerevisiae". Doctoral thesis, Universitat Autònoma de Barcelona, 2004. http://hdl.handle.net/10803/3523.
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La fosfo-defosforilación de aminoácidos es uno de los procesos de regulación reversible más extendidos en los seres vivos. Este tipo de reacciones es llevado a cabo por la acción opuesta de las proteínas quinasas y las proteínas fosfatasas. A pesar de ello, el genoma de la levadura codifica un número mayor de Ser/Thr quinasas. Por ello, algunas fosfatasas han desarrollado un mecanismo de regulación distinto, que se basa en la unión de la subunidad catalítica a una o más subunidades reguladoras. Éstas determinan la localización subcelular, la especificidad por el sustrato y, en definitiva, la función de la fosfatasa. Éste es el caso de la fosfatasa de tipo 1, que está codificada por GLC7 en la levadura Saccharomyces cerevisiae, un enzima esencial que desarrolla funciones muy importantes en la biología de la célula y cuya función está regulada por la unión de la subunidad catalítica a multitud de subunidades reguladoras distintas.El genoma de la levadura codifica dos fosfatasas, conocidas con el nombre de Ppz1 y Ppz2 y cuya mitad carboxiterminal está relacionada estructuralmente con Glc7. A pesar de no ser esenciales, estas fosfatasas desarrollan un papel importante en la biología de la levadura Saccharomyces cerevisiae. En concreto, ejercen un control negativo sobre la tolerancia salina y la progresión del ciclo celular y positivo en el mantenimiento de la integridad celular. En el momento de iniciar este trabajo sólo se conocía una subunidad reguladora de estas fosfatasas, conocida con el nombre de Hal3 o Sis2. Esta proteína actúa como inhibidora de todas las funciones conocidas de Ppz1. Sin embargo, su mecanismo de actuación es desconocido.
El primer objetivo de este trabajo ha sido intentar profundizar en la regulación que lleva a cabo la fosfatasa Ppz1 en el control del ciclo celular de la levadura. Para ello, se ha generado un doble mutante condicional sit4 hal3 que presenta una parada entre las fases G1 y S del ciclo. Esta cepa se ha transformado con dos genotecas multicopia diferentes. Gracias a ello se han podido aislar una serie de genes que, en multicopia son capaces de suprimir el fenotipo de esta cepa. Entre ellos se encuentran elementos reguladores del ciclo celular ya conocidos, algunas fosfatasas, elementos involucrados en homeostasis salina y tres genes cuya función era desconocida por el momento, que han sido renombrados VHS1, VHS2 y VHS3.
En segundo lugar, se ha investigado el mecanismo de acción de Hal3 sobre la fosfatasa Ppz1. Para ello se han estudiado elementos hallados en la secuencia de Hal3 posiblemente relevantes para la función de esta proteína. Además, se ha realizado una estrategia de mutagénesis al azar de la zona más conservada de Hal3 seguida de una búsqueda de pérdida de función. Esto ha permitido hallar una serie cambios aminoacídicos únicos que interfieren en la función de Hal3. La caracterización bioquímica posterior ha permitido determinar dos regiones en la secuencia de Hal3 importantes para la unión a Ppz1 y una tercera región importante para inhibir a la fosfatasa.
Finalmente, se ha estudiado el papel biológico de YFR003c, un ORF cuya función era desconocida por el momento. Estos estudios han demostrado que codifica una proteína con características afines a los inhibidores de la fosfatasa de tipo 1 humana. Además, se ha podido demostrar que Yfr003c es capaz de unirse e inhibir in vitro a Glc7 y, con menor eficiencia, a Ppz1. Estudios posteriores in vivo refuerzan la hipótesis del papel de Yfr003c como inhibidor de Glc7. Por este motivo, ha recibido el nombre de Ypi1 (Yeast Phosphatase 1 Inhibitor) y se ha propuesto como el primer inhibidor conocido de Glc7 en la levadura Saccharomyces cerevisiae.
Phospho-dephosphorylation of amino acids is one of the most extended mechanisms of reversible regulation found in living organisms. This kind of reactions is driven by the opposite action of kinases and phosphatases. In spite of this fact, yeast genome encodes more Ser/Thr protein kinases. For this reason some phosphatases have developed a different regulatory mechanism based on the union of the catalytic subunit to one or more regulatory proteins which in turn regulate the subcellular location, the specificity for substrate and thus, the function of the phosphatase. This is the case of Protein Phosphatase 1, encoded by the essential gene GLC7 in the yeast Saccharomyces cerevisiae. This enzyme plays an important role in the yeast and it is tightly regulated by the union of the catalytic subunit to different regulatory proteins.
The genome of the yeast also encodes two phosphatases, named Ppz1 and Ppz2 that are structurally related to Glc7. Although they are not essential, they also play an important role in the biology of the yeast. These phosphatases act negatively in the control of the progression of cell cycle and in the maintenance of the salt tolerance and positively in the control of osmotic integrity. In the early steps of this work there was described only one regulatory subunit of these phosphatases named Hal3 or Sis2. Although its specific mechanism of function is still unknown it has been described that this protein acts as an inhibitor for all the known functions of Ppz1.
In this work we tried to deepen in the regulation of the cell cycle progression driven by Ppz1. For this reason, we created a conditional sit4 hal3 double mutant, which presents, in non-permissive conditions, a blockade between the G1 and S phases of the cell cycle. This strain was transformed with two different multicopy libraries and colonies able to grow in non-permissive conditions were selected and plasmidic DNA extracted and sequenced. This procedure allowed us to identify a total of 13 genes that, when overexpressed, were able to suppress the phenotype of this strain under non-permissive conditions. They include well-known cell cycle regulatory elements but also some phosphatases, some genes that develop different roles in the control of salt tolerance and three genes with no known function until now that we renamed VHS1, VHS2 and VHS3.
We also investigated the regulatory interaction between Hal3 and Ppz1. For this reason, we studied some elements found in the sequence of Hal3 that could be relevant for the function of this protein. Furthermore, we developed a strategy of random mutagenesis of the most conserved region of Hal3 followed by a screening of loss of function of the protein. This procedure allowed us to identify several amino acidic changes that affect Hal3 function in vivo. Biochemical characterization shows the presence of two regions in Hal3 that are important for the union to Ppz1 and a third one that is relevant in the inhibition of the phosphatase activity.
Finally, we studied the biological role of YFR003c, an uncharacterized ORF with no known function. Our work demonstrated that YFR003c encodes a protein that shares characteristics commonly found in some inhibitors of the human protein phosphatase 1. We have shown that YFR003c is able to interact with Glc7 and Ppz1 in vitro. Furthermore, YFR003c acts as an inhibitor of Glc7 and, in a lesser degree, of Ppz1. Overexpression studies have shown that YFR003c can act in vivo as an inhibitor of Glc7. For this reason we renamed it YPI1 (for Yeast Phosphatase 1 Inhibitor) and it has been proposed to be the first endogenous inhibitor of Glc7 in the yeast Saccharomyces cerevisiae.
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Bentham, Matthew John. "Generation and characterisation of a human cytomegalovirus mutant lacking the virion trans-activator pp71". Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621338.
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Manlan, Olivier. "Initiative PPTE et allègement de la dette de la Côte d'Ivoire : une évaluation économétrique". Paris 10, 2003. http://www.theses.fr/2003PA100172.
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Le premier objectif de cette thèse est de montrer à l'aide d'un modèle économétrique que le niveau d'allégement de la dette déterminé pour la Côte d'Ivoire dans le cadre de l'Initiative PPTE, n'est pas cohérent avec ses besoins de désendettement. Le second objectif de cette thèse est de contribuer aux fondements théoriques de l'implication à part entière de la société civile dans l'élaboration des programmes de lutte contre la pauvreté, et de manière plus générale, dans la mise en oeuvre des programmes de développement durables. Dans ce contexte, les outils de l'analyse économique que sont la théorie des jeux et l'approche économique des conflits sont utilisésThe first aim of this thesis was to demonstrate through an econometric model that the amount of Debt Relief computed by World Bank and IMF for Côte d'Ivoire, is no longer consistent with the country economic growth structure and outlook. The second point was to show that the economic backbone of Poverty Reduction Strategy with an accent on the Empowerment of Civil Society is weak with regard to economic analysis. In this regard, a Gaine Theory analysis is proposed to support involvement and commitment of civil society within the Poverty Reduction Strategy Paper (HIPC Initiative)
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Freitas, Maria João Martinho de. "Addressing sperm motility regulation through characterization and modulation of the GSK/PPP1R2/PPP1 signaling pathway". Doctoral thesis, Universidade de Aveiro, 2018. http://hdl.handle.net/10773/22772.
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Doutoramento em BiologiaSperm motility acquisition and maintenance is a fundamental process for oocyte fertilization and consequently conception. The signaling events underling sperm motility acquisition have been studied for decades. However, many questions are still unanswered. Also, the limited options currently available for male contraception (condom, vasectomy and withdrawal) reflect the necessity of a new group of male contraceptives based on sperm motility modulation. GSK3/PPP1R2/PPP1 signaling pathway is involved in sperm motility acquisition during epididymis transit. The main goal for this work was to deepen the knowledge on the signaling events involved in human sperm motility by focusing on the characterization and modulation of the signaling pathway GSK3/PPP1R2/PPP1. We first designed, synthetized and characterized a disruptive bioportide based on cell penetrating peptide technology. In vitro studies revealed that the disruptive bioportide interferes with PPP1R2/PPP1CC2 interaction and restores PPP1CC2 activity. We also demonstrated that when exposed to the disruptive bioportide, sperm motility is significantly reduced. Aiming to identify sperm protein-protein interactions suitable for pharmacological intervention, we turn our attention to GSK3, a modulator of PPP1R2/PPP1CC2 interactions in sperm. We provide for the first time GSK3 human testis and sperm interactomes. We reported an isoforms specific role for GSK3 in human sperm motility and an in silico analysis revealed GSK3 and GSK3 interactions involved in sperm motility and potential targets for pharmacological intervention. In conclusion, we demonstrated that it is possible to target protein-protein interactions and modulate sperm complexes involved in motility using bioportides. Moreover, we identified new potential protein interactions involved in sperm motility and showed that the development of new type of male contraceptive based on inhibiting sperm motility is now achievable.
A aquisição e manutenção da mobilidade do espermatozoide é fundamental para a fertilização do oócito e consequentemente conceção. Durante décadas, as vias de sinalização necessárias à aquisição de mobilidade por parte do espermatozoide foram alvo de intensos estudos. Contudo, este processo ainda não é inteiramente conhecido. Ademais, as limitadas opções disponíveis para contraceção masculina (preservativo, vasectomia e coito interrompido) refletem a necessidade de desenvolver um contracetivo masculino baseado na modulação da mobilidade do espermatozoide. A via de sinalização GSK3/PPP1R2/PPP1 está envolvida na aquisição de mobilidade do espermatozoide ao longo do transito do epidídimo. O objetivo principal deste trabalho é enriquecer o conhecimento dos eventos celulares necessários na mobilidade do espermatozoide através da caracterização e modulação da via de sinalização GSK3/PPP1R2/PPP1 em espermatozoides humanos. Desenhámos, sintetizámos e caracterizámos um bioportide que quebra interações proteicas baseado em tecnologia de cell penetrating peptides. Estudos in vitro revelaram que o bioportide de ruptura interfere com a interação PPP1R2/PPP1CC2 e é capaz de restabelecer a atividade da PPP1CC2. Também demonstramos que o bioportide reduz significativamente a mobilidade do espermatozoide. Com o objetivo de identificar interacções proteína-proteína adequadas à intervenção farmacológica, focámos a nossa atenção na proteína GSK3, um modulador da interação PPP1R2/PPP1CC2 em espermatozoides. Descrevemos pela primeira vez o interactoma da GSK3 no testículo e espermatozoide humanos e reportamos um papel específico da isoforma GSK3 na mobilidade do espermatozoide. Uma análise in silico revelou interatores da GSK3 e GSK3 que estão envolvidos na mobilidade do espermatozoide e potencialmente poderão ser alvos de intervenção farmacológica para um novo contraceptivo masculino. Em conclusão, demonstramos que é possível provocar a quebra de interações proteína-proteína e modular a mobilidade do espermatozoide usando de bioportides. Também identificamos potenciais novas interações proteicas envolvidas na mobilidade do espermatozoide. Finalmente, mostramos que é possível idealizar um novo tipo de contracepção masculina baseado na inibição da mobilidade do espermatozoide.
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Laia, Marcelo Luiz de. "Clonagem e caracterização de análogos de genes de resistência e desenvolvimento de um marcador SCAR Iigado ao gene Ppr1 em Eucalyptus grandis". Universidade Federal de Viçosa, 2001. http://www.locus.ufv.br/handle/123456789/10102.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
A maioria dos genes de resistência (genes R) cIonados codifica proteínas que tém dominios conservados. Estas proteinas sao classificadas em classes ou subclasses de acordo com a presenca ou ausência desses domínios. Domínios do tipo LRR (Leucine rich repeat) e NBS (Nucleotide binding site) são os mais freqüentes em produtos dos genes R de mono e dicotiledôneas. A presença desses domínios conservados permite o uso da técnica de PCR com vistas ao isolamento e a clonagem de sequências análogas a genes de resistência (RGA - ReS/Stance Gene Ana/og), mediante o uso de oligonucleotídeos degenerados específicos para regiões conservadas. Associado a mapeamento genético, essa abordagem pode facilitar a clonagem e caracterização de novos genes R. Visando a clonagem e caracterização do gene Ppr1, que confere resistência a ferrugem em E. grandis, procurou-se neste trabalho identificar RGA’s que cossegregassem com este gene. Seqüências análogas a genes R foram amplificadas a partir do DNA isolado de uma matriz de E. grandis resistente a ferrugem, com o uso de oligonucleotídeos degenerados que aneIam a seqüências que codificam domínios conservados de proteínas de resistência. Os fragmentos amplificados (~6OO pb) foram clonados no vetor pGEMT-Easy, transformados em E. coli e seqüênciados. Utilizando-se como critérios o tamanho, o rendimento e a qualidade do DNA, 13 clones, dentre noventa obtidos, foram selecionados (ML 5, ML 21, ML 23, ML 25, ML 27, ML 28, ML 29, ML 31, ML 32, ML 33, ML 34, ML 35 e ML 38) para seqüenciamento. Os clones foram comparados entre si e com outros genes de resistência já caracterizados, com base nas suas seqüências de aminoácidos deduzidas. A análise com o algoritmo Blastx revelou que os clones ML27 e ML34 são similares ao gene N de Nicotiana glutinose e RPSS de Arabidopis thaliana, respectivamente. Além disso, o clone ML27 também foi similar a um homólogo do gene N presente em Pinus radiata. Os clones ML28, ML21, ML23, ML29 e ML38 não mostraram similaridade significativa com seqüências depositadas em bancos de dados e o clone ML05 possui NBS similar a de um provável transportador ABC de arabidopsis. Embora estes clones tenham revelado vários locos RFLP polimórficos entre progenitores da população estudada, nenhum desses polimorfismos cossegregou com o gene Ppr1. Uma segunda fase do trabalho consistiu no desenvolvimento de marcadores SCAR (Sequence Characterized Amplified Region) a partir do marcador RAPD AT9917. Para tal, o DNA da matriz G21 (E. grandis) foi amplificado com o oIigonucIeotídeo AT9 e o fragmento de 917pb foi purificado do gel, cIonado em vetor apropriado e multiplicado em E. coli DH5α. A partir da seqüência de nucleotídeos deste fragmento, desenharam-se quatro oligonucIeotídeos no sentido direto e três no sentido reverso. Os oligonucleotídeos foram combinados entre si a fim de identificar um par que gerasse polimorfismo entre o genótipo resistente (G21) e o genótipo suscetível (G38). Não foi possível obter polimorfismo a partir da amplificação, mas após clivagem do produto da PCR com a enzima de restrição Cfo I, obteve-se uma banda de aproximadamente 800 pb que cossegregou com o fenótipo de resistência. Todavia, esta banda não cossegregou com o fenótipo de resistência em outras famílias oriundas do mesmo genitor G21.
The majority of cloned resistance genes (R) codes for proteins which have conserved LRR (Leucine Rich Repeat) and NBS (Nucleotide Binding Site) domains. The presence of these conserved domains allows the use of the specific degenerated primers for these conserved regions and PCR technique to amplify resistance gene analogue sequences (RGAs). Associated to genetic mapping, this approach may facilitate the cloning and characterization of new R genes. Aiming to clone and characterize the Ppr1 gene, which confers resistance to rust in Eucalyptus grandis, this work sought to identify RGAs that would cosegregate with this gene. Degenerated oligonucleotides were used to amplify RGAs from a rust -resistant E. grandis clone G21. The amplified fragments (~6OO bp) were cloned into the vector pGEMT-EASY, transformed into E. coli Using DNA size, yield and quality criteria, 13 clones, among the 90 obtained, were selected (ML 5, ML 21, ML 23, ML 25, ML 27, ML 28, ML 29, ML 31, ML 32, ML 33, ML 34, ML 35 and ML 38) for sequencing. The clones were compared to each other and to other resistance genes already characterized, based on their deduced amino acid sequences. The Blastx searches revelead that clones ML27 and ML34 are similar to the N (Nicotiana glutinosa) and RPS5 (Arabidopsis thaliana) genes, respectively. Besides, clone ML27 was also similar to a homologue of the N gene present in P/nus racfiata. Clones ML28, ML21, ML23, ML29 and ML38 did not show significant similarity with sequences in the Genebank and clone MLo5 has a NBS similar to a putative Arabidopsis ABC transporter. Southern analyses using these clones revealed several polymorphic RFLP loci in the progenitors of the Ppr1 mapping population. However, none of them co-segregated with the Ppr1 gene. The other objective of this work was to develop SCARs (Sequence Characterized Amplified Region) markers from the RAPD AT9 marker. G21 DNA was amplified with the primer AT9 and the 917pb band obtained was gel-purified and cloned into pGEMT-EASY vector. Based on the nucleotide sequence of this fragment were designed four primers that align in the forward and three in the reverse orientation. Several combinations of forward and reverse primers were tested in order to obtain a pair that could amplify the expected fragment of 917 bp and generate polymorphism between the resistant (G21) and susceptible (G38) genotypes, respectively. It was not possible to obtain polymorphism directly from the amplification; however, after cleavage of the PCR product with the restriction enzyme CfoI, a band of approximately 800 bp was obtained, which co-segregated with the resistance phenotype in the mapping population. However, this band did not co-segregate with the resistance phenotype in other families originated from the same G21 progenitor.
Dissertação importada do Alexandria.
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Haddley, Kate. "Transcriptional regulation of the preprotachykinin-A (PPT-A) gene in neuronal cells". Thesis, University of Liverpool, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408571.
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Ruiz, Garzón Amparo. "Nuevos elementos reguladores y funciones celulares de la proteína fosfatasa Ppz1 en la levadura Saccharomyces cerevisiae". Doctoral thesis, Universitat Autònoma de Barcelona, 2006. http://hdl.handle.net/10803/3552.
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Las proteínas Ppz1 y Ppz2 de Saccharomyces cerevisiae son Ser/Thr fosfatasas involucradas en diferentes procesos celulares, tales como el mantenimiento de la integridad celular, la tolerancia salina y la regulación del ciclo celular en la transición G1/S. La mayor parte de los fenotipos asociados con la ausencia o la sobreexpresión de las fosfatasas Ppz son la consecuencia de la inhibición que ejercen estas fosfatasas sobre los transportadores de potasio de alta afinidad Trk1/2. En las primeras fases de este trabajo, sólo se había identificado una subunidad reguladora para estas fosfatasas, la proteína Hal3, y aunque el mecanismo mediante el cual ejerce esta inhibición se desconocía, sí se había descrito que esta proteína actúa como inhibidora de Ppz1 y Ppz2 en todas las funciones conocidas de Ppz1 En este trabajo presentamos nuevas dianas biológicas de las fosfatasas Ppz, como son la vía de calcio/calcineurina y el sistema de transporte de potasio de baja afinidad. En primer lugar, describimos que células que carecen de Ppz1 son hipertolerantes a altas concentraciones de sodio o litio debido a un incremento en la expresión del gen ENA1, que codifica el componente principal para la tolerancia salina en la levadura. Este efecto en la expresión de ENA1 es independiente de Trk1/2 pero totalmente dependiente de la vía de señalización de la fosfatasa calcineurina, sugiriendo así que Ppz1 inhibe, directa o indirectamente, la actividad de la calcineurina. Respecto a esta función, demostramos que Ppz1 actúa como un regulador negativo de la entrada de calcio que tiene lugar a través de la membrana plasmática. Por otro lado, células que carecen de Ppz1/2 y Trk1/2 necesitan más potasio para sobrevivir que células que carecen sólo de los transportadores de potasio de alta afinidad, hecho que sugiere que las fosfatasas Ppz son reguladores positivos de la entrada de potasio de baja afinidad Trk-independiente.
También hemos identificado una segunda subunidad reguladora de las fosfatasas Ppz, denominada Vhs3. Vhs3 actúa inhibiendo las fosfatasas Ppz de igual modo a como lo hace su homólogo Hal3, aunque este último parece ser más eficiente. Además, demostramos que Hal3/Vhs3 participan en el proceso celular de la floculación. Ambas regulan negativamente la vía de señalización de la PKA que, a través de la activación del factor de transcripción Flo8, induce la expresión de la floculina Flo11 causando la floculación. Este efecto es dependiente de las fosfatasas Ppz y podría ser explicado por la regulación negativa que ejercen éstas sobre los transportadores de potasio Trk1/2.
Finalmente, demostramos que Vhs3 y Hal3, que presentan homólogos en plantas y mamíferos, están involucradas en una función esencial que es independiente de su papel sobre Ppz1/2, ya que el doble mutante hal3 vhs3 y el cuádruple mutante hal3 vhs3 ppz1 ppz2 son letales mientras que el mutante ppz1 ppz2 no lo es. Además, la letalidad de la cepa hal3 vhs3 se suprime por la sobreexpresión de otros homólogos de Hal3, como es el caso de AtHal3a de Arabidopsis thaliana, sugiriendo que las funciones de Hal3 y Vhs3 Ppz-independientes están conservadas en otros eucariotas. Experimentos in vitro revelan que la proteína Hal3 de plantas (AtHal3a) cataliza uno de los pasos de la biosíntesis de coenzima A y que para ello requiere de dos residuos: la His90 y la Cys175. Aunque Vhs3 y Hal3 conservan el residuo histidina, no conservan el residuo equivalente a la Cys175, por tanto, no es evidente que esta nueva función sea participar en la síntesis de CoA.
The Saccharomyces cerevisiae Ppz1 and Ppz2 are Ser/Thr protein phosphatases involved in several cell processes, such as maintenance of cell integrity, salt tolerance and regulation of cell cycle at the G1/S transition. Most of the phenotypes associated with the absence or the overexpression of the Ppz phosphatases are the consequence of the inhibition that these phosphatases exert on the Trk1/2 high-affinity potassium transporters. At the early steps of this work, only one regulatory subunit of these phosphatases, Hal3, have been identified and, although its specific mechanism of function is still unknown, it has been described that this protein acts as an inhibitor of Ppz1 and Ppz2 for all the known functions of Ppz1.
In this work we report evidences about the existence of other biological targets of the Ppz phosphatases, such as the calcium/calcineurin pathway and the low-affinity potassium uptake. First, we describe that cells lacking Ppz1 are hypertolerant to high concentrations of sodium or lithium due to an increased expression of the ENA1 gene, which codifies the major determinant for salt tolerance in yeast. This effect on ENA1 expression is independent of Trk1/2 but is fully dependent on the calcineurin pathway and suggests that Ppz1 inhibits, either directly or indirectly, the calcineurin activity. Concerning to this function, we reveal that Ppz1 acts as a negative regulator of the calcium import across the plasma membrane. On the other hand, cells lacking Ppz1/2 and Trk1/2 need more potassium than cells lacking only the high-affinity potassium transporters to survive, suggesting that the Ppz phosphatases are positive regulators of the Trk-independent low affinity potassium uptake.
We also identify a second regulatory subunit of the Ppz phosphatases, named Vhs3. Vhs3 functions inhibiting the phosphatases Ppz, essentially as its homolog Hal3 does, although the last one seems to work more efficiently. In addition, we demonstrate that Hal3/Vhs3 proteins play a role in the flocculation process. They negatively regulate the PKA signalling pathway which, through the activation of the transcription factor Flo8, induces the expression of the Flo11 flocculine causing the flocculation. This effect is dependent on the Ppz phosphatases and could be explained by their inhibitory activity on Trk1/2.
Finally, we demonstrate that Vhs3 and Hal3, which have homologs in both plants and mammals, are involved in an essential function that is independent of their role in regulating Ppz1/2, as the double mutant hal3 vhs3 and the hal3 vhs3 ppz1 ppz2 are lethal whereas the ppz1 ppz2 is not. The lethality of the hal3 vhs3 strain is suppressed by overexpression of other Hal3 homologs, such as the AtHal3a in Arabidopsis thaliana, suggesting that the Ppz-independent functions of Hal3 and Vhs3 are conserved in eukaryotes. In vitro experiments revealed that the plant Hal3 (AtHal3a) is involved in one step of the coenzyme A biosynthesis and that there are two residues completely necessary for this function: His90 and Cys175. Vhs3 and Hal3 have the histidine equivalent to His90, but neither of them possesses the equivalent Cys175 suggesting that the yeast homologs could not be involved in the biosynthesis of CoA.
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Jesson, Jocelyn Gavin. "The PPTA and the State: from militant professionals to bargaining agent : a study of rational opportunism". Thesis, University of Auckland, 1995. http://hdl.handle.net/2292/2016.
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This thesis using oral historiographic techniques examines the changing role and function of a teachers' union during changes in the nature of a national State which can also be related to changing forms of capitalism. The teachers' union is New Zealand Post-Primary Teachers Association (the secondary school teachers' union) and the period of particular focus is between 1983 and the beginning of 1993. This is the period of a crisis in the New Zealand State during which the character of the national State was moved from what has been described as a 'wage earners' Welfare State towards a more residual form providing a 'modest safety net'. The financial and the labour market were deregulated to become more free of direct State involvement. The administration of education was changed and the individual schools' elected Boards of Trustees were made responsible for the provision of schooling. The role of PPTA in the State is moved structurally in this time. As a professional association before the changes, PPTA had both opportunity for input into the mandate of education, as well as the possibility of creating implementation pressure through political action. As a bargaining agent, the input role of PPTA to decisions in education was limited to addressing members' concerns at the school level. The restructuring of education and of the labour market, PPTA was both an object to be acted on and a participant obstructing the changes. This thesis presents what is a PPTA view of those changes. PPTA formed a central part of the education structures which were to be transformed by the economic liberal project. The survival of PPTA demonstrates the extent to which the project was not completed and the resistance of PPTA was one of the reasons why the project in education could not be completed. The thesis is in three parts. Part one is a regulationist-derived periodisation of the historical development of the New Zealand wage earners Welfare State and education. This is followed by a consideration of the economic-liberal challenge to this State Part two considers the development of PPTA's professional project and the possibilities presented through different arenas. Part three focuses on the changing nature of the State labour market policy and PPTA's activity in that. The changing strategies and tactics of PPTA's 'professional project', the thesis argues, occur under changing political conditions and are an example of Offe's concept of rational opportunism
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Idlemouden, Khadija. "Annulations de la dette extérieure et croissance : Une application au cas des "pays pauvres très endettés" (PPTE)". Phd thesis, Université Paris Dauphine - Paris IX, 2009. http://tel.archives-ouvertes.fr/tel-00419972.
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L'objectif de cette thèse est d'évaluer l'effet des réductions de la dette extérieure consenties dans le cadre de l'initiative PPTE sur la croissance. Après un rappel, dans le premier chapitre, des spécificités de la dette des PPTE, nous présentons, dans le deuxième chapitre, les caractéristiques de l'Initiative PPTE. Elle vise à rétablir la soutenabilité de la dette et à encourager la lutte contre la pauvreté. L'objectif de croissance n'y est pas explicite. Le troisième chapitre établit un lien entre la soutenabilité et la croissance à travers le modèle de fardeau virtuel. Celui-ci met en exergue l'impact néfaste du surendettement sur l'investissement. Toutefois, deux limites apparaissent. La première tient aux hypothèses du modèle. La seconde est liée à la méthode d'estimation empirique. Le quatrième chapitre aborde une des limites. Nous montrons l'importance des préférences intertemporelles dans la relation dette-investissement, lorsque l'accès aux capitaux est limité. La dette ne nuit à l'investissement qu'en cas de faible préférence pour le lissage. Forte dans les PPTE, elle remet en question l'approche du fardeau virtuel. Pour s'en assurer, nous proposons, dans les deux chapitres suivants, deux études centrées sur les PPTE. Elles confirment l'absence de fardeau virtuel puisque le stock de dette n'influe ni sur l'investissement, ni sur la croissance. Le service de la dette exerce un effet positif ce qui corrobore les conclusions du quatrième chapitre. Une évaluation des premiers résultats des réductions est présentée dans le septième chapitre. Une croissance légèrement plus élevée est observée mais ne découle pas de l'investissement mais d'une meilleure gouvernance.
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Faille, Alexandre. "É́tude structurale et fonctionnelle de la polykétide synthase PpsC et de son activateur PptT chez Mycobacterium tuberculosis". Toulouse 3, 2013. http://thesesups.ups-tlse.fr/2805/.
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PpsC est une polykétide synthase mono-modulaire de type I essentielle à la synthèse de facteurs de virulence chez Mycobacterium tuberculosis appelés dimycocérosates de phthiocérol. Afin d'aider à la découverte de nouveaux anti-tuberculeux et de mieux comprendre le mécanisme employé par les polykétide synthases, nous avons entrepris l'étude structurale et fonctionnelle de PpsC par cristallographie des rayons X. Les polykétide synthases de type I sont composées de plusieurs domaines, chacun possédant une activité catalytique différente. Nous avons utilisé la méthode du domain trapping pour identifier des fragments solubles représentant chaque domaine de PpsC. L'étude de ces fragments a permis de déterminer la structure des domaines acyl-transférase, déshydratase, et enoyl-reductase de PpsC. Nous avons aussi caractérisé le mécanisme catalytique du domaine déshydratase en se basant sur la structure d'un complexe enzyme-substrat et de tests enzymatiques in vitro. Les polykétide synthases doivent être activées par le transfert d'un groupe prosthétique sur leur domaine porteur d'acyl. Chez Mycobacterium tuberculosis, PptT est responsable de l'activation et est donc essentielle à la virulence. En utilisant la technologie split-GFP, nous avons mis au point un protocole de production et purification de PptT où des cofacteurs jouent un rôle essentiel à sa stabilité in vivo et in vitro. L'activité de l'enzyme a été caractérisée par le transfert in vitro du groupe prosthétique sur le domaine porteur d'acyl de PpsC. Enfin, nous avons résolu la structure tridimensionnelle de PptT à 1,4 A par cristallographie aux rayons X, permettant ainsi la conception rationnelle d'inhibiteursPpsC is a type-I mono-modular polyketide synthase essential for the synthesis of phthiocerol dimycocerosates virulence factors in Mycobacterium tuberculosis. In an attempt to both help the search for new antituberculosis drugs, and decipher the complex mechanism used by polyketide synthases, we have undertaken the functional and structural study of PpsC using X-ray crystallography. Type-I polyketide synthases are made of several domains, each possessing a different catalytic activity. We have used the recently developed domain trapping method to identify soluble fragments representing each domain of PpsC. The study of these fragments has led to the structural determination of the acyltransferase, dehydratase, and enoylreductase domains of PpsC. In addition, we have deciphered the catalytic mechanism of the dehydratase domain, based on the structure of a substrate-enzyme complex and in vitro enzymatic tests. Polyketide synthases need to be activated by the transfer of a prosthetic group onto their acyl carrier protein domain. In Mycobacterium tuberculosis, PptT is responsible for the activation of polyketide synthases and is thus essential to its virulence. Using the split-GFP technology, we have designed a protocol for the production and purification of PptT, where its co-factors play an essential role for its stability both in vivo and in vitro. Activity of the enzyme was assessed by in vitro transfer of the prosthetic group onto the acyl carrier protein domain of PpsC. Finally, we have solved the structure of PptT at 1. 4 A resolution using X-ray crystallography, which will allow the structure-based design of inhibitors
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Ferreira, Mónica Alexandra dos Santos. "Epigenetic control of the male progenitor germline by the protein phosphatase PP1-NIP". Doctoral thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22814.
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Doutoramento em BiologiaNIPP1, for nuclear inhibitor of protein phosphatase 1 (PP1), is a multifunctional scaffold protein that regulates cell signaling, pre-mRNA splicing and transcription by targeting PP1 to specific nuclear substrates. The global deletion of NIPP1 in mice is embryonic lethal at the onset of gastrulation, precluding its functional analysis in adult tissues. This prompted us to generate a tamoxifen-inducible NIPP1 knockout (iKO) mouse model. Unexpectedly, the deletion of NIPP1 was not efficient in the examined organs except for testis. The loss of NIPP1 caused an age-dependent progressive loss of testicular germ cells, culminating in a Sertoli cells-only phenotype. iKO testis showed a decreased proliferation of (un)differentiated spermatogonia and an increased level of apoptosis. Likewise, neonatal iKO testis exhibited an almost complete loss of gonocyte-derived (un)differentiated spermatogonia during the first wave of spermatogenesis. In addition, GFRA1+ progenitor cells isolated from induced iKO testis displayed a reduced proliferation potential. These data suggest that NIPP1 is required for the maintenance of undifferentiated spermatogonia. We also found that the observed phenotype was associated with the deregulation of genes that are implicated in the control of cell proliferation and survival. At the molecular level, the deletion of NIPP1 was associated with the loss of core components of the Polycomb Repressive Complex 2 (PRC2), which affects gene expression through trimethylation of histone H3 at Lys 27. The loss of PRC2 components could be explained by the hyperphosphorylation and degradation of EZH2, the catalytic subunit of the PRC2 complex, resulting in the destabilization of other PRC2 core components. The testis phenotype of the iKOs could be phenocopied by the chemical inhibition of EZH1/2 in organotypic testis cultures. Overall, our study uncovers a key function for PP1-NIPP1 in the regulation of EZH2 phosphorylation and stability, which is essential for the maintenance of germ cells.
NIPP1, inhibidor nuclear da protein phosphatase 1 (PP1), é uma proteina multifuncional que regula a sinalização celular, splicing do pre-mRNA e transcrição mediante o direcionamento da PP1 para substratos nucleares específicos. A deleção global da NIPP1 é letal durante o desenvolvimento embrionário no início da gastrulação, impedindo assim a sua análise funcional em tecidos de adultos. Este facto incitou-nos a gerar um modelo de ratinho knockout induzível (iKO) para a NIPP1. Inesperadamente, a remoção da NIPP1 não foi eficiente na maioria dos órgãos analisados, com exceção do testículo. A deleção da NIPP1 causou uma perda progressiva de células germinativas do testículo dependente da idade, culminando num fenótipo denominado Sertoli cells-only phenotype. O testículo adulto nos ratinhos iKO apresentaram uma diminuição na proliferação das espermatogónias (in)diferenciadas e aumento dos níveis de apoptose. De modo análogo, o testículo dos neonatos exibiu uma perda quase completa das espermatogónias (in)diferenciadas derivadas de gonócitos, durante o primeiro ciclo de espermatogénese. Adicionalmente, culturas celulares enriquecidas em células progenitoras GFRA1+ isoladas do testículo dos ratinhos iKO apresentaram uma diminuição do seu potencial proliferativo. Estes resultados sugerem que a NIPP1 é necessária para a manutenção das espermatogónias indiferenciadas. Demonstrámos também que fenótipo observado está associado à desregulação de genes implicados no controlo da proliferação e viabilidade celular. No que concerne o mecanismo molecular, a deleção da NIPP1 resultou na perda dos componentes centrais do complexo PRC2 (Polycomb Repressive Complex 2), o que afetou a expressão genética através da trimetilação da histone H3 no resíduo Lys27 (H3K27me3). A perda dos componentes integrantes do complexo PRC2 foi explanada pela hiperfosforilação e degradação da proteína EZH2, o componente catalítico central do complexo PRC2, resultando na subsequente destabilização de outros componentes deste complexo. Em conformidade, o fenótipo foi reproduzido através da inibição química da proteína EZH1/2 em culturas organotípicas de testículos. De modo geral, este estudo revela a importância da fosfatase PP1-NIPP1 para a regulação da fosforilação e estabilização da proteína EZH2, essencial para a manutenção das células germinativas.
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Lenne, Astrid. "Caractérisation moléculaire et fonctionnelle de deux nouveaux partenaires potentiels de la protéine phosphatase de type 1 (PP1) chez plasmodium falciparum". Thesis, Lille 2, 2016. http://www.theses.fr/2016LIL2S016/document.
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Plasmodium falciparum (Pf) est un parasite intracellulaire capable d’infecter l’Homme. Dans les 48h après l’invasion des érythrocytes, il passe par le stade d’une cellule géante multinucléée qui se divise en 16 à 32 parasites. Cette multiplication rapide nécessite des mécanismes spécifiques de régulation très subtilement orchestrés. Parmi les modifications post-traductionnelles décrites chez les cellules eucaryotes, la phosphorylation réversible des protéines par les kinases/phosphatases est une des voies majeures dans la transduction des signaux cellulaires. Chez Plasmodium, des études de génétique inverse ont démontré que PfPP1, phosphatase majeure du parasite, est essentielle pour sa survie. L’activité de PP1 est connue pour être contrôlée par divers régulateurs endogènes. Cependant, malgré leur importance dans le ciblage de l’holoenzyme à un compartiment subcellulaire spécifique et/ou dans la régulation de son activité, peu de recherches ont été consacrées à l’identification de partenaires de PP1 chez Pf.Dans le but d’approfondir nos connaissances sur la régulation de PfPP1 et son impact sur la biologie de Pf, nous étudions les partenaires de cette enzyme qui seraient impliqués dans le contrôle de la phosphatase. Nos recherches récentes, basées sur des études de génomique comparative, ont permis d’identifier 4 régulateurs de PfPP1 : PfLRR1, PfI3, PfI2 et PfeiF2β. Au-delà de la capacité de ces protéines à contrôler la fonction de PP1 in vitro, nous avons montré par génétique inverse que leur rôle est vital pour Pf. En parallèle, nous avons entrepris une démarche plus globale pour la recherche de nouveaux partenaires/régulateurs de PfPP1. Nous avons notamment réalisé un criblage par double hybride de levure (Y2H) où PfPP1 est utilisé comme appât.Dans la 1ère partie de ma thèse, nous avons analysé les clones obtenus en criblage Y2H et initié la caractérisation de plusieurs d’entre eux. Notre choix d’étude s’est porté par la suite sur PF3D7_091900 et PF3D7_1202600, retrouvées 8 et 10 fois lors du criblage et présentant une interaction forte avec PP1. Mon projet de thèse avait pour but de caractériser au niveau moléculaire et fonctionnel le rôle de ces protéines sur l’activité de PP1 et de déterminer les régions/motifs de ces potentiels régulateurs pouvant intervenir au niveau de la relation structure/fonction du complexe qu’ils forment avec PfPP1.Dans une 2ème partie, nous avons étudié la séquence de ces protéines. PF3D7_0919900, protéine spécifique du parasite, possède le motif RVxF, souvent impliqué dans l’interaction avec PP1 et présente des motifs RCC1 connus pour interagir avec des protéines. Ainsi elle sera nommée RCC-PIP pour Regulator of Chromosome Condensation - Phosphatase Interacting Protein. PF3D7_1202600 est, quant à elle, un orthologue de Caliban chez la drosophile, et sera nommée CLP pour Caliban-Like Protein. Elle présente 17 motifs RVxF dont 7 se situent dans le fragment obtenu suite au criblage. Différentes approches ont confirmé que ces 2 protéines sont des partenaires de PfPP1. Cependant, la réalisation d’un test pNPP in vitro a mis en évidence une fonction activatrice de RCC-PIP, alors que CLP ne présente pas d’effet.Dans une 3ème partie, l’objectif était d’étudier plus en détails RCC-PIP. Nous avons démontré que le motif RVxF est impliqué dans l’interaction avec PfPP1. Puis nous avons étudié les motifs RCC1 et leur interactome par la réalisation d’un criblage Y2H en utilisant ces motifs comme appât. Une kinase a été trouvée (PfCDPK7) suggérant que RCC-PIP aurait un rôle de plateforme capable d’interagir avec 2 enzymes antagonistes. L’étude du rôle de RCC-PIP chez le parasite est actuellement en cours. La réalisation d’un knock-in a démontré l’accessibilité du locus. Un knock-out a également été effectué, mais l’absence d’intégration du plasmide indique que RCC-PIP serait essentielle au parasite. Pour confirmer cette observation, un knock-out conditionnel chez P. berghei est en cours de réalisationPlasmodium falciparum is an intracellular parasite that evolves in several stages of development in the vertebrate host. Within 48 hours after the invasion of erythrocytes, it goes through the stage of a multinucleated giant cell which divides into as many parasites as nuclei (16-32 parasites). This growth/fast division requires specific regulatory mechanisms subtly orchestrated. Among the post-translational modifications described in eukaryotic cells, the reversible phosphorylation of proteins by kinases/phosphatases is one of the major pathways in the cellular signal transduction. In Plasmodium, PP1 is predicted to catalyze the majority of protein dephosphorylation events, and has been shown to be essential in its survival using reverse genetic approaches. The activity of PP1 is known to be tightly controlled by various endogenous regulators. However, despite their importance in targeting the holoenzyme to a specific subcellular compartment and/or regulating its activity, little has been devoted to identify PP1 partners in the parasite.In order to deepen our understanding of the regulation of PfPP1 and its impact on the biology of Plasmodium, we study the partners of this enzyme that may be involved in the control of its location, its specificity and activity. Our recent research, based on comparative genomic studies, have identified 4 regulators of PfPP1: PfLRR1, PfI3, PfI2 and PfeiF2β. In parallel, since Plasmodium has a particular cell cycle and the function of PP1 should be adapted, we have undertaken a more global approach to the search for new partners/regulators of PfPP1 using different approaches including a yeast two-hybrid screening where PfPP1 was used as bait.In the first part of my thesis, the clones obtained in Y2H screening were analyzed and 2 clones were selected for further characterization. These clones, corresponding to PF3D7_091900 and PF3D7_1202600 were identified 8 and 10 times during the screening, a good indication about their expression in blood stages and their interactions with PfPP1 can be still detectable under high stringency conditions. Hence, my thesis project aimed to characterize molecularly and functionally of the role of these proteins on PfPP1. _x000D_In the second part, we have analysed the sequence of these two proteins. PF3D7_0919900, a parasite-specific protein, shows the canonical binding motif « RVxF », known to be involved in the interaction with PP1, and present on the fragment obtained following the screening. The sequence also has RCC1 motifs known to interact with proteins. Thereafter, this protein was designated as RCC-PIP for Regulator of Chromosome Condensation - Phosphatase Interacting Protein. As far as PF3D7_1202600 is concerned, it seems to be an ortholog of Caliban expressed by Drosophila, and designated Pf Caliban CLP-Like Protein. It has 17 potential RVXF binding motif, of which 7 are located in the fragment obtained following the screening. Different approaches such as GST pull-down or ELISA, identified these two proteins as partners of PfPP1. Using pNPP in vitro assay, we showed a slight activation of PfPP1 by RCC-PIP, while CLP had no effect.In the third part, the objective was to study in more detail the RCC-PIP. We showed that the RVxF motif is involved in the interaction with PfPP1. We then tied to identify the partners of RCC1 by screening a cDNA library of P. falciparum using RCC1 containing protein as bait. We showed that RCC-PIP is able to interact with a kinase (PfCDPK7) suggesting that RCC-PIP may act as a platform since it is able to interact with 2 enzymes with opposed activities. Analysis of the role of RCC-PIP in the parasite is currently underway. The production of a knock-in demonstrated the accessibility of the locus. A knock-out was also carried out, but the lack of integration of the plasmid suggests that RCC-PIP is essential to the parasite. To confirm this observation, a conditional knock-out in P. berghei is in progress
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Hollin, Thomas. "Analyse de l’interactome de la Ser/Thr protéine phosphatase de type 1 (PP1) chez plasmodium falciparum : caractérisation moléculaire et fonctionnelle de Gametocyte EXported Protein 15". Thesis, Lille 2, 2017. http://www.theses.fr/2017LIL2S020/document.
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L’un des obstacles majeurs au développement de nouveaux antipaludiques est notre connaissance limitée de la biologie parasitaire et la rareté des cibles thérapeutiques potentielles identifiées. Les interactions protéines-protéines sont impliquées et essentielles dans divers processus biologiques incluant les modifications post-traductionnelles. Les interactions substrats-kinases ou phosphatases sont considérées comme une liaison transitoire et jouent un rôle central et essentiel dans le cycle cellulaire de Plasmodium. La Ser/Thr Protéine Phosphatase de Type 1 (PP1), l’une des phosphatases majeurs des eucaryotes, est essentielle à la survie du parasite Plasmodium falciparum, responsable du paludisme. Elle est régulée par diverses sous-unités régulatrices dont plus de 200 ont été identifiées chez l’Homme, mais seulement 4 chez Plasmodium.Afin d’explorer le réseau de régulation de la P. falciparum PP1 (PfPP1), nous avons utilisé trois approches complémentaires pour caractériser l’interactome de la PfPP1. La purification par co-affinité suivie d'une analyse par spectrométrie de masse a identifié 6 protéines interagissant avec la PfPP1 dont 3 contenaient le motif consensus d’interaction RVxF, 2 autres le motif Fxx[RK]x[RK], également connu pour interagir avec la phosphatase et une protéine avec les deux motifs de liaison. Le criblage par double hybride chez la levure a identifié 134 protéines dont 30 présentent le motif RVxF et 20 ont le motif de liaison Fxx[RK]x[RK]. Le criblage in silico du génome de P. falciparum en utilisant une séquence consensus du motif RVxF a révélé 55 partenaires potentiels de la PfPP1. Afin de confirmer l’interaction de certaines protéines, 35 partenaires candidats ont été validés par un test d’interaction de type ELISA. Les résultats ont permis de détecter aussi bien des partenaires conservés de la PP1 qu'un nombre élevé d'interacteurs spécifiques à la PP1 du parasite et montrent une grande diversité dans les fonctions biologiques impliquant la PP1 chez Plasmodium. Parmi ces candidats, un partenaire appelé Gametocyte EXported Protein 15 (GEXP15) a été confirmé comme un réel partenaire de la PfPP1 par différentes approches. De plus, GEXP15 est surexprimé chez les gamétocytes, stade responsable de la transmission du parasite chez le moustique. Ces résultats ainsi que des études fonctionnelles seront présentésA major obstacle in the development of novel anti-malarials is our limited knowledge of basic parasite biology and the paucity of identified potential intervention targets. Protein-protein interactions are involved and essential in broad range of biological processes including the post-translational modifications. Substrate-kinase or phosphatase interactions are considered as transient binding and play a central and essential role in Plasmodium cell cycle. The Ser/Thr Protein Phosphatase Type 1 (PP1), one of the main contributors of eukaryotic phosphatase activity, is essential to malaria parasite Plasmodium falciparum and is highly regulated by many regulatory subunits. In humans, there are about 200 distinct regulators, however, only 4 have been so far reported in Plasmodium.To explore the P. falciparum PP1 (PfPP1) regulatory network as complete as possible, we carried out three complementary approaches to characterize the PfPP1 interactome. Co-affinity purification followed by Mass Spectrometry-based proteomics identified 6 PfPP1 interacting proteins (PIPs) of which 3 contained the RVxF consensus binding motif, 2 PIPs with a Fxx[RK]x[RK] binding motif, one with both binding motifs. The Yeast Two-Hybrid screening identified 134 proteins of which 30 have the RVxF binding motif and 20 contain the Fxx[RK]x[RK] binding motif. The in silico screen of the P. falciparum genome using a consensus RVxF motif as template revealed the presence of 55 potential PfPP1 interacting proteins. As further demonstration, 35 candidate partners were validated in an independent ELISA-based assay using recombinant proteins. The data reports several conserved PP1 interacting proteins as well as a high number of specific interactors to PfPP1, indicating a high diversity of biological functions for PP1 in Plasmodium. Among these candidates, one partner assigned as Gametocyte EXported Protein 15 (GEXP15) has been confirmed as a direct interactor of PfPP1 by different approaches. In addition, GEXP15 is over-expressed during gametocyte stage, responsible for the transmission of the parasite in the mosquito. These results as well as functional studies will be presented and discussed
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Serpe, Rossana. "Identification of clock neurons and downstream circuits that are involved in sleep control in Drosophila melanogaster". Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS257.
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Le moment, la qualité et la quantité de sommeil dépendent de l'interaction fine entre l'horloge circadienne et la machinerie homéostatique (Borbely A. et al., 1982, Daan S. et al., 1984, Borbely et Achermann, 1999). Au cours des dernières années, l'utilisation de divers organismes modèles a fourni de nouvelles perspectives sur les mécanismes neuronaux et moléculaires de la régulation du sommeil (Miyazaki S. et al., 2017). Cependant, les bases moléculaires de l'homéostasie du sommeil et les circuits neuronaux sous-jacents à son interaction avec le réseau circadien n'ont pas été établis en détail.Dans ce travail de thèse, j'ai utilisé la mouche Drosophile melanogaster comme système modèle pour étudier la fonction d'un sous-ensemble de neurones d'horloge, les DN1ps, dans la mise en place du sommeil. Des études antérieures ont suggéré un rôle de ces neurones circadiens dans la régulation du sommeil (Kunst et al., 2014, Guo et al 2016, Lamaze et al., 2017, Guo et al., 2017). J’ai ainsi démontré que les cellules d'horloge DN1ps DH31 (+) CRY (+) sont impliquées dans la suppression du sommeil. Par ailleurs, j’ai mis en évidence un circuit en aval des DN1ps, qui comprend le groupe dopaminergique postérieur apparié latéral 1 (PPL1) et les neurones dorsaux en forme d’éventail (dFSB), un centre homéostatique récemment décrit pour la régulation du sommeil chez la drosophile (Donlea JM et al., 2011, Liu S. et al., 2012, Ueno et al., 2012, Donlea JM et al., 2014, Pimentel et al., 2016, Qian et al., 2017, Donlea JM. et al., 2018). Nos résultats indiquent que la suppression du sommeil nocturne nécessite la signalisation DH31-R2 dans une sous-population des neurones dopaminergiques PPL1, qui projette au dFSB. Fait intéressant, la perte de sommeil de jour et de nuit médiée par les DN1ps dépend de l'inhibition du dFSB. Néanmoins, nous suggérons que les neurones DN1ps CRY (-) favoriseraient le sommeil, en concordance avec d'autres travaux (Guo et al., 2016; Guo et al., 2017).Ces résultats fournissent de nouvelles données sur le lien entre l'horloge circadienne et l'homéostasie du sommeil, impliqué dans la régulation du comportement sommeil-éveil chez Drosophile melanogasterThe timing, quality and quantity of sleep depend on the fine interaction between circadian clock and homeostatic machinery (Borbely A. et al., 1982; Daan S. et al., 1984; Borbely and Achermann, 1999). In the recent years, the employment of various model organisms has provided new insights into the neuronal and molecular mechanisms of sleep regulation (Miyazaki S. et al., 2017). However, the molecular basis of the sleep homeostat and the neuronal circuitry underlying its interaction with the circadian network haven’t been established in details.In this work, I use the fruit fly Drosophila melanogaster as a model system to investigate the sleep function of a subset of clock neurons, the DN1ps. Previous studies have already suggested a sleep-regulating role for these circadian neurons (Kunst et al. 2014, Guo et al. 2016; Lamaze et al., 2017; Guo et al. 2017). Here, we report the DH31-positive CRY-positive DN1ps as sleep suppressing clock cells. Furthermore, we identify a sleep-relevant circuit downstream of the DN1ps which includes the paired posterior lateral 1 (PPL1) dopaminergic cluster and the dorsal Fan-shaped body projecting (dFSB) neurons, a recently described homeostatic center for sleep regulation in Drosophila (Donlea JM. et al., 2011; Liu S. et al., 2012; Ueno et al., 2012; Donlea JM. et al., 2014; Pimentel et al., 2016; Qian et al., 2017; Donlea JM. et al., 2018). Our results indicate that the night-time sleep suppression requires DH31-R2 signaling in the PPL1-to-dFSB dopaminergic neurons. Interestingly, both day and night-time DN1ps-mediated sleep loss rely on the inhibition of the dFSB. Nevertheless, we suggest the CRY-negative DN1ps as sleep promoting clock neurons, in concordance with other works (Guo et al. 2016; Guo et al. 2017).These findings provide a novel link between circadian clock and sleep homeostat, in the regulation of sleep-wake behavior in Drosophila melanogaster
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Velázquez, Sánchez Diego. "Exploring the regulatory mechanisms of the S. cerevisiae Ppz1 protein phosphatase and the molecular basis for its toxicity". Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/667958.
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Uno de los más notables componentes en la homeostasis de cationes en levaduras es la proteína fosfatasa Ppz1. El papel de Ppz1 es doble: inhibe la entrada de potasio mediante el control de la actividad de los transportadores Trk1 y Trk2 y atenúa la expresión del gen de la Na+-ATPasa ENA1. Ppz1, está controlada negativamente por dos subunidades reguladoras estructuralmente relacionadas, Hal3 y Vhs3, siendo la primera la más relevante funcionalmente. A parte de esto se ha demostrado que niveles altos de Ppz1 son extremadamente perjudiciales para la célula.
El primer objetivo de este trabajo ha sido estudiar la interacción entre Ppz1 y Hal3. A pesar de las numerosas evidencias de que ambas proteínas interaccionan físicamente, se desconoce que regiones de cada proteína están involucradas en dicha interacción y hasta qué punto es un proceso dinámico. Para ello se han diseñado dos aproximaciones experimentales, por un lado se han creado una serie de cepas con etiquetas fluorescentes en Ppz1 y Hal3, para seguir su interacción in vivo gracias al fenómeno de la transferencia de energía por resonancia de fluorescencia (FRET) mediante citometría de flujo. Estas fusiones de Hal3 y Ppz1 con proteínas fluorescentes, además de ser completamente funcionales, han demostrado la interacción in vivo de ambas proteínas y permiten estudiar posibles variaciones en la interacción. Por otro lado, mediante experimentos de crosslinking in vitro de ambas proteínas y un posterior análisis por LC-MS/MS se han mostrado dos regiones del dominio C-terminal de Ppz1 y tres regiones de Hal3, que podrían ser las responsables de llevar a cabo esta interacción.
A pesar de ser regulada negativamente por Hal3 y Vhs3, la región más N-terminal de Ppz1 es muy rica en residuos fosforilables. De hecho, el 30% de este dominio son serinas o treoninas, y algunas de ellas han sido identificadas como fosforiladas. Sin embargo, apenas ha sido estudiado que efectos funcionales pueden dar lugar cambios en el estado de fosforilación de Ppz1. En este trabajo hemos conseguido demostrar que la MAPK Hog1 fosforila a Ppz1 en la posición S265, pero este hecho no es suficiente para variar el comportamiento de la fosfatasa. Además, hemos conseguido demostrar que el estado de fosforilación de Ppz1 puede variar dependiendo de factores externos como alta concentración de sal o deficiencia de potasio.
Recientemente se ha demostrado que Ppz1 es la proteína más tóxica de levadura cuando se sobredosifica. Recientemente, en nuestro laboratorio se ha verificado que la actividad fosfatasa de Ppz1 es necesaria para causar dicha toxicidad, ya que la sobreexpresión de una versión catalíticamente inactiva no causa problemas de crecimiento en las células. Sin embargo, las bases moleculares de esta toxicidad son desconocidas. En nuestro caso hemos realizado experimentos para conocer el estado del proteoma y del fosfoproteoma de S. cerevisiae cuando se sobreexpresa Ppz1, y hemos descrito una variación en el estado de fosforilación en numerosas proteínas de la levadura. De hecho se han identificado cambios importantes en proteínas relacionadas con la traducción, polarización celular, la transición G1/S del ciclo celular o con el metabolismo de carbohidratos. Además, algunos experimentos paralelos han reforzado los resultados obtenidos mediante espectrometría de masas, como la defosforilación de Mig1 y Rps6, o una hiperfosforilación de eIF2α en la Ser-51, indicando una posible inhibición en la iniciación de la traducción.
En resumen los resultados obtenidos en esta tesis han puesto a punto herramientas para conocer más sobre el dinamismo de la interacción Ppz1-Hal3, han aportado datos sobre la fosforilación de Ppz1, y han generado una gran cantidad de datos de fosfoproteómica cuyo estudio detallado podría llevar a esclarecer bases moleculares de la toxicidad de Ppz1 en S. cerevisiae.The protein phosphatase Ppz1 is one of the most relevant components in cation homeostasis in yeast. It has two major roles: on one hand, it inhibits the potassium influx by controlling the activity of the Trk1 and Trk2 potassium transporters and, on the other hand, it represses the expression of the ENA1 gene, coding for a Na+-ATPase. Ppz1 is negatively regulated by two structurally related subunits, Hal3 and Vhs3, being Hal3 the most functionally relevant. Previous studies have shown that high levels of Ppz1 are extremely detrimental for yeast cell growth. The first objective of this work has been to study the interaction between Ppz1 and Hal3. Although there is evidence of both proteins interacting physically, the regions of each protein involved in the interaction, as well as its dynamism of the interaction, are still unknown. Therefore, two different experimental approaches have been designed. The first approach is based on the detection of fluorescence resonance energy transfer (FRET) by flow-cytometry. Several strains have been constructed with fluorescent tags on Ppz1 and Hal3, to monitor the interaction in vivo. These tagged versions of Ppz1 and Hal3, in addition to be fully functional, have been shown suitable to demonstrate the interaction in vivo between both proteins and to allow the study of possible variations on the interaction. The second approach, based on in vitro crosslinking of both proteins, followed by LC-MS/MS analysis, has shown two regions of Ppz1 C-terminal and three from Hal3 that could be involved in the interaction. Even though Ppz1 is negatively regulated by Hal3 and Vhs3, the most N-terminal region of Ppz1 is very rich in phosphorylable residues (in fact, 30 % of the N-terminal residues are serine or threonine). Some of them have been identified as phosphorylated in several studies. However, how phosphorylation could affect Ppz1 function remains unknown. In this work we show that the MAPK1 Hog1 phosphorylates Ppz1 at S265, but this is not enough to alter the behaviour of the phosphatase. Moreover, we demonstrated that the phosphorylation state of Ppz1 can change depending on external factors, such as high salt or low potassium concentrations. It has been recently demonstrated that Ppz1, when overexpressed, is the most toxic protein for yeast cells. We described in our laboratory that the phosphatase activity of Ppz1 it is crucial for this toxicity, since overexpression of a catalytically inactive version does not negatively affect cell growth. However, the molecular basis of this toxicity is still unknown. To address this question our approach was to characterize the proteomic and phosphoproteomic landscape of S. cerevisiae cells overexpressing Ppz1. We have found changes in the phosphorylation state of numerous proteins, including proteins related to translation, polarized cell growth, G1/S transition and carbon metabolism. Moreover, we provide additional evidence that reinforce the results obtained by mass-spectrometry, such in the case of Mig1 and Rps6 dephosphorylation, or hyperphosphorylation of eiF2α at Ser-51, pointing to a possible inhibition of translation initiation. Overall, the results obtained in this thesis have set tools to dynamically study in vivo the Ppz1-Hal3 interaction, have provide information on the phosphorylation of Ppz1, and have generated a large database of proteomic and phosphoproteomic data whose future analysis might provide the clues about the toxicity of Ppz1 in S. cerevisiae.
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Johns, Anna. "Towards the development of novel Aspergillus fumigatus targeted antifungals with an in-depth analysis of Sfp-PPTase, PptA". Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/towards-the-development-of-novel-aspergillus-fumigatus-targeted-antifungals-with-an-indepth-analysis-of-sfppptase-ppta(5f2e1052-01b3-4fb2-84b9-5871538351db).html.
Texto completoResumen
Humans are continuously confronted by the threat of fungal infection. Estimates put the number of individuals infected by superficial fungal disease at about 1.7 billion. Additionally there are a significant proportion of invasive infections which are difficult to treat and lead to an estimated 1.5 million deaths each year. Aspergillus fumigatus is an opportunistic, fungal pathogen which can lead to a variety of disease manifestations, generally termed aspergillosis and account for more than 200,000 life threatening infections annually with mortality rates of up to 95%. The occurrence of fungal disease has increased significantly due to the expansion of the immune deficient population, particularly those who receive immunosuppressive therapies. This combined with the problems surrounding current therapeutic options for fungal disease such as a rise in the incidence of antifungal resistance, adverse side effects in patients and drug-drug interactions has led to an urgent need to discover and develop an innovative class of antifungal agents. This thesis will address this requirement by validating a potential drug target, PptA, for combating A. fumigatus infection and explore methods to identify novel antifungal target identification. PptA is a Sfp-type 4′-Phosphopantetheinyl transferase required for transfer and covalent tethering of 4’-phosphopantetheine from coenzyme A to a conserved serine residue within a peptidyl carrier domain of a protein substrate. This project outlines the many critical roles PptA plays within A. fumigatus, such as; involvement in the production of many virulence factors as well as the biosynthesis of the essential amino acid, lysine. Furthermore, it is demonstrated that PptA is vital for secondary metabolite production in A. fumigatus, growth in iron limiting conditions and virulence. Finally, the design of a high-throughput screening assay capable of identifying inhibitors of PptA enzymatic activity further demonstrate the suitability of this target. It is the combined effect of all these characteristics that makes PptA such a suitable candidate for an antifungal target. Additionally two validation methods are included which can be used to identify novel drug targets. The first method utilises chemically induced haploinsufficiency profiling, a technique which has been proven successful for drug target identification and determining mode of action of drugs in S. cerevisiae and C. albicans. The project has shown this technology can be successfully used in A. fumigatus. Furthermore, a new technique for in vitro parallel fitness screening using next generation sequencing was validated with a library of phosphatase deletion mutants. Bioinformatic analysis against strains exhibiting severe fitness defects allowed the identification of three phosphatases that have the potential of becoming successful antifungal drug targets.
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Gokhan, Ezgi. "The Repo-Man/PP1 complex role in chromatin remodelling, nuclear structure and cancer progression". Thesis, Brunel University, 2016. http://bura.brunel.ac.uk/handle/2438/14731.
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Repo-Man is a chromatin-associated PP1 targeting subunit that coordinates chromosome re-organisation and nuclear envelope reassembly during mitotic exit. At the onset of mitosis, Repo-Man association with the chromosomes is very dynamic; at anaphase, Repo-Man targets to the chromatin in a stable manner and recruits PP1 to de-phosphorylate histone H3 at Thr3, Ser10 and Ser28. Previous studies have suggested that CDK1 and AuroraB are the kinases responsible for the inactivation of the complex and for its dispersal at the onset of mitosis respectively. We have previously shown that the binding of Repo-Man to PP1 is decreased in mitosis and we have identified a region adjacent to the RVTF motif that contains multiple mitotic phosphosites (RepoSLIM). This region is conserved only in another PP1 targeting subunit: Ki-67. In order to understand the importance of this region for the complex formation and stability, we have conducted mutational analyses on several residues, and addressed their contribution towards Repo-Man chromosome targeting and PP1 binding in vivo. We have identified new sites in Repo-Man that, when phosphorylated, contribute to the weakening of the binding between Repo-Man and PP1. Interestingly, our results also indicate that several kinases are involved in the mitotic regulation of the complex. We have also identified Lamin A/C as a Repo-Man substrate and introduced a new model for Lamin A/C regulation at interphase. Furthermore, we identified Repo-Man as a marker of malignancy in tripe egative breast cancer, which controls cell movement and levels of important oncogenic markers Aurora A and C-Myc, and propose Repo-Man/PP1 complex as a therapeutic target for the treatment of triple negative breast cancer through the newly identified RepoSLIM.