Oppgaven presenterer teknikker og implementasjon for sammenlikning av naboskap av proteiner i protein-protein interaksjons-nettverk.

Post-traumatic stress disorder (PTSD) is a psychiatric condition that can develop when people experience a stressful and life-threatening event. Clinical research indicates that the presence of a state of hyperarousal after a traumatic experience is the best predictor of a subsequent diagnosis of PTSD. The role of arousal peptides called orexins (hypocretins) in a PTSD-like condition produced by exposing rats to a single episode of footshocks (5× 2 s episodes of 1.5 mA) was investigated in this thesis. The first part of my thesis involves the characterization of the footshock model of PTSD and the second part examines the involvement of orexins in this footshock model. The following findings are reported. First, shock rats that exhibited a high level of anxiety to a novel tone (high responders, HR) the day after the footshock exposure subsequently displayed more avoidance when compared to shock rats that exhibited a low level of anxiety (low responders, LR). These results highlight the importance of individual differences in the reaction to a strong fear-inducing experience. Second, the orexin precursor peptide prepro-orexin (ppOX) mRNA was found to be elevated in rats at 6 and 14 days after exposure to footshocks. In addition, ppOX mRNA levels were found to be positively correlated with anxiety at 14 days post-shock. Third, pre-shock injections of the corticotropin releasing factor receptor antagonist antalarmin were found to attenuate the anxiety expressed to the shock chamber and eliminate the correlation between ppOX mRNA levels and anxiety. Fourth, systemic injections of the nonselective orexin receptor antagonist TCS-1102 was found to attenuate the anxiety expressed in rats at 14 days post-shock. Fifth, TCS-1102 was found to have anxiolytic effects that were specific for the HR. The results of these experiments provide evidence linking the orexin system to the anxiety produced by exposure of rats to footshocks. They also provide preclinical evidence in support of the use of orexin antagonists for the treatment of anxiety in PTSD.

Post-traumatic stress disorder (PTSD) is a psychiatric condition that can develop when people experience a stressful and life-threatening event. Clinical research indicates that the presence of a state of hyperarousal after a traumatic experience is the best predictor of a subsequent diagnosis of PTSD. The role of arousal peptides called orexins (hypocretins) in a PTSD-like condition produced by exposing rats to a single episode of footshocks (5× 2 s episodes of 1.5 mA) was investigated in this thesis. The first part of my thesis involves the characterization of the footshock model of PTSD and the second part examines the involvement of orexins in this footshock model. The following findings are reported. First, shock rats that exhibited a high level of anxiety to a novel tone (high responders, HR) the day after the footshock exposure subsequently displayed more avoidance when compared to shock rats that exhibited a low level of anxiety (low responders, LR). These results highlight the importance of individual differences in the reaction to a strong fear-inducing experience. Second, the orexin precursor peptide prepro-orexin (ppOX) mRNA was found to be elevated in rats at 6 and 14 days after exposure to footshocks. In addition, ppOX mRNA levels were found to be positively correlated with anxiety at 14 days post-shock. Third, pre-shock injections of the corticotropin releasing factor receptor antagonist antalarmin were found to attenuate the anxiety expressed to the shock chamber and eliminate the correlation between ppOX mRNA levels and anxiety. Fourth, systemic injections of the nonselective orexin receptor antagonist TCS-1102 was found to attenuate the anxiety expressed in rats at 14 days post-shock. Fifth, TCS-1102 was found to have anxiolytic effects that were specific for the HR. The results of these experiments provide evidence linking the orexin system to the anxiety produced by exposure of rats to footshocks. They also provide preclinical evidence in support of the use of orexin antagonists for the treatment of anxiety in PTSD.

No description available.

L’homeòstasi cel·lular depèn, en part, de la proteòlisi regulada pel sistema Ubicuitina-Proteasoma (UPS). Aquesta proteòlisi inclou la degradació de les proteïnes defectuoses o innecessàries per la cèl·lula i consta de dues etapes. En un primer pas, molècules d’ubicuitina s’uneixen covalentment a un grup amino (ε-NH2) d’alguna lisina del substrat diana. Aquest procés depèn de l’activitat enzimàtica de tres enzims, E1, E2 i E3. Tot seguit, el proteasoma s’encarrega de la seva degradació. El proteasoma és un complex multiproteic en forma de túnel de 2,5 MDa present en el nucli i el citoplasma de totes les cèl∙lules eucariotes i Archaebacteria. La senyal típica de degradació és una cadena d’ubicuitines que es forma a través d’enllaços isopeptídics entre la glicina G76 a la part C-terminal de la molècula i la lisina K48 de la següent ubicuitina. Avui dia, s’han descrit tres receptors proteasomals de substrats poliubicuitinats, Rpn10, Rpn13 i Rpn15. La subunitat Rpn10 (Rpn10 en llevat de gemmació i S5a en humans), que també es troba en fraccions citosòliques, s’uneix a les cadenes de poliubicuitina mitjançant el domini UIM (Ubiquitin-interacting motif). Aquest domini és essencial per l’ubicuitinació del propi receptor. Els enzims involucrats en tal modificació són Uba1 (E1), Ubc4 (E2) i Rsp5 (E3). La poliubicuitinació de Rpn10 depèn de la participació d’una quarta ubicuitina lligasa, Hul5, i comporta la seva degradació proteasomal. Alhora, s’ha vist que una fracció de Rpn10 està conjugada a una sola molècula d’ubicuitina. Aquest tipus de modificació post-traduccional s’anomena monoubicuitinació. Quan Rpn10 està monoubicuitinat, deixa d’interaccionar amb substrats poliubicuitinats, afectant l’activitat proteolítica del proteasoma. Desxifrar els mecanismes pels quals Rpn10 es monoubicuitina ha estat l’objectiu d’aquesta tesi. La hipòtesi inicial és que la proteïna monoubicuitinada pateix un plegament provocat per l’interacció entre l’ubicuitina unida a Rpn10 i l’UIM. Aquest plegament bloquejaria l’UIM i no permetria l’addició de noves ubicuitines per part de la lligasa Rsp5. Per intentar validar la hipòtesi, hem començat optimitzant la reacció de monoubicuitinació de Rpn10 in vitro i hem trobat la seqüència mínima indispensable per obtenir tal monoubicuitinació. Hem observat que la meitat N-terminal de Rsp5, és dispensable per l’ubicuitinació de Rpn10 i hem vist que l’augment de la monoubicuitinació de Rpn10 causa un defecte en el creixement de les cèl·lules. Finalment, hem aconseguit poliubicuitinar Rpn10 in vitro, sense la participació de Hul5, mitjançant una mutació en bloc de la seqüència precedent l’UIM. Hem vist que aquesta seqüència està intrínsecament desestructurada i que la seva flexibilitat podria ser la raó per la qual l’UIM quedés inaccessible quan Rpn10 està monoubicuitinat. Un coneixement en profunditat del sistema ubicuitina-proteasoma és fonamental per trobar la cura de les patologies humanes derivades de seu mal funcionament. La feina que fem al nostre laboratori és la base per a que en un futur malalties que semblen estar relacionades amb l’UPS -malalties autoinmunes, neurològiques, càncer, cardiopaties...- tinguin un millor tractament. / La homeostasis celular depende, en parte, de la proteólisis regulada por el sistema Ubicuitina-Proteasoma (UPS). Esta proteólisis incluye la degradación de las proteínas defectuosas o innecesarias para la célula y consta de dos etapas. En un primer paso, moléculas de ubicuitina se unen covalentemente al grupo amino (ε-NH2) de alguna lisina del sustrato diana. Este proceso depende de la actividad enzimática de tres enzimas, E1, E2 y E3. Acto seguido, el proteasoma se encarga de su degradación. El proteasoma es un complejo multiproteico en forma de túnel de 2,5 MDa presente en el núcleo y el citoplasma de todas las células eucariotas y Archaebacteria. La señal típica de degradación es una cadena de ubicuitinas que se forma a través de enlaces isopeptídicos entre la glicina G76 en la parte C-terminal de la molécula y la lisina K48 de la siguiente ubicuitina. Hoy día, se han descrito tres receptores proteasomales de sustratos poliubicuitinados, Rpn10, Rpn13 y Rpn15. La subunidad Rpn10 (Rpn10 en levadura de gemación y S5a en humanos), que también se encuentra en las fracciones citosólicas, se une a las cadenas de poliubicuitina mediante el dominio UIM (Ubiquitin-interacting motif). Este dominio es esencial para la ubicuitinación del propio receptor. Las enzimas involucradas en tal modificación son Uba1 (E1), Ubc4 (E2) y Rsp5 (E3). La poliubicuitinación de Rpn10 depende de la participación de una cuarta ubicuitina ligasa, Hul5, y conlleva su degradación proteasomal. Asimismo, se ha visto que una fracción de Rpn10 está conjugada a una única molécula de ubicuitina. Este tipo de modificación post-traduccional se llama monoubicuitinación. Cuando Rpn10 está monoubicuitinada, deja de interaccionar con sustratos poliubicuitinados, afectando la actividad proteolítica del proteasoma. Descifrar los mecanismos por los cuales Rpn10 se monoubicuitina ha sido el objetivo de esta tesis. La hipótesis inicial es que la proteína monoubicuitinada sufre un plegamiento provocado por la interacción entre la ubicuitina unida a Rpn10 y el UIM. Este plegamiento bloquearía el UIM y no permitiría la adición de nuevas ubicuitinas por parte de la ligasa Rsp5. Para intentar validar la hipótesis, hemos empezado optimizando la reacción de monoubicuitinación de Rpn10 in vitro y hemos encontrado la secuencia mínima indispensable para obtener tal monoubicuitinación. Hemos observado que la mitad N-terminal de Rsp5 es dispensable para la ubicuitinación de Rpn10 y hemos visto que el aumento de la monoubicuitinación de Rpn10 causa un defecto en el crecimiento de las células. Finalmente, hemos conseguido poliubicuitinar Rpn10 in vitro, sin la participación de Hul5, mediante una mutación en bloc de la secuencia que precede al UIM. Hemos visto que esta secuencia está intrínsecamente desestructurada y que su flexibilidad podría ser la razón por la que el UIM quedaría inaccesible cuando Rpn10 está monoubicuitinada. Un conocimiento en profundidad del sistema ubicuitina-proteasoma es fundamental para encontrar la cura de las patologías humanas derivadas de su mal funcionamiento. El trabajo que hacemos en nuestro laboratorio es la base para que en un futuro enfermedades que parecen estar relacionadas con el UPS -enfermedades autoinmunes, neurológicas, cáncer, cardiopatías...- tengan un mejor tratamiento. / Cellular homeostasis depends, partially, on the proteolysis regulated by the Ubiquitin-Proteasome System (UPS). This proteolysis includes the degradation of misfolded, damaged or unnecessary proteins for the cell and comprises two phases. In the first stage, molecules of Ubiquitin are attached covalently to a target substrate at the amide group (ε-NH2) of some lysine residues. This process depends on the enzymatic activity of three enzymes, E1, E2 and E3. Afterwards, the proteasome takes care of the degradation of the substrate. The proteasome is a barrel-shaped multiprotein complex of 2,5 MDa present in the nucleus and cytosol of eukaryotic and Archaebacteria cells. The canonical degradation signal is a chain of ubiquitins, which is built via isopeptide bonds between the glycine G76 at the C-terminus of the molecule and lysine K48 of the next Ubiquitin. So far, three Ubiquitin proteasomal receptors, Rpn10, Rpn13 and Rpn15, have been described. The Rpn10 subunit that can also be found in an extraproteasomal pool, binds Ubiquitin chains by means of the Ubiquitin-binding motif (UIM). This domain is also essential for the ubiquitination of Rpn10 itself. The enzymes involved in this modification are Uba1 (E1), Ubc4 (E2) and Rsp5 (E3). Rpn10 polyubiquitination requires an additional Ubiquitin ligase, Hul5, and promotes Rpn10 degradation. However, a fraction of Rpn10 has been shown to be conjugated to only one Ubiquitin molecule, a type of post-translational modification that is called monoubiquitination. When Rpn10 is monoubiquitinated, its capacity to bind polyubiquitinated substrates is impaired, affecting the catalytic activity of the proteasome. The goal of this thesis is to decipher the mechanisms by which Rpn10 is monoubiquitinated. The initial hypothesis was that the monoubiquitinated protein undergoes a change in its conformation caused by the interaction between the Ubiquitin bound to Rpn10 and the UIM. This change would block the UIM and would not allow the conjugation of new ubiquitins by the ligase Rsp5. To validate the hypothesis, we started by optimizing the reaction of monoubiquitination of Rpn10 in vitro and we have determined the minimal sequence of Rpn10 required for this ubiquitination. We have shown that the N-terminus of Rsp5 is not essential for Rpn10 ubiquitination and observed that an increase in the levels of monoubiquitinated Rpn10 causes a slow-growth defect. Finally, we have been able to polyubiquitinate Rpn10 in vitro, independently of Hul5, via a mutation en bloc of the sequence that precedes the UIM. We have seen that this sequence is intrinsically unstructured and that its flexibility could be the reason why the UIM could be blocked when Rpn10 is monoubiquitinated. A detailed knowledge of the Ubiquitin-Proteasome System is essential to find the cure for the pathologies that derive from its malfunction. The work we do in our lab is the base for that, in a future, diseases that seem to be related to the UPS -autoimmune, heart or neurological diseases, cancer,…- have a better treatment.

No description available.

Abstract Molecular oxygen is a prerequisite for essential metabolic processes in multicellular organisms. However, the supply of oxygen can be disturbed and tissue aerobic metabolism becomes compromised in several pathophysiological conditions. In prolonged hypoxia, cells initiate cell type-specific adaptation processes, which are typically mediated by alterations in gene expression. Changes are mainly driven by a transcription factor called hypoxia-inducible factor 1 (HIF-1). Heart muscle is a highly oxidative tissue and HIF-1 activation turns on myocardial adaptation mechanisms for enhanced survival in oxygen-deprived conditions. The aim of this study was to characterize myocardial gene expression changes during chronic hypoxia and couple the adaptational changes to cardiomyocyte function. The role of hypoxia and HIF-1 activation was studied by using in vitro mouse and rat heart cell culture models, tissue perfusions and in vivo infarction models. In this study, apelin, sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) and G protein-coupled receptor 35 (GPR35) were characterized as novel functionally important myocardial HIF-1 target genes. Apelin and GPR35 were induced in hypoxia, while SERCA2a expression was reduced HIF-1 dependently. HIF-1 activation also altered cardiac myocyte contractility through modulation of SERCA2a and GPR35 expression, leading to impairment of the cellular calcium metabolism. Reduced contractility was suggested to serve as an adaptive mechanism for reduced aerobic ATP production in hypoxic conditions. This study presents novel information about the plasticity of myocardial adaptation to prolonged hypoxia. The role of a conserved transcription factor, HIF-1, was shown to be essential in the adaptation process in the myocardial cells. / Tiivistelmä Riittävä hapensaanti on välttämätöntä monisoluisten eliöiden elintoiminnoille. Hapensaanti voi kuitenkin häiriintyä erilaisissa tautitiloissa, jolloin happea käyttävät prosessit estyvät. Hapenpuutteen (hypoksia) pitkittyessä elimistön solut aloittavat sopeutumisen tilanteeseen muuttamalla toimintaansa geenien ilmentymismuutosten kautta. Adaptaatiota ohjaa pääasiassa hypoksia-indusoituva tekijä 1 (HIF-1). Sydän käyttää runsaasti happea energiantuotannossaan. Hapenpuutteen aikana HIF-1-transkriptiotekijä muuttaa sydämen geenien ilmentymistä siten, että sydänsolut selviävät paremmin happivajaissa olosuhteissa. Tämän tutkimuksen tavoitteena oli määrittää sydämen geenien ilmentymisen hapenpuutevasteita ja yhdistää muutokset sydänsolujen toiminnallisiin muutoksiin. Hapenpuutteen ja HIF-1:n merkitystä sopeutumisessa tutkittiin käyttäen malleina rotan ja hiiren sydänsoluviljelmiä, in vitro-kudosperfuusiomalleja sekä in vivo-sydäninfarktimalleja. Tässä työssä havaittiin apeliinin, sarkoplasmisen kalvoston Ca2+-ATPaasin (SERCA2a) sekä G-proteiinikytketyn reseptori 35:n olevan toiminnallisesti tärkeitä HIF-1:n säätelemiä geenejä sydämessä. Apeliinin sekä GPR35:n ilmentyminen lisääntyi hypoksian aikana, mutta SERCA2a:n ilmentyminen sen sijaan väheni HIF-1 –aktivaation seurauksena. HIF-1 –aktivaation osoitettiin myös vähentävän sydänsolujen supistustoimintaa muuttuneiden SERCA2a:n ja GPR35:n ilmentymisten kautta. Heikentynyt supistustoiminta sopeuttaa soluja vähentyneeseen aerobiseen ATP:n tuottoon hapenpuutteen aikana. Tämä tutkimus antaa lisätietoa sydämen sopeutumiskyvyn mukautumisesta pitkittyneeseen hapenpuutteeseen. Lisäksi tutkimus osoittaa HIF-1:n roolin olevan oleellinen myös sydänsolujen hypoksia-adaptaatioprosesseissa.

Proefschr. Rijksuniversiteit Groningen. / Auteursnaam op omslag: Leonie Los. Met lit. opg.-Met samenvatting in het Nederlands.

Proefschrift Universiteit van Amsterdam. / Auteursnaam op omslag: Ilse van Grunsven. Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Proefschrift Universiteit van Amsterdam. / Auteursnaam op omslag: Annette van Kalkeren. Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.