Chlamydien durch die Modulation spezifischer Wirtszellproteine verschiedene Wege der Apoptose verhindern können. Mcl-1 und cIAP-2 erwiesen sich als bedeutende Faktoren, die durch die Infektion hochreguliert und absolut notwendig für die Inhibierung der Apoptose durch Chlamydien waren. Hochregulation der Mcl-1 Expression führte zu einem Block im apoptotischen Weg oberhalb der Mitochondrien. cIAP-2 zusammen mit anderen Inhibitor of Apoptosis Proteins (IAP) verhinderten die Aktivierung von Caspase-3, denfinalen Schritt in der apoptotischen Kaskade. Weiterhin wurde beobachtet, dass die Aktivierung des MAPKinase-Signalweges durch die Infektion wichtig war für die Hochregulierung von Mcl-1 und cIAP-2. Ein Hochdurchsatz-Screen wurde durchgeführt, um andere Wirtszellfaktoren, die für die Apoptoseinhibierung durch die Chlamydien verantwortlich sind, zu identifizieren. Neben Mcl-1 waren die identifizierten Faktoren hauptsächlich Mitglieder des MAPKinase-Signalweges. Dabei wurde deren Rolle für die Apoptoseresistenz bestätigt. Eine weiterführende Analyse der im Screen ermittelten Faktoren identifizierte eine Funktion von HIF-1a bei der Modulation der Expression anti-apoptotischer Faktoren während der Infektion. Es wurde beobachtet, dass HIF-1a stabilisiert und zum Nukleus transloziert wird. Es ist bekannt, dass HIF-1a HIF-1a im Nukleus binden kann, um den funktionalen Transkriptionsfaktor HIF zu bilden. Dieser reguliert die Expression verschiedener Überlebensfaktoren, unter anderem Mcl-1. HIF-1a Knockdown inhibierte die Chlamydien-induzierte Hochregulation von Mcl-1 mRNA-Expression. / chlamydial infection blocked the apoptotic pathway at multiple levels by modulation of specific host cell proteins. Mcl-1 and cIAP-2 were two most prominent factors that were up-regulated during the infection, and absolutely required for apoptosis inhibition. Increased expression of Mcl-1 led to a block in the apoptotic pathway upstream of the mitochondria. cIAP-2, together with other inhibitor of apoptosis proteins (IAPs), blocked the activation of caspase-3 at the final step of the apoptosis cascade. Further, it was observed that the activation of the MAPK pathways during infection was needed for the up-regulation of Mcl-1 and cIAP-2. A high throughput RNAi screen was performed to identify other host factors required for the apoptosis resistance during the infection. Besides Mcl-1, the targets from the screen prominently included members of the MAPK pathways, confirming their role in the apoptosis resistance. Pathway analysis of the targets identified the role of HIF-1a in modulating the expression of the anti-apoptotic factors during infection. It was observed that during infection, HIF-1a gets stabilized and translocates to the nucleus. It is known that HIF-1a can bind to HIF-1a in the nucleus to form the functional transcription factor HIF, which can regulate the expression of survival factors like Mcl-1. This was seen to be the case, because knock down of HIF-1a abrogated the infection induced up-regulation of Mcl-1 at the mRNA levels.

CIAP1 (cellular Inhibitor of Apoptosis 1) possède une activité E3-ubiquitine ligase et présente des propriétés oncogéniques. Récemment, notre équipe a montré que cIAP1 pouvait réguler l’activité du facteur de transcription E2F1. L’objectif de mon travail de thèse était d’approfondir les mécanismes de cette régulation et d’évaluer l’importance de la coopération cIAP1-E2F1 dans l’activité oncogénique de cIAP1. J’ai démontré une interaction d’E2F1 avec la poche hydrophobe du domaine BIR3 de cIAP1. J’ai par ailleurs démontré l’importance de la première hélice α de ce domaine pour l’interaction de cIAP1 avec E2F1 et avec les autres protéines partenaires de cIAP1 capables de lier la poche hydrophobe du domaine BIR3. De plus, j’ai participé au travail montrant pour la première fois une régulation d’E2F1 par une ubiquitinylation non dégradative. cIAP1 permet la conjugaison de chaînes d’ubiquitines de type K63 sur les lysines 161 et 164 d’E2F1. Cette modification post-traductionnelle est indispensable à la stabilisation de la protéine lors d’un stress génotoxique et elle permet le recrutement du facteur de transcription sur les promoteurs des gènes cibles. Enfin, l’analyse des propriétés oncogéniques de cIAP1 n’ont pas permis, à ce jour, d’évaluer l’importance de la coopération cIAP1-E2F1. Cependant, nous avons montré l’importance du domaine BIR1 pour les propriétés oncogéniques de cIAP1 (domaine nécessaire à l’interaction de cIAP1 avec l’adaptateur moléculaire TRAF2). / The cellular inhibitor of Apoptosis 1 (cIAP1) behaves as an E3 ubiquitin ligase and has oncogenic properties. Previously, our team has shown that cIAP1 can regulate the E2F1 transcription factor activity. My research project has been focused on deepening our current knowledge on this interaction. Firstly, we characterized the E2F1-cIAP1 interaction, then we analyzed the regulation of E2F1 by cIAP1 and finally assessed the importance of the cIAP1-E2F1 interaction for the oncogenic properties of cIAP1. I have demonstrated a interaction of E2F1 with the hydrophobic pocket of the BIR3 domain of cIAP1. Moreover, I highlighted that the alpha 1 helix of the BIR3 domain is mandatory for the stability of this pocket. Moreover, we discovered an ubiquitination on lysine 161 and 164 of E2F1 by cIAP1. This ubiquitination is essential for the stability and transcriptional activity of E2F1. Finally, it appears that the cIAP1 BIR1 domain that is required for the interaction with TRAF2 is involved in its oncogenic properties.

<p>Multiple myeloma (MM) is a hematological tumor of plasma blast/plasma cell origin heterogeneous with respect to the morphological differentiation stage of the tumor cells, genetic alterations and course of disease. A challenge in MM research is to overcome resistance to therapy, which inevitably arises. In this thesis, we have used different strategies to sensitize MM cells to apoptosis and explored possible mechanisms of apoptotic control by the insulin-like growth factor-1 receptor (IGF-1R) survival pathway.</p><p>mTOR is a key molecule in the regulation of translation activated by survival signaling pathways in MM. We demonstrate that the mTOR-inhibitor rapamycin alone induced apoptosis in primary MM cells. In addition, rapamycin sensitized MM cells to apoptosis induced by dexamethasone, a glucocorticoid frequently used in MM therapy. MM survival factors IGF-1 and IL-6 could neither restore phosphorylation of the mTOR target p70S6K, nor cell growth inhibited by rapamycin and dexamethasone.</p><p>To study the regulation of inhibitors of apoptosis (IAP), we induced apoptosis and cell cycle arrest with dexamethasone and simultaneously abrogated IGF-1R signaling using the antagonistic antibody αIR3 or the selective IGF-1R inhibitor picropodophyllin (PPP). Dexamethasone transiently up-regulated c-IAP2. The subsequent down-regulation of c-IAP2 and XIAP was associated with the onset of apoptosis. c-IAP2 and XIAP levels further decreased when enhancing dexamethasone-induced apoptosis using αIR3 or PPP indicating a role for IAPs in regulating resistance to apoptosis in MM.</p><p>Finally, we explored glycogen synthase kinase (GSK)3 as a possible pro-apoptotic molecule and its role in regulating sensitization to apoptosis. We show that inhibition of GSK3 counteracts growth inhibition induced by dexamethasone alone and in combinatorial treatments with inhibitors against PI 3-kinase, mitogen-activated protein kinase (MEK), mTOR and IGF-1R. CT99021 also reversed cell cycle arrest induced by LY294002 or rapamycin. Importantly, the GSK3 inhibitor CT99021 sustained viability in untreated and dexamethasone-treated primary MM cells.</p>

Multiple myeloma (MM) is a hematological tumor of plasma blast/plasma cell origin heterogeneous with respect to the morphological differentiation stage of the tumor cells, genetic alterations and course of disease. A challenge in MM research is to overcome resistance to therapy, which inevitably arises. In this thesis, we have used different strategies to sensitize MM cells to apoptosis and explored possible mechanisms of apoptotic control by the insulin-like growth factor-1 receptor (IGF-1R) survival pathway. mTOR is a key molecule in the regulation of translation activated by survival signaling pathways in MM. We demonstrate that the mTOR-inhibitor rapamycin alone induced apoptosis in primary MM cells. In addition, rapamycin sensitized MM cells to apoptosis induced by dexamethasone, a glucocorticoid frequently used in MM therapy. MM survival factors IGF-1 and IL-6 could neither restore phosphorylation of the mTOR target p70S6K, nor cell growth inhibited by rapamycin and dexamethasone. To study the regulation of inhibitors of apoptosis (IAP), we induced apoptosis and cell cycle arrest with dexamethasone and simultaneously abrogated IGF-1R signaling using the antagonistic antibody αIR3 or the selective IGF-1R inhibitor picropodophyllin (PPP). Dexamethasone transiently up-regulated c-IAP2. The subsequent down-regulation of c-IAP2 and XIAP was associated with the onset of apoptosis. c-IAP2 and XIAP levels further decreased when enhancing dexamethasone-induced apoptosis using αIR3 or PPP indicating a role for IAPs in regulating resistance to apoptosis in MM. Finally, we explored glycogen synthase kinase (GSK)3 as a possible pro-apoptotic molecule and its role in regulating sensitization to apoptosis. We show that inhibition of GSK3 counteracts growth inhibition induced by dexamethasone alone and in combinatorial treatments with inhibitors against PI 3-kinase, mitogen-activated protein kinase (MEK), mTOR and IGF-1R. CT99021 also reversed cell cycle arrest induced by LY294002 or rapamycin. Importantly, the GSK3 inhibitor CT99021 sustained viability in untreated and dexamethasone-treated primary MM cells.

This thesis is focused on multiple myeloma (MM), a haematological malignancy that still remains incurable. The pathogenesis of MM is not fully understood and there is a large intra-tumour and interclonal genetic variation in MM patients. One of the most challenging areas in MM research is to find mechanisms for initiation and progression of MM, but also to overcome the arising resistance to therapy. In paper I, a signature of under-expressed genes in MM was found to significantly correlate with already defined Polycomb target genes. In selected genes from the profile we found an enrichment of H3K27me3, a repressive mark catalysed by Polycomb repressive complex 2 (PRC2), in MM patients and MM cell lines. Treatment with LBH589 (HDAC inhibitor) and DZNep (methyltransferase inhibitor) reactivated the H3K27me3 target genes and induced apoptosis in MM cell lines. LBH589 reduced tumour load and increased overall survival in the 5T33MM mice. These results suggest an important role for Polycomb complex in MM development and highlight PRC2 as a drug target in MM. In paper II, the insulin-like growth factor type 1 receptor tyrosine kinase (IGF-1RTK) inhibitor picropodophyllin (PPP) in combination with LBH589 synergistically inhibited cell proliferation and enhanced the apoptotic effect in MM. Since the bone marrow microenvironment has an important role in MM disease and also contributes to drug-resistance, we therefore evaluated the drug combination in the immunocompetent 5T33MM murine model. The drug combination significantly prolonged the survival of the 5T33MM mice compared to single drug treatment. We conclude that the combination of PPP and LBH589 has a therapeutic potential in MM. In paper III, the role of the cellular inhibitor of apoptosis protein 2 (cIAP2) was evaluated in MM cells harbouring TRAF3 deletion/mutation. By overexpressing cIAP2 in these cells we found an increased resistance to proteasome inhibitors. cIAP2 over-expression by lentiviral constructs led to decreased caspase activation, activation of the canonical NF-κB pathway, and down-regulation of tumour suppressor genes and genes that contribute to apoptosis. Supporting the role of cIAP2 mediated drug-resistance, we here demonstrate that inhibiting cIAP2 using an IAP antagonist, increased the sensitivity to the proteasome inhibitor, bortezomib.

Apoptosis represents one of the cell death mechanisms which is realized after the application of taxanes in breast cancer cell lines. Apoptosis induction can be principally triggered either by outer or inner pathway. The aim of the diploma thesis is to contribute to the elucidation of role and mechanisms of the inner mitochondrial pathway of apoptosis induction after taxane application (paclitaxel and SB-T-1216) employing a model of breast carcinoma cell lines SK- BR-3 (nonfunctional p53, functional capase-3) and MCF-7 (functional p53, nonfunctional caspase-3). Specifically, we tested the effect of both employed taxanes on mitochondrial membrane potential, ROS level and the expression and localization of proteins regulating inner mitochondrial pathway. Taxane application resulted in mitochondrial membrane dissipation in SK-BR-3 cell line. However, this was not shown in MCF-7 cell line. We found no changes in Bax and Smac/DIABLO expression after taxane application in both tested cell lines. There was a decrease of Bid expression after taxane application in SK-BR-3 line, but not in MCF-7 line. Taxane application did not lead to the translocation of Bax and Bid (tBid) proteins from cytosol to mitochondria in both tested cell lines. Similarly, there was no Smac/DIABLO release from mitochondria to...

In patients affected by acute respiratory failure or acute respiratory distress syndrome (ARDS) the leading cause of death is failure of different vital organs other than the lungs, so called multiple organ dysfunction syndrome (MODS). The abdominal organs have a crucial role in the pathogenesis of this syndrome. There is a lack of knowledge regarding the mechanisms by which mechanical ventilation can affect the abdominal compartment. One hypothesis is that mechanical ventilation can interfere with abdominal fluid balance causing edema and inflammation. We addressed the question whether different levels of ventilatory support (mechanical ventilation with different levels of positive end-expiratory pressure, PEEP, and spontaneous breathing with or without PEEP) can influence abdominal edema and inflammation in both healthy and endotoxin-exposed animals. The effect on lymphatic drainage from the abdomen exerted by different degrees of ventilatory support was evaluated (paper I). We demonstrated that endotoxin increases abdominal lymph production, that PEEP and mechanical ventilation increase lymph production but also impede lymphatic drainage; spontaneous breathing improves lymphatic drainage from the abdomen. By adapting a non-invasive nuclear medicine imaging technique and validating it (paper II), we have been able to evaluate extravascular fluid accumulation (edema formation) in the abdomen over time (paper III) demonstrating that edema increases during endotoxemia, mimicking a sepsis-like condition, and that spontaneous breathing, compared to mechanical ventilation, reduces extravascular fluid. Pro-inflammatory cytokines TNF-α and IL-6 in intestinal biopsies are reduced during spontaneous breathing compared to mechanical ventilation. Abdominal edema results in increased intra-abdominal pressure (IAP): in paper IV we analyzed the effect of increased intra-abdominal pressure on the respiratory system. Pulmonary shunt fraction increased with high IAP both in healthy and LPS animals, resulting in decreased level of oxygenation. These changes are only partially reversible by reducing IAP. In conclusion, mechanical ventilation is a life-saving tool but the possible side effect at the extra-pulmonary level should be considered, and the introduction of some degree of spontaneous breathing when clinically possible is a suggested choice.

All trunk muscles are important contributors to spine stability. However, the deepest abdominal muscle, transversus abdominis (TrA), with its characteristically horizontal fibre orientation seems to serve a unique function in trunk motor control. The main mechanical role of TrA is believed to be to contribute to vertebral alignment during imposed moments on the trunk, executed mainly via either regulating the pressure level within the abdominal cavity and/or transmit forces to the spine via the thoracolumbar fascia. However, the complete function of TrA and what factors affect its activation are still not fully understood. The purpose of the present thesis was to investigate the role of TrA in trunk motor control, specifically in relation to the presence or absence of postural demand on the trunk. The timing and magnitude of TrA activation were investigated, in relation to other trunk muscles, with intramuscular fine-wire electrodes in different loading situations and body positions with varying postural demand. In a side-lying position, with no postural demand of keeping the trunk upright, the activation of TrA was delayed relative the superficial abdominal muscles compared to previous experiments performed in a standing position. The timing and magnitude of activation of TrA did not depend upon the direction of perturbation. In the standing position, different static arm positions revealed that the activation of TrA co-varied with variations in the degree of postural demand on the trunk and also the imposed moments, regardless of moment direction. Finally, a study on rapid arm flexion movements confirmed that TrA is part of the pre-programmed anticipatory response in advance of known perturbations. The activation magnitude of TrA was the same regardless if the arm movement induced flexion or extension  moments on the trunk. In conclusion, the activation of TrA is associated with the upright postural demand on the trunk and with balancing imposed moments acting on the spine, regardless their direction. The findings are in support of the beliefs that TrA act as a general, direction non specific, stabilizer of the lumbar spine.

A bactéria Listeria monocytogenes é reconhecida como um importante patógeno humano estando amplamente distribuída no ambiente e é responsável pela contaminação de alimentos crus e processados. O mecanismo de patogenicidade é determinado pela presença de genes no cromossomo da bactéria e entre eles estão os genes iap e hly que são essenciais para o mecanismo de invasão e atividade hemolítica do microorganismo, respectivamente. O obejtivo do presente estudo foi confirmar as cepas de L. monocytogenes usando a ténica de PCR para o gene hly e análise da variação nucleotídica do domínio central do gene iap, que é caracterizado pela presença de seqüências repetidas dos aminoácidos treonina e asparagina. Vinte e seis cepas de L. mnocytogenes, previamente isoladas de produtos lácteos e identificdas por métodos clássicos, se mostraram positivas para a PCR espécie-específico e então submetidas à determinação da seqüência nucleotídica. Os resultados mostraram variações na seqüência nucleotídica contendo substituições, inserções, deleções e um número de seqüências similares entre as cepas isoladas e a cepa controle EGD-e. Vinte e três cepas exibiram a mesma deleção que compreende 24 pares de bases dentro da seqüência de repetição e alterações similares na proteína traduzida. Apenas três cepas mostraram tamanhos diferentes de deleções e diferentes alterações na proteína. De acordo com estes resultados, a maioria das cepas apresentou uma característica molecular comum, diferentes da cepa padrão e este perfil predominante pode ser considerado como a característica das L. monocytogenes isoladas de produtos lácteos no Sul do Brasil. / The bacterium Listeria monocytogenes is recognized as an important human pathogen being omnipresent in the environment and is responsible for contamination in raw and processed foods. The mechanism of pathogenicity is established by presence of some genes on chromosome of bacteria between them, iap and hly genes that are essential to the invasion mechanism and hemolytic activity of microorganism, respectively. The aim of present study was confirm the strain L. monocytogenes using PCR to hly gene and analysis the nucleotide variability of central domain of iap gene characterized by the presence of similar sequences of threonine and asparagine amino acids. Twenty-six strains previously isolated from dairy products and classified by classic methods to L. monocytogenes were positive to specie-specific PCR and than submitted to nucleotide sequence determination. The results showed a sequence variation containing nucleotide substitutions, insertions, deletions and a number of repeated sequences among the isolates and control strain EGD-e. Twenty-three strains exhibited the same gap that includes a deletion of 24 base pairs inside of the repeated sequence and similar alterations in the translated protein. Just three strains showed different sizes of gaps and different protein alterations. According to these results, the majority strains displayed a common molecular characteristic different from the strain pattern and this predominant profile can be considerate as characteristic to L. monocytogenes isolated from dairy products in South Brazil.

<p>Multiple myeloma (MM) is a virtually incurable B cell malignancy of the bone marrow. One important part of tumor progression and an obstacle for successful therapy is resistance to apoptosis. To combat this resistance, the mechanisms of apoptosis and survival in MM must be better defined. </p><p>In this thesis, we identified Fas up-regulation as a mechanism underlying interferon (IFN)-mediated sensitization to Fas-induced apoptosis in the MM cell line U-266-1970. IFN treatment induced activation of signal transducer and activator of transcription (Stat)1 but, intriguingly, also attenuated activation of MM survival factor Stat3. </p><p>Exploring the role of Stat1 further, we established sub-lines of U-266-1970 with a stable over-expression of Stat1 and of its active mutant Stat1C. These sub-lines displayed a decreased expression and activation of Stat3, and an altered expression of apoptosis-related genes Harakiri, Bcl-2 and Mcl-1. In a drug library screening, Stat1 over-expression was associated with an increased sensitivity to Fas-induced apoptosis and, conversely, an increased resistance to several drugs, including the cyclin dependent kinase (cdk)1 inhibitor CGP74514A. We conclude that Stat1 over-expression does not confer a general resistance or sensitivity to apoptosis in MM, but may strongly affect the response to some specific drugs.</p><p>We also explored the effects of picropodophyllin (PPP), an inhibitor of the insulin-like growth factor I (IGF-I) receptor tyrosine kinase (RTK), in MM. PPP selectively inhibited the IGF-I RTK activity without inhibiting the insulin RTK activity. Furthermore, PPP potently induced cell cycle arrest and apoptosis in all MM cell lines and patient samples tested, also in the presence of survival factors IGF-I and IL-6. We conclude that PPP has great therapeutic potential in MM </p><p>Finally, we examined the expression and regulation of the inhibitors of apoptosis proteins (IAPs) in a panel of MM cell lines and patient samples. The glucocorticoid dexamethasone, which is used in MM therapy, induced a transient up-regulation and a subsequent down-regulation of c-IAP2, as well as a down-regulation of XIAP, possibly influencing the sensitivity to apoptosis induced by this drug. Supporting this notion, abrogation of IGF-IR signaling by PPP, which sensitizes MM cells to dexamethasone-induced apoptosis, enhanced the down-regulation of c-IAP2 and XIAP.</p>