Proefschrift Universiteit van Amsterdam. / Met lit. opg. - Met samenvatting in het Nederlands en Portugees.

Dengue fever is an arthropod-borne transmit disease, caused by dengue virus.The principal vectors are Aedes aegypti and Aedes albopictus. Induce Dengue fever (DF) and a more severe form of dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS). Dengue virus is the most prevalent arbovirus in tropical and subtropical regions. There is no specific drug and vaccine available for treatment and prevention. Therefore, DF is an important disease among transmit diseases in humans. For the effective control and prevention of DF transmission, rapid quantitative molecular biological methods are very important for the diagnosis of dengue fever. At present, there are many methods to isolate the RNA of Dengue virus; however, the new developed magnetic method has not been used for the Dengue virus isolation yet. At first, we evaluated various methods for Dengue virus isolation. The result indicate that the best method of RNA extraction for dengue virus is the QIAamp® Viral RNA kit manual extraction. There are no apparent differences of the effect for Dengue virus RNA isolation between filting film and magnetic bead method. Furthermore, DHF caused by Dengue virus is a very serious disease and the pathologic mechanism of DHF has not been elucidated completely. Both clinical and experimental trials have confirmed that the liver cell is one of the target infected by Dengue virus. And, the mechanism of Dengue virus-induced liver cell apoptosis remains poorly understood.Furthermore, there are free radical and cytokines production in patient¡As serum in the acute phase of DF. Therefore, the role of antioxidant and p21 in the mechanisms should be elucidcited. Our preliminary data show that p21 mRNA expression increase in HepG2 after Dengue virus infection. NAC, GSH, and DPI all can attenuate Dengue-induced cell apoptosis. Howerer, the relationship between p21 expression and liver cell apoptosis should be further clarified in the near future.

Platelets are involved in many processes ranging from fighting microbial infections and triggering inflammation to promoting tumor angiogenesis and metastasis. Nevertheless, the primary physiological function of platelets is to act as essential mediators in maintaining homeostasis of the circulatory system by forming hemostatic thrombi that prevent blood loss and maintain vascular integrity. The p21-activated kinases (PAKs) are a family of serine/threonine kinases known to be the downstream effectors of GTPases, Cdc42 and Rac1. PAKs are the key regulators of actin polymerization and have been shown to play an important role in platelet spreading and aggregation in thrombin-stimulated platelets. Whereas several signaling cascades downstream of heterotrimeric G proteins that regulate platelet functions have been characterized, little attention is paid towards the signaling cascades that involve small G proteins effectors such as PAK. A few studies have characterized the role of PAK, downstream of the Rho family of small G proteins, in outside-in signaling, but its role in the regulation of platelet functional responses by inside-out signaling events have not been elucidated. PAK is reported to interact with numerous proteins including Akt, PDK1 and PI3-kinase in different cell lines. PAK’s function as a scaffolding protein expands the role of this protein in cellular functions. Although PAK is known to have non-catalytic scaffolding functions and is shown to associate and translocate Akt in other cell systems, the catalytic and possible non-catalytic scaffolding role in platelet functions are not clearly defined. In this dissertation we propose to elucidate the scaffolding function of PAK and also its role platelet functional responses using molecular genetics approach. Akt is an important signaling molecule regulating platelet aggregation. Akt is phosphorylated upon translocation to the membrane through Gi signaling pathways by a PIP3-dependent mechanism. However, Akt is more robustly phosphorylated by thrombin compared to ADP in platelets. In this study, we investigated the mechanisms of Akt translocation as a possible explanation for this difference. Stimulation of washed human platelets with protease-activated receptor (PAR) agonists caused rapid translocation of Akt to the membrane, whereas Akt phosphorylation occurred later. The translocation of Akt was abolished in the presence of a Gq-selective inhibitor or in Gq-deficient murine platelets, indicating that Akt translocation is regulated downstream of Gq signaling pathways. Interestingly, PI3-kinase inhibitors or P2Y12 antagonist abolished Akt phosphorylation without affecting Akt translocation to the membrane, suggesting that Akt translocation occurs through a PI3-kinase/PIP3/ Gi-independent mechanism. An Akt scaffolding protein, PAK, translocates to the membrane upon stimulation with PAR agonists in a Gq-dependent manner with the kinetics of translocation similar to that of Akt. Co-immunoprecipitation studies showed constitutive association of PAK and Akt, suggesting a role of PAK in Akt translocation. These results show for the first time an important role of the Gq signaling pathway in mediating Akt translocation to the membrane in a novel Gi/PI3-kinase/PIP3-independent mechanism. PAK contains an autoinhibitory domain that suppresses the catalytic activity of its kinase domain. This autoregulatory domain found within PAK kinase provides a unique target for chemical inhibitors. IPA3, a small molecule allosteric inhibitor of PAK activation, binds covalently to the PAK regulatory domain and prevents binding to its upstream activators. IPA3 has been used in various cells, including platelets, to evaluate the role of PAK in signaling. Herein, we investigated the specificity and selectivity of IPA3 as a PAK inhibitor in the human platelets. Stimulation of platelets pretreated with IPA3 using a PAR-4 or GPV1 agonist resulted in a concentration dependent inhibition of aggregation, as was suggested by earlier studies. Interestingly, we found that incubation of washed human platelets with IPA3 leads to a non-specific increase in phosphorylation of several proteins in absence of any agonist. However, this phosphorylation is not sufficient for aggregation of platelets by IPA3. In summary, we demonstrate that IPA3 by itself can phosphorylate several proteins in human platelets and thus its use is not an appropriate strategy for investigating PAK function in platelets. PAKs are classified into two groups based on their structural differences. Human platelets have been shown to express both group I (PAK1, PAK2, and PAK3) and group II PAKs (PAK4). Previous studies showing the role of PAK were performed with nonspecific inhibitors of PAK, such as IPA3, that do not distinguish isoforms. Thus, we propose to evaluate the function of specific PAK isoforms in platelets using knockout murine platelets, which are more selective tools to study the role of individual isoforms of PAK. We observed that Pak2 null mice showed enhanced secretion responses upon stimulation with 2MeS ADP and collagen, and delayed clot retraction. Interestingly, Pak1 null murine platelets did not have any functional defects, suggesting redundancy with other PAK isoforms. The studies proposed in my thesis will provide further insights into the molecular mechanisms of platelet activation and hence provide a basis for development of PAK as novel antithrombotic therapeutic targets. Furthermore, PAK inhibitors are currently being developed by pharmaceutical companies to treat malignancies, although this enzyme is ubiquitously expressed in the body. A thorough understanding of the role of PAK in platelets can predict the effect of these drugs on hemostatic functions, which helps during clinical trials. In the future, targeted inhibition of signaling molecules in platelets could be developed and that would solely target platelet signaling pathways. / Organ Systems & Translational Medicine

Telomere attrition is a natural process that occurs due to inadequate telomere maintenance. Once at a critically short threshold, telomeres signal the cell to cease division and enter a cell fate termed senescence. Telomeres can be elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Mutations in the telomerase complex or telomere-related genes give rise to the premature aging disorder Dyskeratosis Congenita (DC). DC provides a unique model system to study human aging in relation to telomerase insufficiency and the subsequent accelerated telomere attrition. In this thesis, skin fibroblasts as well as keratinocytes and T-cells were analyzed from patients with Autosomal Dominant Dyskeratosis Congenita (AD DC) caused by a single allele mutation in the telomerase RNA component (TERC) that leads to telomerase haploinsufficiency. These cells were determined to have a severe proliferative defect and extremely short telomeres. It is demonstrated that the short telomeres in AD DC cells initiate a DNA damage response transduced by the p53/p21WAF/CIP pathway which mediate an elevation in steady-state levels of mitochondrially-derived superoxide and oxidative stress. Exogenous expression of the catalytic reverse transcriptase component of telomerase (TERT) activated telomerase in DC fibroblasts but resulted in reduced activity (~50% compared to control fibroblasts); however telomeres were successfully maintained, albeit at a short length. Simultaneous expression of both TERT and TERC led to robust telomerase activity and elongation of telomeres, indicating that TERC haploinsufficiency is a rate-limiting step in telomere maintenance in DC cells. Reconstitution of telomerase activity in AD DC cells ameliorated the proliferative defects, reduced the p53/p21WAF/CIP response and decreased oxidative stress. Increased superoxide and slow proliferation found in DC cells could also be mitigated by inhibiting p21WAF/CIP or by decreasing the oxygen tension to which the cells are exposed. Together, these results support the hypothesis that the insufficient telomerase leads to critically short telomeres which signal the activation of p21WAF/CIP, leading to increased steady-state levels of mitochondrial superoxide and metabolic oxidative stress as a means to engage senescence. These studies provide insight into mechanisms whereby shortened telomeres lead to premature aging in a humans and point to potential strategies to reduce the effects of tissue dysfunction in DC patients.

Head and neck cancer is diagnosed in more than 50,000 Americans each year, resulting in roughly 11,000 deaths. For this disease, a typical therapeutic regimen involves cisplatin, a radiosensitizer, given alongside targeted irradiation. While technological advances such as IMRT have been useful in sparing normal tissues from radiotherapy, the salivary glands occupy much of the head and neck and surround several lymph nodes, and thus, non-diseased salivary glands are often damaged. This causes reduced salivary output, damaged oral mucosa, dysphagia, malnutrition and tooth decay. Often, these side-effects are so severe that patients discontinue treatment, however, in many cases, salivary gland damage is permanent, and treatment options are palliative. Specifically, muscarinic-cholinergic agonists are used to enhance secretion from remaining salivary cells, although due to non-specific action, these drugs have a number of ill-effects. It is clear that therapies are needed to prevent radiation-induced salivary gland damage, as well as to restore glandular function in patients who are already suffering.Previous work from our group has shown that salivary gland dysfunction results from loss of acinar cells to radiation-induced apoptosis. Importantly, a single intravenous dose of IGF1 can prevent apoptosis and preserve salivary output when given immediately prior to irradiation. Because of its broad effects, however, IGF1 may never be a viable clinical option. Instead, our goal is to identify signaling events that mediate the radioprotective effects of IGF1 downstream of Akt. Because radiation-induced apoptosis in salivary glands is p53-dependent, we assessed the contributions of the p53 homologs p63 and p73 to the DNA damage response. Here, we show that IGF1 enhances cell cycle arrest following irradiation by reducing inhibitory binding of deltaNp63 to the p21 promoter. We hypothesize that IGF1-induced cell cycle arrest may allow time for DNA repair, thus preventing apoptosis and maintaining salivary function. In addition, we indicate chronic signaling events downstream of p63 that may contribute to permanent loss of salivary function by blocking differentiation of salivary progenitor cells. Together, these results indicate that p63 may be a valid therapeutic target for both prevention of damage and restoration of function in irradiated salivary glands.

TBX2, a member of the T-box family of transcription factors, plays important roles in embryonic development. Aberrant expression of TBX2 is observed in many cancers, and serves as an oncogene to maintain tumor cell proliferative and malignant properties. We found that TBX2 was expressed in both embryonic myoblasts and adult proliferative satellite cells, but was quickly down regulated during muscle differentiation in mouse models, which suggests an important function of TBX2 in the early myogenesis. Using molecular and cellular biology approaches we showed that TBX2 forms complex with myogenin and MyoD, and then recruits HDAC1 to muscle-specific promoters to repress the myogenin and MyoD dependent differentiation of myoblasts. In rhabdomyosarcoma (RMS), which is typically referred to as a muscle derived cancer, we found TBX2 was over expressed in both major subtypes of RMS. The deregulated TBX2 repressed the expression of cell cycle regulators, such as p21 and p14/p19, and the tumor suppressor PTEN in RMS tumor cells. Knock down of TBX2 significantly decreased the proliferation rate of RMS cells. We also found that loss of TBX2 significantly inhibited tumorigenesis of RMS cells by decreasing cell proliferation, mobility, migration, anchorage-independent growth and xenograft formation. To determine why TBX2 was deregulated in RMS cells, we performed cellular biological experiments to understand how TBX2 is regulated by cell signaling pathways and growth factors in both normal muscle myoblasts and RMS tumor cells. In normal murine myoblasts and primary murine ARMS tumor cells TBX2 was up regulated by FGF-2 treatment, but in primary murine ERMS cells TBX2 expression showed no response to FGF-2 stimulation. In human RMS cell lines a modest up regulation of TBX2 was detected by treatment of FGF-2. RMS cells constitutively express PAX3 and PAX7 which are expressed and function in myogenic precursors, but are quickly degraded in myoblasts and during myogenesis. We found that TBX2 was a downstream target of PAX3 in RMS cells, as well as the ARMS specific fusion proteins PAX3/7-FOXO1. Our novel findings on TBX2 highlight the significant roles of TBX2 in muscle development and adult muscle regeneration, where TBX2 represses MRF activities to inhibit myogenic differentiation and promote proliferation of myoblasts. Also, our work establishes essential oncogene effects of TBX2 in driving and maintaining RMS proliferation and tumorigenesis by repressing cell cycle regulatory factors, p21 and p19/p14, and tumor suppressor of PTEN. Therefore, this work provides an exciting opportunity for development of new therapeutic treatments for TBX2 driven RMS cancer.

The extracellular signal-regulated protein kinase 5 (ERK5), also known as big mitogen-activated protein (MAP) kinase 1 (BMK1), is a non-redundant mitogen-activated protein kinase (MAPK) implicated in mediating the response of cells to mitogens, oxidative and osmotic stresses. The molecular complexity of the ERK5 cascade has been mostly delineated by over-expression studies. For example, like other MAPKs, ERK5 activity increases upon phosphorylation by a MAPK/ERK kinase, namely MEK5. However, the physiological role of ERK5 is not rigorously established by these data. Furthermore, in comparison to the other members of the family, little is known about the downstream targets of ERK5. This constitutes an obstacle for the molecular understanding of the signalling mechanisms that account for the effect of ERK5 activation in vivo. To clarify these issues, I have tested the effect of the conditional loss of ERK5 in primary mouse embryonic fibroblasts (MEFs). My results indicate that ERK5 is required for the proliferation of MEFs, at least in part, by promoting the entry into S phase of the cell cycle. ERK5 suppressed the expression of the cyclin-dependent protein kinase (CDK) inhibitors, p21 and p27. As a result, low-level CDK2 activity detected in ERK5-deficient MEFs correlated with hypo-phosphorylation of the retinoblastoma (Rb) protein and with a defect in G1 to S phase transition of the cell cycle. ERK5 blocks p21 expression by decreasing the stability of the p21 transcript. This process might, at least partially, involve a mechanism implicating c-Myc-induced transcriptional up-regulation of the miR-17-92 cluster. Concerning p27, ERK5 decreases p27 protein stability. The stabilisation of p27 in the absence of ERK5 resulted in the accumulation of the protein in the nucleus. To examine the relevance of my findings in cancer, I tested the effect of pharmacological inhibition of ERK5 in two human breast cancer cell lines, MCF7 and MDA- MB-231, using XMD8-92, a novel potent and selective inhibitor of ERK5. My results show that these cells are dependent on ERK5 to proliferate. Furthermore, I found that incubation of MDA- MB-231 cells with XMD8-92 compromised their ability to invade. In both breast cancer cell lines, ERK5 down-regulates p21 and p27 expression. Together with evidence that cancer patients with poor prognosis display a high-level of expression of components of the ERK5 signalling pathway, these findings support the hypothesis that ERK5 can be a potential target for cancer therapy.

Orientador: Cleslei Fernando Zanelli / Resumo: eIF5A é um fator de elongação da tradução conservado em arqueias e eucariotos, que interage com a subunidade maior do ribossomo 80S. É a única proteína que possui o aminoácido hipusina, formado pós-traducionalmente, essencial para sua função. eIF5A auxilia na tradução de proteínas que contêm motifs ricos em prolina, pois sequência repetida deste aminoácido pode causar parada do ribossomo em tradução devido à rigidez estrutural que forma. Na ausência de eIF5A funcional ocorre parada do ciclo celular em G1, na transição de G1 para S, tanto em Saccharomyces cerevisiae quanto em mamíferos, sugerindo papel essencial desta na tradução de proteínas importantes para a progressão do ciclo celular. Neste trabalho foi proposta a identificação das proteínas relevantes para a fase G1 que se encontram diferencialmente presentes durante desativação de eIF5A em S. cerevisiae. Foram utilizados dados de larga escala de perfil proteômico diferencial entre linhagens selvagens e mutantes de eIF5A, previamente obtido por nosso grupo, como ponto de partida para identificação das proteínas relevantes. A confirmação de níveis diferenciais destas proteínas foi realizada utilizando metodologia de Western blot após indução da desativação de eIF5A, utilizando o mutante sensível a temperatura hyp2-3. A desativação foi feita através do cultivo da linhagem mutante em temperatura semi-permissiva, que induz perda da função de eIF5A, porém mantendo viabilidade celular. Foi observado que a proteína Cka2 está di... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: eIF5A is a translation elongation factor conserved in archaea and eukaryotes, that interacts with the major subunit of the 80S ribosome. It is the only protein to contain the amino acid hypusine, formed post-translationally, essential to its function. eIF5A helps the translation of proteins that contain proline enriched motifs, as repeated sequence of this amino acid can cause ribosome stall during translation due to the rigid structure that it forms. In the absence of functional eIF5A occurs cell cycle arrest in G1, on the transition of G1 to S, both in Saccharomyces cerevisiae and mammals, suggesting an essential role of it on the translation of important proteins to the progression of the cell cycle. In this work, it was proposed the identification of relevant proteins to the G1 phase that are present differentially during deactivation of eIF5A in S. cerevisiae. It was used data of high-throughput differential proteomic profile between wild type strains and eIF5A mutant strains, previously obtained by our group, as a starting point to the identification of the relevant proteins. The confirmation of the differential levels of these proteins was made using Western blot methodology after induction of eIF5A deactivation, using the temperature-sensitive mutant hyp2-3. The deactivation was performed through cultivation of the mutant strain in semipermissive temperature, that induces loss of function of eIF5A but maintain cell viability. It was observed that the protein Cka2 is d... (Complete abstract click electronic access below) / Mestre

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京都大学 / 新制・論文博士 / 博士(医学) / 乙第13572号 / 論医博第2298号 / 新制||医||1069(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田 恭之, 教授 武藤 学, 教授 伊藤 貴浩 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM