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1 Page 1 of 13 Concursos de Projectos de I&D Proposals for R&D Projects Voltar à descrição do projecto Back to project description Instruções para consultar e imprimir esta página Instructions to view and print this page Imprimir esta página Print this page Visão global da candidatura Application form overview Ocultar todos os formulários da candidatura Hide all forms for this application - Referência do projecto Project reference PTDC/BIA-BCM/71920/ Identificação do projecto 1. Project description - Financiamento solicitado Requested funding ,00 Euros Área científica principal Main Area Ciências Biológicas - Biologia Celular e Molecular Área científica Secundária Secondary area Ciências da Saúde - Epidemiologia, Saúde Pública e Ambiente Título do projecto (em português) Project title (in portuguese) O papel do SR-BI na infecção de malária Título do projecto (em inglês) Project title (in english) The role of SR-BI in malaria infection Palavra-chave 1 Keyword 1 Plasmodium Plasmodium Palavra-chave 2 Keyword 2 Hepatócito Hepatocyte Palavra-chave 3 Keyword 3 Molécula receptora Receptor molecule Palavra-chave 4 Keyword 4 Profilaxia Prophylaxis Objectivos sócio-económicos Socio-economic objectives Saúde Humana Data de início do projecto Duração do projecto em meses Starting date Duration in months

2 Page 2 of Instituições participantes 2. Participating institutions - Instituição Proponente Principal Contractor Instituto de Medicina Molecular (IMM/FM/UL) Avenida Professor Egas Moniz Lisboa Instituições Participantes Participating Institutions Cenix Bioscience GmbH (Cenix) Tatzberg Dresden Unidade de Investigação Principal Research Unit Instituto de Medicina Molecular (IMM/FM/UL) Avenida Professor Egas Moniz Lisboa Instituição de Acolhimento Host Institution Instituto de Medicina Molecular (IMM/FM/UL) Avenida Professor Egas Moniz Lisboa 3. Orçamento 3. Budget - Instituição Proponente Principal Contractor Instituto de Medicina Molecular DESCRIÇÃO DESCRIPTION TOTAL Recursos Humanos Human resources Missões Missions Consultores Consultants Aquisição de serviços e manutenção Acquisition of services and maintenance Outras despesas correntes Other current expenses Despesas gerais Overheads Equipamento Equipment TOTAL Instituições Participantes Participating Institutions Cenix Bioscience GmbH

3 Page 3 of 13 DESCRIÇÃO DESCRIPTION TOTAL Recursos Humanos Human resources Missões Missions Consultores Consultants Aquisição de serviços e manutenção Acquisition of services and maintenance Outras despesas correntes Other current expenses Despesas gerais Overheads Equipamento Equipment TOTAL Orçamento Global Global budget DESCRIÇÃO DESCRIPTION TOTAL Recursos Humanos Human resources Missões Missions Consultores Consultants Aquisição de serviços e manutenção Acquisition of services and maintenance Outras despesas correntes Other current expenses Despesas gerais Overheads Equipamento Equipment TOTAL Plano de financiamento Finance plan DESCRIÇÃO DESCRIPTION TOTAL Financiamento solicitado à FCT Requested funding Financiamento próprio Own funding Outro financiamento público Other public-sector funding Outro financiamento privado Other private funding Total do Projecto Total of the project do orçamento 4. Budget justification dos recursos humanos 4.1. Human resources justification

4 Page 4 of 13 Tipo Nº de pessoas Duração Custo envolvido ( ) (BTI) Bolsa de Técnico de Investigação (Lic. ou Bacharel) In view of the the significant amount of research work planned in this proposal, two full-time technicians/researchers will be hired throughout the duration of the project. It is expected that both these elements will participate actively in dicussions regarding all the tasks proposed in this project. Nevertheless, the specific functions and tasks of each element will be clearly defined so as to avoid overlap and maximise productivity. Custo total: de missões 4.2. Mission justification Tipo Local Nº de deslocações Custo envolvido ( ) Outro (other) Germany Researcher(s) involved in the project will be expected to travel to the collaborating laboratory in Germany several times during the duration of the project. Tipo Local Nº de deslocações Custo envolvido ( ) Participação em congressos International It is expected that the results obatined will be presented in International conferences after the initial 6 months of this project. Custo total: de consultores 4.3. Consultants justification Nome Instituição Fase do projecto Custo envolvido ( ) Dr. Christophe Echeveri Cenix Bioscience GmbH Several 3000 Regular meetings between the researchers in Portugal and the collaborators in Germany will take place. It is expected that the latter will be invited to Portugal at least once per project year. Nome Instituição Fase do projecto Custo envolvido ( ) Dr. Michael Hannus Cenix Bioscience GmbH Several 3000 Regular meetings between the researchers in Portugal and the collaborators in Germany will take place. It is expected that the latter will be invited to Portugal at least once per project year. Custo total: de aquisição de serviços e manutenção 4.4. Acquisition of services and maintenance justification Tipo Custo envolvido ( ) Computer 4000 Two computers will be acquired for data and image processing Tipo Custo envolvido ( ) Services 7500 It is expected that services unavailable in the host laboratory will be subcontracted whenever necessary. This will include, for example, sequencing, proteomics analyses, raising of specific antibodies, etc.. Tipo Custo envolvido ( ) Software 2500 It is expected that specific software and updates will be acquired throughout the duration of the project. Participation in the acquisition of a Spotfire license is contemplated in this item. Custo total: de outras despesas correntes 4.5. Current expenses justification Tipo de despesa Custo envolvido ( )

5 Page 5 of 13 Antibodies 5000 Antibodies against SR-BI or relevant parasite proteins will be acquired Tipo de despesa Custo envolvido ( ) Biological material Both wild-type and transgenic mice will be used throughout the project. Plasmodium-infected mosquitoes will also be required in order to obtain sufficient amounts of the Plasmodium parasite. Tipo de despesa Custo envolvido ( ) Molecular biology / biochemistry related products (non-kits) Cloning of relevant genes will involve significant expenses in terms of restriction enzymes, PCR-related reagents, protein expression and purification material, etc.. Tipo de despesa Custo envolvido ( ) Molecular biology kits RNA extraction, cdna synthesis and real-time PCR kits will be routinely used throughout the project Tipo de despesa Custo envolvido ( ) Two-hybrid system Settting-up a functioning yeast two-hybrid system in the host laboratory will require a significant investment both in terms of expertise and reagents. A library of Plasmodium-expressed genes will be constructed and the experimental procedure optimised to meet our requirements. Custo total: do Equipamento 4.6. Equipment justification Equipamento já disponível para a execução do projecto Available equipment Tipo de equipamento Fabricante Modelo Ano Fast flow cell sorter DakoCytomation MoFlo color flow cytometer BD Biosciences FACScalibur 2002 Real-Time PCR Thermal Cycler ABI Prism 7000 Sequence detection system 2003 Optical Microscope Zeiss Axioskop Confocal Microscope Zeiss LSM Beta-plate counter Packard Matrix Laminar flow cabinet Heraeus HS CO2 incubator Sanyo MCO-17AI 2002 Confocal Microscope Zeiss 510 Meta 2006 Inverted Flourescence Microscope Zeiss Axiovert 200M Discriminação do equipamento a adquirir List of new equipment requested Tipo de equipamento Fabricante Modelo Custo envolvido ( ) Set of multichannel electronic pipettes Eppendorf Research Pro 4000 Multichannel electronic pipettes will be used during the transfection, immunofluorescence staining, quantitative realtime PCR and two-hybrid system procedures that will be routinely carried out in multi-well plates. A set of 4 pipettes covering all relevant dispensing volumes and a 4-pipette holder will be purchased. Plate Washer Tecan Power Washer

6 Page 6 of 13 A Plate Washer will be purchased to be used during the staining procedures for immunofluorescence microscopy and the procedures regarding the two-hybrid system. The washer allows up to 3 wash channels and both a 96-well and a 384-well wash head will be acquired, to allow the use of both these types of cell plates. A dedicated computer will be purchased to program and operate this equipment. The Plate Washer will be available to all other Units of the IMM and will be used by a significant number of researchers that regularly utilize 96- and 384-well plates in experiments such as ELISAs. Custo total: Equipa de investigação 5. Research team Lista de membros (4) 5.1. Members list (4) Nome Name Função Role Grau académico Academic degree %tempo %time Miguel Prudêncio Inv. Responsável DOUTORAMENTO 100 Cristina Dias Rodrigues Investigador LICENCIATURA 25 Maria Manuel Dias da Mota Investigador DOUTORAMENTO 5 João Pedro Monteiro e Louro Machado d... Investigador DOUTORAMENTO 5 (O curriculum vitae de cada membro da equipa está disponível clicando no nome correspondente) (Curriculum vitae for each research team member is available by clicking on the corresponding name) 5.2. Lista de membros a contratar durante a execução do projecto (2) 5.2. Members list to hire during project's execution (2) Membro da equipa Team member Função Role Duração Duration %tempo %time (BTI) Bolseiro Técnico de Investigação (Lic. ou Bacharel) 2 Bolseiro (BTI) Bolseiro Técnico de Investigação (Lic. ou Bacharel) 3 Bolseiro Projectos financiados 6. Funded projects - (Sem projectos financiados) (No funded projects) 7. Indicadores previstos 7. Expected indicators - Indicadores de realização previstos para o projecto Expected output indicators DESCRIÇÃO DESCRIPTION Total A - Publicações Publications Livros Books Artigos em revistas internacionais Papers in international journals Artigos em revistas nacionais Papers in national journals B - Comunicações Communications Comunicações em encontros científicos internacionais Communications in international meetings Comunicações em encontros científicos nacionais Communications in national meetings C - Relatórios Reports D - Organização de seminários e conferências

7 Page 7 of 13 Organization of seminars and conferences E - Formação avançada Advanced training Teses de Doutoramento PhD theses Teses de Mestrado Master theses Outras Others F - Modelos Models G - Aplicações computacionais Software H - Instalações piloto Pilot plants I - Protótipos laboratoriais Prototypes J - Patentes Patents L - Outros Other Acções de divulgação da actividade científica Scientific activity spreading actions In compliance with the requirements of the European Commission s Science and Society Action Plan, specific communication actions concerning all Research and Technological Development projects taking place at the Institute of Molecular Medicine (IMM) will be developed for placement in IMM s website ( The Science & Society component of IMM s website will be used as an experimental model to explore novel approaches into the delineation of an Internet-based science communication strategy with the direct participation of the audiences. The outcome will be a fully interactive website on biomedical sciences and technology and public health a live platform for the discussion and debate of related ethical, social and S&T policy issues combining high quality graphics, innovative web engineering technologies and written content in a format that clearly points out the extent to which medicine and biomedical R&T are a part of everyday experience. The ultimate objective of IMM s Science & Society Online project is to promote public awareness, encourage engagement in dialogue and foster a trust-based relationship between scientists and society. Several R&D projects directly related to the objectives of the Science and Society Action Plan and the promotion of RTD Culture and Public Understanding of Science have been submitted for Community funding and a PhD research fellowship, namely for 1) the assessment of public attitudes to biomedical research and technology throughout Portugal, with emphasis on the study of differences in public perceptions between rural, industrial and urban areas; 2) the development of an effective Internet-based science communication strategy; and 3) optimising two-way communication about science and technology on the Internet. Dados temporariamente indisponíveis 8. Anexo técnico 8. Technical addendum Resumo 8.1. Abstract Resumo (em português) Abstract (in portuguese) A malária é, hoje em dia, uma das doenças mais prevalentes no mundo. Responsável por 500 milhões de infecções e cerca de 3 milhões de crianças mortes por ano a malária é, sem dúvida, uma das maiores causas de morbilidade e mortalidade em muitos países, em especial na África sub-sariana. A proposta agora apresentada visa contribuir significativamente para diminuir o impacto da malária, revelando aspectos cruciais mas até agora desconhecidos da interacção entre o parasita da malária e o seu hospedeiro humano.

8 Page 8 of 13 A malária é causada por um parasita protozoário do género Plasmodium e é transmitido através da picada de mosquitos Anopheles fêmea. Quando um mosquito infectado pica um hospedeiro mamífero, deposita esporozoítos de Plasmodium entram na circulação sanguínea e chegam ao fígado onde invadem hepatócitos. Após vários dias de desenvolvimento intra-hepatocitário, milhares de merozoítos são libertados para a corrente sanguínea onde invadem glóbulos vermelhos, dando início à fase sintomática da doença. A fase hepática da malária é assintomática e, por essa razão, constitui um alvo ideal para vacinas ou drogas profiláticas. Embora a invasão de células do fígado e o desenvolvimento do parasita dentro de hepatócitos constituam passos obrigatórios de qualquer infecção natural de malária, pouco sabe muito acerca das moléculas do hospedeiro que intervêm neste processo. Estudos de RNA de interferência levados a cabo pelo nosso laboratório em colaboração com investigadores da Cenix Bioscience, GmbH conduziram à identificação do SR-BI, uma molécula que se demonstrou ter um papel crucial na invasão de hepatócitos por esporozoítos. Interessantemente, esta molécula foi previamente implicada no processo de invasão de células pelo vírus da hepatite C. As nossas observações mostraram que a subexpressão do gene SR-BI leva a um decréscimo significativo no número de parasitas de Plasmodium em desenvolvimento dentro dos hepatócitos. Estudos subsequentes, descritos em detalhe na secção state-of-the-art, confirmaram a importância do SR-BI durante a fase hepática do ciclo de vida de Plasmodium e enquanto potencial alvo de intervenção profilática. Fisiologicamente, o SR-BI está envolvido na incorporação de colesterol extracelular em células. Este projecto pretende elucidar os detalhes moleculares do papel do SR-BI durante a infecção de células hepáticas por Plasmodium. Mais concretamente, faremos uso da nossa experiência e da dos nossos colaboradores para determinar se o SR-BI actua durante a invasão e/ou durante o desenvolvimento intra-hepatocitário dos parasitas. Utilizaremos técnicas de microscopia de fluorescência para discernir se e de que modo o SR-BI faz parte da estratégia utilizada pelo parasita para invadir as células hospedeiras e e/ou para satisfazer as suas necessidades de colesterol durante o processo de replicação que ocorre dentro daquelas. Para estudar este aspecto, faremos uso da experiência dos nossos colaboradores na Alemanha em biosíntese e metabolismo de colesterol e utilizaremos anticorpos apropriados em estudos de microscopia de células vivas. Uma importante parte do projecto diz respeito à identificação das moléculas do parasita que interagem com o SR-BI, o que será feito recorrendo à técnica de two-hybrid, a qual foi previamente utilizada com sucesso num dos laboratórios que colaboram neste projecto. Em paralelo, utilizaremos o domínio extracelular clonado de SR-BI, presentemente a ser produzido, para investigar aquela interacção com base em técnicas bioquímicas. Finalmente, investigaremos o potencial do SR-BI enquanto alvo para profilaxia antimalárica. Levaremos a cabo estudos in vivo com ratinhos que não expressam SR-BI e estudos in vitro com hepatócitos primários humanos. Em conjunto com os nossos colaboradores na Alemanha, participaremos no desenvolvimento de drogas destinadas a bloquear este receptor e em testes dos seus efeitos na infecção por Plasmodium. Resumo (em inglês) Abstract (in english) Malaria is one of the most prevalent infectious diseases worldwide. With 500 million people becoming infected and up to 3 million children dying every year, malaria is one of the most overwhelming causes of morbidity and mortality in many countries, especially in sub-saharan Africa. The proposal hereby submitted is intended to contribute towards decreasing the burden of malaria, by unveiling crucial but hitherto unknown aspects of the biology of the interaction between the malaria parasite and its human host. Malaria is caused by a protozoan parasite from the genus Plasmodium and is transmitted through the bite of the female Anopheles mosquito. Plasmodium sporozoites are deposited when an infected mosquito bites a mammalian host and, after entering the circulatory system, eventually reach the liver and invade hepatocytes. After several days of intra-hepatocytic development, thousands of merozoites are released into the bloodstream where they invade red blood cells, initiating the symptomatic erythrocytic stage of the disease. The liver stage of malaria is asymptomatic and, therefore, constitutes an ideal target for vaccines or prophylactic drugs. Although the invasion of liver cells by Plasmodium sporozoites and their development inside hepatocytes are obligatory steps of any natural malaria infection, not much is known about the host molecules that mediate these processes. RNA interference screens undertaken by our laboratory with the collaboration of researchers at Cenix Bioscience, GmbH, led to the identification of SR-BI as a crucial molecule in hepatocyte invasion by sporozoites. Interestingly, this molecule was previously shown to be implicated in cell invasion by the hepatitis C virus (HCV). Our observations showed that knock-down of the expression of the SR-BI gene led to a significant decrease in the number of Plasmodium parasites developing inside hepatocytes. Subsequent findings, described in detail in the state-of-the-art section, clearly confirmed the importance of SR-BI during the liver stage of Plasmodium s life cycle

9 Page 9 of 13 and as a potential target for prophylactic intervention. The physiological role of SR-BI regards the uptake of extracellular cholesterol into cells. This project is aimed at unveiling the molecular details of why SR-BI is important during the infection of liver cells by Plasmodium parasites. In particular, we will make use of our expertise and that of our collaborators to investigate whether SR-BI plays a role during invasion of and/or development inside hepatocytes. We will employ high-content fluorescence microscopy techniques to dissect if and how SR-BI is involved in the parasite s strategies for invasion of the host cells and/or in fulfilling the parasite s requirements for cholesterol during the replication process that takes place intracellularly. In studying the latter, we will make use of the expertise of our collaborators in Germany in the area of cholesterol biosynthesis and metabolism and appropriate antibodies will be used in live fluorescence microscopy studies. An important part of the project will regard the identification of the parasite s molecules that interact with SR-BI by using the yeast two-hybrid system, a technique that has extensively been used by one of our collaborating laboratories. In parallel, we will make use of the cloned extracellular domain of SR-BI, currently being produced, to investigate that interaction by biochemical techniques. Finally, the potential relevance of SR-BI as a target for antimalarial prophylaxis will be investigated. In vivo studies with SR-BI-deficient mice and in vitro studies with human primary hepatocytes will be performed. With our collaborators in Germany we will participate in the development of drugs aimed at blocking this receptor protein and testing their effects in terms of Plasmodium infection Objectivos 8.2. Objectives Descrição dos Objectivos do Projecto Project Objectives (description) Preliminary results indicate that SR-BI plays a crucial role in this stage of the life cycle of Plasmodium, the causative agent of malaria. Since invasion of and development within hepatocytes are obligatory steps in any natural malaria infection, there are great potential implications in understanding the parasite s strategies to accomplish this. Thus, the objectives of the proposed work are: (i) to unveil novel fundamental aspects of the cell biology of liver infection by Plasmodium; (ii) to understand the specific role(s) of SR-BI in cell invasion by the parasite and its intracellular development; (iii) to investigate the molecular interactions that occur between Plasmodium and hepatocytes during malaria infection; (iv) to define possible strategies to block infection during its asymptomatic liver stage, by interfering with SR-BI s function and its exploitation by the invading parasite. Overall, the proposed work will undoubtedly help clarify crucial aspects of the liver stage of malaria and, hopefully, provide important clues for antimalarial intervention. Descrição dos Objectivos do Investigador Responsável Principal Investigator Objectives (description) The PI of this project has initiated his scientific career investigating the function and structure of various biologically relevant proteins in bacterial systems. During this period, he acquired a vast expertise in biochemical, molecular biology and spectroscopic techniques. His research interests then shifted towards the biomedical sciences, where he expected to acquire new skills that, in conjunction with those acquired as a protein biochemist, could ensure a different perspective of tackling important biomedical issues. A particular interest in the so-called neglected diseases, led the PI to undertake research in the field of malaria with Dr. Maria M. Mota, one of the leading malariologists in Portugal. The PI aims at pursuing his career as an independent malaria researcher at the IMM, with the support and collaboration of Dr. Mota s laboratory Estado da Arte 8.3. State of the Art Descrição do Estado da Arte State of the Art (description) Introduction. Malaria is one of the most devastating diseases in the world today. Efforts to eradicate it have, so far, been unsuccessful. To a significant extent, this can be attributed to the strategies evolved by the malaria parasite to evade the host s defences and to gain resistance to anti-malarial drugs (Cunha-Rodrigues et al., 2006). Malaria is caused by Plasmodium, a parasite transmitted through the bite of a female Anopheles mosquito. When an infected mosquito bites a mammalian host, it deposits Plasmodium sporozoites under the skin. After entering the circulatory system, sporozoites reach the liver where they migrate through several hepatocytes by breaching their plasma membranes before infecting a final cell with the formation of a parasitophorous vacuole (Mota et al., 2001). Inside the hepatocytes, each sporozoite develops into exoerythrocytic forms (EEFs), formed by thousands of merozoites that will eventually be released into the bloodstream and invade red blood cells, initiating the symptomatic erythrocytic stage of the disease (Prudêncio et al., 2006a). Thus, the hepatic stage of a Plasmodium infection constitutes an appealing target for the development of an anti-malarial vaccine or prophylactic drug since it would act before the onset of pathology. However, although the discovery of Plasmodium liver stages dates back to the late 19th century, it has merited relatively little attention from malaria researchers and important gaps subsist in our understanding of some fundamental processes that occur during this period (Prudêncio et al., 2006b). A more

10 FCT : PROJECTOS DE INVESTIGAÇÃO CIENTÍFICA E DESENVOLVIMENTO T... Page 10 of 13 detailed knowledge about these processes holds immense potential for anti-malarial intervention and merits further exploitation. Background. So far, the only hepatocyte surface molecule known to be involved in sporozoite invasion is CD81 (Silvie et al., 2003), a transmembrane protein that has been shown to act as a receptor for the hepatitis C virus (HCV) (Pileri et al., 1998; Cormier et al., 2004; Wakita et al., 2005). Scavenger receptor B-I (SR-BI) is a receptor molecule expressed in multiple tissues, including the liver, where it serves as a receptor for several lipoproteins, including HDL and LDL. In its physiological role, SR-BI is a central component of the transport of cholesterol between different tissues, particularly the reverse cholesterol transport from peripheral tissues to the liver and the gut (van der Velde et al., 2005). Recently, SR-BI has also been implicated in cell invasion by HCV (Scarselli, et al., 2002; von Hahn et al., 2006). In collaboration with Cenix, Bioscience, GmbH, we have used RNA interference (RNAi) to try and gain an understanding about the influence of host factors during the liver stage of malaria infection. Interestingly, this work showed that knock-down of the expression of the SR-BI gene led to a significant decrease in the number of Plasmodium parasites developing inside hepatocytes. This observation triggered a more detailed investigation of the role of SR-BI in malaria and its potential use as a target for anti-malarial intervention. Our preliminary findings, described below, clearly confirmed the importance of SR-BI during the infection of hepatocytes by Plasmodium and constitute the basis for the work proposed in this project. Preliminary data. The SR-BI gene was targeted by 3 independent short interfering RNAs (sirnas) that were shown to lead to a marked decrease in Plasmodium infection of Huh7 cells, a human hepatoma cell line. To confirm that the infection phenotype observed was due to the knockdown of SR-BI expression, quantitative real-time PCR was performed in cells transfected with the same sirnas in parallel experiments. The results clearly show that a knockdown of at least 70% was achieved for the 3 sirnas, thus unequivocally implicating the SR-BI receptor in the observed decrease in infection (Fig. 1-A in attached file). A more careful analysis of the fluorescence microscopy data on cells that were infected following SR-BI expression knock down revealed that both the number and the size of the EEFs obtained was significantly decreased (Fig. 1-B in attached file). This observation constitutes an indication that SR-BI is not only involved in cell invasion by Plasmodium but also in the intracellular development of the parasite. A screen for small molecule inhibitors of SR-BI yielded a class of molecules termed BLTs (Blocker of Lipid Transport) which efficiently block selective uptake from HDL by cells expressing SR-BI (Nieland et al., 2002). To test whether those chemical inhibitors of SR-BI-mediated selective uptake also inhibited the liver stage infection, BLT-1, -2, -4 and -5 were added to Huh7 cells 10 minutes prior to sporozoite addition. Consistent with the effect of RNAimediated inhibition of SR-BI activity, all compounds reduced the infection rate with different IC50s. Interestingly, the relative efficiencies by which the different BLTs inhibited sporozoite infection and selective cholesterol uptake (published) were identical, with BLT-1 being 5 times more efficient than BLT-2 whilst BLT-4 and BLT-5 show considerably higher IC50s than both BLT-1 and BLT-2 (Fig 2-A in attached file). Addition of BLT-1 at various timepoints relative to sporozoite addition indicated that this compound affects both parasite invasion and its development (Fig 2-B in attached file). In addition to constituting further proof of a role for SR-BI in malaria liver stage, these results open extremely interesting possibilities in terms of interfering with sporozoite infection of hepatocytes by targeting SR-BI with appropriate drugs. Conclusion and prospects. The data obtained so far clearly point towards a pivotal role for SR-BI in malaria liver infection. For this reason, two patents on the potential anti-malarial applications of SR-BI have been filled in a partnership between our laboratory and that of our German collaborators. Moreover, our preliminary data pave the way for a number of studies that can shed light on the molecular details of SR-BI s function. Both from a fundamental cell biology point of view and from the point of view of its potential applications, it seems clear that this molecule merits further attention. This project is designed to build upon existing data and obtain the complete picture of SR-BI s role in malaria, including its interaction with the Plasmodium parasite and its potential use as a target for anti-malarial intervention. Attached file: MPrudencio_Malaria_SR-BI_PrelimData.pdf References Cormier EG, Tsamis F, Kajumo F, Durso RJ, Gardner JP, Dragic T (2004) CD81 is an entry coreceptor for hepatitis C virus, Proc. Natl. Acad. Sci U S A, 101, Cunha-Rodrigues M, Prudêncio M, Mota MM, Haas W (2006) Antimalarial drugs - host targets (re)visited, Biotechnol. J., 1, von Hahn T, Lindenbach BD, Boullier A, Quehenberger O, Paulson M, Rice CM, McKeating JA Oxidized low-density lipoprotein inhibits hepatitis C virus cell entry in human hepatoma cells, Hepatology, 43, Mota MM, Pradel G, Vanderberg JP, Hafalla JC, Frevert U, Nussenzweig RS, Nussenzweig V, Rodriguez A. (2001)

11 FCT : PROJECTOS DE INVESTIGAÇÃO CIENTÍFICA E DESENVOLVIMENTO T... Page 11 of 13 Migration of Plasmodium sporozoites through cells before infection, Science, 291, Nieland TJ, Penman M, Dori L, Krieger M, Kirchhausen T (2002) Discovery of chemical inhibitors of the selective transfer of lipids mediated by the HDL receptor SR-BI, Proc. Natl. Acad. Sci. U S A, 99, Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, Petracca R, Weiner AJ, Houghton M, Rosa D, Grandi G, Abrignani S (1998) Binding of hepatitis C virus to CD81, Science, 282, Prudêncio M, Rodrigues CD, Mota MM (2006a) The relevance of host genes in Malaria, In: Parrington, J. and Coward, K. (Eds) Comparative Genomics and Proteomics in the Identification of New Drug Targets, Taylor & Francis, Oxford, UK, in press Prudêncio M, Rodriguez A, Mota MM (2006b), The path to thousands of merozoites: how Plasmodium sporozoites reach and infect the liver, Nat. Rev. Microbiol., 2006, in press Scarselli E, Ansuini H, Cerino R, Roccasecca RM, Acali S, Filocamo G, Traboni C, Nicosia A, Cortese R, Vitelli A (2002) The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus, EMBO J., 21, Silvie O, Rubinstein E, Franetich JF, Prenant M, Belnoue E, Renia L, Hannoun L, Eling W, Levy S, Boucheix C, Mazier D (2003) Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity, Nat. Med., 9, van der Velde AE, Groen AK (2005) Shifting gears: liver SR-BI drives reverse cholesterol transport in macrophages, J. Clin. Invest., 115, Wakita T, Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, Murthy K, Habermann A, Krausslich HG, Mizokami M, Bartenschlager R, Liang TJ (2005) Production of infectious hepatitis C virus in tissue culture from a cloned viral genome, Nat. Med., 11, Resultados e Repercussões 8.4. Results and Repercussions Divulgação de Resultados (descrição) Diffusion of Results (description) Data arising from this project is expected to have with implications for both the fundamental knowledge of molecular aspects of liver stage malaria infection and the potential use of SR-BI as a drug target. These results will be relevant for parasitologists in general and for the malaria community in particular. Their diffusion will be based on publications in high-impact journals and presentations in international conferences. If and when relevant, diffusion of results in the media will be considered. Additionally, in compliance with the requirements of the European Commission s Science and Society Action Plan, specific communication actions concerning all RTD projects taking place at the IMM will be included in the institute s website ( The Science & Society component of this website will be used as a fully interactive website on biomedical sciences and technology and public health. Moreover, the PI is a member a non-governmental organization for scientific spreading called "Viver a Ciência", whose President, Dr. M.M. Mota, is also part of the research team. Repercussões (descrição) Repercussions (description) Efforts to eradicate malaria have, so far, been unsuccessful. To a significant extent, this can be attributed to the strategies evolved by the malaria parasite to evade the host s defences and to gain resistance to anti-malarial drugs. However, one cannot ignore the failure of developed countries to invest seriously in malaria research, perhaps because this is a disease that seldom (although with increasing frequency) affects those living in these countries. Fortunately, this scenario is changing and malaria is slowly but surely loosing its status of neglected disease, having recently integrated the European Union list of infectious diseases linked to poverty ( ), which are the priority for disease control at both Union and international level. Knowledge about the molecular details of liver stage malaria and, particularly, about the interactions that occur between the Plasmodium sporozoite and the host s hepatocytes is, at present, quite limited. This project will improve our understanding of the molecular mechanisms at play during sporozoite invasion and development inside hepatocytes. The exact role of the host SR-BI receptor molecule will be dissected and the mechanisms used by the parasite to interact with it will be revealed. It can be expected that our findings will uncover novel parasite invasion and survival strategies as well as novel host-parasite interactions. These will be of relevance not only for researchers in the malaria field but also for other parasitologists and microbiologists. Furthermore, our preliminary data (please see state-of-the-art section) suggest that SR-BI may be an anti-malarial target molecule that can be of use in prophylaxis or vaccine development. A patent on the potential anti-malarial applications of SR-BI (EPO ) has been filed in a partnership between our laboratory and that of our German collaborators. If this molecule proves, indeed, to be a useful target for intervention against malaria, the repercussions of the present project can hardly be overestimated. The outcome of the research contained in the present proposal may, thus, hold enormous impact not only for the scientific community but also for the populations of malaria endemic areas and/or people travelling to those regions.

12 FCT : PROJECTOS DE INVESTIGAÇÃO CIENTÍFICA E DESENVOLVIMENTO T... Page 12 of Regionalização 8.5. Regionalization Região Percentagem Region Percent Norte 10 Centro 10 Lisboa e Vale do Tejo 40 Alentejo 10 Algarve 10 Região Autónoma dos Açores 10 Região Autónoma da Madeira 10 Descrição Description Research and development in the Life Sciences plays a central role in generating knowledge and in the application of this knowledge to improving the quality of life. It is therefore in Europe s strategic interest to provide optimal support for its Life Scientists, irrespective of the region where laboratories are located. Stimulating creativity and encouraging scientific progress is the pipeline to produce a breakthrough that can open up a whole new set of applications leading to improvements in the quality of live and developing the national and European economy. Scientific investigation in the Life/Health Sciences such as that proposed in this project leads to unexpected results but will certainly have transnational impact. Science thrives on interaction between individuals spurring each other on. Theories and ideas need to be tested against the best in the field if they are to be accepted. In Portugal, the size of the scientific community inevitably means that contacts need to be made throughout the entire country and across borders in order to reach critical mass and multidisciplinarity. Providing support to this research project will ensure that national researchers recruited among the best University students all over the country will have the opportunity to work at the leading edge in this field. National researchers will also have the opportunity to generate new knowledge working closely with their counterparts in other countries by means of international co-operating programmes currently in progress in the host laboratory. The Project team is committed to sharing its resources and to collaborating with research groups from any other region. The scientific knowledge generated by the project will reinforce our current collaborations and open possibilities for new interactions with other groups. This research project will also contribute to foster general scientific education and breed new forms of transfer of knowledge in the field of Biomedicine. Impacts such as the contribution of the project s results to science communication activities are not specific of any region. Efforts will be made to bring any positive result to the general public through the media and through the IMM Web site. Our team will contribute to public understanding of the scientific work on this area organizing activities which involve participants from any region of the country, such as conferences and workshops for teachers and students from secondary schools, oriented visits for students to our laboratory and seminars held at secondary schools. The application for recruitment of scientists to work on the project will be opened at national level and there will be no bias on the selection of candidates, which will be solely based on merit. The same applies for suppliers of materials and services, quality and price being the only criteria of choice Tarefas 8.6. Tasks Lista de tarefas (5) Task list (5) Designação da tarefa Data de início Data de fim Pessoas * mês Task denomination Start date End date Person * months Task 1 - Involvement of SR-BI in Plasmod Task 2 - Cholesterol requirements by Pla Task 3 - Interaction of Plasmodium with Task 4 - Development of drugs targeting Task 5 - Relevance of SR-BI for malaria (Os detalhes de cada tarefa estão disponíveis clicando na designação correspondente) (Details for each task are available by clicking on the corresponding denomination)

13 FCT : PROJECTOS DE INVESTIGAÇÃO CIENTÍFICA E DESENVOLVIMENTO T... Page 13 of Referências Bibliográficas 8.7. Bibliographic references Ano Publicação Year Publication Prudêncio M, Rodriguez A, Mota MM (2006), The path to thousands of merozoites: how Plasmodium 2006 sporozoites reach and infect the liver, Nat. Rev. Microbiol., 2006, in press Cunha-Rodrigues M, Prudêncio M, Mota MM, Haas W (2006) Antimalarial drugs - host targets (re)visited, 2006 Biotechnol. J., 1, Mota MM, Hafalla JC, Rodriguez A (2002), Migration through host cells activates Plasmodium sporozoites for 2002 infection, Nat Med., 2002, 11, Madureira PA, Matos P, Soeiro I, Dixon LK, Simas JP, Lam EW (2005), Murine gamma-herpesvirus 68 latency 2005 protein M2 binds to Vav signaling proteins and inhibits B-cell receptor-induced cell cycle arrest and apoptosis in WEHI-231 B cells, J Biol Chem., 280, Greco V, Hannus M, Eaton S (2001), Argosomes: a potential vehicle for the spread of morphogens through 2001 epithelia, Cell, 106, Artigos Anteriores 8.8. Previous Articles Ano Year Artigo (endereço na Internet - URL) Paper (Link in the Internet - URL) Ficheiros Anexos 9. Attachments - 1:45:37 Financiamento de Fundos Estruturais e de Fundos Nacionais do MCTES

14 Page 1 of 2 Concursos de Projectos de I&D Proposals for R&D Projects Tarefa Task Fechar esta janela Close this window Referência do projecto Project reference PTDC/BIA-BCM/71920/2006 Membros da equipa de investigação que participam nesta tarefa Members of the research team who participate in this task Nome Name Miguel Prudêncio (BTI) Bolseiro Técnico de Investigação (Lic. ou Bacharel) 2 (BTI) Bolseiro Técnico de Investigação (Lic. ou Bacharel) 3 Cristina Dias Rodrigues Maria Manuel Dias da Mota Descrição da tarefa Task decription Designação da tarefa Task denomination Task 1 - Involvement of SR-BI in Plasmodium invasion and development Data de início Start date Data de fim End date Duração (em meses) Duration (in months) Pessoas * mês nesta tarefa Person * month in this task Resultados esperados Expected results Conceptually, the entire process of infection at the liver stage can be divided into an initial invasion phase and a subsequent phase of development. Upon completion of this task, it will be clear whether SR-BI is involved in cell invasion by sporozoites, in intracellular parasite development or in both. Our previous results strongly point towards the possibility that SR-BI has a role in both invasion and development and, in all likelihood, the experiments carried out in the context of this task will confirm these preliminary findings. Nevertheless, the possibility exists that SR-BI is only involved in one of those two processes. Such a finding would have no implications for the overall aims of the project, which remain the elucidation of the exact role(s) of SR-BI during the liver stage of malaria. Building up on our preliminary data and on the initial results obtained in this task, each of the invasion and development processes will be studied per se in more detail. As far as invasion is concerned, it is presently unclear whether SR-BI acts as a gate for sporozoite entry or whether it influences invasion in a more indirect fashion, perhaps by providing cholesterol to the invading parasite. The proposed experiments will attempt to discriminate between these two possibilities. In what regards intracellular parasite development, it is possible that SR-BI is involved in providing the developing EEF with cholesterol, necessary for extensive membrane biosynthesis for the forming merozoites. The proposed experiments will test this hypothesis. Overall, this task will contribute towards building the complete in vitro molecular picture of SR-BI s involvement in sporozoite entry and development in the host s hepatocytes. Descrição da tarefa Task description Invasion/Development. Huh7 hepatoma cells will be incubated with an SR-BI blocking antibody (absrbi) at different periods during infection by P. berghei sporozoites, namely, from 1 hour before to 2 hours after sporozoite addition and from 2 to 24 hours post-sporozoite addition. Cells incubated with the same antibody from 1 hours before to 24 hours after sporozoite addition will be used as controls. Invasion takes place during 1 to 2 hours after sporozoites are added to cells. They then become developing EEFs. Therefore, determining the number of EEFs obtained in each of the conditions described will reveal whether absrbi has an effect during invasion, development, or both. High-

15 Page 2 of 2 content immuno-fluorescence microscopy will be used to determine whether addition of absrbi leads to a decrease in EEF size, as was observed when BLT-1 was added to cells before infection (please refer to state-of-the-art section for details). SR-BI s involvement in invasion will also be addressed by fluorescence activated cell sorting (FACS), an approach that is made possible by the availability in our laboratory of GFP-expressing P. berghei parasites. This technique will be employed to measure infection 2 hours after sporozoite addition to Huh7 cells in which SR-BI expression was knocked-down by RNAi. Invasion. In order to test whether SR-BI can be used by Plasmodium sporozoites for their initial docking to and, possibly, internalization into the host cell, competition experiments with selected lipoproteins and chemically modified lipoproteins will be performed. Human Huh7 hepatoma cells will be shifted to lipoprotein deficient medium shortly before sporozoite addition, which will then be carried out in the presence of defined amounts of LDL, LDLox, LDLac, HDL and VLDL. Development. As an efficient HDL receptor, SR-BI is involved in the transport of cholesterol between different tissues, particularly the reverse transport from peripheral tissues to the liver and to the gut. Published data sugest that Plasmodium requires cholesterol for liver stage Plasmodium infection. In order to test this hypothesis, Huh7 cells will be infected under cholesterol starvation conditions. Cells will be shifted to lipoprotein deficient medium (thereby depleting the cells of their extracellular cholesterol source) containing 1µM hydroxy-cholesterol (which will block intracellular cholesterol biosynthesis) 24 h before sporozoite infection. A decrease in infection levels in these conditions can thus be expected and will indicate that cholesterol is indeed required for liver stage Plasmodium infection. Then, we will test whether this decrease in infection can be reversed by addition of specific lipoproteins 2 hours after sporozoite addition, i.e., after the invasion step is completed. This will reveal whether cholesterol is required during the intracellular development phase of the parasite. Performing the same experiment in cells where SR-BI levels were knocked-down by RNAi will reveal whether SR-BI is involved in providing the developing parasite with cholesterol. Financiamento de Fundos Estruturais e de Fundos Nacionais do MCTES

16 Page 1 of 2 Concursos de Projectos de I&D Proposals for R&D Projects Tarefa Task Fechar esta janela Close this window Referência do projecto Project reference PTDC/BIA-BCM/71920/2006 Membros da equipa de investigação que participam nesta tarefa Members of the research team who participate in this task Nome Name Miguel Prudêncio (BTI) Bolseiro Técnico de Investigação (Lic. ou Bacharel) 2 Cristina Dias Rodrigues Maria Manuel Dias da Mota Descrição da tarefa Task decription Designação da tarefa Task denomination Task 2 - Cholesterol requirements by Plasmodium during malaria liver stage Data de início Start date Data de fim End date Duração (em meses) Duration (in months) Pessoas * mês nesta tarefa Person * month in this task Resultados esperados Expected results Task 2 will build upon the results of task 1 to uncover the molecular aspects of cholesterol delivery to the intracellular Plasmodium parasite. Our collaborator, Dr. Michael Hannus, has extensive expertise in cholesterol and lipoprotein biology, having carried out a druggable genome wide RNAi screen for new genes involved in the regulation of LDL uptake, performed in collaboration with Bayer Healthcare. Thus, task 2 will employ that expertise in order to identify which apolipoproteins are involved in Plasmodium-related cholesterol delivery and what role SR- BI plays in this process. It can be expected that the results of this task will provide new insights not only in what regards cholesterol involvement in Plasmodium development inside hepatocytes but also about more general aspects of hepatocytic cholesterol transport. Descrição da tarefa Task description Immunofluorescence microscopy and live microscopy techniques will be employed to fulfil the objectives outlined for this task. Both our laboratory and that of our collaborators in Germany have extensive expertise in these techniques and our model of infection has been optimised, ensuring the feasibility of the proposed approach. Antibodies against specific apolipoproteins will be used to stain infected cells at specific time-points following sporozoite addition. An antibody against Plasmodium is available in our laboratory and will be used to identify the intrahepatocytic EEF. In parallel, chemically modified, fluorescent lipid molecules will be used in similar fluorescence microscopy experiments. Both these strategies will help identify the lipoproteins that may be relevant during the intrahepatocytic development of the parasite. As a complement to the experiments outlined above, live fluorescence microscopy will be used to track lipoproteins in Plasmodium-infected cells. Lipoproteins covalently attached to fluorescent dyes will be used in these experiments, in conjunction with a GFP-expressing Plasmodium strain available in our laboratory. The use of antibodies against the SR-BI receptor will help shed light onto the role of the latter in this process. Taken together, these results will provide a more detailed picture of Plasmodium s requirements for cholesterol during the hepatocytic stage of malaria infection. The likely role of SR-BI in this process will also be elucidated, providing a rationale for a potential SR-BI-based anti-malarial approach.

17 Page 2 of 2 Financiamento de Fundos Estruturais e de Fundos Nacionais do MCTES

18 Page 1 of 2 Concursos de Projectos de I&D Proposals for R&D Projects Tarefa Task Fechar esta janela Close this window Referência do projecto Project reference PTDC/BIA-BCM/71920/2006 Membros da equipa de investigação que participam nesta tarefa Members of the research team who participate in this task Nome Name Miguel Prudêncio (BTI) Bolseiro Técnico de Investigação (Lic. ou Bacharel) 2 Cristina Dias Rodrigues Maria Manuel Dias da Mota João Pedro Monteiro e Louro Machado de Simas Descrição da tarefa Task decription Designação da tarefa Task denomination Task 3 - Interaction of Plasmodium with SR-BI Data de início Start date Data de fim End date Duração (em meses) Duration (in months) Pessoas * mês nesta tarefa Person * month in this task Resultados esperados Expected results This is an ambitious, yet undoubtedly feasible, task that aims at understanding the molecular details of Plasmodium s interaction with the SR-BI receptor. Protein-protein interactions are intrinsic to virtually every cellular process. Thus, it can be expected that one or more proteins expressed on the surface of the Plasmodium sporozoite bind SR-BI and, possibly, use it as an attachment/entry port into the hepatocyte. Experiments will be carried out in order to identify this(ese) putative protein(s) and to determine whether its(their) expression is specific to the liver stage of the Plasmodium life cycle. Upon completion of this task, the nature of the molecular interactions that occur between the Plasmodium parasite and the SR-BI molecule on hepatocytes will have been elucidated. An understanding of these interactions, at the molecular and physical levels, will have important implications for the rational design of compounds that can specifically inhibit SR-BI. It will also provide an insight into the biology of Plasmodium, by revealing whether the putative protein(s) involved in its interaction with SR-BI is(are) specifically expressed during the liver stage of the parasite or whether it(they) is(are) expressed in other stage(s) of its life cycle. Descrição da tarefa Task description Several alternative approaches have been used for the identification of protein partners and no single technique can, a priori, ensure success. Different complementary approaches will be employed in order to meet the objectives stated in this task. The procedures described below will be carried out, as much as possible, in parallel, in an attempt to try and maximise the amount of data obtained and facilitate interpretation of the results. Once the SR-BI Plasmodium sporozoite protein(s) has(ve) been identified, the interaction between binding partners will be characterised from the physical point of view. Yeast two-hybrid system. The "two-hybrid" technique exploits the fact that the DNA-binding domain of a given transcription factor is incapable of activating transcription unless physically, but not necessary covalently, associated with an activating domain. To use this technique to determine whether two proteins X and Y interact, they are

19 Page 2 of 2 expressed as fusion proteins with a transcriptional activating domain (prey), and a DNA-binding domain (bait), respectively. In this task, the gene encoding SR-BI will be cloned and used as bait. A Plasmodium cdna library will be prepared from the sporozoite stage of the parasite, where the target protein is expected to be biologically relevant. Dr. Simas, one of the collaborators in this project, has successfully used this technique in the past and will contribute with his expertise towards the design of the cdna library and the overall experimental setup. Binding assays. Dr. Hannus, our collaborator from the German laboratory, is currently producing a construct containing a fusion between the extracellular domain of SR-BI (ecdsr-bi) and the fc domain of human IgG, to be expressed using a mammalian expression system. This recombinant ecdsr-bi will be employed to raise an antibody against the protein. In addition, it will be used in binding assays, using a variety of biochemical methods such as protein affinity chromatography, affinity blotting, immunoprecipitation and cross-linking. Surface Plasmon Resonance. Surface plasmon resonance (SPR) is a phenomenon which occurs when light is reflected off thin metal films. In order to detect an interaction, one molecule (the ligand) is immobilised onto a sensor surface and its binding partner (the analyte) is injected in aqueous solution under continuous flow. The binding of the analyte to the ligand can be monitored in real-time and provide information about the binding affinity between the two. During this task, the recombinant ecdsr-bi (see above) will be used as ligand and its Plasmodium sporozoite binding partner(s) will be used as analyte. This will enable the determination of the affinity constant of the binding between the two partners. This experimental setup will also be used in studying compounds that can be used as inhibitors of SR-BI (see Task 5). Financiamento de Fundos Estruturais e de Fundos Nacionais do MCTES

20 Page 1 of 2 Concursos de Projectos de I&D Proposals for R&D Projects Tarefa Task Fechar esta janela Close this window Referência do projecto Project reference PTDC/BIA-BCM/71920/2006 Membros da equipa de investigação que participam nesta tarefa Members of the research team who participate in this task Nome Name Miguel Prudêncio (BTI) Bolseiro Técnico de Investigação (Lic. ou Bacharel) 2 (BTI) Bolseiro Técnico de Investigação (Lic. ou Bacharel) 3 Cristina Dias Rodrigues Maria Manuel Dias da Mota Descrição da tarefa Task decription Designação da tarefa Task denomination Task 4 - Development of drugs targeting SR-BI for malaria prophylaxis Data de início Start date Data de fim End date Duração (em meses) Duration (in months) Pessoas * mês nesta tarefa Person * month in this task Resultados esperados Expected results SR-BI has great potential as a target for anti-malarial action. Our preliminary results, described in the state-of-theart section of the present proposal, showed that compounds known as Blockers of Lipid Transport (BLTs) significantly reduce in vitro infection of hepatoma cells by Plasmodium. It is expected that completion of this task will result in the design and production of a small number of highly effective compounds whose efficiency in preventing liver-stage infection has been established in vitro (hepatoma cell lines), in vivo (rodent models) and in human primary hepatocytes. It is expected that the most effective of these compounds will be ready to undergo human trials. Descrição da tarefa Task description Our preliminary data show that the different BLT molecules tested yield distinct effects in terms of infection decrease. This observation provides useful information about the relationship between the structure and the efficiency of these molecules. A project by our collaborators at Cenix to design compounds with improved drug efficiency has recently been funded by a German grant. The aim is to build up on existing BLT structures to create a drug library whose anti-malarial efficiency can be tested by a variety of methods. This information can then be used to iteratively generate compounds with increasing efficiency in preventing liver infection by Plasmodium. We will actively collaborate with the German laboratory in testing the anti-malarial efficiency of these componds. This task will be articulated with tasks 3 and 5 that will generate, respectively, structural information about Plasmodium s natural interaction with SR-BI and information about the drugs efficiency in vivo. In vitro studies. High-throughput immunoflurescence microscopy will be used to screen libraries of available as well as new compounds for their efficiency in decreasing infection of hepatoma cell lines. The method is well established by our laboratory and the results obtained will constitute the basis for the experiments detailed below. In vivo studies. Molecules identified in the in vitro screen as good antimalarial candidates will be tested in vivo, using