Made available in DSpace on 2016-03-28T12:42:11Z (GMT). No. of bitstreams: 2 janaiara_cunha_ioc_mest_2014.pdf: 5340118 bytes, checksum: d30f5606e8d7731758e5708a036d73ec (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016-01-13 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Os primatas neotropicais dos gêneros Saimiri e Aotus são modelos recomendados pela OMS para estudos experimentais da malária humana, pois são susceptíveis à infecção por plasmódios humanos e reproduzem de forma relativamente confiável a patologia e a imunidade observadas em humanos. Eles apresentam, entretanto, uma importante limitação: a necessidade de se esplenectomizar o animal para que as parasitemias sejam elevadas e consistentes. O objetivo desse estudo foi avaliar a influência da administração de lipossomo-clodronato (LC), utilizado para depletar monócitos/macrófagos em vários modelos experimentais, na infecção por Plasmodium falciparum em Saimiri sciureus. Realizou-se um experimento in vitro utilizando cultivo de esplenócitos de Saimiri, incubados em presença ou não de LC, quantificando-se a depleção de macrófagos por citometria de fluxo. Primeiro experimento: foram utilizados seis animais não esplenectomizados divididos em dois grupos que receberam 1mL PBS ou de LC (5mg/mL) a partir do dia 0 de infecção com inóculo contendo 106 hemácias parasitadas com P. falciparum (cepa FUP). Segundo experimento: foram utilizados 14 animais não esplenectomizados divididos em seis grupos - três grupos não infectados, com dois animais cada, que receberam 1mL PBS, ou 0,5mL ou 1mL de LC e três grupos infectados no dia 0 que receberam as mesmas administrações; sendo que o grupo que recebeu PBS tinha dois animais e os grupos que receberam LC tinham três animais cada. Em ambos os experimentos as administrações foram por via intravenosa duas vezes por semana a partir de dia 0. Após eutanásia, foram realizados exames histopatológicos e ensaio de expressão de citocinas em células esplênicas No ensaio in vitro o LC induziu uma citotoxicidade dose-dependente de monócitos/macrófagos. No primeiro experimento o grupo tratado com LC apresentou aumento na parasitemia alcançando valores superiores a 20% no d11 e requerendo tratamento. O grupo tratado com PBS apresentou parasitemia de 0,029% a 8,15% e foi capaz de controlar espontaneamente a infecção no d18. No segundo experimento os animais infectados que receberam 0,5mL LC apresentaram parasitemias mais elevadas (pico entre 16,1% e 26,7% entre d10 e d14) do que os outros grupos (grupo PBS - picos entre 6,3% e 17,7% entre d12 e d13, e grupo 1mL LC \2013 pico entre 4,8% e 8,8% entre d11 e d15). Em todos os animais infectados, a temperatura foi relacionada com a presença de parasitemia e a hemoglobina e o hematócrito diminuíram alcançando valores mínimos quando parasitos já não são mais detectáveis na circulação. Os animais toleraram clinicamente a administração de LC, mas apresentaram sinais histopatológicos de toxicidade hepática. Em conclusão, o LC é capaz de promover parasitemias mais altas em infecções de P. falciparum em primatas S. sciureus. A infecção esteve associada a evidências de depleção parcial de macrófagos como diferenças no tamanho dos baços e menor presença de ferro nos baços e fígados dos animais que receberam LC. Ensaios ainda precisam ser realizados para se estabelecer volumes mínimos funcionais e superar o problema da aparente toxicidade hepática / The WHO recommends the Neotropical primates of the genus Saimiri and Aotus as models for experimental studies of human malaria. The se monkeys are susceptible to infection by human P lasmodi a and reproduce relatively reliably the pathology and immunity observed in man . However, the model has a s limitation the need of splenectomy for the obtention of high and consistent parasitemias . The aim was to evaluate the influence of administration of clo dronate - liposome (CL), used to deplete monocytes/macrophages in several experimental models, in Plasmodium falciparum infected Saimir i sciureus . We conducted an experiment using in vitro culture of spleen cells from Saimiri incubated in the presence or abs ence of C L and quantifying the depletion of macrophages by flow cytometry. Other experiment s were: Exp. 1 : six non splenectomized animals were divided into two groups receiv ing 1 mL of PBS or 1 mL of C L (5mg/mL) from the d ay 0 of infection with 10 6 P. falc iparum (FUP strain) parasitized erythrocytes . Exp. 2 : 14 non - splenectomized animals were divided in six groups - three non - infected groups , with two animals each, receiv ing 1mL PBS, 0.5 mL or 1 mL CL ; and three infected groups receiving the same injections . The group that received PBS had two animals each; and the groups that received CL had three animals each . In both experiments the injections were intravenous, two times a week from d0. After euthanasia, histopathological examination and testing of cytoki ne expression in splenic cells were performed. In vitro assay : the CL induced a dose - dependent monocyte / macrophage cytotoxicity. In Exp 1, the CL treated group showed an increase in parasitemia reaching values higher than 20% at d11 and requir ed treatm ent. T he group treated with PBS showed parasitemia from 0.029 % to 8.15% and was able to spontaneously control the infection by d18. In Exp 2, the infected animals receiv ing 0.5 ml CL showed higher parasitaemia (peak s between 16.1% and 26.7% between d10 and d14) than the other groups (PBS group - peaks between 6.3% and 17 7% between d12 and d13, and 1mL CL group - peak between 4.8% and 8.8% between d11 and d15). In all infected animals, the temperature was related to the presence of parasitaemia and hemoglob in and hematocrit decreased , reaching minimum values during or after clearance of the parasite. The animals tolerated clinically the CL administration but showed histopahological signs of liver toxicity. Animals receiving CL showed less iron in the spl ee n , suggesting a decreased erythrophagocitosis . In conclusion, the CL is capable of promoting higher parasitemia in P. falciparum infect ed S. sciureus primates. The infection was associated with evidence s of partial depletion of macrophages , such as differe nces in the spleen size s and decreased presence of iron in the spleens and livers of animals receiv ing CL. However , more tests still need to be conducted to define the lower volum es of LC that are still functional , to overcome the problem of the apparent l iver toxicity.

Made available in DSpace on 2014-06-12T17:35:11Z (GMT). No. of bitstreams: 2 arquivo4409_1.pdf: 676822 bytes, checksum: 4a1153b3912f6483c79f20c422b118f1 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2002 / Oligonucleotídeos foram construídos com base na sequência primária do gene codificando o rRNA de Plasmodium para amplificar DNA de P. falciparum, P. vivax, P. malariae e P. ovale, de maneira gênero-específica. Três sistemas de PCR foram utilizados: PCR simples, hemi-nested PCR convencional e hemi-nested PCR em um único tubo, desenvolvidos em nosso laboratório. Na PCR simples, composta de 30 ciclos, foram utilizados os oligonucleotídeos GJ1 e HR842 (20 pmol/50μl), já testados por nosso grupo. Na hemi-nested PCR convencional utilizou-se três oligonucleotídeos (GJ1, PGFO3 e HR842), em duas reações sequenciais, sendo o PGFO3 construído durante o desenvolvimento do presente trabalho, visando a detecção do gênero Plasmodium. O par GJ1 e HR842 foram utilizados como oligonucleotídeos externos na primeira reação, e o PGFO3 como interno, ancorado ao HR842 na segunda reação. A hemi-nested PCR em um único tubo consistiu em 60 ciclos (92ºC, 30s; 58ºC, 30s e 72ºC, 45s), e concentrações limitantes de oligonucleotídeos externos (4 pmols/50μl) participavam da PCR sem competição com os oligonucleotídeos internos durante os primeiros 15 ciclos da reação e 40 pmol/50μl de primers internos (imobilizados na face interna da tampa do microtubo) foram introduzidos na PCR no 16º ciclo. As concentrações dos outros componentes da reação foram as mesmas utilizadas nas reações convencionais de PCR. Observou-se que a quantidade mínima de DNA genômico detectada pela PCR simples, hemi-nested PCR e hemi-nested PCR em um único tubo foi de 10 pg; 0,01 pg e 0,1 pg, respectivamente. Apesar da hemi-nested PCR em um único tubo ter sido menos sensível que a heminested PCR convencional, é muito mais simples e conveniente, já que o risco de contaminação cruzada é bem menor. Esses sistemas moleculares de diagnóstico podem ser usados em situações quando se requer uma alta sensibilidade e especificidade, tais como na avaliação da eficiência de quimioterapia, detecção precoce de infecção e prevenção de transmissão a partir de pacientes hipoparasitêmicos

Pour assurer ses besoins de croissance, multiplication, et survie, Plasmodium modifie sa cellule hôte, l'érythrocyte, après l'invasion. Le parasite met en place ainsi un système d'échanges (import/export) avec sa cellule hôte et le milieu extérieur. Nous avons identifié dans la base de données de Plasmodium berghei, le parasite de rongeurs, une famille de gènes, sep, correspondant à la famille etramp chez Plasmodium falciparum. Cette famille de gènes code pour des petites protéines exportées, et conservées dans tout le genre Plasmodium. Les protéines SEP (13?16 kDa) contiennent en N-terminal un peptide signal prédit, un domaine hydrophobe interne, et elles diffèrent au niveau des régions C-terminal et 3' UTR. Toutefois, les protéines SEP sont exprimées à différents moments du cycle de Plasmodium. Durant le cycle érythrocytaire, PbSEP1 et PbSEP3 sont exprimées à partir du stade trophozoïte, et la même quantité de protéine est détectée au stade schizonte et gamétocyte, pendant que PbSEP3 est hautement détectée dans les trophozoïtes mûrs et les gamétocytes. Chez le moustique, PbSEP1 et PbSEP3 sont détectées seulement chez les ookinètes, alors que PbSEP2 est très abondante dans les ookinètes, oocystes, et sporozoïtes des glandes salivaires. Les protéines SEP ont également des localisations différentes. Dans l'érythrocyte, PbSEP1 est localisée dans la membrane de la vacuole parasitophore, alors que PbSEP2 et PbSEP3 sont exportées au-delà de cette vacuole, et sont ainsi localisées dans la cellule hôte, en association avec des structures vésiculaires. Dans cette étude, nous avons identifié les signaux d'adressage des protéines SEP dans la vacuole parasitophore et dans la cellule hôte, chez Plasmodium berghei. L'autre partie du travail, effectuée à l'Université de Montpellier II, a consisté à étudier la localisation de deux protéines du squelette sous- membranaire de l'érythrocyte, la dématine, et l'adducine, durant le développement intra-érythrocytaire de Plasmodium falciparum. Le but de cette étude étant d'identifier un mécanisme potentiel d'internalisation des composants du squelette sous-membranaire de l'érythrocyte dans le parasite. Des études d'immuno-localisation ont montré que la dématine et l'adducine sont internalisées à partir du stade trophozoïte, et sont localisées probablement à la vacuole parasitophore (membrane et/ou lumière). Cette internalisation a été confirmée par des études de fractionnement cellulaire et d'accessibilité à la protéinase K, montrant que la dématine est totalement internalisée, alors l'adducine ne l'est que partiellement, suggérant une localisation de la protéine à la périphérie du parasite. / Plasmodium endurance depends on the ability of the parasite to reorganize the cytosol of the erythrocyte, a terminally differentiated cell, and remodel its skeleton membrane immediately after invasion. In this way the parasite can organize the import/export of the molecules necessary to its survival. The comprehension of cellular trafficking mechanisms which occur during Plasmodium infection is a very important step and fundamental contribute to understand the biology of the malaria parasite.We identified in database of the rodent malaria parasite Plasmodium berghei the gene family sep, corresponding to etramp in P. falciparum, encoding small exported proteins conserved in the genus Plasmodium. SEP proteins (13?16 kDa) contain a predicted signal peptide at the NH2-terminus, an internal hydrophobic region while they differ in their C-terminal region; the genes share the upstream regulative region while differ in the 3' UTR. Despite this, we showed that SEPs have a different timing of expression and a different localization: in the erythrocytic cycle PbSEP1 and PbSEP3 start to be expressed at trophozoite and the same amount of protein is detected also in schizonts and gametocytes, while PbSEP2 is highly detected in mature trophozoites and even more in gametocytes. In mosquitoes stages PbSEP1 and PbSEP3 are expressed only in ookinetes, while PbSEP2 is very abundant in ookinetes, oocysts and in sporozoites of the salivary glands. SEPs also have a different localization in the iRBC: PbSEP1 is targeted to the membrane of the parasitophorous vacuole, while PbSEP2 and 3 are exported beyond the parasite membrane and translocated to the host cell compartment in association with vesicle-like structures. In this study we identified the specific signals necessary for the correct timing of expression and to direct SEP proteins to the vacuolar membrane and to the host cell compartments.The second part of the work was carried out in Montpellier II University and aims to identify the localization of two RBC membrane skeleton components, dematin and adducin, during Plasmodium falciparum infection. Our purpose is to recognize a possible mechanism of internalization of host cytoskeleton components to the parasite compartments. In fact, IFA experiments carried on iRBCs showed that dematin and adducin start to be internalized at trophozoite stage and localize at the periphery of the parasite, most probably at the parasitophoruos vacuole (PV) membrane/lumen. Dematin and adducin internalization during Plasmodium infection is also demonstrated by subcellular fractionation and proteinase K assay: while dematin is fully internalized, adducin is partially protected and suggesting a localization of the protein at the periphery of the parasite where it can be exposed to PK degradation.

Malaria affects the daily lives of more than 2 billion people worldwide and has been estimated to result in 300-500 million clinical cases annually leading to approximately 2 million deaths, mainly caused by the most virulent malaria species, Plasmodium falciparum. The lack of a vaccine and the rapid emergence and spread of drug resistant strains of P. falciparum, necessitate the development of new antimalarials and the identification and validation of new parasite-specific therapeutic targets. Numerous studies directed at interfering with the polyamine biosynthetic pathway in P. falciparum have shown its potential as a target for the development of a new class of antimalarials. The essential nature of P. falciparum spermidine synthase (PfSpdSyn), an enzyme in the polyamine pathway of the parasite warranted the further investigation to find novel lead compounds. The high cost and attrition rate of drug discovery has resulted in the implementation of smart drug discovery platforms in both academia and industry. The strategy implemented in this study involved the development of a dynamic receptor-based pharmacophore model (DPM) of PfSpdSyn complemented by a knowledge-based rational design strategy. The use of pharmacophore models to identify lead compounds has become increasingly popular over the last decade and has been shown to be a reliable method in the drug discovery process. The development of a DPM allows for the incorporation of protein exibility within the drug design process. This methodology results in a wealth of information of the chemical space of the active site and was incorporated in designing new inhibitors against PfSpdSyn using a knowledge-based rational design strategy. The active site of PfSpdSyn was subdivided into four binding regions (DPM1-DPM4) to allow for the identi cation of fragments binding within these speci c binding regions. DPMs representative of the chemical characteristics of each binding region were constructed and subsequently screened against the drug-like subset of the ZINC database. From the screens a total of nine compounds were selected for in vitro testing, complementing each other in exploring specific active site binding characteristics. From these compounds a new lead compound N-(3-aminopropyl)-cyclohexylamine (NAC; Ki 2.8 μM) was identified for PfSpdSyn. NAC was specifically designed to bind in both the putrescine and decarboxylated adenosylmethionine cavities by chemically bridging the catalytic center and was confirmed by kinetic studies. NAC shows great potential for lead optimization to increase its binding affinity. This study then paves the way for lead optimization and possibly the development of a novel antimalarial. The development of a DPM for PfSpdSyn has seen the establishment of this methodology in the Bioinformatics and Computational Biology Unit, Department of Biochemistry at the University of Pretoria. It can be concluded that the development of a DPM complemented by a knowledge-based rational design strategy is an effective approach for the identification of novel lead compounds in the presence of a 3D target structure. This paves the way for more studies on both malaria and other drug targets using DPMs. Copyright / Thesis (PhD)--University of Pretoria, 2009. / Biochemistry / unrestricted

La doxycycline est l’une des molécules recommandées par l’OMS en prophylaxie pour les voyageurs dans les zones d’endémie palustre, en particulier dans les zones de multirésistance. Une étude récente avait suggéré que les isolats de P. falciparum présentaient différents niveaux de sensibilité à la doxycycline et que l’augmentation du nombre de copies de deux gènes, pfmdt ou pftetQ, pouvait être associée à une baisse de sensibilité.Le premier objectif de ce travail a consisté à valider ce modèle à partir d’un nouvel échantillonnage d’isolats africains. Le second objectif était d’évaluer le nombre de copies de ces deux gènes sur des isolats originaires de Thaïlande. Le troisième objectif a consisté à rechercher d’autres sources de résistance en investiguant le polymorphisme des gènes codant l’ARN ribosomal plasmodial potentiellement impliqués dans la résistance in vitro à la doxycycline.Les résultats nous ont permis de confirmer que le nombre de copies des gènes pfmdt ou pftetQ pouvait être impliqué dans la résistance in vitro à la doxycycline en Afrique. Les résultats concernant les isolats Thaï n’ont pas permis de corréler le nombre de copies des gènes pfmdt et pftetQ au phénotype CI50. Ces éléments montrent que ce mécanisme de résistance seul est insuffisant pour expliquer la résistance à la doxycycline ; les résultats sont en faveur d’une résistance médiée par plusieurs gènes.La recherche de points de mutation sur le gène pfssrRNA codant pour la petite sous-unité ribosomale de l’ADN plasmodial n’a pas abouti. D’autres cibles moléculaires sont en cours d’étude pour expliquer les mécanismes de résistance de P. falciparum à la doxycycline. / Doxycycline is currently one of the recommended chemoprophylactic regimens for travellers visiting malaria-endemic, particularly in countries with a high prevalence of resistance to chloroquine and multiresistance. A previous study suggested that increased pfmdt or pftetQ copy number could be associated with a lower susceptibility to doxycycline.The first aim of this study was to validate the pre-established model involving these two molecular markers with other African isolates. The second was to evaluate these markers in P. falciparum isolates coming from a multiresistance area in Thaïland. The third was to investigate the eventual association between the polymorphism in genes encoding ribosomal rRNA and in vitro resistance to doxycycline.The results confirm that pfmdt or pftetQ copy numbers should be involved in in vitro susceptibility to doxycycline in African P. falciparum isolates. The results concerning the Thai isolates indicate that there is no correlation between the pfmdt and pftetQ genes copy numbers and the belonging to the high doxycycline IC50 phenotype; this implies that this mechanism of resistance is not enough by itself to explain resistance to doxycycline; it augurs that the resistance to doxycycline should be controlled by multiple genes, and that these genetic markers could be continent-dependent. The search for points of mutation in isolates from the different doxycycline IC50 phenotypic groups has not resulted with pfssrRNA. Other therapeutic targets are being considered to explain P. falciparum resistance to doxycycline.

The in vitro antimalarial activity of clofazimine and seven of its analogues, all TMP(tetramethyl-piperidyl group)-derivatives except 8669, against the R8-1 and pfUP-1 laboratory strains of Plasmodium falciparum was investigated using a flow cytometric procedure. The flow cytometric method was compared with microscopy and radiometry for efficiency in quantitating the level of parasitemia in malaria cultures. The flow cytometric method compared well, as determined by the 81and and Altman measure of agreement, with both microscopy and radiometry and was chosen for use in this study due to its speed, precision and convenience (includes a fixing step that allows samples to be evaluated at anyone time). The riminophenazine agents were found to exhibit antimalarial action of varying degrees: B669, B4100, B4103, B4112 and B4158 showed the best activity followed by B4121 and B4169. Clofazimine did not exhibit any activity at concentrations up to 2µg/ml in this system. Their effective concentrations in vitro were comparable to that of standard antimalarial agents such as chloroquine. The agents B4103 and B4112 exhibited additive antimalarial activities when combined with chloroquine. The inclusion of the TMP group and extent of halogenation of six of the riminophenazines tested indicate that it is these structural properties which are the major determinants of the antiplasmodial activity. This is the first study to establish an antiplasmodial activity of riminophenazines and further tests are necessary to establish their antiparasitic mode of action and therapeutic potential in animal models of experimental chemotherapy. / Dissertation (MSc)--University of Pretoria, 2010. / Pharmacology / unrestricted

Please read the abstract in the section 00front of this document / Dissertation (MSc(Biochemistry))--University of Pretoria, 2004. / Biochemistry / unrestricted

Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine Johannesburg, 2004 / Malaria is the most prevalent parasitic disease in the world and the emergence of drug resistant strains of Plasmodium falciparum has made the search for new antimalarial drugs important. Protein kinases play an important role in cellular function and the phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is implicated in diverse cellular processes such as glucose transport, cell survival and proliferation. A homology based approach identified an open reading frame (ORF) coding for the catalytic region of part of the 6.4 Kb ORF of PFE0765w gene sequence found at plasmoDB. The ORF consisted of 1 758 base pairs which coded for a 586 amino acid protein with a molecular weight of 68.5 KDa. The PfPI3K ORF was amplified from P.falciparum DNA, subcloned into an expression vector and the sequence verified. Analysis of the expressed protein obtained by Western blotting and probing with anti-His monoclonal antibody showed a protein of 68.5 KDa as well as some smaller products. / IT2018

Malaria is a terrible disease caused by a protozoan parasite within the Plasmodium genus, claiming the lives of hundreds of thousands of people yearly, the majority of whom are children under the age of five. Of the five species of Plasmodium causing malaria in humans, P. falciparum is responsible for most of the death toll. An increase in malaria cases was detected between the years 2016 to 2017 according to the World Malaria Report of 2017, despite control efforts. The rapid development of resistance within P. falciparum against antimalarials has led to the use of artemisinin combinational therapy as the current gold standard for malaria treatment. Yet decreased parasite clearance demonstrates that using combination therapy is insufficient in maintaining current antimalarials’ effectiveness against these resistant parasites. Hence, novel compounds with a mode of action (MoA) different than current antimalarials are required. Though phenotypic screening has delivered thousands of promising hit compounds, hit-to-lead optimisation is still one of the rate-limiting steps in pre-clinical antimalarial drug development. While knowing the exact target or MoA is not required to progress a compound in a medicinal chemistry program, identifying the MoA early can accelerate hit prioritization, hit-to-lead optimisation and preclinical combination studies in malaria research. In this study, we assessed machine learning (ML) approaches for their ability to stratify antimalarials based on transcriptional responses associated with the treatments. From our results, we conclude that it is possible to identify biomarkers from the transcriptional responses that define the MoA of compounds. Moreover, only a limited set of 50 genes was required to build a ML model that can stratify compounds with similar MoA with a classification accuracy of 76.6 ± 6.4%. These biomarkers will help stratify new compounds with similar MoA to those already defined with our strategy. Additionally, the biomarkers can also be used to monitor if the MoA of a compound has changed during hit-to-lead optimisation. This work will contribute to accelerating antimalarial drug discovery during the hit-to-lead optimisation phase and help the identification of compounds with novel MoA. / Dissertation (MSc)--University of Pretoria, 2019. / Biochemistry / MSc / Unrestricted

ABSTRACT Malaria is considered by many to be the most important infectious diseases a of humans in the world. Since the dawn of civilization, malaria has left it detrimental fingerprints all over history. The World Health Organization reports an estimated 300 million people infected with the protozoan parasite worldwide and a mortality rate that claims more than one million victims annually. Of the four species known to infect huinans with malaria, Plasmodium falciparum is by far the most pathogenic and efforts to inhibit its ~idespread epidemiology have been minimal. Only a handful of antimalarials are currently available to combat the disease and even they are loosing efficacy due to the increased prevalence of drug resistance. Therefore, it is important to discover new modes of treatment. Inhibitors of protein prenylation have been identified by our laboratory to have the potential to be a new class of antimalarials. Protein prenylation is a relatively new research interest that studies the functional significance of famesyl (C15) and geranylgeranyl (C20) posttranslational modifications in a variety of cellular regulatory processes. These prosthetic groups are thought to be absolutely essential for the activity and cellular localization of prenylated proteins. Inhibitors of protein prenylation act through perturbation of the subcellular localization of these important proteins. In an effort to understand the mechanism of action of prenyl transferase inhibitors, it is important to characterize malarial prenylated proteins. This thesis initiated studies in characterizing putative prenylated proteins and has focused on three P.falciparum proteins, PfPTP, PtRab6, and PfDnaJ. Attempts have been made to clone and over-express recombinant proteins and generate antibodies for functional analysis.