Lectins are a class of proteins that bind to carbohydrates with a high degree of specificity. They are involved in various cellular processes such as, host - pathogen interactions, targeting of proteins within cells, cell - cell interaction, cellular segregation and development. They serve as important tools for probing the carbohydrate structures in biological systems such as cell membranes and also as model systems for elucidating protein - carbohydrate interactions. Lectins are distributed ubiquitously in nature ranging from microorganisms to the plants and animals. Plant lectins are a group of proteins that according to a recently updated definition comprise all plant proteins possessing at least one non-catalytic domain that binds reversibly to specific mono- or oligosaccharide. The majority of all currently known plant lectins may be classified into four major groups - (1) Legume lectins, (2) Chitin-binding lectins, (3) Type 2 Ribosome inactivating proteins and the (4) Monocot mannose binding lectins. The monocot mannose binding lectins are an extended superfamily of structurally and evolutionarily related proteins. Till now these proteins have been isolated from the following families, namely, Amaryllidaceae, Affiaceae, Araceae, Orchidaceae, Iridaceae and Li/iaceae. They exhibit marked sequence homology and a unique specificity for mannose. At present there is a wide interest in the monocot mannose-binding lectins because of: (1) their exclusive specificity towards mannose, (2) their anti - retroviral activity and (3) their potent entomotoxic properties. Of particular interest are lectins from the bulbs of garlic (Allium sativum) and ramson (A. ursinum), which contain more than one type of lectin. The first report of the presence of lectins in the bulbs of garlic {Allium sativum agglutinin, ASA) was made by Van Damme et al in 1991. Bulbs of garlic are known to accumulate two types of mannose binding lectins, the heterodimeric, ASAI and the hornodimeric, ASAII. Though these two lectins differ in the lengths of their polypeptide chains, they exhibit marked similarities with respect to their primary sequence, post translational modifications, serological properties, immunochemical attributes as well as carbohydrate binding properties. This thesis describes the successful cloning of the ASAI gene from the garlic genomic DNA and expression of the functional recombinant protein in insect cell lines. ASAI was subsequently characterized for its carbohydrate binding specificity by means of a sensitive enzyme based assay. Finer insights into this sugar binding topology of ASAI for its complementary ligands was obtained from the surface plasmon resonance studies. Lastly, the folding behaviour as well as an estimate of its conformational stability was investigated by differential scanning calorimetric and equilibrium solution denaturation studies. Chapter 1 provides a comprehensive review on lectins pertaining to their definition, historical background, occurrence in nature, three dimensional structure and architecture, modes of bonding, biological functions and implications as well as their applications in biomedical research. Chapter 2 describes the isolation and purification of the heterodimeric lectin, ASAI in two steps using affinity chromatography followed by gel filtration chromatography from the bulbs of garlic. The purified ASAI was then characterized for their serological, physico- and immuno-chemical properties by means of capillary electrophoresis, hemagglutination activity and generation of antisera against ASAI in rabbits. Chapter 3 revolves around the cloning of the gene encoding ASAI by PCR amplification from garlic genomic DNA. The authenticity of the ASA gene was established by means of gene sequencing, which in turn provided us with the primary sequence of this lectin. With the ASAI clone established innumerable attempts, as highlighted in the chapter, were made to express the functional protein in bacteria. All attempts yielded pure recombinant garlic lectin with no detectable activity. This prompted us to shift our efforts into expression of the recombinant protein in the baculovirus expression system using the Sf21 insect cell lines and the Autographa californica nuclear polyhedrosis virus (AcNPV). The choice of this system proved beneficial as we obtained functional recombinant garlic lectin with its hemagglutinating activity comparable to the native protein. Chapter 4 highlights the design of an elegant coupled enzyme-based colorimetric assay (Enzyme Linked Lectin Adsorbent Assay) for elucidation of the carbohydrate binding specificity of ASAI. This expansive and extensive study involved the assay of a wide range of mannooligosaccharides in order to gain an insight into the sugar binding details of ASAI. ASAI recognizes monosaccharides in the mannosyl configuration. The potencies of the ligands for ASAI is shown to increase in the following order: Mannobiose < Mannotriose Mannopentaose Man9 oligosaccharide. Mannononase glycopeptide (Man9GlcNAc2Asn), the highest oligomer studied exhibited the greatest binding affinity suggesting ASAI to possess a preference for cluster of terminal αl-2-linked mannosyl residues at the non-reducing end. This kind of exquisite specificity is unique in the lectins described so far. Among the glycoproteins assayed, invertase, soyabean agglutinin and ovalbumin displayed high binding affinity. Chapter 5 unravels the fine specificity of the mannose containing carbohydrate moieties for binding to ASAI with emphasis on their kinetics of binding. This has been achieved by invoking the principle of surface plasmon resonance allowing measurement of bimolecular interactions in real time. This investigation corroborates our earlier study about the special preference of garlic lectin for terminal a α1-2 linked mannose residues. Increase in binding propensity can be directly correlated to the addition of αl-2 linked mannose to the mannooligosaccharide at its non-reducing end. An analyses of these data reveals that the α1-2 linked terminal mannose on the α1-6 arm to be the critical determinant in the recognition of mannooligosaccharides by the lectin. While kI increases progressively from Man3 to Man7 derivatives, and more dramatically so for Man8 and Man9 derivatives, k-1 decreases relatively much less gradually from Man3 to Man9 structures. An unprecedented increase in the association rate constant for interaction with ASAI with the structure of the oligosaccharide ligand constitutes a significant finding in protein-sugar recognition. Chapter 6 deals with the thermal unfolding of ASAI, characterized by differential scanning calorimetry and circular dichroism which shows it to be highly reversible and can be defined as a two-state process in which the folded dimer is converted directly to the unfolded monomers (A2 2U). Moreover, its conformational stability has been determined as a function of temperature; GdnCl concentration and pH using a combination of thermal and isothermal GdnCl induced unfolding monitored by DSC, far-UV CD and fluorescence, respectively. Analysis of these data yielded the heat capacity change upon unfolding (∆CP) as also the temperature dependence of the thermodynamic parameters, namely, ∆G, ∆H, ∆S. The protein appears to attain a completely unfolded state irrespective of the method of denaturation. The absence of any folding intermediates suggests the quaternary interactions to be the major contributor to the conformational stability of the protein, which correlates very well with its X-ray structure. The final chapter summarizes the findings reported in the thesis.

Snake venoms are a complex mixture of polypeptide and other molecules that adversely affect multiple homeostatic systems within their prey in a highly specific and targeted manner. Amongst the most potently toxic venoms in the world are those of the Australian venomous snakes, which belong almost exclusively to the elapid family. Their venoms posses a number of unique properties by which they target the mammalian cardiovascular and neuromuscular systems and are the focus for the identification of novel pharmacologically interesting compounds which may be of diagnostic or therapeutic benefit. Although much is known about the biochemical properties of Australia snake venoms as a whole, little research attention has focused upon individual components at the molecular level. This thesis describes the cloning, characterisation and comparative analysis of a number of unique toxins from the venom gland of the coastal taipan (Oxyuranus scutellatus) and a total of seven other related Australian snakes. These include the factor X- and factor V-like components of a prothrombin activator that causes a highly coagulable state in mammals. Comparative analysis of the sequences identified in this study, along with recombinant expression of an active form of the factor X-like component, provides important information on the structural, functional and evolutionary relationships of these molecules. Numerous other toxins were similarly identified and characterised including a pseudechetoxin-like protein, multiple phospholipase A2 enzymes and neurotoxin isoforms as well as vasoactive venom natriuretic peptides. Identified transcripts included not only toxin sequences but also other cellular peptides implicated in toxin processing, including a calglandulin-like protein. This thesis is the first description of the majority of these molecules at either the cDNA or protein level, and provides a means to study the activity of individual components from snake venoms and probe their function within the systems they specifically target. This study represents the most detailed and comprehensive description to date of the cloning and characterisation of different genes associated with envenomation from Australian snakes.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-12-15T16:53:37Z No. of bitstreams: 2 Dissertação - Luiz Augusto Pereira - 2004.pdf: 6566857 bytes, checksum: 144c5557e8a138c6a21c528ad0262aa8 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2014-12-15T17:01:19Z (GMT) No. of bitstreams: 2 Dissertação - Luiz Augusto Pereira - 2004.pdf: 6566857 bytes, checksum: 144c5557e8a138c6a21c528ad0262aa8 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-12-15T17:01:19Z (GMT). No. of bitstreams: 2 Dissertação - Luiz Augusto Pereira - 2004.pdf: 6566857 bytes, checksum: 144c5557e8a138c6a21c528ad0262aa8 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2004-05-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / An antigen of Paracoccidioides brasiliensis (Pb) was gel isolated and characterized. Endoproteinase Lys-C digested peptides of the purified protein which presented, molecular mass of 29-kDa and pI of 5.8, were subjected to sequence analysis of its amino acids. Search at databases comparing the sequence of amino acids from the three peptides of the native protein, revealed strong homology to triosephosphate isomerase (TPI: E.C. 5.3.1.1) from several sources. The complete cDNA and gene encoding PbTPI were obtained and both contained an open reading frame predicted to encode a 249 amino acid protein that presented all the peptides characterized in the native PbTPI. The Pbtpi gene contained 6 exons interrupted by 5 introns. Analysis performed with the deduced PbTPI suggested its usefulness in providing phylogenetic relatedness, as well as evidenced the correlation between the phylogeny provided by the deduced proteins and introns positions in the cognate genes. The immunological reactivity of PbTPI was examined. The complete coding cDNA of PbTPI was over expressed in an Escherichia coli host to produce high levels of recombinant fusion protein with glutathione S-transferase (GST) that has been purified by affinity chromatography. The purified recombinant TPI was recognized by sera of patients with confirmed Paracoccidioidomycosis and not by sera of healthy individuals. Thus, recombinant PbTPI can be a valuable addition to the still small arsenal of P. brasiliensis immunoreactive proteins, which could be tested for incorporation in assays for serodiagnosis of the disease. / Um antígeno de Paracoccidioides brasiliensis (Pb) foi isolado do gel e caracterizado. Os peptídeos digeridos por Endoproteinase Lys-C da proteína purificada, que apresentou uma massa molecular de 29-kDA e pI de 5.8, foram submetidos à análise da seqüência de aminoácidos. Uma busca em bancos de dados de seqüências de aminoácidos comparados com os três peptídeos da proteína nativa revelou forte homologia com triose fosfato isomerase (TPI: E.C. 5.3.1.1) de vários organismos. O cDNA e o gene completos que codificam para PbTPI foram obtidos, e ambos contém uma provável ORF que codifica para uma proteína com 249 aminoácidos que apresenta todos os peptídeos caracterizados na PbTPI nativa. O gene Pbtpi apresentou 6 exons interrompidos por 5 introns. Análises realizadas com a PbTPI deduzida sugerem sua utilidade em prover relações filogenéticas, como também evidenciou a correlação entre a filogenia gerada pelas proteínas deduzidas e as posições dos introns nos genes cognatos. A reatividade imunológica da PbTPI foi examinada. O cDNA completo que codifica para PbTPI foi altamente expresso no hospedeiro Escherichia coli, produzindo altos níveis de uma proteína recombinante fundida a glutathione S-transferase (GST), que foi purificada por cromatografia de afinidade. A TPI recombinante purificada foi reconhecida por soros de pacientes com paracoccidioidomicose confirmada e não por soros de indivíduos saudáveis. Assim, PbTPI recombinante pode ser uma adição valiosa para o pequeno arsenal de proteínas imunoreativas de P. brasiliensis, que poderiam ser testadas por incorporação em ensaios de sorodiagnóstico da doença.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-04-04T13:14:56Z No. of bitstreams: 1 diegopandelojose.pdf: 913814 bytes, checksum: 7e149c7c8e0bee3253c4894426a6892c (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-04-04T15:36:35Z (GMT) No. of bitstreams: 1 diegopandelojose.pdf: 913814 bytes, checksum: 7e149c7c8e0bee3253c4894426a6892c (MD5) / Made available in DSpace on 2017-04-04T15:36:35Z (GMT). No. of bitstreams: 1 diegopandelojose.pdf: 913814 bytes, checksum: 7e149c7c8e0bee3253c4894426a6892c (MD5) Previous issue date: 2009-03-03 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / O gênero Lippia pertence à família Verbenaceae, inclusa no clado Asteridaee, ordem Lamiales, compreendendo aproximadamente 175 gêneros e 2800 espécies, onde muitos gêneros apresentam plantas com propriedades medicinais e ornamentais. A espécie Lippia alba, originária da América do Sul, também ocorre no Brasil e é uma das mais estudadas do gênero Lippia. Ela floresce durante o ano todo e recebe grande destaque no gênero, devido às suas inúmeras propriedades medicinais. O óleo essencial de Lippia alba é composto basicamente por sesqui e monoterpenos, que são as substâncias responsáveis por suas propriedades medicinais. O objetivo central do presente trabalho foi clonar e analisar a expressão de dois potenciais genes codificadores de terpeno sintases em Lippia alba. Através do alinhamento de genes codificadores de monoterpeno sintases caracterizadas, primers degenerados foram desenhados dentro de regiões conservadas e utilizados para se obter a clonagem de genes codificadores de terpeno sintases em Lippia alba. Dois potenciais genes codificadores de terpeno sintases foram clonados, LaTPS12 e LaTPS23. Após a clonagem, técnicas de RT-PCR semiquantitativo foram empregadas para análises de expressão desses dois genes em diferentes estágios foliares e em três diferentes quimiotipos de Lippia alba. Os resultados mostraram que em folhas situadas no quarto segmento nodal o gene LaTPS12 apresenta maior nível de expressão. A diferença na expressão do gene LaTPS23 foi menos acentuada nos três quimiotipos analisados em relação ao gene LaTPS12, que apresentou uma expressão diferencial. Análises filogenéticas foram realizadas comparando-se as seqüências desses dois genes com outros genes codificadores de terpeno sintases já caracterizadas de diferentes espécies de plantas. De acordo com essas análises, LaTPS12 e LaTPS23 pertencem à classe TPS-b, que é composta principalmente por monoterpeno sintases de angiospermas. / The genus Lippia belongs to Verbenaceae family, Asteridaee, order Lamiales. This family comprises about 175 genus and 2800 species, and many of them have medicals and ornamentals proprierties. Lippia alba is native from South America, and is also found in Brazil and is the most studied species of the genus Lippia. This plant blooms throughout the year and has great importance due to its medicinal properties. The Lippia alba essential oils are composed by sesquiterpenes and monoterpenes conferring its medicinal properties. The aim of this work was to clone and to analize gene expression of putative terpene synthases genes (TPS) in Lippia alba. Alignment of TPS genes was used to design degenerate primers into conserved domains for cloning of these genes in Lippia alba. We have cloned two putative TPS genes, LaTPS12 and LaTPS23. After cloning, semiquantitative RT-PCR was employed to expression analysis of these two genes in different leaf stages and among three different chemotypes of Lippia alba. The result of expression level showed that LaTPS12 occurred at higher level in leaves located in fourth nodal segment and showed a marked differential expression among the chemotypes. The difference of expression of the LaTPS23 was less prominent comparing the three studied chemotypes. We performed a phylogenetic analysis in order to compare the LaTPS12 and LaTPS23 to others TPS genes in different plant species. The results showed that these LaTPS12 and LaTPS23 belong to the class TPS-b, which comprises mainly angiosperms monoterpene synthases genes.

Les abcès cérébraux sont des infections potentiellement mortelles, entraînant souvent des séquelles graves. La prise en charge médicale en reste empirique en raison d’un manque de connaissance approfondie des microorganismes responsables de cette condition. Dans la plupart des laboratoires microbiologiques, le diagnostic d’abcès cérébral est basé sur la culture du pus recueilli chirurgicalement. Malheureusement, cette procédure a de nombreuses limites et ne permet l’identification que d’une petite partie de la population microbienne en cause. L’amplification par PCR et le séquençage du gène codant la fraction 16S de l’ADN ribosomal ont récemment été utilisées pour surmonter les limites de la culture, et ont été démontré leur efficacité dans la documentation des infections bactériennes. Malheureusement, cette procédure présente un degré de discrimination limité en cas d’infection polymicrobienne. Des études métagénomiques de flores complexes de l’homme, basées sur une combinaison de PCR, clonage et séquençage des produits de PCR se sont avérées utiles pour évaluer la diversité bactérienne des flores dentaires, vaginales et intestinales. Nous avons appliqué cette technique à des échantillons d’abcès cérébral pour étudier la flore associée à cette maladie. Dans une première étape, nous avons réalisé une enquête en utilisant la culture et les techniques moléculaires. Le but de cette étude était d’analyser et d’évaluer les bactéries de la flore responsable des abcès cérébraux, en comparant la culture à trois techniques moléculaires basées sur le gène 16S rDNA, incluant le séquençage direct, le clonage suivi de séquençage par méthode de Sanger, et le séquençage direct des produits de PCR par pyroséquençage. Cette enquête a déterminé que la variété des espèces bactériennes associée aux abcès cérébraux est beaucoup plus grande que précédemment décrite, et inclut de nombreuses bactéries anaérobies et des bactéries incultivables de la flore buccale. Cette étude préliminaire a identifié 49 agents bactériens différents, et a permis l’identification de 27 bactéries jamais détectées auparavant dans des abcès du cérébraux, dont 15 n’avaient jamais été cultivées. Un tel nombre d’espèces bactériennes impliquées dans les abcès cérébraux a motivé l’étude de 51 nouveaux spécimens dans le but de décrire plus en détail la flore associée aux abcès cérébraux en fonction de leurs étiologies. Ainsi, nous avons effectué une analyse métagénomique, basé sur le gène 16S rDNA, de 51 patients ayant développé un abcès cérébral. Notre stratégie a été beaucoup plus discriminatoire et a permis à l’identification d’un plus grand nombre de bactéries que la culture et l’amplification et le séquençage direct de l’ANRr 16S. La combinaison des données de 71 patients (20 de la première étude et 51 de la deuxième étude) a permis l’identification de plusieurs associations à l’aide de la méthode de data mining.En outre, notre étude a permis l’identification de deux nouvelles bactéries, la première étant une nouvelle espèce de genre Staphylococcus (Staphylococcus massiliensis) et la seconde étant une bactérie anaérobie qui représente une nouvelle espèce dans un nouveau genre au sein du phylum des Bacteroidetes (Phocaeicola abscesses). En outre, nous avons décrit deux cas inhabituels d’abcès du cerveau, à Mycoplasma hominis après curetage utérin, et à Nocardia carnea chez un greffé rénal. Malgré les limites inhérentes à la procédure de clonage, nos résultats suggèrent que le clonage et le séquençage de gène DNAr 16S est une méthode très performante pour identifier les agents bactériens associés aux abcès cérébraux. / Brain abscess is a life-threatening infection with frequent serious sequelae. The medical management remains empirical due to a lack of comprehensive knowledge of the microorganisms responsible for this condition. In most microbiology laboratories the diagnosis of brain abscess is based on culture from pus collected surgically. Unfortunately, this procedure has many limitations and reveals only a small portion of the true microbial population. PCR-amplified 16S rDNA sequencing has recently been used to overcome the limitations of culture-based bacterial detection in brain abscess pus, and it was demonstrated to be effective in the documentation of monomicrobial infections. Unfortunately, this procedure failed to discriminate among polymicrobial floras.Metagenomic studies of complex human floras using a combination of 16S rDNA PCR and cloning-sequencing of PCR products proved useful to evaluate the bacterial diversity of dental, vaginal and intestinal floras. Thus, we applied this technique to brain abscess samples to study the flora associated with this condition. In a first step, we performed an investigation using culture and molecular techniques. The purpose of this investigation was to analyze and evaluate the bacterial flora responsible for brain abscess by comparing standard culture technique to three techniques using 16S rDNA amplification, that is, direct sequencing, multiple sequencing following cloning, and multiple sequencing via high throughput pyrosequencing. This investigation has determined that the variety of brain abscess-associated bacterial species is much larger than previously reported, and it includes many anaerobes and uncultured bacteria from the oral cavity flora. This preliminary study identified 49 distinct brain abscess bacterial agents, and enabled the identification of 27 bacteria never detected before in brain abscess, 15 of which were uncultured.Such a high number of bacterial species involved in brain abscess prompted the study of 51 new specimens in an effort to describe further the flora associated with brain abscesses and their etiologies. Thus, we performed a 16S rDNA-based metagenomic analysis of cerebral abscesses from 51 patients. Our strategy was significantly more discriminatory and enabled the identification of greater number of bacterial taxa, than culture and conventional 16S rDNA PCR/sequencing, respectively. The combination of data from 71 patients (20 from the first study and 51 from the second study) enabled the identification of several associations using the data mining analysis. Also, these studies permitted the identification of two novel bacteria, the first being a novel Staphylococcus species (Staphylococcus massiliensis) and the second being a novel anaerobic bacterium that represents a novel species in a new genus within the phylum Bacteroidetes (Phocaeicola abscesses). In addition, we reported tow unusual cases of brain abscess, the first case was a Mycoplasma hominis brain abscess following uterus curettage and the second case was a Nocardia carnea infection in a kidney transplant recipient patient.Despite limitations inherent to the cloning procedure, our results suggest that cloning and sequencing of PCR-amplified 16S rDNA is a highly valuable method to identify bacterial agents of brain abscesses.

Hepatit-B är en leversjukdom som orsakas av hepatit-B-virus (HBV) vilket är en kapslad DNA-virus. Kapsidprotein (Cp) har stor betydelse i virusets livscykel exempelvis DNA-replikation, interaktion med värdceller och andra virala glykoproteiner. HBV, som många andra virus, tar sig in i cellen genom att binda till cellreceptorer. Transferrinreceptor är en välkänd receptor som mögliggör virus inträde i cellen genom att binda till virusproteiner, intraktionen sker i apikala domänen i TfR. Båda Cp149, kapsidsammansättnings domänen i Cp, och apikala domänen i TfR är betydelsefulla ändamål för utveckling av antivirala läkemedel. Syftet med arbetet var att klona och uttrycka olika varianter av ett nytt modifierat Cp149, där Cp149 har modifierats med AP01 (lösliga formen av apikala dömanen),  och analysera intraktioner mellan proteinerna och viralt glykoprotein, MGP1. Modifierade proteingener klonades i plasmid (pET-11a) med hjälp av rekombinant DNA-teknik och användning av restriktionsenzymer NdeI och BamHI. Agarosgelelektrofores och DNA-sekvensering användes för att  kontrollera förekomst av eftersökta DNA-sekvenser. Nya plasmider fördes över till bakterieceller, Escherichia coli, och  proteinutrycket inducerades i bakteriecellerna genom kemiskbehandling. Framrenade proteiner från respektive provlösning analyserades med Sodium dodecyl sulphate polyakrylamid gel electrophoresis (SDS-PAGE) och proteinernas funktion undersöktes med flödescytometri genom att besämma bindningsförmågan till MGP1, som uttrycktes på jästceller, i närvaro av TfR. Rekombinant plasmid innehållande proteingen kodande Cp149 för varianter A-D samt F lyckades att framställas. Resultatet från SDS-PAGE påvisade inga tydlyga protein-band och flödescytometri resultatet var svårt att bedömma, troligen då ytterliggare proteinupprening behövdes för att isolera kapsidproteinerna. Syftet med arbetet har erhållits delvis och fortsatt undersökningar på nya proteiner förslås. / Hepatitis B is one av the major worldwide health problems that is caused by enveloped DNA virus, Hepatitis B virus (HBV). HBV’s capsid protein (Cp) has an important role in the virus life cycle, för example DNA replication, intraction with host cells, and other viral glycoproteins. HBV, like many other viruses, enters the cells by binding to cell receptors. Transferrin receptor (TfR) is a well-known receptor that enables virus entry into the cell by binding to viral proteins. The interaction takes place in the apical domain of TfR. Both Cp149, the capsid’s composition domain of Cp, and the soluble form of the apical domain, AP01, from TfR are important builing parts to be explored as starting building blocks for the development of antiviral therapeutics. Modification of Cp149 with AP01 is an interesting combination to produce new protein-based drugs to prevent viral infections. The aim of this project was to clone and express six different variants of a AP01 modified Cp149 protein as a starting points to analyze interactions between the proteins and Machupo virus glykoprotein 1 (MGP1). All target DNA templates and plasmids (pET-11a) were digested with restriction enzymes, NdeI and BamHI, and ligated by T4 DNA ligase. Agarose gel electrophoresis and DNA sequencing were used as validation methods to confirm the presence of desired and corrected DNA sequences, respectively, during gene cloning. DNA transformation and induction of Escherichia coli cells was used to express the desired proteins. The purified proteins were validated for their binding ability to MGP1, expressed on yeast cells by flow cytometry in a competition assay with TfR. Recombinant plasmid including the expected DNA sequence encoding Cp149 for variants A-D and F was successfully produced. There was no clear detection of protein bands on Sodium dodecyl sulphate polyakrylamid gel electrophoresis (SDS-PAGE) gel and flow cytometry results were difficult to interpret due to insufficient protein purification during ammonium sulphate percipitation. The purpose of the project has been obtained partially and more studies have to be carried out to produce pure proteins that can be used for further analysis.

O Diabetes mellitus (DM) do tipo 1 é uma doença causada pela destruição, por mecanismo auto-imune, das células beta das ilhotas pancreáticas, produtoras de insulina. O tratamento convencional da doença é realizado por meio de injeções diárias de insulina exógena. O transplante de ilhotas pancreáticas inclui-se, atualmente, como uma das alternativas terapêuticas à insulinoterapia. Entretanto, para atingir a insulino-independência, é necessário transplantar um grande número de ilhotas por paciente. O conhecimento do mecanismo de proliferação das células beta pode possibilitar a realização do transplante a partir da expansão celular ex vivo. A glicose é um dos principais indutores da proliferação de células beta. Neste trabalho, foi estabelecida e executada a tecnologia de isolamento e purificação de ilhotas pancreáticas humanas, visando sua estimulação com glicose. Para identificar genes regulados por glicose nestas ilhotas, foi utilizada a técnica de hibridização subtrativa SSH, associada ao rastreamento da biblioteca através de macroarranjos de DNA. Num primeiro rastreamento, foram identificados dois fragmentos gênicos induzidos pela glicose. Um destes apresentou homologia com uma proteína hipotética humana de função desconhecida e o segundo com o receptor de polipetídeo pancreático. Este trabalho permitiu a identificação de novos genes regulados pela glicose em ilhotas pancreáticas humanas, os quais podem estar relacionados à proliferação celular deste tecido. / Type 1 Diabetes mellitus (T1DM) is caused by autoimmune destruction of the insulin-producing pancreatic islet b-cells. Treatment is generally approached by daily subcutaneous injections of exogenous insulin. Nowadays, pancreatic islet transplantation is considered as an effective alternative treatment to insulin therapy. However, in order to reach insulin-independence, a large number of islets is required for each patient. Knowledge of the mechanisms regulating islet b-cell proliferation may allow ex-vivo b-cell expansion prior to transplant. Glucose is considered one of the main inducers of islet b-cells proliferation. We established and executed the technology of human islet isolation and purification. The islets were then stimulated in culture with glucose. In order to identify glucose-regulated genes in cultured human islets, we utilized the suppression subtractive hybridization (SSH) method, followed by cDNA library screening by DNA macroarrays. Preliminary screening allowed us to isolate two cDNAs displaying glucose regulation, one of which is similar to a human hypothetical protein of unknown function and the other shows similarity to the pancreatic polypeptide receptor. This work allowed identification of glucose-regulated genes in human pancreatic islets, which may be related to cell proliferation in this tissue.

O Diabetes mellitus (DM) do tipo 1 é uma doença causada pela destruição, por mecanismo auto-imune, das células beta das ilhotas pancreáticas, produtoras de insulina. O tratamento convencional da doença é realizado por meio de injeções diárias de insulina exógena. O transplante de ilhotas pancreáticas inclui-se, atualmente, como uma das alternativas terapêuticas à insulinoterapia. Entretanto, para atingir a insulino-independência, é necessário transplantar um grande número de ilhotas por paciente. O conhecimento do mecanismo de proliferação das células beta pode possibilitar a realização do transplante a partir da expansão celular ex vivo. A glicose é um dos principais indutores da proliferação de células beta. Neste trabalho, foi estabelecida e executada a tecnologia de isolamento e purificação de ilhotas pancreáticas humanas, visando sua estimulação com glicose. Para identificar genes regulados por glicose nestas ilhotas, foi utilizada a técnica de hibridização subtrativa SSH, associada ao rastreamento da biblioteca através de macroarranjos de DNA. Num primeiro rastreamento, foram identificados dois fragmentos gênicos induzidos pela glicose. Um destes apresentou homologia com uma proteína hipotética humana de função desconhecida e o segundo com o receptor de polipetídeo pancreático. Este trabalho permitiu a identificação de novos genes regulados pela glicose em ilhotas pancreáticas humanas, os quais podem estar relacionados à proliferação celular deste tecido. / Type 1 Diabetes mellitus (T1DM) is caused by autoimmune destruction of the insulin-producing pancreatic islet b-cells. Treatment is generally approached by daily subcutaneous injections of exogenous insulin. Nowadays, pancreatic islet transplantation is considered as an effective alternative treatment to insulin therapy. However, in order to reach insulin-independence, a large number of islets is required for each patient. Knowledge of the mechanisms regulating islet b-cell proliferation may allow ex-vivo b-cell expansion prior to transplant. Glucose is considered one of the main inducers of islet b-cells proliferation. We established and executed the technology of human islet isolation and purification. The islets were then stimulated in culture with glucose. In order to identify glucose-regulated genes in cultured human islets, we utilized the suppression subtractive hybridization (SSH) method, followed by cDNA library screening by DNA macroarrays. Preliminary screening allowed us to isolate two cDNAs displaying glucose regulation, one of which is similar to a human hypothetical protein of unknown function and the other shows similarity to the pancreatic polypeptide receptor. This work allowed identification of glucose-regulated genes in human pancreatic islets, which may be related to cell proliferation in this tissue.