Bacteriophage Lambda (λ) has played a historical role as an essential model contributing to our current understanding of molecular genetics. Lambda’s major capsid protein “gpD” occurs on each capsid at 405 to 420 copies per phage in homotrimeric form and functions to stabilize the head and likely to compact the genomic DNA. The interesting conformation of this protein allows for its exploitation through the genetic fusion of peptides or proteins to either the amino or carboxy terminal end of gpD, while retaining phage assembly functionality and viability. The lytic nature of λ and the conformation of gpD in capsid assembly makes this display system superior to other display options. Despite previous reports of λ as a phage display candidate, decorative control of the phage remains an elusive concept. The primary goal of this study was to design and construct a highly controllable head decoration system governed by two genetic conditional regulation systems; plasmid-mediated temperature sensitive repressor expression and bacterial conditional amber mutation suppression. The historical λ Dam15 conditional allele results in a truncated gpD fragment when translated in nonsuppressor, wild-type E. coli cells, resulting in unassembled, nonviable progeny. I sequenced the Dam15 allele, identifying an amber (UAG) translational stop at the 68th codon. Employing this mutant in combination with a newly created isogenic cellular background utilizing the amber suppressors SupD (Serine), SupE (Glutamine), SupF (Tyrosine) and Sup— (wild type), we sought to control the level of incorporation of undecorated gpD products. As a second dimension, I constructed two separate temperature-inducile plasmids whereby expression of either D or D::eGFP was governed by the λ strong λ CI[Ts]857 temperature-sensitive repressor and expressed from the λ PL strong promoter. Our aim was to measure the decoration of the λ capsid by a D::gfp fusion under varying conditions regulated by both temperature and presence of suppression. This was achieved utilizing this controllable system, enabling the measurement of a variable number of fusions per phage based on diverse genetic and physical environments without significantly compromising phage viability. Surprisingly, both SupE and SupF showed similar levels of Dam15 suppression, even though sequencing data indicated that only SupE could restore the native gpD sequence at amino acid 68 (Q). In contrast, SupD (S), conferred very weak levels of suppression, but imparted an environment for very high decoration of gpD::eGFP per capsid, even at lower (repressed) temperatures. The presence of albeit few wild-type gpD molecules allowed for an even greater display than that of the perceived “100%” decoration scenario provided by the nonsuppressor strain. It appears that the lack of wild-type gpD does not allow for the space required to display the maximum number of fusions and in turn creates an environment that affects both phage assembly and therefore phage viability. Finally, the use of Western blotting, confirmed the presence of gpD::eGFP fusion decoration by employing a polyclonal anti-eGFP antibody. The significance of this work relates to the unique structure of λ’s capsid and its ability to exploit gpD in the design of controlled expression, which is guiding future research examining the fusion of different therapeutic peptides and proteins. Furthermore this approach has important implications specifically for the design of novel vaccines and delivery vehicles for targeted gene therapy in which steric hindrance and avidity are important concerns. The execution of this project employed basic bacterial genetics, phage biology and molecular biology techniques in the construction of bacterial strains and plasmids and the characterization of the phage display system.

The research detailed in this dissertation is focused on the use of biomolecules (i.e., peptides and proteins) to form non-biologically produced materials under mild reaction conditions (i.e, neutral pH, aqueous solutions, and room temperature). The peptides utilized in the studies detailed in this dissertation were identified through the screening of single crystal rutile TiO2 substrates or Ge powder with a phage-displayed peptide library. Twenty-one peptides were identified which possessed an affinity for Ge. Those peptides possessing a basic isoelectric point as well as hydroxyl- and imidazole-containing amino acid residues were found to be the most effective in precipitating amorphous germania from an alkoxide precursor. The phage-displayed peptide library screening of TiO2 substrates yielded twenty peptides. The titania formation activity of these peptides was found to correlate with the number of positive charges they carried. The titania materials generated by the library-identified and designed peptides were found to be composed of amorphous titania as well as <10 nm anatase and/or monoclinic TiO2 crystallites. Four recombinant proteins, derived from the amino acid sequences of proteins (silaffins) associated with biosilicification in diatoms, were also investigated for titania precipitation activity. The two most basic of these recombinant silaffins, rSil1L and rSilC, were able to induce the formation of titania. The titania precipitates generated by rSil1L were found to be similar to those produced by the phage-displayed library identified peptides. The second recombinant silaffin, rSilC, was found to produce hollow spheres of titania, which, following dehydration, were observed to transform into larger, solid spheres composed of radially aligned columns of rutile TiO2. The highly repetitive nature of the rSilC s amino acid sequence is believed to be responsible for the differences in TiO2 polymorph generated by the different recombinant silaffins and peptides. This dissertation also details research conducted on the formation of titania utilizing rSilC conjugated to synthetic and biogenic silica surfaces. These silica surfaces were functionalized with a newly developed drendritic growth technique. The dendritic functional-group amplification process was demonstrated to increase the loading of hexahisitidine tagged proteins on silica surfaces by more than 40%, as compared to traditional immobilization procedures.

The recent development of methods for constructing andhandling large collections (libraries) of proteins, from whichvariants with desired traits can be isolated, hasrevolutionized the field of protein engineering. Key elementsof such methods are the various ways in which the genotypes(the genes) and the phenotypes (the encoded proteins) arephysically linked during the process. In one section of thework underlying this thesis, one such technique (phagedisplay), was used to isolateand identify protein librarymembers based on their catalytic or target molecule-bindingproperties. In a first study, phage display libraries of the lipolyticenzyme Lipolase from Thermomyces lanuginosa were constructed,the objective being to identify variants with improvedcatalytic efficiency in the presence of detergents. Toconstruct the libraries, nine positions were targeted for codonrandomization, all of which are thought to be involved in theconformational change-dependent enzyme activation that occursat water-lipid interfaces. The aim was to introduce two tothree amino acid mutations at these positions per lipase gene.After confirming that the wt enzyme could be functionallydisplayed on phage, selections with the library were performedutilizing a mechanism-based biotinylated inhibitor in thepresence of a detergent formulation. According to rhodamineB-based activity assays, the fraction of active clonesincreased from 0.2 to 90 % over three rounds of selection.Although none of the variants selected using this approachshowed increased activity, in either the presence or absence ofdetergent compared to the wild type enzyme, the resultsdemonstrated the possibility of selecting variants of theenzyme based on catalytic activity. In the following work, phage libraries of the StaphylococcalProtein A (SPA)-derived Z-domain, constructed by randomizationof 13 surface-located positions, were used to isolate Z domainvariants (affibodies) with novel binding specificities. Astargets for selections, the parental SPA domains as well as twopreviously selected affibodies directed against two unrelatedtarget proteins were used. Binders of all three targets wereisolated with affinities (KD) in the range of 2-0.5 µM.One SPA binding affibody (ZSPA-1) was shown to bind to each of the fivehomologous native IgG-binding domains of SPA, as well as theZdomain used as the scaffold for library constructions.Furthermore, the ZSPA-1affibody was shown to compete with one of thenative domains of SPA for binding to the Fc part of humanantibodies, suggesting that the ZSPA-1affibody bound to the Fc-binding surface ofthe Z domain. The majority of the affibodies isolated in theother two selections using two different affibodies as targets,showed very little or no binding to unrelated affibodies,indicating that the binding was directed to the randomizedsurface of their respective targets, analogously toanti-idiotypic antibodies. The structure of the wild type Z domain/ZSPA-1affibody co-complex was determined by x-raycrystallography, which confirmed the earlier findings in thatthe affibody ZSPA-1affibody was shown to bind to the Fc bindingsurface of the Z domain. Further, both the Z domain and the ZSPA-1affibody had very similar three helix-bundletopologies, and the interaction surface involved ten out of thethirteen randomized residues, with a central hydrophobic patchsurrounded by polar residues. In addition, the interactionsurface showed a surprisingly high shape complementarity, giventhe limited size of the library used for selections. The ZSPA-1affibody was further investigated for use invarious biotechnological applications. In one study, the ZSPA-1affibody was successfully recruited as a novelaffinity gene fusion partner for production, purification anddetection of cDNA-encoded recombinant proteins using anSPA-based medium for affinity chromatography. Further, the SPAbinding capability of the ZSPA-1affibody was employed for site-specific andreversible docking of ZSPA-1affibody-tagged reporter proteins onto an SPAfusion protein anchored to a cellulose surface via acellulose-binding moiety. These generated protein complexesresembles the architecture of so-called cellulosomes observedin cellulolytic bacteria. The results suggest it may bepossible to use anti-idiotypic affibody-binding protein pairsas modules to build other self-assembling types of proteinnetworks. Keywords:phage display, selection, mechanism-basedinhibitor, affinity domains, crystal structure, Staphylococcusaureus protein A, affinity chromatography, anti-idiotypicbinding pairs, affibody, combinatorial, protein engineering,lipase, cellulosome, assembly.

Bovine anaplasmosis is caused by Anaplasma marginale and A. centrale. The most pathogenic and important species for cattle production is A. marginale, and is widely distributed in tropical, subtropical and temperate regions of the world. A. marginale is an intra-erythrocyte rickettsia of susceptible ruminants, biological and mechanically transmitted by ticks and hematophagous insects. The tick Rhipicephalus (Boophilus) microplus is the main vector of A. marginale in Brazil. The congenital form of transmission in cattle may occur, causing the neonatal anaplasmosis. The outer membrane of A. marginale includes six well characterized major surface proteins, MSP1a, MSP1b, MSP2, MSP3, MSP4 and MSP5, which play important role in the development of the immune response of infected animals. In this study, we have used the Phage Display technology to identify specific peptides that were immunoreactive to monoclonal antibodies anti-A. marginale proteins. Peptide selection was performed using a subtractive selection of a peptide library with 12 random amino acids, Ph.D.-12, expressed on the surface of the M13 filamentous phage concurrently against the anti-MSP1a and anti-MSP2. After four rounds of selection and validation by ELISA, the selected peptides have recognized only the anti-MSP1. Analysis of bioinformatics identified 45 peptides, which showed the protein consensus sequence STxS that was represented in 78% of selected phages. Due to the multiple motif repeats found in MSP1 protein, the STSSxL motif may become an important biological target, with potential use in diagnostic tests and vaccine for the control of Anaplasma marginale. / Anaplasmose bovina é uma infecção causada por Anaplasma marginale e A. centrale. A espécie mais patogênica e de maior importância para bovinos é a A. marginale e está amplamente distribuída nas regiões tropicais, subtropicais e temperada do mundo. A. marginale é uma rickettsia intraeritrocitária de ruminantes susceptíveis, transmitido biológica e mecanicamente por carrapatos e insetos hematófagos. O carrapato Rhipicephalus (Boophilus) microplus é o principal transmissor de A. marginale no Brasil. A forma congênita de transmissão em bovinos pode ocorrer, ocasionando a anaplasmose neonatal. A membrana externa do A. marginale inclui seis proteínas principais de superfície (MSPs) bem caracterizadas, designadas de MSP1a, MSP1b, MSP2, MSP3, MSP4 e MSP5 e desempenham papel importante no desenvolvimento da resposta imune de animais infectados. Para o desenvolvimento deste estudo, foi utilizada a técnica de Phage Display para identificar peptídeos ligantes a anticorpos monoclonais reativos a proteínas de Anaplasma marginale a partir de bibliotecas de peptídeos recombinantes por meio da tecnologia Phage Display. Para seleção dos peptídeos foi realizado uma seleção subtrativa utilizando uma biblioteca de peptídeos com 12 aminoácidos randômicos, Ph.D.-12, expressa na superfície do fago filamentoso M13 concomitantemente contra os anticorpos anti-MSP1a e anti-MSP2. Após quatro ciclos de seleção e validação por ELISA, o conjunto de peptídeos selecionados apresentou ser unicamente reconhecido pelo anticorpo anti- MSP1. Análises de bioinformática identificaram 45 peptídeos, que apresentaram o motivo proteico consenso STxS, representado em 78% dos fagos seqüenciados. Devido aos múltiplos sítios repetidos encontrados na proteína MSP1, o motivo proteíco STSSxL pode ser um importante alvo biológico, com potencial utilização em ensaios diagnósticos e vacinais para o controle de Anaplasma marginale. / Mestre em Genética e Bioquímica

Les cyanobactéries sont des micro-organismes qui préoccupent les autorités de santé publique dans le monde entier, en raison de la toxicité des cyanotoxines qu'elles produisent. Certaines cyanotoxines dont les microcystines (MC) sont des hépatotoxines inhibitrices de protéines phosphatases à sérine/thréonine. Aujourd'hui, plus de 200 variants de MCs ont été identifiés. Il s'agit d'heptapeptides monocycliques synthétisés par voie non-ribosomale dont la MC-LR (cyclo- (D-Ala-L-Leu-D-érythro-β-méthylAsp-L-Arg-ADDA-D-Glu-N-méthyl-hydro-Ala) est le variant le plus étudié en raison de sa fréquence et de sa forte toxicité. L’objectif de cette étude est le développement d'une méthode d'immunoanalyse rapide, sensible et fiable pour détecter les MCs. Le projet vise donc à développer un outil alternatif de détection de la MC-LR, qui serait mieux adapté aux analyses sur le terrain que les méthodes analytiques, biologiques ou les méthodes d'inhibition d'activité enzymatique actuellement disponibles. L'originalité de ce projet réside dans l'utilisation de deux approches différentes pour sélectionner de nouveaux anticorps spécifiques de la MC-LR. La première repose sur l'immunisation d'animaux de laboratoire, la technologie d'hybridation cellulaire et la sélection d'hybridomes sécréteurs d'anticorps monoclonaux. Si la méthodologie mise en œuvre a effectivement permis d'obtenir des immun-sérums spécifiques, la sélection des hybridomes d'intérêt reste à optimiser. La seconde stratégie mise en œuvre est basée sur la technologie du phage display pour sélectionner des fragments d'anticorps spécifiques de MC-LR à partir d'une banque de taille d’environ 109 phages, exprimant en surface des anticorps sous un format scFv (Shahsavarian et al., 2014). Plusieurs méthodes de criblage ont été développées et trois scFv ont été sélectionnés et étudiés, parallèlement à un quatrième scFv identifié dans une étude précédente (McElhiney et al., 2002), tous spécifiques à la MC-LR. Ces scFv ont été produits sous forme libre, soluble et leur spécificité à la MC-LR a été évaluée par ELISA et résonance plasmonique de surface. Les résultats obtenus montrent que les scFv sélectionnés sont tous capables de reconnaître la MC-LR. Néanmoins, ces résultats sont peu reproductibles et remettent en question le protocole de renaturation utilisé. Un travail de fond sur l’optimisation du protocole de renaturation s’avèrerait nécessaire pour les scFv ici sélectionnés, afin d’identifier les paramètres précis aboutissant à la perte ou au gain de leur fonctionnalité. / Cyanobacteria are ubiquitous microorganisms that present a worldwide concern to public health authorities because of the toxicity of the cyanotoxins they produce. Some cyanotoxins are hepatotoxins such as microcystins (MCs). At least 200 variants of MCs have been identified till today. In our study, we focus on MC-LR, a monocyclic heptapeptide (cyclo-(D-Ala-L-Leu-D-erythro-β-methylAsp-L-Arg-ADDA-D-Glu-N-methyldehydro-Ala), since it is the most frequently detected and one of the most toxic. In our study, we are interested in developing a fast, sensitive and reliable method to detect MCs. The project aims to develop an alternative pollution detection method that would be better suited to field measurements than the physicochemical methods currently available. The originality of this project lies in the use of two different approaches to select a panel of antibodies suitable for the development of immunodetection tests. The first one is based on the hybridoma technology for the production of monoclonal antibodies. The second one is based on phage display technique to select antibody fragments that are specific to MC-LR from a library of approximately 109 phages, expressing on the surface scFv fragments (Shahsavarian et al., 2014). Two monoclonal antibodies were selected using the first approach, and their specificity was evaluated using ELISA technique. Along with three scFvs selected from phage display approach. An additional scFv was added to this list: 3A8, selected from a previous study (McElhiney et al., 2002) and also specific to MC-LR. The scFvs were cloned into an expression vector in order to get each clone in its scFv soluble form. Then, their specificity to MC-LR was evaluated using ELISA technique and Surface plasmon resonance. The results show a potential specificity to MC-LR. Nevertheless, these results are not very reproducible and call into question the refolding protocol used. A thorough work on this protocol optimization would be necessary, in order to find the key parameters that control the loss or gain of their functionality

No description available.

Proteins perform an incredible array of functions facilitated by a diverse set of biochemical properties. Changing these properties is an essential molecular mechanism of evolutionary change, with major questions in protein evolution surrounding this topic. How do new functional biochemical features evolve? How do proteins change following gene duplication events? I used the S100 protein family as a model to probe these aspects of protein evolution. The S100s are signaling proteins that play a diverse range of biological roles binding Calcium ions, transition metal ions, and other proteins. Calcium drives a conformational change allowing S100s to bind to diverse peptide regions of target proteins. I used a phylogenetic approach to understand the evolution of these diverse biochemical features. Chapter I comprises an introduction to the disseration. Chapter II is a co-authored literature review assessing available evidence for global trends in protein evolution. Chapter III describes mapping of transition metal binding onto a maximum likelihood S100 phylogeny. Transition metal binding sites and metal-driven structural changes are a conserved, ancestral features of the S100s. However, they are highly labile at the amino acid level. Chapter IV further characterizes the biophysics of metal binding in the S100A5 lineage, revealing that the oft–cited Ca2+/Cu2+ antagonism of S100A5 is likely due to an experimental artifact of previous studies. Chapter V uses the S100 family to investigate the evolution of binding specificity. Binding specificity for a small set of peptides in the duplicate S100A5 and S100A6 clades. Ancestral sequence reconstruction reveals a pattern of clade-level conservation and apparent subfunctionalization along both lineages. In chapter VI, peptide phage display, deep-sequencing, and machine-learning are combined to quantitatively reconstruct the evolution of specificity in S100A5 and S100A6. S100A5 has subfunctionalized from the ancestor, while S100A6 specificity has shifted. The importance of unbiased approaches to measure specificity are discussed. This work highlights the lability of conserved functions at the biochemical level, and measures changes in specificity following gene duplication. Chapter VII summarizes the results of the dissertation, considers the implications of these results, and discusses limitations and future directions. This dissertation includes both previously published/unpublished and co- authored material.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Early diagnosis of leprosy is an important contribution to reducing the incidence of the disease. For its early detection, the development of new platforms that include the mapping of antigens with potential to be used in immunodiagnostic is of great interest. Among these antigens, the PGL-1 and epitopes derived from specific bacillus proteins have received great attention. Alternatively, due to their versatility to perform the same functions as the protein and non-protein natural antigens, mimetic peptides are considered an important tool. Thus, our goal was to produce mimetic peptides of Mycobacterium leprae antigens that are promising as serological markers, which will be explored in new diagnostic platforms. To produce peptide mimetics, phage display technology was used. In the first case, we used a monoclonal anti-PGL-1 (CS-38) aiming to obtain peptides that mimics the PGL-1. In the second case, the peptides were obtained having purified IgGs from patients with leprosy as target. The sequences of the selected peptides expressed on the phage surface were chemically synthesized. The synthetic peptides were validated by ELISA (case 1 and 2) and by an immunosensor based on Surface Plasmon Resonance (case 1). Aiming to confirm and identify the targets of the mimetic peptides, scFv antibodies were produced by reverse engineering. The PGL-1-M3 peptide that mimics the native PGL-1 had a sensitivity of 89.11% and specificity of 100.00% in the IgM detection, with positivity of 100% in lepromatous (LL). The IgG detection had positivity of 60% for tuberculoid (TT) and 39% for household contacts (HC). This peptide was used in assembling one biophotonics platform, which allowed the differentiation of all forms of leprosy (p <0.05). The anti-scFv-M3 PGL-1 recognized native PGL-1 and accurately detected the M. leprae in immunohistochemistry tests. The MPML11, MPML14 and MPML12 peptides that mimics M. leprae antigens detect IgG and IgA in patients and HC. In IgG detection, MPML11 peptide showed positivity in 52.2% of TT and 35% of HC, and is also a promising marker of type 2 reaction. MPML12 and MPML14 peptides showed a very similar behavior to the PGL-1, with positivity of 100% and 92.85% in LL, respectively. The three peptides detected IgA in the serum of patients, especially multibacillary (MBs); and IgA in saliva of MBs and HC which index case was multibacillary. Mimetic peptides obtained in this work were confirmed as true mimetics of M. leprae antigens and can be applied in the diagnosis of leprosy in different platforms. / O diagnóstico precoce da hanseníase representa uma contribuição importante para diminuir a incidência da doença. Para isso é fundamental o desenvolvimento de novas plataformas, que incluam o mapeamento de antígenos com potencial para o imunodiagnóstico. Dentre estes destaca-se o PGL-1 e epítopos obtidos de proteínas específicas do bacilo. Alternativamente, peptídeos miméticos apresentam-se como uma importante ferramenta, pela versatilidade em desempenhar as mesmas funções que os antígenos naturais proteicos e não-proteicos. Dessa forma, nosso objetivo foi produzir peptídeos miméticos a antígenos do Mycobacterium leprae e que sejam promissores como marcadores sorológicos, os quais serão explorados em novas plataformas diagnósticas. Para produzir os peptídeos miméticos foi utilizada a tecnologia phage display. No primeiro processo utilizou-se um anticorpo monoclonal anti-PGL-1 (CS-38) para obter peptídeos miméticos ao PGL-1. No segundo processo, os peptídeos foram produzidos tendo como alvo IgGs purificadas de pacientes com hanseníase. As sequências dos peptídeos expressos nos fagos foram sintetizadas quimicamente. Os peptídeos sintéticos foram validados por ELISA (processo 1 e 2) e imunosensor baseado em Ressonância Plasmônica de Superfície (processo 1). Por meio de engenharia reversa, anticorpos scFv foram produzidos para confirmar e identificar alvos dos peptídeos miméticos. O peptídeo PGL-1-M3 mimético ao PGL-1 nativo apresentou sensibilidade de 89,11% e especificidade de 100,00% na detecção de IgM, com positividade de 100% em lepromatosos (LL), bem como títulos de IgG detectaram positividade de 60% para tuberculóides (TT) e 39% para contatos domiciliares (HC). Com este peptídeo foi montada uma plataforma biofotônica, que diferencia todas as formas clínicas de hanseníase (p<0,05). O scFv anti-PGL-1-M3 reconheceu PGL-1 nativo e detectou com precisão o M. leprae na imuno-histoquímica. Os peptídeos MPML11, MPML12 e MPML14 miméticos de antígenos do M.leprae detectam IgG e IgA em pacientes e HC. Na detecção de IgG, MPML11 apresentou positividade de 52,2% em TT e 35% em HC e também é um promissor marcador de reação tipo 2. MPML12 e MPML14 apresentaram um comportamento muito similar ao PGL-1, tendo 100% e 92,85% de positividade em LL, respectivamente. Os três peptídeos detectaram IgA nos soros de pacientes, especialmente multibacilares (MBs); bem como IgA na saliva de MBs e HC cujo caso índice era multibacilar. Os peptídeos miméticos obtidos nesse trabalho foram confirmados como miméticos verdadeiros de antígenos do M. leprae e podem ser aplicados no diagnóstico da hanseníase em diferentes plataformas. / Doutor em Genética e Bioquímica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Alzheimer's Disease (AD) is the most important cause of dementia in the world.The involvement of the immune system in the pathogenic process has been shown by several studies including the description of autoantibodies (AAc) directed to targets in the central nervous system.It is not possible yet to further define what their exact involvement in AD is. In this study, using the methodology of Phage Display we sought to identify AAc specific to patients with the disease and to characterize their antigens using bioinformatics tools. We selected 10 patients with AD according to DSM-IV-TR and NINCDS ASRDA criteria and 10 healthy controls matched for age and sex.Using a library of peptides displayed on M13 phages, we performed biopanning, thus selecting 10 peptides recognized by IgG in sera from patients with AD. The probable epitopes were characterized and their involvement with AD was assessed in the literature.We found alignment with Neurexin 3&#946;, PLK4, Neuroserpin, DNAJ, Mint-1/X11, units of the nicotinic acetylcholine receptor, Trombospondin-1, TRAF6, ACE, GDNF &#945;3 receptor, PRUNE2 and CD44. The Phage display technology, combined with bioinformatics tools, has proven to be an interesting research methodology of AAb present in the serum of patients with AD and may provide new insights into disease mechanisms and therapeutic targets. / A Doença de Alzheimer (DA) é a causa mais importante de demência no mundo. O envolvimento do sistema imunológico no processo patogênico tem sido demonstrado em diversos estudos inclusive com a descrição de autoanticorpos (AAcs) presentes no soro dirigidos a alvos no sistema nervoso central. Não é possível definir ainda qual a sua participação exata na DA. Neste estudo, utilizando a metodologia do Phage Display procuramos identificar AAcs específicos de pacientes com a doença e caracterizar seus antígenos utilizando ferramentas de bioinformática. Foram selecionados 10 pacientes com DA segundo os critérios do DSM-IV TR e NINCDS-ASRDA e 10 controles saudáveis pareados por sexo e idade. Utilizando uma biblioteca de peptídeos expostos em fagos M13, foi realizado biopanning selecionando 10 peptídeos reconhecidos por IgGs no soro de pacientes com DA. Os prováveis epítopos foram caracterizados e seu envolvimento com a DA foi avaliado na literatura. Foram encontrados alinhamentos com a Neurexina 3&#946;, PLK4, Neuroserpina, vários membros da família DNAJ, Mint-1/X11, Gene 2 de susceptibilidade para o Autismo, unidades &#945;2, &#945;3, &#945;4 e &#945;6 do receptor nicotínico da acetilcolina, Trombospondina-1, TRAF6, ECA, receptor &#945; 3 do GDNF, PRUNE2 e CD44. A tecnologia de Phage Display, aliada a ferramentas de bioinformática, demonstrou ser interessante metodologia de investigação de AAcs presentes no soro de pacientes com DA podendo fornecer novas pistas sobre mecanismos da doença e alvos terapêuticos. / Mestre em Ciências da Saúde

Introduction: Tuberculosis is an infectious disease that affects more than 9 million people each year worldwide . For tuberculosis control methods for the study of more accurate diagnostic and monitoring treatment , make it essential . Objectives: To identify biomarkers by Phage display technique and evaluate the immunoreactivity of the IG\'s clones selected sera from patients with tuberculosis to monitor treatment . Methods: Blood samples were collected from 61 individuals over 18 years , both sexes , and performed the tuberculin test ( PPD ) as a control . The samples were subjected to the process of selection of clones, the biopanning using a library of random 12 amino acid region of the protein expressed on the phage pIII . After selecting the ELISA assay was performed to analyze the immunoreactivity of selected clones . Results: after the in silico analysis , it was found that the sequence of clone F10 ( VYKTPNSTANRW ) has similarity to the membrane protein MPT64 low molecular weight , 28 to 32 kDa, immunogenicity of the M. tuberculosis complex. In ELISA clone F10 showed reactivity significant ( p < 0.05 ) and monitoring of individuals in treatment (P < 0.005). Conclusion : the reactivity of clone F10 demonstrated that it can be used for monitoring the treatment of tuberculosis , contributing to its control. / Introdução: A Tuberculose é uma doença infectocontagiosa , que acomete mais de 9 milhões de indivíduos a cada ano em todo o mundo . Para o controle da tuberculose o estudo por métodos de diagnósticos mais acurados e o monitoramento ao tratamento, tornam se imprescindíveis. Objetivos: identificar biomarcadores pela técnica de Phage display e avaliar a imunorreatividade dos clones selecionados ás igG\'s de soros de pacientes com tuberculose para o monitoramento do tratamento. Métodos: foram coletadas amostras de sangue de 61 indivíduos maiores de 18 anos, ambos os sexos, e realizada a prova tuberculina (PPD) como controle. As amostras foram submetidas ao processo de seleções de clones, o Biopanning, utilizando uma biblioteca de 12 aminoácidos randômicos expressos na região da proteína pIII do bacteriófago. Após a seleção foi realizado o ensaio ELISA para analisar a imunorreatividade dos clones selecionados. Resultados: após as análises in silico, observou-se que a sequência do clone F10 (V Y K T P N S T A N RW), possui similaridade com a proteína MPT64 de membrana de baixo peso molecular, 28 a 32 KDa, imunogênica do complexo M.tuberculosis . No ensaio ELISA o clone F10 apresentou uma reatividade significante ( p<0,05 ) e no monitoramento dos indivíduos em tratamento ( p<0,005 ). Conclusão: a reatividade do clone F10 demonstrou que pode ser utilizada para o monitoramento do tratamento da tuberculose, contribuindo para seu controle. / Mestre em Ciências da Saúde