A relatively high percentage of porcine <i>Escherichia coli</i> isolates from cases associated with neonatal and post-weaning diarrhea are positive for the gene encoding the adhesin involved in diffuse adherence I (AIDA-I). This gene and its corresponding protein were first identified and characterized in <i>E. coli</i> strain 2787 isolated from human infantile diarrhea. Little is known about the role of the AIDA-I protein in pathogenesis of porcine enteric disease caused by AIDA-I positive E. coli and the properties of AIDA-I protein expressed by porcine AIDA-I positive <i>E. coli</i> isolates and its receptors. <p>In this study, we demonstrated that AIDA-I adhesin isolated from porcine AIDA-I positive <i>E. coli</i> PD20 and PD58 is an acidic protein consisting of five isoforms. It has a molecular weight (100 kDa) similar to the AIDA-I adhesin expressed by human AIDA-I positive <i>E. coli</i> strain 2787 and has a relatively high amino acid homology (78-87%) with it. Immunodetection of AIDA-I positive <i>E. coli</i> strains using polyclonal anti-AIDA-I antibodies had relatively low sensitivity and specificity, accordingly these tests are unlikely to be used for regular diagnostic detection. <p>Using affinity chromatography, we isolated from porcine intestinal mucus proteins that bind to purified AIDA-I adhesin. These proteins were separated by one- and two-dimensional electrophoresis and subjected to overlay Western blot with purified AIDA-I adhesin and AIDA-I positive <i>E. coli</i> to demonstrate 65 and 120 kDa (p65 and p120) proteins as AIDA-I binding proteins. The identity of p65 was not determined based on LCMS/MS data, whereas p120 was matched to two nuclear proteins (namely, DNA damage binding protein and splicing factor 3b) and one cytoplasmic protein, which is an IgG Fc binding protein. Based on similar amino acid homology, molecular weight, structural similarity to mucin and reported evidence of being secreted by goblet cells into the intestinal lumen, we think that the IgG Fc binding protein is the most likely candidate to serve as a potential receptor in intestinal mucus for AIDA-I adhesin.

abstract: Protein affinity reagents have aptly gained profound importance as capture reagents and drugs in basic research, biotechnology, diagnostics and therapeutics. However, due to the cost, labor and time associated with production of antibodies focus has recently changed towards potential of peptides to act as protein affinity reagents. Affinity peptides are easy to work with, non-immunogenic, cost effective and amenable to scale up. Even though researchers have developed several affinity peptides, we are far from compiling library of peptides that encompasses entire human proteome. My thesis describes high throughput pipeline that can be used to develop and characterize affinity peptides that bind several discrete sites on target proteins. Chapter 2 describes optimization of cell-free protein expression using commercially available translation systems and well-known leader sequences. Presence of internal ribosome entry site upstream of coding region allows maximal expression in HeLa cell lysate whereas translation enhancing elements are best suited for expression in rabbit reticulocyte lysate and wheat germ extract. Use of optimal vector and cell lysate combination ensures maximum protein expression of DNA libraries. Chapter 3 describes mRNA display selection methodology for developing affinity peptides for target proteins using large diversity DNA libraries. I demonstrate that mild denaturant is not sufficient to increase selection pressure for up to three rounds of selection and increasing number of selection rounds increases probability of finding affinity peptide s. These studies enhance fundamental understanding of mRNA display and pave the way for future optimizations to accelerate convergence of in vitro selections. Chapter 4 describes a high throughput double membrane dot blot system to rapidly screen, identify and characterize affinity peptides obtained from selection output. I used dot blot to screen potential affinity peptides from large diversity of previously ii uncharacterized mRNA display selection output. Further characterization of potential peptides allowed determination of several high affinity peptides from having Kd range 150- 450 nM. Double membrane dot blot is automation amenable, easy and affordable solution for analyzing selection output and characterizing peptides without ne ed for much instrumentation. Together these projects serve as guideline for evolution of cost effective high throughput pipeline for identification and characterization of affinity peptides. / Dissertation/Thesis / Masters Thesis Biochemistry 2014

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No. of bitstreams: 2 Dissertação - Rívia Aparecida Reinalda Arruda - 2016.pdf: 3000238 bytes, checksum: 221f622157672c4f34ee32f2aed12d0b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-09-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The main purpose of this study was to assess conformational changes in preselected aptamers A03 and C10 against untranslated regions present in the genome of the dengue virus (DENV), by means of molecular biology and physics assays. Aptamers are nucleic acid molecules with potential theranostic applications. In this context, dengue was the target chosen for study because is an important infectious viral disease presenting high rates of morbidity and mortality in the worldwide, especially in tropical and subtropical areas, where there are high incidences of the mosquito vector Aedes aegypti. These aptamers showed, by dot-blot and magnetic beads ligand capture and subsequent qPCR detection, similar affinities and specificities to binding to the DENV 5'UTR. Probably, the analogy of the motifs and secondary structures presented in these ligands, identified by in silico studies, are responsible for these similarities. Data obtained by using Fluorescence Resonance Energy Transfer Assays (FRET) confirmed the affinity of A03 as DENV ligand by conformational changes in the double labeled aptamer (5’-FAM, 3’-TAMRA). Aptamer conformacional changes resulting from its interaction with the target were detected by the modifications in the fluorescence intensities between FAM and TAMRA fluorophores. These conformational changes results in donor-acceptor distance modification that has an effect on FRET efficiency and on fluorescence intensity of these molecules. The ionic strength of the buffer used in the trials had significant influence on the conformation of the aptamer, allowing the detection of interactions between A03 and DENV. The ions in the buffer lead an effect in the conformation of the aptamer, increasing its thermal stability, and the Mg2+ ion has led to major conformational changes on A03 compared to Na+ ion. The data presented herein demonstrated the importance of the studies of oligonucleotides binding affinity and specificity to Dengue virus. It can be concluded that the A03 and C10 aptamers have potential in application for diagnostic and/or therapeutic purposes, considering the diluent buffer, since different salts have influence on the secondary and tertiary conformational structures of aptamers, and on bioavailability of interaction with the target. / O objetivo principal desse trabalho foi avaliar alterações conformacionais nos aptâmeros A03 e C10 previamente selecionados contra regiões não traduzidas presentes no genoma do vírus da dengue (DENV), por meio de testes de biologia e física molecular. Os aptâmeros são moléculas de ácidos nucleicos com potencial aplicação na teranóstica das mais diversas doenças. Nesse contexto, destaca-se a dengue, uma doença infecciosa viral com altos índices de morbi-mortalidade em um cenário mundial, principalmente em regiões tropicais e subtropicais, onde são altas as incidências do mosquito vetor Aedes aegypti. Esses aptâmeros demonstraram, por meio de dot blot e de captura de ligantes por beads magnéticos com detecção por PCR em Tempo Real, afinidades e especificidades semelhantes de ligação à 5’UTR do DENV. Provavelmente, essas semelhanças se deram em função dos motivos e das estruturas secundárias análogas apresentadas por esses ligantes, obtidas por ensaios in silico. Os ensaios de Transferência Ressonante de Energia de Fluorescência (FRET) permitiram a confirmação dessa afinidade de ligação entre A03:DENV, devido às alterações estruturais ocorridas no aptâmero duplamente marcado em sua sequência (5’-FAM; 3’-TAMRA). As alterações conformacionais no aptâmero resultantes de sua interação com o alvo foram detectadas pelas mudanças nas intensidades de fluorescência do FAM e do TAMRA, decorrentes de alterações nas distâncias entre o doador e o aceitador que afetam a eficiência FRET e por conseguinte as intensidades de fluorescência destas moléculas. A força iônica do tampão utilizado nos ensaios apresentou importante influência na conformação do aptâmero, permitindo a detecção das interações entre A03 e o DENV. Os íons presentes na solução tampão afetaram a conformação estrutural do aptâmero e aumentaram sua estabilidade térmica, sendo que o íon Mg2+ levou a maiores alterações estruturais sobre o A03 comparado ao íon Na+. Os dados aqui apresentados demostraram a importância dos estudos de afinidade e especificidade dos oligonucleotídeos ligantes ao vírus da dengue. É possível concluir que os aptâmeros A03 e C10 apresentam potencial de aplicação para fins diagnósticos e/ou terapêuticos, considerando o tampão diluente, uma vez que os diferentes sais presentes exercem influência sobre as estruturas conformacionais secundárias e terciárias dos aptâmeros, e em sua biodisponibilidade de interação com o alvo.

BTX biodegradation rates, biochemical expression patterns and microbial ecology were studied under anoxic (denitrifying), anoxic/microaerobic/aerobic, and anoxic/microaerobic conditions in activated sludge sequencing batch reactors. The studies showed that toluene and m-xylene were denitrified via benzoyl-CoA reductase. Although benzene, o-, and p-xylene were recalcitrant under denitrifying conditions, they were biodegraded under microaerobic (< 0.2 mg/L dissolved oxygen) and nitrate or nitrite (NOx)-supplemented microaerobic conditions. The patterns of the specific enzymes associated with BTX biodegradation under microaerobic conditions indicated that the three compounds were metabolized by oxygen-dependent pathways. The expression levels of catechol 1, 2-dioxygenase and catechol 2, 3-dioxygenase under microaerobic conditions were induced to levels as high as under aerobic conditions (> 4 mg/L dissolved oxygen). Benzene, o-, and p-xylene biodegradation rates were twice as fast under NO_x-supplemented compared to NO_x-free microaerobic conditions, and the specific biodegradation rates under aerobic and NO_x-supplemented microaerobic conditions were comparable. 16S rRNA probes targeting representative toluene-degraders were used to investigate the microbial communities in the three sequencing batch reactors by using a dot blot hybridization technique. The hybridization results suggest that multiple redox environments fostered a more diverse microbial community and the activities of the target organisms in the reactors with multiple redox environments were higher than in the single redox reactor. Additionally, facultative toluene-degraders appeared to play a less significant role than the strict anoxic and aerobic toluene-degraders in all three SBRs. / Ph. D.

L’étude de la variation du génotype et du phénotype fut réalisée sur deux espèces de parasites intestinaux (Cryptosporidium parvum et C. muris) via une infection expérimentale de 10 passages successifs chez le veau (Bos taurus). L’infection avec C. parvum a bien fonctionné alors qu’aucun signe clinique n’a été observé dans le cadre de cette étude avec C. muris. Pour le génotype, deux gènes (HSP70 et GP60) ont été amplifiés par double PCR puis séquencés. Les résultats ont indiqué que ces gènes n’étaient pas modifiés après 10 passages chez des veaux. Cela montre une faible évolution génétique du parasite lorsqu’il passe dans un animal hôte, facilitant ainsi les études épidémiologiques lors d’épisode de cryptosporidiose. Il faut cependant noter que les parasites utilisés ne provenaient pas de l’environnement mais d’une compagnie spécialisée en parasitologie (WaterBorne®). L’étude de la variation du phénotype a été tentée, sans succès, à l’aide d’un immuno-buvardage en point utilisant le sérum des veaux infectés. Des problèmes liés à la concentration des ookystes de C. parvum placés sur la membrane de l’immuno-buvardage en point furent suspectés. / The study of the variation of the genotype and phenotype has been done with 2 species of intestinal parasites (Cryptosporidium parvum and C. muris) by using an experimental infection with 10 successives passages on calves (Bos taurus). Infection with C. parvum worked well but failed for C. muris. The genotype has been studied with two genes (HSP70 and GP60), amplified with a double PCR and sequenced. Results showed that these genes are not changing after 10 passages on calves. It indicates that the genetic evolution of the parasite is very slow even when passing to a susceptible host. That particularity might be useful for epidemiologic studies when an episode of cryptosporidiosis occurs. It’s important to consider that the parasites used were not taken from the environment but from a company specialised in parasitology (WaterBorne®). A Dot Blot using serum from infected calves has been tried without success. Problems related to oocysts concentration were suspected.

Clostridium chauvoei is a gram-positive, spore-forming anaerobic bacterium. It is the pathogenic agent of blackleg, a disease causing serious toxemia and high mortality in cattle, sheep and many other domestic and wild animals. It is considered the most important Clostridium producing economic losses in livestock. Typically, animals infected with blackleg die rapidly without any signs of illness. Animals quickly die within 12 to 48 hours after contracting the disease. Therefore, the control of this disease is done by commercial vaccines consisting of whole formolized cultures. Immunity against C. chauvoei is associated with whole cell, including its somatic and flagellar antigens while in other clostridial diseases, protective immunity is obtained by the use of vaccines containing toxoids. Moreover, it is essential to obtain new information about the somatic antigens of C. chauvoei. Proteomics is the study of the proteome, the protein complement of the genome. The proteome has been defined as the entire complement of proteins expressed by a cell, organism, or tissue type, and accordingly, proteomics is the study of this complement expressed at a given time or under certain environmental conditions. 2-DE with Immobilized pH Gradients (IPGs) combined with protein identification by Mass Spectrometry (MS) is currently the workhorse for proteomics. Much of information about immunogenic component can be derived from proteomics coupled to Western blotting, namely immunoproteomics. Our study constitutes the first immunoproteomic analysis of C. chauvoei to identify candidate immunogenic antigens for development of new vaccines. Analyses were performed by Western blot and dot blot techniques against the whole cell extract proteins of C. chauvoei separated by 2-DE. Firstly, the growth conditions of two different strains, C. chauvoei ATCC 11957 and C. chauvoei 20 were optimized. After mice immunization studies with experimental vaccines prepared, sera were obtained for evaluation of the immunoglobulin G antibody level by ELISA. After high level of antibody response determination, 1-DE, 2-DE and immunoblot studies were performed for the characterization of immunogenic proteins. In the study, a total of 460 protein spots could be detected on the 2-DE gels by the help of Delta2D image analysis software and 30 of them were reacted with polyclonal antibodies against inactivated whole cells of C. chauvoei. Among these 30 spots, and 8 of them could be characterized by MALDI-TOF MS analyses. Of these 8 spots revealed four different gene products (distinct ORFs). Ornithine decarboxylase, methionine adenosyltransferase, glucose-6-phosphate isomerase, and flagellin protein FliB (C) are the characterized proteins. Glucose-6-phosphate isomerase has been identified as an immunogenic protein for a pathogenic microbe and in C. chauvoei for the first time. Methionine adenosyltransferase and ornithine decarboxylation were identified as immunogenic for C. chauvoei for the first time. The last defined protein is the flagellin protein FliB(C) which is known to be major immunogenic protein of C. chauvoei.

This thesis aims to investigate if the endothelial protein C receptor is expressed in the murine kidney. This was done by performing flow cytometry and Western blot analysis on cultivated murine kidney endothelial cells (mKECs) as well as SDS-PAGE and Western blot analysis on murine kidney tissue. Flow cytometry was also performed on cultivated ARPE19 and 4T1 cells for comparison. It was discovered that ≥95,5% of the mKECs, ≥93,6% of the ARPE19 cells and ≥60,9% of the 4T1 cells express the receptor according to the flow cytometry data. A dot blot was performed to validate the primary antibody used for detection of EPCR in Western blot and SDS-PAGE. According to the dot blot, the primary antibody can be visualised in the dilution range from 1:2000 to 1:10. The dot blot also showed that the secondary antibody binds specifically to the primary antibody. Yet, Western blot analysis did not detect the receptor neither in mKECs nor tissue lysate. This was likely due to the fact that the primary antibody used did not bind specifically to the receptor, and may not be applicable for this method. SDS-PAGE did not show any indication that the receptor was present in the kidney tissue. In conclusion, it was discovered that the EPCR was expressed in the murine kidneys endothelial cells through flow cytometry, but the presented methods for Western blot and SDS-PAGE could not confirm the expression of the receptor.

Huntington’s disease (HD) and Spinocerebellar ataxia (SCA) are incurable neurodegenerative diseases that affect the central nervous system. Amyloids, highly organized protein aggregates, are a hallmark for many neurodegenerative diseases. The presence and accumulation of amyloids are toxic and constitute the major cause of neuron cell death. Both genetic and environmental factors contribute to the onset and progression of these diseases. However, despite intensive research, the underlying cause remains unclear. The role of viral infection as an environmental factor in the context of neurodegenerative diseases has not received much attention. The purpose of this study is to investigate the effects of Herpes Simplex Virus 1 (HSV-1) infection on nuclear amyloid aggregation in model cell lines of HD and SCA. The research process consists mainly of laboratory work which involved the use of several molecular techniques used in the field of biotechnology. The work comprises cultivating cells, infecting cells with HSV-1, Fluorescence microscopy, Western Blot and isolation and detection of amyloids. Western Blot is used for the analysis of specific proteins associated with protein aggregation in HD and SCA. The techniques used for detecting amyloids are Dot Blot and Antibody-staining of amyloids in cells. The results from Western Blot showed that aggregates changed in the presence of the virus. This pattern is observed for both HD and SCA1 cell lines. A big effort is done in this study to optimize Dot Blot as it is method that could be applied in every lab. Normalization of samples proved to be the most challenging part with Dot Blot. No definitive conclusions can be drawn from the Dot Blot results as reproducibility and sensitivity were lacking. This work addresses some of the difficulties encountered when working with detection of amyloids especially Dot Blot. Antibody-staining of amyloids showed that amyloids were formed in the presence of virus in comparison to non-infected. To conclude, aggregates changed, and amyloids were formed in the presence of virus. These results point to the fact that HSV-1 infection could be involved in the process of nuclear amyloid aggregation. The data presented in this thesis will need further investigation and characterization to identify the precise role of viral-induced amyloid formation in HD and SCA patient cells. / Huntingtons sjukdom (HD) och Spinocerebellära ataxier (SCA) är obotliga neurodegenerativa sjukdomar som påverkar det centrala nervsystemet. Amyloid, proteinaggregat som har en viss konformation är ett kännemärke för många neurodegenerativa sjukdomar. Ackumulering av dessa amyloider är toxiskt och är den främsta orsaken till att nervceller dör. Både genetiska faktorer och miljöfaktorer bidrar till uppkomsten och progressionen av dessa sjukdomar. Trots intensiv forskning är den bakomliggande orsaken emellertid fortfarande oklar. Virusinfektion som en potentiell miljöfaktor har i detta sammanhang inte fått mycket uppmärksamhet. Syftet med denna studie är att undersöka effekterna av Herpes Simplex Virus 1 (HSV-1) infektion på amyloid aggregering i modellcellinjer av HD och SCA. Forskningsarbetet bestod i huvudsakligen av experimentellt arbete med hjälp av flera molekylära tekniker inom bioteknikområdet som cell odling, infektering av celler med HSV-1, fluorescensmikroskopi, Western Blot och isolering och detektion av amyloider. Western Blot användes for att analysera specifika proteiner associerade med protein aggregering i HD och SCA. Amyloider detekterades med Dot Blot och med antikroppar specifika för amyloider. Resultat från Western Blot visade att amyloiderna förändras i virusinfekterade celler. Detta mönster observerades i både HD and SCA1 cellinjer. En stor bemöda görs i denna studie för att optimera Dot Blot eftersom det är en metod som kan användas i alla laboratorier. Normalisering visade sig vara det svåraste med detektion av amyloider. Inga definitiva slutsatser kan dras från dessa experiment, eftersom reproducerbarhet och känslighet var bristande. Detta arbete tar upp några av de svårigheter som uppstod vid arbetande med detektion av amyloider speciellt Dot Blot. Detektion av amyloider med antikropp visade att amyloider bildades till stor utsträckning i infekterade cellinjer i jämförelse med icke-infekterade. Sammanfattningsvis, amyloider förändrades och amyloider bildades i närvaro av virus. Dessa resultat indikerar på att HSV-1 infektion skulle kunna vara involverad i processen av amyloid aggregering. De presenterade uppgifter i detta examensarbete är preliminära och behöver följas upp med ytterligare studier för att identifiera virusens exakta roll i amyloid bildning i HD och SCA patient celler.

Urinary bladder cancer is a heterogeneous disease appearing in different forms, e.g. non-muscle invasive and muscle invasive. For all variants, the expression of proteins is interesting to analyze for diagnostic, predictive, prognostic and drug targeting purposes, since it reflects the altered gene expression causing the cancer. Since urothelial cells of the bladder are in direct contact with urine it is likely that this body fluid contains cancer-related proteins. In Paper I, unbiased analysis of proteins in urine from urinary bladder cancer patients and controls, using label-free quantification by mass spectrometry, was applied and four interesting proteins APOE, FGB, LRG and SERPINA1 were selected and further analyzed with western and dot blot. In Paper II, two more proteins, POLR1E and TOP2A, were validated as relevant proteins in bladder cancer urine. In Paper III and IV, the proteins GAL1 and STMN1 were investigated for their prognostic and therapeutic target potential in bladder cancer. In Paper II, III and IV, the expression of seven of the proteins were analyzed on tissue microarrays representing tumour tissue from 360 patients with different tumour stages. For the proteins identified by the urine screening approach, their protein expressions were confirmed in bladder cancer tissue. The expression level in tissue of five of the proteins, APOE, FGB, POLR1E (Paper II), GAL1 (Paper III) and STMN1 (Paper IV), increased with tumour stage, showing diagnostic relevance and three of the proteins, SERPINA1 (Paper II), STMN1 (Paper IV) and GAL1 (Paper III) had prognostic potential in urinary bladder cancer. In addition, GAL1 and STMN1 were demonstrated to be highly expressed in metastatic disease and inhibition of STMN1 reduced cell growth (Paper III and IV), indicating that these proteins are promising drug targets in urinary bladder cancer. In conclusion, the approach of this thesis has generated several candidate protein biomarkers in urine and tissue, validated with independent methods, which have the potential to improve the care for bladder cancer patients.

SANTOS, C. D. G. Caracterização molecular e biológica de um begomovírus isolado de tomateiro, Lycopersicon esculentum Mill., no estado de Goiás e sua interação com o vetor Bemisia argentifolii Bellows & Perring. 2001. 174 f. Tese (Doutorado em Agronomia/Fitotecnia) - Centro de Ciências Agrárias, Universidade Federal do Ceará, Fortaleza, 2001. / Submitted by Francisco Lacerda (lacerda@ufc.br) on 2014-06-30T19:54:23Z No. of bitstreams: 1 2001_tese_cdgsantos.pdf: 2566273 bytes, checksum: 11a904889bfaa1c1d29f07be24985442 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa(jairo@ufc.br) on 2014-07-02T17:55:18Z (GMT) No. of bitstreams: 1 2001_tese_cdgsantos.pdf: 2566273 bytes, checksum: 11a904889bfaa1c1d29f07be24985442 (MD5) / Made available in DSpace on 2014-07-02T17:55:18Z (GMT). No. of bitstreams: 1 2001_tese_cdgsantos.pdf: 2566273 bytes, checksum: 11a904889bfaa1c1d29f07be24985442 (MD5) Previous issue date: 2001 / The whitefly-transmitted viruses from the family Geminiviridae, genus Begomovirus, have been reported as an economically important pathogen group that affect important crops in tropical and subtropical countries. Since the beginning of the 1980 decade, the occurrence of the whitefly associated to Begomovirus infection has drastically increased worldwide. In Brazil, these pathogens have been responsable for severe economical losses in tomato (Lycopersicon esculentum) orchards and the production has hampered since 1994. In this work, infected tomato plants showing symptoms, such as mosaic, intervein clearing, leaf curling and growth reduction were collected in tomato orchards in Anápolis, State of Goiás. The virus was identified as a member of the genus Begomovirus by PCR reaction, using specific primers to amplify fragments of A and B components of the virus DNA genome. The Chapter I of this thesis presents the results of the molecular characterization of the virus and the Chapter II shows the determination of its host range and the relationship with its natural vector Bemisia argentifolii. The virus isolate denoted GO-ANPL was cloned and partially sequenced. Part of the sequenced genome (2.180 nucleotides long) corresponded to the coat protein and Rep genes and comprised the entire intergenic region. Sequence comparison revealed that the GO-ANPL isolate is distantly related to the begomoviruses found in Asia, Europe and Africa, and it is related to other begomoviruses reported in Brazil. The virus isolate showed to be more closely related to viruses found in the State of Minas Gerais (TRMV isolate) and in the Federal District (isolate DF-Br2). The highest homology was observed with the isolate DF-Br2 and it may represent a new specie of the genus Begomovirus. In order to determine the virus host range, 46 plant species from nine different botanical families were mechanically and using the virus vector inoculated. The GO-ANPL isolate preferentially infected plants of the family Solanaceae as Nicotiana benthamiana, Datura stramonium and Nicandra physalodes. The number of infected plants was higher when they were inoculated by the virus vector, and the results were distinct from those obtained for other begomoviruses reported in Brazil. Viruses infections were all confirmed by dot blot hybridization using specific molecular probes to the virus. 4 To study virus/vector interaction, the acquisition access period (AAP), inoculation access period (IAP), and the latent period were determined transfering five whiteflies per plant and using tomato cv. Santa Clara as the host. For the AAP and IAP, nine different time periods were tested: 0.25, 0.5, 1, 2, 4, 8, 16, 20 and 24 h. The minimal AAP determined was 0.25 h, after which, 6% of the tested plants became infected. The number of infected plants increased to 65% with an AAP of24h.Afteran IAP of 0.5 h, 18% of the plants were infected and their number increased to 67% after an IAP of 24 hours. The latent period was considered to be 16 h, after which, 3% of the inoculated plants became infected. The results of AAP, IAP and latent period seem to indicate an early interaction between virus and vector starting at early stages of vector development. The presence of the GOANPL was determined in all stages of the vector (eggs, 1st to 4th instar and adults) in infected plants, in adults under different AAPs, in the progenies of viruliferous females, and in adults originated from nymphs developed from infected plants. More than 2.500 insects were tested by PCR to detect the GO-ANPL isolate. The virus was detected in nymphs from the 1st to 4th instar that had fed in infected plants and no virus was found in eggs from aviruliferous female that had been laid in infected plants. Transmission to the progenies was observed, since the virus was detected in all stages of insect development from eggs to adults. High level transmission was also observed in newly emerged adults that had acess to virus-infected plants as immatures. This fact, in addition to the transmission to the progenies, suggests that virus retention is an important part of virus/vector interaction. No transmission was observed from adults originated from viruliferous females. However, 33% of virus transmission was obtained when adults that retained virus from their early larval stages were employed. 2001. / Os begomovírus, vírus da família Geminiviridae transmitidos por mosca branca, têm emergido como sérios patógenos de culturas agronômicas e hortícolas em regiões tropicais e subtropicais de muitos países em todo o mundo. A partir da década de 80, têm aumentado os relatos da disseminação da mosca branca, Bemisia argentifolii, e de begomovírus provocando impacto devastador nas regiões em que ocorrem. No Brasil, estes patógenos têm sido limitantes para a produção de tomate (Lycopersicon esculentum) em várias áreas de cultivo com incidência crescente desde 1994. No presente trabalho, plantas de tomate exibindo sintomas de infecção provocada por vírus como mosaico, clorose internerval, enrolamento do limbo foliar e redução do crescimento, foram coletadas em lavouras de tomate indústria em Anápolis-GO. O vírus foi identificado como pertencente ao gênero Begomovirus mediante técnica de PCR usando oligonucleotídeos específicos que amplificaram fragmentos dos componentes A e B do genoma viral. No capítulo I são apresentados os resultados da caracterização molecular e no capítulo II, os dados da determinação do círculo de hospedeiros e da investigação da relação do begomovírus com o vetor Bemisia argentifolii. O isolado denominado GOANPL, foi clonado e parcialmente seqüenciado tendo sido obtidas as seqüências nucleotídicas dos genes da capa proteica, Rep e de toda a região intergênica, em um total de 2.130 nucleotídeos. A análise comparativa das seqüências revelou que, em geral, o GOANPL possui relacionamento genético distante com begomovírus da Ásia, Europa e África sendo mais próximo das espécies do Brasil, particularmente, com os begomovírus identificados em Minas Gerais (TRMV) e no Distrito Federal (DF-BR2). Com este último, apresentou alta homologia em todo o genoma podendo vir a constituir, com o mesmo, uma nova espécie. A determinação do círculo de hospedeiros do GO-ANPL foi realizada inoculando-se 46 espécies vegetais pertencentes a nove famílias botânicas, sob duas modalidades de inoculação: mecânica e com a mosca branca. Constatou-se que o GO-ANPL infecta, preferencialmente, plantas da família Solanaceae como Nicotiana benthamiana, Datura stramonium e Nicandra physalodes. O número de espécies infectadas com o inseto vetor foi superior ao obtido pela inoculação mecânica e diferiu dos resultados obtidos para outros isolados de begomovírus de tomate no Brasil. Os testes foram todos confirmados com hibridização com sondas moleculares, em \"dot blot\"No estudo da relação vírus-vetor, foram investigados o período de acesso de aquisição do vírus (PAA), o período de acesso de inoculação do vírus (PAI), e o período de latência do vírus na fase adulta do vetor, empregando-se cinco moscas/planta de tomate \'Santa Clara\' em todos os tratamentos. Para a definição do PAA e do PAI, foram testados nove diferentes períodos de tempo: 0,25, 0,5, 1, 2, 4, 8, 16, 20 e 24 horas. Nos testes para determinação do PAA, após cada um desses períodos seguiu-se uma inoculação de 48 horas e para definição do PAI, antes de cada período antecedeu-se um período de acesso de aquisição fixo de 72 horas. Constatou-se que o PAA mínimo da mosca branca foi de apenas 0,25 hora, com o qual foram obtidas 6% de plantas infectadas. O percentual de plantas aumentou de 6 para 65% com a extensão do PAA de 0,25 para 24 horas. Com relação ao período de acesso de inoculação do vírus, foram registrados 18% de plantas infectadas com o PAI de 0,5 hora. O percentual elevou-se para 67% quando 24 horas de PAI foram concedidos. Valores isolados de 90 e 100% na transmissão viral, também foram observados. O término do período latente do vírus no vetor ocorreu 16h após a aquisição do mesmo em planta infectada, considerando os 3% de infecção observados nas plantas inoculadas. Os dados obtidos indicam que a interação vírus-vetor é estabelecida desde a fase inicial de desenvolvimento do inseto. Como parte do estudo dessa interação, avaliou-se a presença do begomovírus GO-ANPL em todas as fases de desenvolvimento do inseto vetor (ovo, 1º ao 4º ínstar e adulto) na planta infectada, em adultos com diferentes PAA, na progênie de fêmeas virulíferas e em adultos cujos estágios ninfais desenvolveram-se em tomateiro infectado. A técnica PCR foi empregada para a detecção do GO-ANPL em mais de 2.500 espécimens testados. O vírus foi detectado em ninfas do 1º ao 4º ínstar que se alimentaram em plantas de tomate infectada, contudo, em ovos provenientes de avirulíferas, os quais foram ovipositados em planta infectada e coletados após sete dias, o vírus não foi detectado. A transmissão à progênie foi constatada pela detecção do vírus em ovos, ninfas e adultos que se desenvolveram em planta não hospedeira do vírus. A transmissão transestadial ocorreu com índice elevado e, ao lado da transmissão à progênie, indica que a retenção do vírus é uma etapa importante da interação vírus–vetor. A transmissão do vírus para mudas de tomate, a partir de adultos da progênie de fêmeas virulíferas, não foi constatada. Contudo, transmissão para tomateiro em um percentual de 33% foi verificado nos casos em que a inoculação das plantas foi realizada pelos adultos que retiveram o vírus da sua fase imatura (transestadial).