Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / Title page: Dept. of Genetics, Faculty of Science / ENGLISH ABSTRACT: Grapevine leafroll-associated virus 3 (GLRaV-3) is the type strain for the genus Ampelovirus, family Closteroviridae. There has been only one report that claimed the complete nucleotide sequence of GLRaV-3 (isolate NY-1, AF037268). Here we report the complete sequence of the South African GLRaV-3, isolate GP18 (EU259806) and show a significantly extended 5’ end. We used RLM-RACE to determine the 5’ end of GP18 and found the 5’ UTR to be 737 nt compared to 158 nt in the NY-1 sequence. This extended UTR was found in all other South African isolates of GLRaV-3 that were tested. In two collaborative studies the existence of the extended 5’ UTR was confirmed and further investigated. In the first study (Coetzee et al., 2010), metagenomic data generated by next generation sequencing (Illumina Genome Analyzer II) was analysed for GLRaV-3 specific sequences. Sequences similar to the GP18 isolate confirmed the sequence of the extended 5’ UTR. In the second study (Jooste et al., 2010), three genetic variants were identified and their respective 5’ UTRs studied. Great diversity was observed between the 5’ UTRs of the different genetic variants, however within a variant the 5’ UTR was found to be highly conserved. Grapevine leafroll-associated virus 3 is a positive sense, single stranded RNA virus that has been shown, like other closteroviruses, to produce subgenomic (sg) RNAs during replication. These sgRNAs are deployed for the expression of the ORFs on the 3’ half of the genome. In this study a dsRNA blot confirmed the presence of three, 3’ coterminal sgRNAs species [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] in GLRaV-3-infected plant material when using a probe directed at the coat protein gene. The specific 5’ terminal nucleotides for these sgRNAs as well as four additional sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] were determined by RLM-RACE for GLRaV-3 isolate GP18. The construction of a GLRaV-3 mini-replicon, analogous to RNA1 of Lettuce infectious yellows virus, for the evaluation of putative sg-promoters is also described. / AFRIKAANSE OPSOMMING: Grapevine leafroll-associated virus 3 (GLRaV-3) is ‘n lid van die Closteroviridae familie en die hooflid vir die genus Ampelovirus. Tot dusver was daar net een studie wat die volledige nukleïensuurvolgorde van GLRaV-3 gerapporteer het (isolaat NY-1, AF037268). In hierdie studie rapporteer ons die volledige volgorde van ‘n Suid-Afrikaanse GLRaV-3, isolaat nl. GP18 (EU259806) wat noemenswaardig langer is aan die 5’ kant. RLM-RACE is gebruik om die 5’ eindpunt van GP18 te bepaal en daar is gevind dat die 5’ ongetransleerde streek (UTR) 737 nt lank is in vergelyking met die 158 nt van die NY-1 volgorde. Die verlengde 5’ UTR is gevind in alle Suid-Afrikaanse monsters wat getoets is. Die verlengde 5’ UTR is bevestig en verder bestudeer tydens twee samewerkingsprojekte. In die eerste studie (Coetzee et al., 2010), is metagenomiese data gegenereer deur volgende-generasie volgordebepaling (Illumina Genome Analyzer II) en geanaliseer vir GLRaV-3 spesifieke volgordes. Volgordes soortgelyk aan die GP18 isolaat het die verlengde 5’ UTR volgorde bevestig. In die tweede studie (Jooste et al., 2010), is drie genetiese variante van GLRaV-3 geidentifiseer en hulle onderskeie 5’ UTR volgordes bepaal en bestudeer. Daar is groot diversiteit tussen die 5’ UTRs van die verskillende genetiese variante gevind, maar tussen isolate van dieselfde variant is die volgordes gekonserveerd. Grapevine leafroll-associated virus 3 is ‘n positiewe-sin, enkelstring RNA virus wat al voorheen bewys is om, soos ander closterovirusse, subgenomiese (sg) RNAs te produseer tydens replisering. Hierdie sgRNAs word ingespan vir die uitdrukking van die ORFs op die 3’ helfte van die virusgenoom. In hierdie studie is ‘n dsRNA klad gebruik om die voorkoms van 3’ ko-terminale sgRNAs [sgRNA(ORF3/4), sgRNA(ORF5) and sgRNA(ORF6)] te bevestig in GLRaV-3 geinfekteerde plantmateriaal deur gebruik te maak van ‘n peiler teen die kapsiedproteïengeen. Die spesifieke 5’ terminale nukleotiedes vir hierdie sgRNAs sowel as vier additionele sgRNAs [sgRNA(ORF7), sgRNA(ORF8), sgRNA(ORF9) and sgRNA(ORF10-12)] is bepaal deur gebruik te maak van RLM-RACE op die GLRaV-3 isolaat GP18. Die konstruksie van ‘n GLRaV-3 mini-repliserings konstruk, analoog aan die RNA1 van Lettuce infectious yellows virus, vir die evaluasie van moontlike sg-promotors word ook beskryf.

The European eel, Anguilla anguilla, has a complex life-cycle involving migrations between the Sargasso Sea and the river systems of Europe and North Africa. The requirement to move across large salinity gradients presents a significant physiological challenge and the developmental stages of the eel are closely linked to these migrations. Microarrays were created to elucidate gene expression changes occurring during; i. The transition from juvenile yellow to the adult sexually maturing, migrating silver eel and; ii. Salinity adaptation during the migration from freshwater to seawater. Groups (n = 6) of freshwater-acclimated yellow or silver eels were transferred to seawater for between 6 hours and 5 months and complementary control groups were transferred to freshwater. Brain, kidney, intestine and gill cDNA libraries were constructed using suppression subtractive hybridisation (SSH) techniques and a novel protocol based on Invitrogen's Gateway cloning system. The latter technique produced a low redundancy (~4 %) EST bank with a wide range of insert sizes (0.5 – 10 kb). Two microarray types were produced; one comprised 5760 clones from the two brain libraries whilst the other was a multi-tissue microarray incorporating 6144 clones from the SSH libraries. Pooled RNA samples were probed against the microarrays to highlight differentially expressed genes. Real-time quantitative PCR (QPCR) was used to validate the observed expression changes of selected genes in the tissues of individual fish. Following yellow to silver transformation of freshwater-adapted eels, the expression of tyrosine 3-mono-oxygenase/tryptophan 5-mono-oxygenase activation protein (14-3-3) and vaccinia related kinase 3 was shown to be consistently elevated. Prolactin expression increased in the brains of silver eels following two-day seawater-acclimation but QPCR analysis revealed high variation amongst freshwater-adapted eels. This is the first eel microarray study and the expression profiles highlighted herein will provide new avenues for research into the sexual development and salinity acclimation of A. anguilla.

This thesis is a part of the project aimed at sequencing the genome and transcriptome of the bank vole (Clethrionomys glareolus). The role of natural selection in the evolution of mitochondrial DNA (mtDNA) has been subject to much discussion; while some studies did not provide evidence that selection affected the phylogeography of the studied species, other considered adaptive evolution important. The bank vole is the key model we use to study the adaptation to climate change. As with other species, the phylogeography of the bank vole has been based on the variation of a small part of mtDNA. The goal of the thesis was to sequence the entire mitochondrial genome for representatives of all main mtDNA lineages of the bank vole using the Sanger and Illumina technologies, and to assess the role of selection and adaptation in the evolution and phylogeography of this species. The adaptive evolution in mtDNA probably was not the main driving force during the postlacial colonization of Europe. However, signatures of adaptive evolution have been found - an amino acid change with possible functional consequences in one gene and an excess of radical changes in physical- chemical properties of amino acids in populations at the latitudinal (northern and southern) extremes of the bank vole distribution. Key...

p83 é uma proteína com massa molecular aparente de 83 kDa, supostamente ainda não descrita, específica de sistema nervoso, e desenvolvimento regulada. p83 interage fortemente com laminina, Tau, tubulina e heat shock protein 90. p83 foi inicialmente detectada por imunohistoquímica e western blot usando-se um anticorpo anti-lectina KM+ purificado por afinidade. Sua purificação a partir de cérebro de rato está em progresso. Identificar o envolvimento de p83 em processos do Sistema Nervoso Central humano é um passo necessário em direção à compreensão de sua função biológica. Uma biblioteca de expressão de cDNA de cerebelo de rato (Lambda ZAP II, Stratagene) foi submetida ao screening, usando-se um anticorpo específico para isolar o cDNA de p83. O anticorpo anti-KM+ foi pré-adsorvido contra proteínas de E. coli XL1 Blue MRF, antes de ser usado no screening. As membranas foram reveladas por imunodetecção cromogênica (fosfatase alcalina e NBT/BCIP). A análise de todos os clones Lambda ZAP II foi feita por excisão in vivo do fagomídeo pBluescript, subclonagem em E. coli XL1 Blue MRF, purificação do DNA plasmidial e digestão com Eco RI. A seqüência correspondente ao clone isolado foi analisada usando-se ferramentas e bancos de dados do NCBI. A seqüência nucleotídica mostrou identidade com as isoformas A e E de drebrina. As isoformas A e E de drebrina foram detectadas em adulto e embrião, respectivamente. Drebrina A é uma proteína sistema nervoso-específica, desenvolvimento regulada e associa-se com F-actina. Embora drebrina e p83 compartilhem propriedades em comum, nossos dados de western blot indicaram que parecem não se tratar da mesma proteína. Nós investigamos a expressão de drebrina em Displasia Cortical Focal tipo IIB, comparando com córtex normal. As secções de tecido foram coradas com hematoxilina-eosina e prata (Bielchowsky). Secções foram processadas por imunohistoquímica usando-se os anticorpos anti-drebrina M2F6 e o DAS2, e recuperação antigênica. A detecção foi feita usando-se um anticorpo biotinilado, e DAB como cromógeno. Os tecidos displásicos (13 casos) foram obtidos cirurgicamente de tecidos exibindo epilepsia droga-resistente. Os controles foram obtidos de necrópsia de 15 pacientes sem história prévia de doenças neurológicas ou alterações patológicas. Nossos resultados sugerem uma associação entre drebrina e DCF IIB, um distúrbio do desenvolvimento cortical. / p83 is 83 kDa protein supposedly not yet described, nervous system specific, and developmentally regulated. p83 strongly interacts with laminin, Tau, tubulin and heat shock protein 90. It was initially detected by immunohistochemistry and western blot using an affinity-purified anti-lectin KM+ antibody. Its purification from rat brain is in progress. Identifying the involvement of p83 in human Central Nervous System processes is a required step towards understanding its biological roles. A premade cDNA rat cerebellum expression library (Lambda ZAP II, Stratagene) has been screened, using a specific antibody to isolate p83 cDNA. Anti-KM+ antibody was pre-adsorbed against E. coli XL1 Blue MRF proteins, before using in screening. Membranes were revealed by cromogenic immunodetection (alcaline fostase and NBT/BCIP). The analysis of all positive Lambda ZAP II clones was carried out by in vivo excision of pBluescript, subcloning in E. coli XL1 Blue MRF, plasmidial DNA purification and Eco RI digestion. The sequence corresponding to the clone isolated was analyzed using the NCBI tools and database. The nucleotide sequence showed identity with drebrin A and E isoforms. Drebrin A and E isoforms were detected in adults and embryos. Drebrin A is a neuron-specific, development-regulated F-actin-binding protein. It participates in growth cone extension and dendritic spine formation. Although have same drebrin and p83 properties in common, they not seem to be the same protein. We have investigated the expression of drebrin in Focal Cortical Dysplasia type IIB (FCD IIB) as compared to normal cortex. Tissue sections were stained with hematoxylin-eosin and silver (Bielchowsky). Sections were processed for immunohistochemistry using anti-drebrin antibodies M2F6 and DAS2, and an antigen retrieval technique. Detection was carried out using a biotinylated antibody, using DAB as chromogen. Dysplastic tissues (13 cases) were obtained at surgery for drug-resistant epilepsy. Controls were obtained at autopsy from 15 patients without history of neurological disorder and gross pathological changes. A specific drebrin labeling in dysplastic tissue was more intense than in controls. Indeed, most control cases exhibited at most a slightly higher staining than the background. Balloon, clear and undetermined cells, and giant, dysmorphic neurons, showed a conspicuous labeling by anti-drebrin. These cells showed a thin rim labeling of the nuclear membrane, and a finely punctate nuclear labeling. In contrast, a coarse nuclear, but a faint cytoplasm labeling was observed in autopsy cases. Our data suggest an association between Drebrin expression and the FCD IIB, a disturbance of cortical development.

During this PhD project three technical platforms were either improved or newly established in order to identify interesting genes involved in SNF, validate their expression and functionally characterise them. An existing 5.6K cDNA array (Colebatch et al., 2004) was extended to produce the 9.6K LjNEST array, while a second array, the 11.6K LjKDRI array, was also produced. Furthermore, the protocol for array hybridisation was substantially improved (Ott et al., in press). After functional classification of all clones according to the MIPS database and annotation of their corresponding tentative consensus sequence (TIGR) these cDNA arrays were used by several international collaborators and by our group (Krusell et al., 2005; in press). To confirm results obtained from the cDNA array analysis different sets of cDNA pools were generated that facilitate rapid qRT-PCR analysis of candidate gene expression. As stable transformation of Lotus japonicus takes several months, an Agrobacterium rhizogenes transformation system was established in the lab and growth conditions for screening transformants for symbiotic phenotypes were improved. These platforms enable us to identify genes, validate their expression and functionally characterise them in the minimum of time.<br> The resources that I helped to establish, were used in collaboration with other people to characterise several genes like the potassium transporter LjKup and the sulphate transporter LjSst1, that were transcriptionally induced in nodules compared to uninfected roots, in more detail (Desbrosses et al., 2004; Krusell et al., 2005). Another gene that was studied in detail was LjAox1. This gene was identified during cDNA array experiments and detailed expression analysis revealed a strong and early induction of the gene during nodulation with high expression in young nodules which declines with the age of the nodule. Therefore, LjAox1 is an early nodulin. Promoter:gus fusions revealed an LjAox1 expression around the nodule endodermis. The physiological role of LjAox1 is currently being persued via RNAi.<br> Using RNA interference, the synthesis of all symbiotic leghemoglobins was silenced simultaneously in Lotus japonicus. As a result, growth of LbRNAi lines was severely inhibited compared to wild-type plants when plants were grown under symbiotic conditions in the absence of mineral nitrogen. The nodules of these plants were arrested in growth 14 post inoculation and lacked the characteristic pinkish colour. Growing these transgenic plants in conditions where reduced nitrogen is available for the plant led to normal plant growth and development. This demonstrates that leghemoglobins are not required for plant development per se, and proves for the first time that leghemoglobins are indispensable for symbiotic nitrogen fixation. Absence of leghemoglobins in LbRNAi nodules led to significant increases in free-oxygen concentrations throughout the nodules, a decrease in energy status as reflected by the ATP/ADP ratio, and an absence of the bacterial nitrogenase protein. The bacterial population within nodules of LbRNAi plants was slightly reduced. Alterations of plant nitrogen and carbon metabolism in LbRNAi nodules was reflected in changes in amino acid composition and starch deposition (Ott et al., 2005). These data provide strong evidence that nodule leghemoglobins function as oxygen transporters that facilitate high flux rates of oxygen to the sites of respiration at low free oxygen concentrations within the infected cells. / Pflanzen der Ordnung der Leguminosen sind von weltweiter Bedeutung für Landwirtschaft und die allgemeine Nährstoffzusammensetzung von Böden. Die physiologische Besonderheit der Leguminosen liegt in ihrer Fähigkeit begründet, zusammen mit Bakterien, den sogenannten Rhizobien, eine Symbiose einzugehen, im Zuge derer es möglich wird, molekularen Luftstickstoff zu binden. Dieser biochemische Prozess findet in neu gebildeten Pflanzenorganen, den sogenannten Wurzelknöllchen statt.<br> In den Pflanzenwissenschaften werden Gene, die im Zuge der Infektion von Leguminosen mit Rhizobien reguliert werden und für den Entwicklungsprozess der Knöllchen eine wichtige Rolle zu spielen scheinen, als Noduline bezeichnet. Mit Hilfe von sogenannten Hochdurchsatzverfahren ist es in den letzten Jahren möglich geworden, die differentielle Expression von Tausenden von Genen gleichzeitig zu beobachten. Zu diesen Verfahren gehören sogenannte cDNA Arrays. Im Zuge dieser Doktorarbeit wurden die weltweit zweitgrößten cDNA Arrays für die Modell-Leguminose Hornklee (Lotus japonicus), der in unserer Gruppe als Untersuchungsobjekt verwendet wird, entwickelt. Mit Hilfe dieser Methode ist es uns möglich, die Regulation von etwa 15.000 Genen gleichzeitig zu untersuchen. Im Zuge von Untersuchungen, die sich mit der Entwicklung von Wurzelknöllchen in Lotus japonicus beschäftigten wurde ein Nodulin, dessen Existenz früher schon einmal beschrieben wurde, noch einmal bestätigt und die Funktion dieses Genes genauer untersucht. Es kodiert für das Enzym Vitamin C Oxidase, das unter Verwendung von molekularem Sauerstoff reduziertes Vitamin C zu einer anderen Form, dem Dehydroascorbat, oxidiert. Dabei wird Wasserstoffperoxid gebildet. Es konnte gezeigt werden, dass sich die Transkription dieses Gens in infizierten Wurzeln kontinuierlich im Verlauf der Symbiose erhöht, jedoch ist die Transkription in jungen Wurzelknöllchen höher als in alten. Darüber hinaus ist es in nur einer Zellschicht der Wurzelknöllchen, die sehr wichtig für die Entwicklung und tatsächliche Funktion der Knöllchen ist, aktiv. Aus den Beobachtungen kann geschlossen werden, dass dieses Gen eine wichtige Funktion in der Entwicklung der Knöllchen zu spielen scheint und vermutlich zur Zellstreckung und Zellteilung in dieser speziellen Zellschicht beiträgt. In einem zweiten Teil der Arbeit wurde sich einem zweiten und dem wohl wichtigsten Nodulin der Leguminosen, dem Leghämoglobin, gewidmet. Leghämoglobin ist dem menschlichen Blutbestandteil Hämoglobin sehr ähnlich und erfüllt dieselbe Aufgabe: es bindet Sauerstoff. Dieser Prozess ist für Leguminosen von erheblicher Bedeutung, da die bereits beschriebene Fixierung von molekularem Luftstickstoff durch ein bakterielles Enzym katalysiert wird, das extrem sauerstoffempfindlich ist. Leghämoglobine gelten unbestritten als die am besten charakterisierten Einweiße aus Wurzelknöllchen und Wissenschaftler behaupten seit fast 40 Jahren, dass sie essentiell für die Funktion der Knöllchen sind. Doch dies wurde bis jetzt nie bewiesen.<br> Mit Hilfe einer neuen Methode, die die spezifische Bildung von Eiweißen verhindert, war es uns möglich, die Synthese von Leghämoglobin in Lotus japonicus vollkommen zu unterdrücken. In Folge dessen zeigen die transgenen Pflanzen deutliche Nährstoffmangelerscheinungen, wenn sie ohne zusätzlichen Stickstoff aber zusammen mit Rhizobien angezogen werden. Sie können zwar Wurzelknöllchen bilden, jedoch sind diese kleiner und haben nicht die charakteristische rötliche Farbe, die bei unveränderten Pflanzen gefunden wird. Der Phänotyp dieser transgenen Pflanzen wird ganz eindeutig durch ihre Unfähigkeit hervorgerufen, Luftstickstoff fixieren zu können. Der Grund dafür ist das Fehlen des bakteriellen Enzyms, das für die Fixierung verantwortlich ist. Dieser Verlust wird durch erhöhte Sauerstoffgehalte in den Knöllchen verursacht. Außerdem konnten durch weitere Untersuchungen eine der vermuteten Funktionsmechanismen von Leghämoglobin bestätigt werden. Diese hier präsentierten Untersuchungen beweisen erstmalig die jahrzehnte alte Hypothese, dass Leghämoglobine essentiell für die Stickstofffixierung in Leguminosen sind.

科学研究費補助金 研究種目:一般研究(B)(2) 課題番号:07457240 研究代表者:宮田 敏男 研究期間:1995-1996年度

Made available in DSpace on 2014-06-11T19:32:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-07-24Bitstream added on 2014-06-13T20:43:03Z : No. of bitstreams: 1 colombo_j_dr_sjrp.pdf: 949474 bytes, checksum: 856a4b47ea1a9aa6adebae3e2e97b6a0 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os tumores de cabeça e pescoço ocupam, mundialmente, a quinta posição na lista das neoplasias mais freqüentes. O tipo histológico predominante é o carcinoma de células escamosas, que acomete a cavidade oral, a orofaringe, a hipofaringe e a laringe. O tumor de laringe é um dos tipos mais comuns, correspondendo a 25% dos casos, com alto índice de mortalidade e prognóstico reservado. O principal fator etiológico para o seu desenvolvimento é o consumo combinado de álcool e fumo. O desenvolvimento do câncer de cabeça e pescoço é um processo multipasso acompanhado por mudanças genéticas e epigenéticas. Recentemente, estudos envolvendo a tecnologia microarray têm identificado genes específicos, cuja expressão está alterada em câncer de cabeça e pescoço quando comparado ao tecido normal. No entanto, a maioria dos estudos são realizados usando tumores de diferentes sítios. Neste estudo, foi analisado somente amostras de carcinoma de laringe para minimizar as diferenças genéticas. Dessa forma, os objetivos do presente projeto foram identificar e validar possíveis biomarcadores moleculares envolvidos na carcinogênese de laringe. Para tanto, foi construído um cDNA microarray com 340 genes previamente identificados pelo Head and Neck Annotation Consortium. A expressão desses genes foi analisada em 8 amostras de tecido tumoral e em 4 amostras de tecido histologicamente normal de laringe pela técnica de microarray. Foram identificados 35 genes diferencialmente expressos (SNR ½1.0½, p-value 0.001), os quais estão envolvidos em diversos processos celulares como adesão celular, apoptose, ciclo celular, inibição de proteases, metabolismo, proteólise, reparo de DNA, regulação da transcrição e transdução de sinal. A robustez da assinatura dos 35 genes diferencialmente expressos foi confirmada em um conjunto adicional de 5 amostras tumorais e 6 amostras... / Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer world-wide. More than 90% of this cancer type has a squamous origin and common sites include oral cavity, oropharynx, hypopharynx and larynx Laryngeal squamous cell carcinoma is very common in head and neck cancer, corresponding to 25% of cases, with high mortality rates and poor prognosis. Tobacco use and/or alcohol consumption are the two principal risk factors involved in development of HNSCC. The development of head and neck cancer is a multistep process accompanied by genetic and epigenetic changes. In recent years, studies involving microarrays have identified specific genes whose expression has changed in head and neck cancer compared with normal tissue. However, most microarray studies are performed using tumors from different sites in head and neck. In our study, we analyzed only larynx carcinoma samples to minimize the genetic differences. Thus, the purpose of this work was to identify and to validater molecular biomarkers involved in larynx carcinogenesis. Therefore, we constructed a cDNA microarray containing 340 genes previously identified by Head and Neck Annotation Consortium. Expression analysis was applied to 8 larynx tumor samples and 4 larynx normal samples. We identified 35 differentially expressed genes between tumor and non-tumor adjacent tissue of larynx (SNR ½1.0½, p-value 0.001). Genes detected were involved in processes as apoptosis, cell adhesion, cell cycle, DNA repair, metabolism, protease inhibition, proteolysis, signal transduction and transcription regulation. The robustness of the 35-gene signature was confirmed using data from an additional set of 5 larynx tumor samples and 6 adjacent non-tumor larynx tissues. For real time RT-PCR validation we selected fourteen genes, of which 10 (ADCY6, AES, AL2SCR3, CRR9, CSTB, DUSP1, MAP3K5, PLAT, UBL1 and ZNF706) were validated... (Complete abstract click electronic access below)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Porcine reproductive and respiratory syndrome virus (PRRSV) was isolated for the first time in 1991 and since then it has been associated with significant economic losses to the pig industry worldwide. Although vaccination against PRRSV is widely used, an important advance would be the development of marker vaccines allowing serologic discrimination between vaccinated and naturally infected animals. The present study aimed to identify immunogenic and conserved regions dispensable to viral replication in different PRRSV proteins, which could be used as negative serologic markers in a new generation of liveattenuated vaccines. A fine mapping of B-cell linear epitopes in different PRRSV proteins by Pepscan is presented in the first part of this thesis. The results indicated the presence of several B-cell linear epitopes in the non-structural protein 2 (Nsp2) and in all structural proteins encoded by PRRSV, which were consistently recognized by antibodies raised in pigs experimentally infected with a North American strain of the virus (NVSL97-7895). The Nsp2 was found to harbor the highest frequency of immunodominant epitopes (n=18) when compared to structural proteins. In the structural proteins, epitopes consistently recognized by immune sera were located in all studied proteins. Overall, the highest degree of immunogenicity and conservation was exhibited by two epitopes identified in the C-terminal end of the M protein (ORF6). The antibodies recognizing the immunodominant epitopes of each protein were detected as early as days 7 to 15 post-infection (p.i.) and remained detectable until the end of the experiment (day 90 p.i). Based on their immunodominance and level of amino acid (aa) conservation, two target epitopes were selected to serve as serological marker candidates in each of the following PRRSV proteins: Nsp2, GP3 and M. These epitopes were deleted in the wild-type cDNA infectious clone (FL-12) by site-directed mutagenesis. The results of this study are presented in the second part of this thesis. A Nsp2 mutant virus (FLdNsp2/44) was successfully rescued following RNA transfection in MARC 145 cells. This epitope deletion mutant fulfilled the requirements for a differential vaccine virus such as efficient growth in vitro and in vivo and induction of active seroconversion as measured by a commercial ELISA kit associated with the absence of a marker-specific peptide-ELISA response in 100% (n=15) of the vaccinated animals. In vitro and in vivo characterization of the mutant virus clearly showed that removal of a 15-mer Nsp2 epitope had no effect on the immunogenicity, growth properties or virulence when compared to the wild type virus. On the other hand, deletions of previously identified peptide marker candidates within GP3 and M genes were shown to be lethal for virus viability in vitro. Alternatively, by substitution of 5aa at a time within a M peptide marker candidate, a viable mutant virus could be recovered although it still resulted in a positive marker virus. In summary, our results provide proof of concept that PRRSV marker vaccines can be developed using such methodology. Taken together, these data indicate that the combination of a mutant virus carrying a deletion of an immunodominant epitope and the corresponding peptide ELISA represents an attractive approach for the development of PRRSV differential modified-live vaccines. / O vírus da síndrome respiratória e reprodutiva dos suínos (PRRSV) foi isolado pela primeira vez em 1991 e, desde então, tem sido associado a perdas significativas para a suinocultura mundial. Apesar da vacinação contra o PRRSV ser amplamente utilizada, um grande avanço seria alcançado com a elaboração de vacinas diferenciais que permitam a discriminação sorológica entre animais vacinados e naturalmente infectados. O presente estudo teve como objetivo a identificação de regiões imunogênicas, conservadas e dispensáveis a replicação viral, em diferentes proteínas do PRRSV, que pudessem ser utilizadas como marcadores sorológicos negativos em uma nova geração de vacinas atenuadas. Na primeira parte desta tese estão apresentados os resultados de um mapeamento de epítopos lineares de linfócitos B em diferentes proteínas do PRRSV, pelo uso da tecnologia de Pepscan. Os resultados indicam a presença de diversas regiões imunodominantes na proteína não estrutural 2 (Nsp2) e em todas as proteínas estruturais do vírus. Essas regiões foram consistentemente reconhecidas pelo soro de suínos experimentalmente infectados com uma cepa norte-americana do PRRSV (NVSL97-7895). A maior freqüência de epítopos imunodominantes foi identificada na Nsp2 (n=18) e o mais alto grau de imunogenicidade e nível de conservação de aminoácidos foi observado em dois epítopos identificados na extremidade carboxi-terminal da proteína M (ORF6). Anticorpos reagentes com epítopos imunodominantes de cada proteína foram detectados inicialmente entre os dias 7-15 pós-infecção (pi), permanecendo em altos títulos até o final do experimento (dia 90 pi). Com base na imunodominância e nível de conservação de amino ácidos (aa) das seqüências mapeadas, dois epítopos alvos foram selecionados como candidatos a marcadores sorológicos negativos em cada uma das proteínas Nsp2, Gp3 e M. Esses epítopos foram então deletados em um clone infeccioso de cDNA (FL12) por mutagênese sítio-direcionada. Os resultados desses experimentos encontram-se descritos na segunda parte da tese. Um vírus mutante carreando a deleção de um epítopo imunodominante da Nsp2 (FLdNsp2/44) foi obtido após transfeccção de RNA viral em células MARC145. A caracterização in vitro e in vivo do vírus mutante demonstrou que a remoção dos 15 aa da Nsp2 não produziu efeito sobre a imunogenicidade, replicação ou virulência quando comparado ao vírus parental. Além disso, observou-se indução de soroconversão contra o PRRSV em animais infectados, detectada pelo uso de um teste ELISA comercial. Por outro lado, não foi detectada resposta humoral específica contra a região deletada nos animais imunizados com o FLdNsp2/44, conforme resultados de um teste ELISA contendo como antígeno um peptídeo sintético correspondente a seqüência removida. Por outro lado, deleções dos epítopos previamente identificados na Gp3 e proteína M foram letais à viabilidade viral in vitro. Alternativamente, um outro vírus mutante foi gerado pela substituição de 5 aa do epítopo identificado na proteína M, embora a alteração de resíduos não tenha sido suficiente para eliminar a imunogenicidade da região. Em resumo, os resultados do presente estudo se constituem em uma prova de conceito no sentido do desenvolvimento de vacinas diferenciais contra o PRRSV. A utilização de um vírus mutante carreando a deleção de um epítopo imunodominante, associado com um teste de ELISA baseado no peptídeo sintético correspondente a região deletada, representam uma alternativa para o desenvolvimento de vacinas diferenciais atenuadas contra o PRRSV.

Made available in DSpace on 2014-12-17T14:03:38Z (GMT). No. of bitstreams: 1 RenataKSE_DISSERT.pdf: 1992793 bytes, checksum: 77e111c6a7208973429d97e0cf1a51eb (MD5) Previous issue date: 2011-09-09 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Flowering is a fundamental process in the life cycle for plant. This process is marked by vegetative to reproductive apical meristem conversion, due to interactions between several factors, both internal and external to plant. Therefore, eight subtractive libraries were constructed using apical meristem induced or not induced for two contrasting species: Solanum lycopersicum cv. Micro-Tom and Solanum pimpinellifolium. Several cDNAs were identified and among these, were selected two cDNAs: one homologous cDNA to cyclophilin (LeCYP1) and the other to Auxin repressed protein (ARP). It has observed that LeCYP1 and ARP genes are important in the developmental process to plants. In silico analysis, were used several databases with the exclusion criterion E-value <1.0x10-15. As a result, conservation was observed for proteins analyzed by means of multiple alignments and the presence of functional domains. Then, overexpression cassettes were constructed for the ARP cDNA in sense and antisense orientations. For this step, it was used the CaMV35S promoter. The cDNA orientation (sense or antisense) in relation to the promoter was determined by restriction enzymes and sequencing. Then, this cassette was transferred to binary vector pZP211 and these cassettes were transferred into Agrobacterium tumefaciens LBA4404. S. lycopersicum cv. Micro-Tom (MT) and MT-Rg1 plants were transformed. In addition, seedlings were subjected to hormone treatments using a synthetic auxin (&#61537;- naphthalene acetic acid) and cyclosporin A (cyclophilin inhibitor) treatments and it was found that the hormone treatment there were changes in development of lateral roots pattern, probably related to decreases in auxin signaling caused by reduction of LeCYP1 in MT-dgt plants while cyclosporin A treatments, there was a slight delay in flowering in cv. MT plants. Furthermore, assay with real-time PCR (RT-qPCR) were done for expression level analysis from LeCYP1 and ARP in order to functionally characterize these sequences in tomato plants. / A flora??o ? fundamental no ciclo de vida de uma planta. Este processo ? marcado pela convers?o do meristema apical vegetativo em reprodutivo, devido a intera??es entre diversos fatores, tanto internos quanto externos ? planta. Diante disso, foram constru?das oito bibliotecas subtrativa utilizando ?pices meristem?ticos induzidos e n?o induzidos para a flora??o de duas esp?cies de tomateiro: Solanum lycopersicum cv Micro-Tom e S. pimpinellifolium. In?meros cDNAs foram identificados e dentre estes, foram selecionados o cDNA hom?logo a ciclofilina (LeCYP1) e a Prote?na Reprimida por Auxina (ARP) para a caracteriza??o in silico e funcional em esp?cie de tomateiro. Tem sido observado em outros sistemas vegetais que os genes LeCYP1 e ARP s?o importantes no processo de desenvolvimento da planta. Na an?lise in silico, foram utilizados diversos bancos de dados, tendo como crit?rio de exclus?o o E-value <1.0x10-15. Uma conserva??o para as prote?nas analisadas foi observada por meio dos alinhamentos m?ltiplos e a presen?a de dom?nios funcionais. Em seguida, foram realizadas constru??es de cassetes de superexpress?o para o cDNA hom?logo ? ARP em orienta??o senso e antissenso. Para esta etapa, foi utilizado o promotor forte CaMV35S presente no vetor intermedi?rio pBC (Stratagene). A orienta??o do cDNA (senso ou antissenso) em rela??o ao promotor foi determinada por meio de enzimas de restri??o e sequenciamento dos potenciais clones. Em seguida, este cassete foi transferido para o vetor bin?rio pZP211 e os clones obtidos foram confirmados pelo padr?o de restri??o utilizando diferentes enzimas. Subsequentemente, estes cassetes foram transferidos para a Agrobacterium tumefaciens LBA4404 e posteriormente transformado em plantas de S. lycopersicum cv. Micro-Tom (MT) e MT-Rg1. Em adi??o, pl?ntulas foram submetidas a tratamentos hormonais utilizando uma auxina sint?tica (?cido &#61537;-naftaleno ac?tico) e com ciclosporina A (inibidor de ciclofilina) e foi constatado que no tratamento hormonal houve altera??es no padr?o de desenvolvimento das ra?zes laterais, provavelmente relacionada ao d?ficit na sinaliza??o da auxina ocasionada pela redu??o de LeCYP1 em plantas MT-dgt, enquanto que no tratamento com ciclosporina A, ocorreu um atraso no florescimento de plantas cv MT. Al?m disso, ensaios de PCR em tempo real (RT-qPCR) foram efetuados para an?lise de express?o dos cDNAs hom?logos a LeCYP1 e ARP de modo a caracterizarmos funcionalmente estas sequ?ncias em plantas de tomateiro.

p83 é uma proteína com massa molecular aparente de 83 kDa, supostamente ainda não descrita, específica de sistema nervoso, e desenvolvimento regulada. p83 interage fortemente com laminina, Tau, tubulina e heat shock protein 90. p83 foi inicialmente detectada por imunohistoquímica e western blot usando-se um anticorpo anti-lectina KM+ purificado por afinidade. Sua purificação a partir de cérebro de rato está em progresso. Identificar o envolvimento de p83 em processos do Sistema Nervoso Central humano é um passo necessário em direção à compreensão de sua função biológica. Uma biblioteca de expressão de cDNA de cerebelo de rato (Lambda ZAP II, Stratagene) foi submetida ao screening, usando-se um anticorpo específico para isolar o cDNA de p83. O anticorpo anti-KM+ foi pré-adsorvido contra proteínas de E. coli XL1 Blue MRF, antes de ser usado no screening. As membranas foram reveladas por imunodetecção cromogênica (fosfatase alcalina e NBT/BCIP). A análise de todos os clones Lambda ZAP II foi feita por excisão in vivo do fagomídeo pBluescript, subclonagem em E. coli XL1 Blue MRF, purificação do DNA plasmidial e digestão com Eco RI. A seqüência correspondente ao clone isolado foi analisada usando-se ferramentas e bancos de dados do NCBI. A seqüência nucleotídica mostrou identidade com as isoformas A e E de drebrina. As isoformas A e E de drebrina foram detectadas em adulto e embrião, respectivamente. Drebrina A é uma proteína sistema nervoso-específica, desenvolvimento regulada e associa-se com F-actina. Embora drebrina e p83 compartilhem propriedades em comum, nossos dados de western blot indicaram que parecem não se tratar da mesma proteína. Nós investigamos a expressão de drebrina em Displasia Cortical Focal tipo IIB, comparando com córtex normal. As secções de tecido foram coradas com hematoxilina-eosina e prata (Bielchowsky). Secções foram processadas por imunohistoquímica usando-se os anticorpos anti-drebrina M2F6 e o DAS2, e recuperação antigênica. A detecção foi feita usando-se um anticorpo biotinilado, e DAB como cromógeno. Os tecidos displásicos (13 casos) foram obtidos cirurgicamente de tecidos exibindo epilepsia droga-resistente. Os controles foram obtidos de necrópsia de 15 pacientes sem história prévia de doenças neurológicas ou alterações patológicas. Nossos resultados sugerem uma associação entre drebrina e DCF IIB, um distúrbio do desenvolvimento cortical. / p83 is 83 kDa protein supposedly not yet described, nervous system specific, and developmentally regulated. p83 strongly interacts with laminin, Tau, tubulin and heat shock protein 90. It was initially detected by immunohistochemistry and western blot using an affinity-purified anti-lectin KM+ antibody. Its purification from rat brain is in progress. Identifying the involvement of p83 in human Central Nervous System processes is a required step towards understanding its biological roles. A premade cDNA rat cerebellum expression library (Lambda ZAP II, Stratagene) has been screened, using a specific antibody to isolate p83 cDNA. Anti-KM+ antibody was pre-adsorbed against E. coli XL1 Blue MRF proteins, before using in screening. Membranes were revealed by cromogenic immunodetection (alcaline fostase and NBT/BCIP). The analysis of all positive Lambda ZAP II clones was carried out by in vivo excision of pBluescript, subcloning in E. coli XL1 Blue MRF, plasmidial DNA purification and Eco RI digestion. The sequence corresponding to the clone isolated was analyzed using the NCBI tools and database. The nucleotide sequence showed identity with drebrin A and E isoforms. Drebrin A and E isoforms were detected in adults and embryos. Drebrin A is a neuron-specific, development-regulated F-actin-binding protein. It participates in growth cone extension and dendritic spine formation. Although have same drebrin and p83 properties in common, they not seem to be the same protein. We have investigated the expression of drebrin in Focal Cortical Dysplasia type IIB (FCD IIB) as compared to normal cortex. Tissue sections were stained with hematoxylin-eosin and silver (Bielchowsky). Sections were processed for immunohistochemistry using anti-drebrin antibodies M2F6 and DAS2, and an antigen retrieval technique. Detection was carried out using a biotinylated antibody, using DAB as chromogen. Dysplastic tissues (13 cases) were obtained at surgery for drug-resistant epilepsy. Controls were obtained at autopsy from 15 patients without history of neurological disorder and gross pathological changes. A specific drebrin labeling in dysplastic tissue was more intense than in controls. Indeed, most control cases exhibited at most a slightly higher staining than the background. Balloon, clear and undetermined cells, and giant, dysmorphic neurons, showed a conspicuous labeling by anti-drebrin. These cells showed a thin rim labeling of the nuclear membrane, and a finely punctate nuclear labeling. In contrast, a coarse nuclear, but a faint cytoplasm labeling was observed in autopsy cases. Our data suggest an association between Drebrin expression and the FCD IIB, a disturbance of cortical development.