Η πλειοτροπίνη είναι αυξητικός παράγοντας 18kDa ο οποίος παρουσιάζει υψηλή συγγένεια με την ηπαρίνη και εμφανίζει μεγάλη ομολογία με τη midkine, με την οποία σχηματίζουν μια διακριτή οικογένεια αυξητικών παραγόντων που έχουν υψηλή χημική συγγένεια με την ηπαρίνη. Η πλειοτροπίνη εκφράζεται σε διάφορες μορφές καρκίνου έχοντας είτε επαγωγικό είτε ανασταλτικό ρόλο, με τα διάφορα τμήματα της να εμφανίζουν διακριτές δράσεις σε σχέση με το μητρικό μόριο. Στην παρούσα εργασία υποκλωνοποιήθηκε η κωδική περιοχή της πλειοτροπίνης αλλά και τμημάτων της σε φορείς έκφρασης. Έγινε μετασχηματισμός βακτηρίων με τους φορείς που κατασκευάστηκαν και βρέθηκαν οι βέλτιστες συνθήκες έκφρασης των πεπτιδίων. Έγινε απομόνωση των τμημάτων που αντιστοιχούν στα αμινοξέα 9-59 (ΡΤΝ9-59) και 60-110 (ΡΤΝ60-110) με δυο διαφορετικά συστήματα και έγινε αξιολόγηση αυτών των συστημάτων ως προς την απόδοση και την καθαρότητα του προϊόντος. Η καθαρότητα των προϊόντων που προέκυψαν ήταν πολύ καλή, η απόδοση όμως ήταν χαμηλή σε όλες τις περιπτώσεις μικρότερη από 1 mg/ml. Λόγω της χαμηλής απόδοσης, οι ποσότητες των προϊόντων που προέκυψαν δεν είναι δυνατό να χρησιμοποιηθούν σε μελέτες διαμόρφωσης, xρησιμοποιήθηκαν όμως σε βιολογικές μελέτες. Από τη μελέτη των τμημάτων ΡΤΝ9-59 και ΡΤΝ60-110 που απομονώθηκαν, καθώς και ενός συνθετικού πεπτιδίου που αντιστοιχεί στα 26 τελευταία αμινοξέα της πλειοτροπίνης (ΡΤΝ111-136) προέκυψαν τα εξής συμπεράσματα: 1) Για την πρόσδεση της πλειοτροπίνης στην ανβ3 αλλά και στον RPTPβ/ζ δεν ευθύνονται οι δυο κεντρικές περιοχές της πλειοτροπίνης ΡΤΝ9-59 και ΡΤΝ60-110, 2) το πεπτίδιο ΡΤΝ111-136 έχει την ικανότητα να επάγει τη φωσφορυλίωση της υπομονάδας β3 της ιντεγκρίνης ανβ3 στη θέση Υ773 μέσω ενεργοποίησης του RPTPβ/ζ και της κινάσης c-Src, δράση που χάνεται παρουσία ΡΡ1 ή μετά από αποσιώπηση της έκφρασης του RPTPβ/ζ, 3) το πεπτίδιο ΡΤΝ111-136 έχει την ικανότητα να ενεργοποιεί τις ERΚ1/2, επίσης μέσω ενεργοποίησης του RPTPβ/ζ και της κινάσης c-Src. Το γεγονός ότι το πεπτίδιο ΡΤΝ111-136 ενεργοποιεί μόρια μεταγωγής σήματος που ενεργοποιούνται και από την πλειοτροπίνη, ενώ είναι γνωστό ότι αναστέλλει την επαγωγική της δράση στην αγγειογένεση in vivo και την κυτταρική μετανάστευση in vitro, καταδεικνύει ότι οι δράσεις της πλειοτροπίνης περιλαμβάνουν περισσότερους του ενός υποδοχείς ή/και συνυποδοχείς, η λεπτή ρύθμιση των οποίων οδηγεί στην τελική βιολογική της δράση, γεγονός που συνάδει με τις διαφορετικές δράσεις της πλειοτροπίνης που παρατηρούνται σε διαφορετικά συστήματα. Επιπλέον, τα αποτελέσματα αυτά ενισχύουν την άποψη ότι το C-τελικό άκρο της πλειοτροπίνης παίζει σημαντικό ρόλο στις βιολογικές της δράσεις που σχετίζονται με αγγειογένεση και καρκινική ανάπτυξη, ενώ παραμένει άγνωστος ο ρόλος των κεντρικών της περιοχών. / Pleiotrophin is 18kDa growth factor that shows high affinity to heparin. PTN is highly homologous to midkine (MK), forming together a family of growth factors. There has been found that pleiotrophin is expressed in different types of cancer. It is considered a tumour-promoting and angiogenic growth factor, although there are reports that show a negative effect of PTN on tumour growth and angiogenesis. Several data suggest that different regions of PTN may exert distinct, or even opposite effects on tumour growth and angiogenesis. In this study the coding area of pleiotrophin and regions of it were subcloned in expression vectors. Bacteria were transformed with the vectors and the conditions, where the maximum of the expression of the proteins was observed, were established. Regions of the native protein corresponding to aminoacids 9-59 (PTN9-59) and 60-110 (PTN 60-110) were isolated with two different ways and the results were compared for the quantity and the quality of the peptides. Because of the low quantity, the peptides could not be used for structural studies but they were used for biological studies. From the study of the peptides PTN9-59, PTN 60-110, and a synthetic peptide that corresponds to the last 26 aminoacids of pleiotrophin (PTN111-136) there have been emerged the following observations: 1) the two central regions of pleiotrophin seem not to have involvement to the binding of pleiotrophin to ανβ3 and to RPTPβ/ζ, 2) PTN111-136 induces the phosphorylation of β3 subunit at Y773 of the integrin via the activation of RPTPβ/ζ and c-Src kinase, something that is not observed in the presence of PP1 and siRPTPβ/ζ, 3) the peptide activates ERK1/2 via RPTPβ/ζ and c-Src kinase. The fact that the peptide PTN111-136 activates molecules of the signaling pathway of pleiotrophin and at the same time it is known that pauses the positive effect of the native protein to angiogenesis in vivo and the migration in vitro shows that reactions of pleiotrophin involve more than one of receptors or co-receptors and that the different regulation of which results in the different actions. Also the results strengthen the option that the C-terminal region of pleiotrophin has an important role in the biological activity which correlates with angiogenesis in cancer and the development of cancer although the role of the central regions of PTN is not known.

Thesis (PhD (Genetics))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Grapevine (Vitis vinifera L.) is a very important agricultural commodity that needs to be protected. To achieve this several in vivo tools are needed for the study of this crop and the pathogens that infect it. Recently the grapevine genome has been sequenced and the next important step will be gene annotation and function using these in vivo tools. In this study the use of Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, as transient expression and VIGS vector for heterologous protein expression and functional genomics in Nicotiana benthamiana and V. vinifera were evaluated. Full-length genomic sequences of three South African variants of the virus (GTR1-1, GTG11-1 and GTR1-2) were generated and used in a molecular sequence comparison study. Results confirmed the separation of GVA variants into three groups, with group III (mild variants) being the most distantly related. It showed the high molecular heterogeneity of the virus and that ORF 2 was the most diverse. The GVA variants GTG11-1, GTR1-2 and GTR1-1 were placed in molecular groups I, II and III respectively. A collaboration study investigating the molecular divergence of GVA variants linked to Shiraz disease (SD), described two interesting GVA variants of group II, namely GTR1-2 and P163-M5 (Goszczynski et al., 2008). The group II variants were found to be closely linked to the expression of SD. GTR1-2 was isolated from a susceptible grapevine plant that never showed SD symptoms (Goszczynski 2007). The P163-M5 variant that resulted in exceedingly severe symptoms in N. benthamiana and is that used as SD positive control by the grapevine industry, was found to contain a 119 nt insert within the native ORF2. Comparative analysis performed on the complete nt and aa sequences of group II GVA variants suggested that the components in the GVA genome that cause pathogenicity in V. vinifera are more complex (or different) to those that cause pathogenicity in N. benthamiana. The three South African variants (GTR1-1, GTG11-1 and GTR1-2) were assembled into fulllength cDNA clones under control of CaMV 35S promoters. After several strategies were attempted, including a population cloning strategy for GTR1-2, none of the clones generated were able to replicate in N. benthamiana plants. A single amino acid substitution at position 13 (Tyr/Y Cys/C) in ORF 5 of the GTR1-2 cDNA clone was shown to abolish or reduce replication of the virus to below a detectable level. Two infectious clones of Israeli variants of GVA (T7-GVA-GR5 and T7-GVA118, obtained from M. Mawassi) were brought under control of a CaMV 35S promoter (35S-GVA-GR5 and 35S-GVA118). Both clones were infectious, able to replicate, move systemically and induce typical GVA symptoms after agroinfiltration in N. benthamiana. These Israeli clones served as backbone for further experiments in characterisation of transient expression and VIGS vectors. The use of GVA as gene insertion vector (35S-GVA118) and gene exchange vector (35S-GVA-GR5- ORF2+sgMP) in N. benthamiana and V. vinifera was compared. The gene insertion vector, 35S-GVA118 was based on the full-length GVA genome. The gene exchange vector, 35SGVA- GR5- ORF2+sgMP, was constructed in this study by elimination of ORF 2 and insertion of a sgMP and unique restriction sites to facilitate transgene insertion. In N. benthamiana both vectors showed similar GUS expression levels and photobleaching symptoms upon virus-induced NbPDS silencing. In V. vinifera limited GUS expression levels and VIGS photobleaching symptoms were observed for the gene insertion vector, 35SGVA118. No GUS expression was observed for the gene exchange vector 35S-GVA-GR5- ORF2+sgMP in this host. As for silencing, one plant, agroinfiltrated with 35S-GVA-GR5- ORF2-VvPDS+sgMP, developed photobleaching symptoms in 3 systemic infected leaves after 4 months. This study showed that GVA can be used as gene insertion and gene exchange vector for expression and VIGS in N. benthamiana, but in grapevine its use is limited to expression and silencing of genes in the phloem tissue. It is also the first report that ORF 2 of GVA is not needed for long distance movement in grapevine. To investigate the possible role of the P163-M5 119 nt insertion and the GVA ORF 2 (of unknown function), in expression of symptoms in plants, ORF 2 of a 35S-GVA-GR5 cDNA clone was removed and subsequently substituted by the corresponding ORFs of four South African GVA variants. Upon agro-infiltration into N. benthamiana leaves, all chimaeric GVA constructs were able to move systemically through the plant. At this stage no correlation could be found between severity of symptoms, the presence of the P163-M5 insert and the specific GVA ORF 2 present in the chimaeras, indicating that other factors in the viral genome or the host plant probably play a crucial role. This study contributed to the pool of available in vivo tools for study and improvement of the valuable grapevine crop. It also opened several exciting research avenues to pursue in the near future. / AFRIKAANSE OPSOMMING: Wingerd (Vitis vinifera L.) is ‘n baie belangrike landboukundige gewas wat beskerm moet word. Om die rede word verskeie in vivo gereedskap vir die bestudering van die wingerdplant, en die patogene wat dit infekteer benodig. Die wingerd genoom se volgorde is bepaal en dus is die volgende logiese stap om die gene te annoteer en funksie daaraan toe te skryf. In hierdie studie is die gebruik van Grapevine virus A (GVA), genus Vitivirus, familie Flexiviridae, as tydelike uitdrukking- en virus-geinduseerde geenuitdowingsvektor vir heteroloë proteïen uitdrukking en funksionele genoomstudies in Nicotiana benthamiana en V. Vinifera getoets. Vollengte genoomvolgordes van drie Suid-Afrikaanse variante van die virus (GTR1-1, GTG11-1 en GTR1-2) is gegenereer en in ‘n molekulêre volgorde vergelyking studie gebruik. Resultate het die verdeling van GVA variante in drie groepe, waar groep III die verste verwant is, bevestig. Dit het ook gewys dat die virus ‘n baie hoë molekulêre heterogeniteit het en dat oopleesraam 2 (ORF 2) die mees divers is. ‘n Samewerking studie waar die molekulêre diversiteit van GVA variante, gekoppel aan Shiraz siekte (SD), ondersoek is, is twee interessante variante van groep II beskryf, naamlik GTR1-2 en P163-M5 (Goszczynski et al., 2008). Groep II variante is vooraf gevind om nou verwant te wees aan die ontwikkeling van SD in wingerd. Die GTR1-2 variant is uit ’n vatbare wingerd plant, wat nooit SD-simptome vertoon het nie, geïsoleer (Goszczynski et al., 2007). In die ORF 2 van die P163-M5 variant, wat simptome van die ergste graad in N. benthamiana geïnduseer het, en ook deur die industrie as betroubare SD-positiewe kontrole gebruik word, is ’n 119 nt invoeging gevind. Die vergelykende analise wat uitgevoer is, het daarop gedui dat die determinante van patogenisiteit in die GVA genoom moontlik meer kompleks kan wees in V. vinifera as in N. benthamiana. Die drie Suid-Afrikaanse variante (GTR1-1, GTG11-1 en GTR1-2) is in afsonderlike vollengte cDNA klone, onder beheer van CaMV 35S promotors, aanmekaargesit. Nadat verskeie kloneringstrategieë, insluitend ’n populasie kloneringstrategie vir die GTR1-2 kloon, gebruik is, het geen een van die cDNA klone die vermoë besit om in N. benthamiana te repliseer nie. ’n Enkele aminosuur substitusie in posisie 13 (Tyr/Y Cys/C) in ORF 5 van die GTR1-2 kloon, het die replisering van die virus tot laer as ’n opspoorbare vlak verlaag. Twee infektiewe klone van Israeliese GVA variante (T7-GVAGR5 en T7-GVA118, verkry van M. Mawassi) is onder beheer van ‘n CaMV 35S promotor geplaas (35S-GVA-GR5 and 35S-GVA118). Beide klone het na agro-infiltrasie in N. benthamiana plante gerepliseer, sistemies beweeg en tipiese GVA simptome geinduseer. Hierdie twee klone het as raamwerk gedien vir verdere eksperimente in karakterisering van tydelike uitdrukkings- en VIGS vektore. Die gebruik van GVA as geen-insvoegingsvektor (35S-GVA118) en geen-vervangingsvektor (35S-GVA-GR5- ORF2+sgMP) is in N. benthamiana en V. vinifera vergelyk. Die geen-invoegingsvektor 35S-GVA118, was op die vollengte GVA genoom gebasseer. Die geen-vervangingsvektor 35S-GVA-GR5- ORF2+sgMP, was in hierdie studie gekonstrueer. Dit is gemaak eerstens deur eliminasie van ORF 2 in die 35S-GVA-GR5 kloon, en tweedens deur die invoeging van ’n subgenomiese promotor van die beweginsproteïen (sgMP) en unieke beperkings-ensiemsetels om klonering van transgene te fasiliteer. Beide vektore het in N. benthamiana vergelykbare GUS uitdrukkingsvlakke en fotobleikende simptome getoon na virus-geinduseerde NbPDS uitdowing. In V. Vinifera is beperkte GUS uitdrukkingsvlakke en VIGS fotobleikende simptome opgemerk met die geen-invoegingsvektor, 35S-GVA118. Geen GUS uitdrukking is in hierdie gasheerplant met die geen-vervangingsvektor opgemerk nie. Slegs een wingerdplant het fotobleikende simptome, na 4 maande in 3 sistemies geïnfekteerde blare gewys, na agroinfiltrasie van die 35S-GVA-GR5- ORF2-VvPDS+sgMP konstruk. Hierdie studie het bevestig dat GVA as geen-invoeging en geen-vervangingsvektor, vir heteroloë proteïenuitdrukking en VIGS, in N. benthamiana gebruik kan word, maar dit blyk of die gebruik daarvan in wingerd meer tot die floeëm weefsel beperk is. Hierdie studie wys vir die eerste keer dat ORF 2 nie nodig is vir langafstand beweging van die virus in wingerd nie. Om die moontlike rol van die P163-M5 119 nt invoeging en die GVA ORF 2 (met onbekende funksie), in die uitdrukking van simptome in plante te ondersoek, is ORF 2 van die 35SGVA- GR5 cDNA kloon verwyder en daaropvolgens vervang met die ooreenstemmende ORFs van vier Suid-Afrikaanse GVA variante. Na agro-infiltrasie in N. benthamiana blare, het al die chimeras die vermoë gehad om te repliseer, sistemies te beweeg en simptome te induseer. Geen korrelasie kon gevind word tussen die graad van simptome, die teenwoordigheid van die P163-M5 insersie en die spesifieke GVA ORF 2 teenwoordig in die chimeras nie, wat dus daarop dui dat ander faktore in die virusgenoom of die gasheerplant `n moontlike belangrike rol kan speel. Hierdie studie het bygedrae tot die beskikbare poel van in vivo gereedskap vir die bestudering en verbetering van die kosbare wingerdgewas. Dit het ook talle interessante navorsingsgeleenthede oopgemaak om in die nabye toekoms te betree.

Submitted by Luciane Willcox (luwillcox@gmail.com) on 2016-09-12T17:13:48Z No. of bitstreams: 1 Nayara Final.pdf: 4140088 bytes, checksum: ab360755584995cab3cab99f56557007 (MD5) / Approved for entry into archive by Luciane Willcox (luwillcox@gmail.com) on 2016-09-12T18:50:42Z (GMT) No. of bitstreams: 1 Nayara Final.pdf: 4140088 bytes, checksum: ab360755584995cab3cab99f56557007 (MD5) / Made available in DSpace on 2016-09-12T18:50:42Z (GMT). No. of bitstreams: 1 Nayara Final.pdf: 4140088 bytes, checksum: ab360755584995cab3cab99f56557007 (MD5) / Fiocruz / Instituto Carlos Chagas, Fiocruz-PR, Curitiba, PR, Brasil / Sistemas convencionais para expressão de anticorpos diméricos consistem em geral na co-transfecção de dois plasmídeos, cada um codificando uma das duas cadeias do anticorpo. Este trabalho teve por objetivo construir novos vetores para expressão simultânea das duas cadeias do anticorpo e outras proteínas oligoméricas a partir de um único plasmídeo, buscando melhorar o nível de expressão de proteínas heterodiméricas. A eficiência desses vetores foi testada para expressão do anticorpo dimérico constituído pelo Fab do anticorpo MEDI-493 fusionado à fração cristalizável de uma IgG1 humana selecionada devido à sua maior estabilidade. A escolha deste anticorpo foi baseada no seu potencial para o tratamento e diagnóstico rápido de infecções pelo Vírus Sincicial Respiratório Humano (hRSV). Os sistemas de expressão desenvolvidos neste projeto foram usados também para expressar partículas tipo-vírus (VLPs) de hRSV. As VLPs foram reconstituídas utilizando a combinação das proteínas G e M, ou F0 e M de hRSV. Sequências de aminoácidos do anticorpo e das VLPs foram usadas para gerar genes sintéticos que foram subclonados nos vetores de expressão. A expressão do anticorpo foi conduzida em células HEK293/HEK293T. Os melhores resultados foram obtidos com o plasmídeo baseado em dois cassetes de expressão. O anticorpo recombinante foi purificado a partir dos sobrenadantes da cultura celular por cromatografia de afinidade utilizando proteína A/G imobilizada em uma fase sólida e a expressão foi confirmada por Western Blot. O reconhecimento da F1, que é a proteína do hRSV que contém o epítopo alvo deste anticorpo, foi demonstrado inicialmente por Western Blot contra a proteína F1 recombinante. A atividade do anticorpo recombinante foi subsequentemente testada utilizando amostras de paciente infectados com o vírus hRSV por Western Blot em também por ensaios de ELISA. O anticorpo recombinante foi capaz de reconhecer as amostras clínicas com especificidade equivalente a do anticorpo comercial MEDI-493, confirmando seu potencial para aplicação em diagnóstico de infecções causadas pelo hRSV. / Conventional systems for expression of dimeric antibodies rely usually on cotransfection of two plasmids, each one coding one of the two antibody chains. The aim of this work was to construct new vectors for simultaneous expression of both antibodies chains and of other oligomeric proteins from a single plasmid, aiming to improve the level of expression of heterodimeric proteins. The efficiency of these vectors was tested for expression of a dimeric antibody constructed by using the Fab fragment of the antibody MEDI-493 with the crystallizable fraction of a human IgG1 selected based on its higher stability. This antibody was chosen based on its potential for treatment and rapid diagnostic of Respiratory Syncytial Virus (hRSV) infections. The expression system developed in this project was also used to express virus-like particles (VLPs) of hRSV. The hRSV VLPs were reconstituted using G and M, or F0 and M protein combinations. The amino acid sequences of this antibody and VLPs were used to generate nucleotide sequences. The respective genes were acquired from a gene synthesis company and subcloned into the new expression vectors. Expression of the recombinant antibody was conducted in HEK293/HEK293T cells in parallel experiments to compare the performance of the three different genetic constructs. Best results were obtained with a plasmid containing two expression cassettes, one for each antibody chain. The recombinant antibody was purified from cell culture supernatants by affinity chromatography using protein A/G immobilized on a solid phase and its presence was confirmed by Western Blot. Interaction of the recombinant antibody with its target on the hRSV F1 protein was determined by Western Blot. The activity of the recombinant antibody was tested using clinical specimens from patients infected with hRSV by Western Blotting and ELISA assays. The recombinant antibody was able to detect the F1 protein the clinical specimens with efficiency similar to the commercial antibody MEDI-493. This result indicates the recombinant antibody can be used for diagnosis of infections caused by hRSV.

Made available in DSpace on 2015-04-20T12:31:30Z (GMT). No. of bitstreams: 1 jonathan.pdf: 3096267 bytes, checksum: 8d8efe41b3d0eaa2b1ee28e28f9ebb5e (MD5) Previous issue date: 2012-09-21 / Plasmids are extrachromosomal genetic elements that generally are not essential for survival of the bacteria , however , provide unique advantages ( as resistance to antibiotics) for the organism. For genetic engineering, naturally occurring plasmids have been modified extensively for the production of vectors with the desired characteristics. Promoters are elements of a vector which may have a profound effect on the strength and duration of transcription and, consequently, the protein yield . The mRNA synthesis starts when RNA polymerase binds the promoter sequence adjacent to the target gene. Sigma factors are proteins that regulate transcription in bacteria , and they can be activated in different environmental conditions . The sigma factor ( S ) is seen as a master regulator of the general stress response , encoded from the gene RpoS in late stationary phase . This factor is the primary regulator of the genes stationary phase . Prosecutors based metabolic regulators of stationary phase represent a class of promoters metabolically controlled and can be exploited for the design of expression vectors . This project were developed three expression cassettes for construction of expression vectors , which have promoters with affinity for sigma factor S , in order to evaluate the expression profile of recombinant proteins in Escherichia coli . To evaluate the strength of promoters and expression profiling , we used the green fluorescent protein ( GFP ) as a reporter gene . The three vectors constructed, which were named p26 , p53 and PFS expressed GFP in the correct formation of the fluorophore , and the vector p26 and p53 showed overexpression of the recombinant protein when compared to the vector pGS21a . Furthermore , it was observed that the vectors constructed here are contributing to the reduction in the rate of growth and filamentation of E. coli. This gives the morphological plasticity bacterium higher storage capacity of the recombinant protein expressed . / Plasmídeos são elementos genéticos extracromossomais, que no geral não são essenciais para a sobrevivência da bactéria, entretanto, conferem vantagens peculiares (como resistência à antibióticos) para o organismo. Para engenharia genética, plasmídeos de ocorrência natural têm sido modificados extensivamente para produção de vetores com as características desejadas. Promotores são elementos de um vetor que podem ter um profundo efeito na força e duração da transcrição, e consequentemente, no rendimento proteico. A síntese de mRNA inicia quando a RNA polimerase se liga a sequência promotora , adjacente ao gene alvo. Fatores sigma são proteínas que regulam a transcrição em bactérias, e os mesmos podem ser ativados em diferentes condições ambientais. O fator sigma (S) é visto como um regulador mestre de resposta ao estresse geral, codificado a partir do gene RpoS na fase estacionária tardia. Este fator é o regulador primário dos genes de fase estacionária. Promotores baseados em reguladores metabólicos de fase estacionária representam uma classe de promotores controlados metabolicamente e que podem ser explorados para o design de vetores de expressão. Neste projeto foram desenvolvidos três cassetes de expressão para a construção de vetores de expressão, os quais possuem promotores com afinidade pelo fator sigma S, com o objetivo de avaliar o perfil de expressão de proteínas recombinantes em Escherichia coli. Para a avaliação da força dos promotores e do perfil de expressão, utilizou-se a proteína verde fluorescente (GFP) como gene repórter. Os três vetores construídos, os quais foram denominados de p26, p53 e pFS expressaram a GFP com a correta formação do fluoróforo, sendo que os vetores p26 e p53 apresentaram superexpressão da proteína recombinante, quando comparados ao vetor pGS21a. Ademais, observou-se que os vetores aqui construídos estão contribuindo para a redução da taxa de crescimento e filamentação da E. coli. Esta plasticidade morfológica confere à bactéria maior capacidade de armazenamento da proteína recombinante expressa.

The species of Brucella were isolated and characterized almost 120 years ago and their genomes sequenced for almost 4 years. Compared to other bacterial pathogens relatively, little is known about the factors contributing to their persistence in hosts and multiplication within phagocytic cells. Also, many aspects of the interactions between Brucella and its host remain unclear. Molecular characterization of intracellular survival processes of Brucella will provide guidance for additional prevention and control measures. One of the features that distinguishes Brucella is that they do not express classic virulence factors. Thus identification of virulence factors has been elusive and some of the identified virulence genes are putative. Disruption of putative virulence genes and studying the consequent effect on attenuation in cell lines or mouse models is a widely used method. However, in most cases it is not apparent whether the mutated genes encode virulence factors or merely affect normal metabolic or biological functions. Some mutations in Brucella can be compensated by redundancy or backup mechanisms. One method for identifying putative virulence genes involved in pathogenesis is to express these genes in a nonpathogenic host and isolate recombinants with increased virulence or survival ability either in cell culture or animal model. We hypothesize that over-expression of Brucella putative virulence genes in the non-pathogenic and close phylogenic relative Ochrobactrum anthropi should enhance its survival in infection models in vivo. O. anthropi is one of the closest Brucella relatives based on DNA, rRNA, and protein analyses but it is unable to establish chronic infection and considered as opportunistic pathogen that, under certain circumstances, may produce disease in immunocompromised humans. Therefore, we established enhanced expression system in Brucella and Ochrobactrum to identify B. suis virulence genes. We created an enhanced expression system that can be used for cloning and expression of heterologous genes in Brucella and Ochrobactrum. We studied the transcriptional activity of several promoters and created some tools to enhance the expression, detection and purification of Brucella recombinant protein in Ochrobactrum. The presumable importance of alkyl hydroperoxide reductases encoded by ahpC and ahpD genes and their contribution to intracellular survival of Brucella were studied by over-expressing them. The recombinant O. anthropi expressing B. suis ahpC and ahpD genes were able to resist in vitro killing by H2O2 and or cumene hydroperoxide and survived longer in the macrophage J774 A.1 cell line. The control O. anthropi was cleared from BALB/c mice in five days while the recombinants were recovered from spleens, livers and lungs of infected mice up to eight days post-infection. We tested the contribution of B. suis cyclic glucan synthetase gene (cgs) to virulence by over-expressing it in O. anthropi. We studied the ability of the recombinant O. anthropi to resist killing in vitro and in vivo. We generated evidence that B. suis cgs when over-expressed in O. anthropi increased the amount of cyclic glucans synthesized and accumulated in the periplasmic space. This accumulation changed the virulence of the microorganism from a soil bacterium that cleared from mice in less than five days into a pathogenic organism that could survive up to 9 days and at higher doses killed the mice. In summary, several vectors have been constructed for gene expression and protein purification in Brucella and Ochrobactrum. Novel useful tools for enhancement of heterologous gene expression were created and demonstrated to work in Brucella and Ochrobactrum. Brucella putative virulence genes were studied in Ochrobactrum using the newly constructed vectors and tools. Ochrobactrum as a gain of function model for studying putative virulence genes of intracellular pathogens in general and for Brucella in particular proved to be a very useful model. / Ph. D.

O prion celular (PrPc) é uma glicoproteína ligada à membrana plasmática por uma âncora de GPI (glycosylphosphatidylinositol). A sua isoforma anormal (PrPsc) é uma molécula infecciosa que causa várias doenças neurodegenerativas em mamíferos. A etiologia dessas doenças está associada a uma mudança conformacional pós-traducional de PrPc que ocorre após sua internalização (Prusiner, 1998). Na tentativa de desvendar as funções fisiológicas de PrPc, nosso grupo tem identificado e caracterizado as interações celulares que PrPc participa. A primeira delas é a interação entre PrPc e STI1 (Stress Inducible Protein 1). Essa interação transduz sinalização por cAMP e PKA levando a neuroproteção contra morte celular programada (Chiarini e cols, 2002; Zanata e cols, 2002). A segunda é a interação específica que existe entre PrPc e as proteínas da matriz extracelular, laminina e vitronectina, contribuindo para os processos neuronais, tais como crescimento, manutenção (Graner e cols., 2000 a e b) e regeneração dos neuritos (Hajj e cols., submetido), além da formação de memória de curta e longa duração (Coitinho e cols., submetido). Na primeira parte deste trabalho, procuramos investigar os genes regulados pelos sinais resultantes dessas interações e também pela remoção de PrPc usando a técnica de \"differential display\'\' RT-PCR. Na segunda parte do trabalho, caracterizamos que a interação PrPc - laminina é capaz de induzir uma sinalização transitória de cálcio, a qual ocorre mesmo na ausência de cálcio do meio extracelular. PrPc é uma molécula que cicla continuamente entre a membrana plasmática e os compartimentos intracelulares. Estudos recentes têm correlacionado o processo de internalização de PrPc com alguns dos seus papeis fisiológicos, tais como, homeostase de Cu2 + (Brown, 2001 ), interação com receptor de laminina (Gauczynski e cols, 2001) e até na conversão de PrPc para PrPsc (McKinley e cols, 1991; Arnold e cols, 1995). Portanto, na terceira parte deste trabalho, caracterizamos a localização e o tráfego celular de PrPc mostrando que PrPc está localizado na membrana plasmática e em compartimentos intracelulares e que trafega pelo Golgi, membrana plasmática, endossomos iniciais e de reciclagem. Foram mapeados ainda domínios na região amino-terminal responsáveis pela internalização de PrPc e na região carboxi-terminal como participantes da via secretora. Este trabalho contribuiu para o esclarecimento de alguns eventos biológicos relacionados à sinalização e ao tráfego de PrPc. Estes achados são de grande importância para a determinação das funções celulares de PrPc e ainda dos mecanismos envolvidos com as doenças relacionadas com esta molécula. / The cellular prion protein (PrPc) is a glycoprotein anchored to the plasma membrane by GPI (Glycosyl-phosphatidylinositol). Its abnormal isoform (PrPsc) is the infectious protein responsible for several neurodegenerative diseases. The main etiology of the prion diseases is related to conformational changes in the PrPc molecule, which occur after its internalization (Prusiner, 1998). In order to elucidate the physiological functions of PrPc, our group identified and characterized interactions between PrPc and other cellular molecules. The first is the interaction between PrPc and STI 1 (Stress Inducible Protein 1). This interaction has an important role in the neuroprotection against apoptosis through cAMP and PKA signaling (Chiarini et al., 2002; Zanata et al., 2002). PrPc also interacts with proteins of the extracellular matrix such as laminin and vitronetin. These interactions contribute for neurite outgrowth, maintenance and regeneration (Graner et al., 2000 a and b; Hajj et al., submitted) and also in memory formation (Coitinho et al., submitted). In the first part of this work we have applied the differential dysplay RTPCR technique in order to identify genes that are regulated by PrPc - STI 1 interaction and also by the deletion of PrPc. In the second part we have demonstrated that PrPc-laminin interaction induces transient calcium signaling in neuronal cells, which occurs even in the absence of extracellular calcium. PrPc cycles continuously between the plasma membrane and intracellular compartments. This mechanism is associated with some of the physiological function of PrPc, such as Cu2+ homeostasis (Brown, 2001 ), interaction with laminin receptor (Gauczynski et al., 2001 ), and PrPc conversion into PrPsc (McKinley et al., 1991; Arnold et al., 1995). Thus, in the third part of this project, we have characterized the PrPc localization at the cell surface and in intracellular compartments. The protein trafficking through Golgi apparatus, plasma membrane, early and recycling endosomes was also defined. Moreover, we have determinated that N-terminus PrPc domain is responsible for its internalization while C-terminus participates in PrPc delivery. Therefore, this work has contributed to elucidate biological events related to the cell signaling and trafficking of PrPc, which are important for the characterization of PrPc physiological functions and to understand the pathological mechanisms related to this molecule.

O prion celular (PrPc) é uma glicoproteína ligada à membrana plasmática por uma âncora de GPI (glycosylphosphatidylinositol). A sua isoforma anormal (PrPsc) é uma molécula infecciosa que causa várias doenças neurodegenerativas em mamíferos. A etiologia dessas doenças está associada a uma mudança conformacional pós-traducional de PrPc que ocorre após sua internalização (Prusiner, 1998). Na tentativa de desvendar as funções fisiológicas de PrPc, nosso grupo tem identificado e caracterizado as interações celulares que PrPc participa. A primeira delas é a interação entre PrPc e STI1 (Stress Inducible Protein 1). Essa interação transduz sinalização por cAMP e PKA levando a neuroproteção contra morte celular programada (Chiarini e cols, 2002; Zanata e cols, 2002). A segunda é a interação específica que existe entre PrPc e as proteínas da matriz extracelular, laminina e vitronectina, contribuindo para os processos neuronais, tais como crescimento, manutenção (Graner e cols., 2000 a e b) e regeneração dos neuritos (Hajj e cols., submetido), além da formação de memória de curta e longa duração (Coitinho e cols., submetido). Na primeira parte deste trabalho, procuramos investigar os genes regulados pelos sinais resultantes dessas interações e também pela remoção de PrPc usando a técnica de \"differential display\'\' RT-PCR. Na segunda parte do trabalho, caracterizamos que a interação PrPc - laminina é capaz de induzir uma sinalização transitória de cálcio, a qual ocorre mesmo na ausência de cálcio do meio extracelular. PrPc é uma molécula que cicla continuamente entre a membrana plasmática e os compartimentos intracelulares. Estudos recentes têm correlacionado o processo de internalização de PrPc com alguns dos seus papeis fisiológicos, tais como, homeostase de Cu2 + (Brown, 2001 ), interação com receptor de laminina (Gauczynski e cols, 2001) e até na conversão de PrPc para PrPsc (McKinley e cols, 1991; Arnold e cols, 1995). Portanto, na terceira parte deste trabalho, caracterizamos a localização e o tráfego celular de PrPc mostrando que PrPc está localizado na membrana plasmática e em compartimentos intracelulares e que trafega pelo Golgi, membrana plasmática, endossomos iniciais e de reciclagem. Foram mapeados ainda domínios na região amino-terminal responsáveis pela internalização de PrPc e na região carboxi-terminal como participantes da via secretora. Este trabalho contribuiu para o esclarecimento de alguns eventos biológicos relacionados à sinalização e ao tráfego de PrPc. Estes achados são de grande importância para a determinação das funções celulares de PrPc e ainda dos mecanismos envolvidos com as doenças relacionadas com esta molécula. / The cellular prion protein (PrPc) is a glycoprotein anchored to the plasma membrane by GPI (Glycosyl-phosphatidylinositol). Its abnormal isoform (PrPsc) is the infectious protein responsible for several neurodegenerative diseases. The main etiology of the prion diseases is related to conformational changes in the PrPc molecule, which occur after its internalization (Prusiner, 1998). In order to elucidate the physiological functions of PrPc, our group identified and characterized interactions between PrPc and other cellular molecules. The first is the interaction between PrPc and STI 1 (Stress Inducible Protein 1). This interaction has an important role in the neuroprotection against apoptosis through cAMP and PKA signaling (Chiarini et al., 2002; Zanata et al., 2002). PrPc also interacts with proteins of the extracellular matrix such as laminin and vitronetin. These interactions contribute for neurite outgrowth, maintenance and regeneration (Graner et al., 2000 a and b; Hajj et al., submitted) and also in memory formation (Coitinho et al., submitted). In the first part of this work we have applied the differential dysplay RTPCR technique in order to identify genes that are regulated by PrPc - STI 1 interaction and also by the deletion of PrPc. In the second part we have demonstrated that PrPc-laminin interaction induces transient calcium signaling in neuronal cells, which occurs even in the absence of extracellular calcium. PrPc cycles continuously between the plasma membrane and intracellular compartments. This mechanism is associated with some of the physiological function of PrPc, such as Cu2+ homeostasis (Brown, 2001 ), interaction with laminin receptor (Gauczynski et al., 2001 ), and PrPc conversion into PrPsc (McKinley et al., 1991; Arnold et al., 1995). Thus, in the third part of this project, we have characterized the PrPc localization at the cell surface and in intracellular compartments. The protein trafficking through Golgi apparatus, plasma membrane, early and recycling endosomes was also defined. Moreover, we have determinated that N-terminus PrPc domain is responsible for its internalization while C-terminus participates in PrPc delivery. Therefore, this work has contributed to elucidate biological events related to the cell signaling and trafficking of PrPc, which are important for the characterization of PrPc physiological functions and to understand the pathological mechanisms related to this molecule.

O principal controle do ciclo celular de mamíferos, que é dividido em G0/G1/S/G2/M, ocorre na transição G0→G1→S. Nesta tese mostramos que a proteína ciclina D1 desempenha um papel fundamental nos circuitos de transdução de sinais que regulam a transição G0→G1→S na linhagem Y-1 de células adrenocorticais de camundongo. Esta conclusão não é surpreendente, uma vez que, ao longo dos últimos anos, muitos laboratórios contribuíram para estabelecer a noção de que a atividade das diversas formas do complexo ciclina/CDK é essencial para a transição G0→G1→S, e também para outras etapas do ciclo celular. Em células Y-1, FGF-2 induz tardiamente (5-6h) a expressão do gene e da proteína ciclina D 1, através de um processo dependente de síntese de proteínas. Peptídeos hipofisários não identificados e vasopressina bloqueiam a indução de ciclina DI, antagonizando FGF-2. Por este mecanismo, vasopressina exerce um efeito anti-mitótico, bloqueando a transição G0→G1→S promovida por FGF-2. ACTH, que também exibe um forte efeito anti-mitótico sobre FGF-2 não afeta a indução de ciclina D1. A transfecção dupla de uma forma induzível de c-Myc (MycER) e constitutiva do cDNA de ciclina D1, em presença de ACTH mimetiza a ação mitogênica de FGF-2 em células Y-1 no estado G0. Estes resultados mostram que, em células adrenocorticais, c-Fos, c-Jun, c-Myc e ciclina D1 agem de forma independente e complementar, sendo necessários para a transição G0→G1→S do ciclo celular. / The main control of mammalian cell cycle, which is divided in G0/G1/S/G2/M, occurs in G0→G1→S transition. In this work we show that cyclin D1 protein plays a key role in signal transduction circuits underlying the G0→G1→S transition of mouse Y-1 adrenocortical cell line. This conclusion is not surprising, once in the last years, many laboratories have contributed to establish the notion that the activity of the distinct forms of cyclin/CDK complexes is essential for the G0→G1→S transition, and also for other phases transition of cell cycle. In Y-1 cells, FGF-2 causes a delayed (5-6h) induction of cyclin D1 gene and protein, through a process dependent on protein synthesis. Hypophisary peptides, not identified, as well as vasopressin, block cyclin D1 induction, antagonizing FGF-2. By this mechanism, vasopressin exert an antimitotic effect, blocking G0→G1→S transition promoted by FGF-2. ACTH, which also exhibit a strong anti-mitotic effect upon FGF-2, does not affect cyclin D1 induction. Double transfection of inducible c-Myc (MycER) and constitutive cyclin D1 cDNA, in the presence of ACTH, mimics the mitogenic action of FGF-2 in G0 Y-1 cells. Altogether, these results show that, in adrenocortical cells, c-Fos, c-Jun, c-Myc and cyclin D1 act in an independent and complementary manner, being necessary for the G0→G1→S transition of cell cycle.