Chicken and pufferfish genomic libraries were screened with the intent of isolating PEA3 orthologues from evolutionarily removed vertebrate species. The chicken PEA3 gene was found to reside within 15 kb of genomic sequence, and approximately 2 kb of promoter sequence has been identified. Although the pufferfish PEA3 genomic sequence has yet to be completed, exons 2, 3, 4, 5, 12 and 13 have been found, and approximately 1 kb of sequence upstream of the putative start codon has been determined. In addition to the genomic sequence that was isolated, 5' RACE using pufferfish heart RNA produced a 334 bp cDNA sequence encompassing exons 2 to 5 of pufferfish PEA3. A pufferfish homologue of the human RNA helicase 1 (HRH1) gene was also found 3' of the PEA3 gene. Given that HRH1 is also found 3' of human PEA3 (E1AF) on chromosome 17q21, this finding would seem to indicate that chromosomal synteny is maintained between the human and pufferfish PEA3 loci. A four-way alignment of the mouse, human, chicken and pufferfish PEA3 promoters revealed that a region spanning from +1 to -260, relative to the transcriptional start site of mouse PEA3, is well conserved across the four promoters. Conserved transcription factor binding sites for SRY, HNF3β, NFY, AP-1, TCF, AP-2, v-myb, δEF1, and c-Ets-1 were found in three, and in some cases four of the promoters. An additional outcome of the pufferfish genomic library screen was the isolation of a pufferfish orthologue of the Ets transcription factor ERM. The relevance of these findings to the issue of transcriptional regulation of PEA3 expression is discussed. / Thesis / Master of Science (MS)

Die Mykorrhiza (griechisch: mýkēs für „Pilz”; rhiza für „Wurzel”) stellt eine Symbiose zwischen Pilzen und einem Großteil der Landpflanzen dar. Der Pilz verbessert durch die Symbiose die Versorgung der Pflanze mit Nährstoffen, während die Pflanze den Pilz mit Kohlenhydraten versorgt. Die arbuskuläre Mykorrhiza (AM) stellt dabei einen beson-dere Form der Mykorrhiza dar. Der AM-Pilz bildet dabei während der Symbiose die namensgebenden Arbuskeln innerhalb der Wurzelzellen als Ort des primären Nährstoff- austausches aus. Die AM-Symbiose (AMS) ist der Forschungsschwerpunkt dieser Arbeit. Als Modellorganismen wurden Medicago truncatula und Glomus intraradices verwendet. Es wurden Transkriptionsanalysen durchgeführt um u.a. AMS regulierte Transkriptions- faktoren (TFs) zu identifizieren. Die Aktivität der Promotoren von drei der so identifizier-ten AMS-regulierten TFs (MtOFTN, MtNTS, MtDES) wurde mit Hilfe eine Reportergens visualisiert. Der Bereich der größten Promotoraktivität waren in einem Fall nur die ar- buskelhaltigen Zellen (MtOFTN). Im zweiten Fall war der Promotor auch aktiv in nicht arbuskelhaltigen Zellen, jedoch am stärksten aktiv in den arbuskelhaltigen Zellen (MtNTS). Ein weiterer Promotor war in arbuskelhaltigen Zellen und den diesen benach-barten Zellen gleich aktiv (MtDES). Zusätzlich wurden weitere Gene als AMS-reguliert identifiziert und es wurde für drei dieser Gene (MtPPK, MtAmT, MtMDRL) ebenfalls eine Promotor::Reporter-Aktivitäts- studie durchgeführt. Die Promotoren der Kinase (MtPPK) und des Ammoniumtrans-porters (MtAmt) waren dabei ausschließlich in arbuskelhaltigen Zellen aktiv, während die Aktivität des ABC-Transporters (MtMDRL) keinem bestimmten Zelltyp zuzuordnen war. Für zwei weitere identifizierte Gene, ein Kupfertransporter (MtCoT) und ein Zucker- bzw. Inositoltransporter (MtSuT), wurden RNA-Interferenz (RNAi)-Untersuchungen durchgeführt. Dabei stellte sich in beiden Fällen heraus, dass, sobald ein RNAi-Effekt in den transformierten Wurzeln vorlag, diese in einem deutlich geringerem Ausmaß wie in der Wurzelkontrolle von G. intraradices kolonisiert worden sind. Im Falle von MtCoT könnte das aus dem selben Grund geschehen, wie im Falle von MtPt4. Welche Rolle MtSuT genau in der Ausbildung der AMS spielt und welche Rolle Inositol in der Aus- bildung der AMS spielt müsste durch weitere Untersuchungen am Protein untersucht werden. Weitere Untersuchen an den in dieser Arbeit als spezifisch für arbuskelhaltige Zellen gezeigten Genen MtAmT, MtPPK und MtOFTN könnten ebenfalls aufschlussreich für das weitere Verständnis der AMS sein. Dies trifft auch auf die TFs MtNTS und MtDES zu, die zwar nicht ausschließlich arbuskelspezifisch transkribiert werden, aber auch eine Rolle in der Regulation der AMS innerhalb von M. truncatula Wurzeln zu spielen scheinen. / The mycorrhiza (Greek: mýkēs for "mushroom"; rhiza for "root") is a symbiosis between fungi and the vast majority of land plants. The fungus improves the nutrient supply of the plant, while the plant provides the fungus with carbohydrates. The arbuscular my-corrhiza (AM) represents a special type of mycorrhiza. The AM forms during the sym-biosis eponymous arbuscules within the root cells as the supposed site of the major nu-trient exchange. The AM symbiosis (AMS) is the research focus of this work. Medicago truncatula and Glomus intraradices were used as model organisms. During the project several transcription analysis were performed to identify AMS re-gulated transcription factors (TFs). The activity of the promoters of three of the identified AMS regulated TFs (MtOFTN, MtNTS, MtDES) were visualised using a reporter gene. Cells with promoter activity were in one case the arbuscle containing cells (MtOFTN). In the another case, the promoter was also weakly active in non arbuscle containing cells, however the major site of activity were the arbuscle containing cells (MtNTS). Another promoter was active in arbuscle containing and adjacent cells (MtDES). In addition, other genes were identified as AMS regulated and for three of these genes (MtPPK, MtAmT, MtMDRL) a promoter::reporter activity study was conducted, too. The promoters of the kinase (MtPPK) and the ammonium transporter (MtAmT) were active exclusively in arbuscle containing cells, whereas the activity of the ABC-transporter (MtMDRL) could not be assigned to a specific cell type. For two other identified genes (a copper transporter (MtCoT) and a sugar/ inositol transporter (MtSuT)) RNA-interference (RNAi) studies were carried out. The studies revealed in both cases that, once an RNAi effect was present in the transformed roots, the roots were colonised by G. intraradices in a much lesser extent as in the vector-control. In the case of MtCoT it maybe has the same basic principle as in the case of the phosphate transporter MtPt4. Which role MtSuT and inositol plays during the fo-rmation of the AMS has to be reviewed. Further examinations on the genes MtAmT, MtPPK and MtOFTN could also be reveal-ing for the understanding of the AMS, as their promotors, as shown in this thesis, are exclusively active in arbuscle containing cells The same can be said for the TFs MtNFTS and MtDES. They are not exclusively transcripted in arbuscle containing cells, but nevertheless seem to play a role in the formation of the AMS within M. truncatula roots.

Made available in DSpace on 2015-03-26T13:36:46Z (GMT). No. of bitstreams: 1 texto completo.pdf: 3795682 bytes, checksum: 12bec11cea96f50faf5ddfa5a06024dc (MD5) Previous issue date: 2007-03-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The soybean SBP2 (sucrose binding protein) promoter is capable to drive vascular tissue-specific expression of reporter genes in tobacco transgenic lines. This vascular-specific activity of the SBP2 promoter is confined to a distal region (-2000 to -700 sequences) designated CRD-A (cis-regulatory domain-A). Here, we first confirmed the tissue-specific activity of CRD-A through gain-of-function experiments, in which the CRD-A sequences were directly fused to 5´end of GUS cDNA, -136pSBP2-GUS and -92pSBP2-GUS constructs. In tobacco, CRD-A was able to reduce GUS activity in all organs analyzed, recapitulating in some cases the tissue-specific pattern of the full promoter. In addition, CRD-A promoted GUS transcription in the absence of the proximal TATA-containing region, which suggests that CRD-A may contain cis-regulatory elements to sustain basal transcription. In fact, this region (-2000 a -700) harbors several TATA box-like sequences, positions -790, -783 and -761, that potentially may function as alternative TATA boxes. To delimit the cis-regulatory elements responsible for the tissue-specific activity of SBP2 promoter, the -2000 to -700 sequence was divided into five fragments, which were fused to -92pSBP2-GUS construct and used to obtain transgenic lines. Histochemical analysis revealed that all the CRDA sub-fragments reduced the SBP2 promoter activity, as their fusion to the 5 end of -92pSBP2 altered its constitutive expression pattern. These results identified the presence of several potentially cis-regulatory domains. The region encompassing the sequences -1765 to -945 may contain strong shoot apex expression-repressing elements, capable to totally abolish expression, whereas the region -944 to -705 may harbor weaker repressing elements that restricted GUS expression to the vascular tissue. We also found several root expression silencers, operating in the root meristem (-1765 to -705) and in the root elongation zone (-1765 to -1485 and -1211 to -945). Furthermore, the region delimited by positions -1765 to -1485 also exhibited a strong root expressionrepressing element whose effect may be attenuated by cis-regulatory elements present in the -1485 to -705 region. Finally, a cis-element that confines GUS expression to the inner phloem of stem was identified in the region delimited by positions -1485 to -1212. The function of the identified cis-elements was evaluated through electrophoretic mobility shift assay (EMSA) that revealed sequence-specific interactions between putative transfactors from soybean and tobacco nuclear extracts and the -1765/-1485 (fragII) fragment from GmSBP2. To determine whether the SBP2 protein accumulation correlated with the tissuespecific promoter activity, a SBP2-GFP fusion was expressed in tobacco transgenic lines under the control of SBP2 promoter. Fluorescence analysis revealed that the SBP2 protein was, indeed, located in the vascular tissue, which was consistent with SBP2 promoter activity and the involvement of SBP2 in physiological process dependent of sucrose translocation. / O promotor do gene SBP2 (sucrose binding protein) de soja é capaz de dirigir a expressão tecido vascular-específica de genes repórteres em plantas transgênicas de tabaco. Esta regulação se deve à presença de domínios cisregulatórios distais (CRD-A, posição -2000 a -700) presentes no promotor. Neste trabalho, a atividade tecido-específica de CRD-A foi confirmada por meio de experimentos de ganho-de-função, nos quais o fragmento CRD-A foi diretamente fusionado ao gene repórter GUS e às construções -136pSBP2-GUS e -92pSBP2-GUS e sua atividade avaliada no sistema heterólogo de tabaco. CRDA foi capaz de reduzir a atividade de GUS em todos os órgãos analisados, restaurando, em alguns casos, o padrão tecido-específico do promotor completo. Além disso, observou-se que CRD-A é capaz de promover a transcrição de GUS, independente de promotor mínimo, indicando a presença de cis-elementos capazes de promoverem a transcrição basal. De fato, nessa região (-2000 a -700) foram identificados vários elementos TATA box, localizados nas posições -790, -783 e -761, que podem potencialmente funcionar como TATA boxes alternativos. No intuito de delimitar os cis-elementos responsáveis pelo padrão tecido-específico do promotor SBP2, a seqüência -2000 a -700 foi dividida em cinco fragmentos, os quais foram inseridos na construção -92pSBP2-GUS, e utilizados para obtenção de plantas transgênicas. Análises histoquímicas revelaram que todos os fragmentos foram capazes de reduzir a atividade do promotor SBP2, uma vez que sua inserção na extremidade 5 de -92pSBP2 alterou o padrão de expressão constitutiva do mesmo. Com base nestes resultados, diversas regiões potencialmente regulatórias foram identificadas. A região compreendida entre -1765 e -945 deve conter fortes elementos repressores para o ápice caulinar, capazes de abolir totalmente a atividade do promotor, enquanto que a região entre -944 e -705 demonstrou conter elementos repressores mais fracos, que restringiram a expressão ao tecido vascular. Foram encontrados vários elementos silenciadores para a raiz, tanto para o meristema radicular (região entre -1765 e -705), quanto para a zona de alongamento (de -1765 a -1485 e de -1211 a -945). Além disso, a região de -1765 a -1485 também apresenta um forte repressor para raiz, cujo efeito deve ser atenuado por ciselementos presentes entre -1485 e -705. Por fim, foi identificado um elemento responsável por restringir a expressão apenas ao floema interno no caule, na região entre -1485 e -1212. A funcionalidade dos cis-elementos identificados foi avaliada através do ensaio de mudança na mobilidade eletroforética (EMSA), tendo sido observada a interação seqüência-específica entre possíveis transfatores presentes em extratos nucleares de soja e de tabaco e o fragmento -1765/-1485 (fragII) de GmSBP2. A fim de verificar se o acúmulo da proteína SBP2 correlaciona-se com a atividade do promotor em tecidos específicos, foi obtida a proteína quimérica SBP2-GFP, sob o controle do promotor SBP2, em tabacos transgênicos. A análise de fluorescência revelou que a proteína SBP2 está, de fato, localizada na região de tecido vascular, consistente com o padrão de atividade do gene repórter e com seu envolvimento nos processos fisiológicos dependentes de translocação de sacarose.

El presente estudio comparó los principales parámetros productivos de pollos de engorde suplementados con promotores de crecimiento con actividad anticlostridial en la dieta, se desarrolló en las instalaciones de la Facultad de Medicina Veterinaria de la Universidad Nacional Mayor de San Marcos, Lima -Perú. Se emplearon 575 aves distribuidas en 5 grupos de 115 animales con 5 repeticiones cada uno. G1 fue el grupo desafiado y tratado con lisozima encapsulada, G2 fue desafiado y tratado con fitobiótico (Humulus lupulus), G3 fue desafiado y tratado con Zinc bacitracina, G4 fue el control positivo sin promotor y desafiado y G5 fue el control negativo sin promotor y sin desafío. Se realizaron dos desafíos con Eimeria vía oral y agua de bebida (14 y 22 días) y un desafío con Clostridium perfringes (108 UFC/mL) vía oral a los 26 días. Los resultados mostraron que las aves de G1 y G2 ,tuvieron 56 y 42 g mas de peso corporal que G4. La mayor ganancia de peso promedio lo obtuvo G2 (2570.82 g). El mayor consumo acumulado lo presentó G4 (4797.82 g), G1 el menor (4676.12 g). La mejor conversión alimenticia la alcanzó G1 (1.78), seguido de G2 (1.79).El índice de eficiencia productivo Europeo arrojó una mejor eficiencia para G1 (349.16).Comparando G1 y G2 con G3 se obtuvo un 4.65% y 3.60% más de eficiencia productiva respectivamente. Sin embargo, el análisis de varianza de los parámetros productivos (peso promedio, ganancia de peso, índice de conversión alimenticia, consumo de alimento y el índice de eficiencia Europeo) en los cinco grupos del estudio no mostraron diferencia significativa entre ellos (p>0.05). Los resultados permiten concluir que la suplementación con productos alternativos a Zinc bacitracina tales como las lisozimas encapsuladas y fitobióticos (Humulus lupulus) mejoran el rendimiento productivo de los pollos de engorde bajo condiciones de reto de clostridios. Palabras clave: promotor de crecimiento, Clostridium perfringens, parámetros productivos, pollos de engorde. / --- This study compared the main productive parameters of broilers supplemented with growthpromoting activity anticlostridial diet, developed on the facilities of the Faculty of Veterinary Medicine of the Universidad Nacional Mayor de San Marcos, Lima-Peru. 575 birds divided into 5 groups of 115 animals with 5 repetitions each were used. Group G1 was challenged and treated with encapsulated lysozyme, G2 was challenged and treated with phytobiotics (Humulus lupulus), G3 was challenged and treated with zinc bacitracin, G4 was the positive control without promoter and challenged and G5 was the negative control without promoter and unchallenged. Two challenges with Eimeria oral and drinking water (14 to 22 days) and Clostridium perfringens challenge (108 CFU / mL) at 26 days oral route were performed. The results showed that birds of G1 and G2 were 56 and 42 g of body weight more than G4. The greater weight gain average was obtained by G2 (2570.82 g). G4 had the highest cumulative consumption introduced (4797.82 g), G1 the lowest (4676.12 g). G1 the best feed conversion (1.78), followed by G2 (1.79) .The European productive efficiency index showed a better efficiency for G1 (349.16) Comparing G1 and G2 against G3, obtained 4.65% and 3.60% over respectively production efficiency. However, analysis of variance of the production parameters (average weight, weight gain, feed conversion rate, feed intake and European efficiency index) in the five study groups showed no significant difference between them (p> 0.05). The results suggest that supplementation with alternative products such as Bacitracin Zinc and phytobiotics encapsulated lysozyme (Humulus lupulus) improve the productive performance of broiler chickens under clostridia challenging conditions. Keywords: growth promoter, Clostridium perfringens, production parameters, broilers.

Das Ziel dieser Arbeit war die Klärung der phänotypischen Konsequenzen struktureller Variationen in den regulatorischen Regionen einiger für psychische Erkrankungen potentiell relevanter Entwicklungsgene. Die Pax-Gene sind Mitglieder einer Familie der Transkriptionsfaktoren, die sowohl mehrere Schritte in der Embryogenese als auch Aufrechterhaltung des Differenzierungszustandes der Zellen einiger adulten Gewebe kontrollieren. Im Rahmen dieser Fragestellung wurden die Promotorregionen der menschlichen PAX3-, PAX6- und PAX7-Gene charakterisiert. Weiterhin wurden funktionelle Folgen der mit diesen Promotoren assoziierten Repeat-Polymorphismen auf die Expression dieser Gene untersucht. Schliesslich wurde die Relevanz für die psychischen Erkrankungen wie die Schizophrenie getestet. / The aim of this study was the elucidation of phenotypical consequences of structural variations in regulatory regions of developmental genes that are potentially relevant for mental disease. The Pax genes are members of a family of evolutionary conserved transcription factors, which control several steps in the embryogenesis as well as maintain the differentiation status of cells in adult tissues. In this context the promoter regions of the human PAX3, PAX6 and PAX7 genes were characterized. Therefore, functional effects of the polymorphic repetitive elements in the gene promoter regions on the expression of these genes were examined. Finally, the relevance for the psychiatric diseases like schizophrenia was tested.

Bisher sind zwei Isoformen des humanen CD23 (CD23a und CD23b) beschrieben. Beide unterscheiden sich lediglich in 6-7 Resten im N-terminalen, zytoplasmatischen Anteil. CD23a wird ausschließlich auf B-Zellen exprimiert, während CD23b sowohl auf B-Zellen als auch auf Monozyten, eosinophilen Granulozyten, Makrophagen und zahlreichen anderen Zelltypen durch Stimulation mit IL-4 induziert werden kann. Die beiden Isoformen vermitteln wahrscheinlich unterschiedliche Funktionen. CD23a gilt als Isoform, welche vornehmlich mit der Endozytose von IgE-Immunkomplexen und der Vermittlung von Antigen-Präsentation auf B-Zellen assoziiert ist. CD23b besitzt ein Phagozytose-Motiv und scheint bei der Phagozytose IgE besetzter Partikel, der Freisetzung von Zytokinen und der Bildung von Peroxiden eine Rolle zu spielen. Frühere Untersuchungen legen die Vermutung nahe, dass die beiden Isoformen zwei getrennte Signalübertragungswege miteinander verbinden. Die Gegenüberstellung von Ereignissen, welche in Zellen, die nur eine einer oder beide Isoformen von CD23 besitzen, stattfinden, legt die Vermutung nahe, dass CD23b cAMP und iNOS hochreguliert, wohingegen CD23a einen Anstieg des intrazellulären Kalziums vermittelt. Im ersten Teil unserer Untersuchungen haben wir die Regulation der B-Zell-spezifischen Expression von CD23a analysiert. Pax-5 ist ein auf B-Zellen beschränkter Transkriptionsfaktor, welcher für die frühe und späte B-Zellentwicklung von entscheidender Bedeutung ist. Mögliche Pax-5 Bindungsstellen wurden in den proximalen Abschnitten des CD23a Promotors vermutet. Die Analyse des CD23a Promotors ergab drei mutmaßliche Pax-5 Bindungsstellen mit mehr als 50% Homologie zur Konsensus-Sequenz. Eine dieser Bindungsstellen, namens CD23-1, kann mit einer hochaffinen Pax-5 Bindungsstelle konkurrieren oder direkt das Pax-5 Protein in Elektromobilitäts Experimenten (EMSA) binden. Das Einfügen von Mutationen an dieser Stelle verhindert die Bindung. Ein weiterer Versuch, bei dem die gesamte Länge des CD23a Promotors durch überlappende Peptide in einem kompetitiven Verfahren gegenüber hoch affinen Bindungsstellen getestet wurde, zeigt ebenso CD23-1 als die einzige Stelle, welche direkt Pax-5 binden kann. In weiteren Experimenten führte die Expression von Pax-5 in 293 Zellen zu einer 7fachen Aktivierung eines CD23a Kernpromotor Konstrukts. Die Kotransfektion zusammen mit STAT6 zeigte, dass Pax-5 mit diesem Transkriptionsfaktor kooperiert, indem es die Transkriptionsrate eines vergrößerten CD23a Promotorkonstrukts erhöht. Von besonderer Bedeutung ist die Tatsache, dass die ektope Expression von Pax-5 in der monozytären Zelllinie U-937, die normalerweise nur die CD23b Isoform exprimiert, dann zu einer Expression von CD23a nach Stimulation mit IL-4 und PMA führte. Unsere Ergebnisse legen nahe, dass Pax-5 in der auf B-Zellen beschränkten Expression der CD23 Isoform eine Schlüsselrolle zukommt. Im zweiten Teil des Projekts haben wir ein “Zwei-Hefen-Hybrid-System“ (Cyto-Trap von Stratagene) verwendet, um nach zytoplasmatischen Interaktionspartnern für den CD23 Rezeptor zu suchen. Das System wurde modifiziert um eine hohe Effizienz an Transformation zu erzielen. Unterschiedliche „Köder“-Vektorkonstrukte wurden hergestellt. Das Screening wurde mittels einer humanen Milzbibliothek mit dem Zielvektor des Systems durchgeführt. Die anfangs benutzten Konstrukte –pSosCD23a und pSosCD23b – exprimierten sehr kurze (22 Aminosäuren) zytoplasmatischen Reste der Isoformen am C-terminalen Ende des Fusionsproteins (humanes SOS). Verbesserte Konstrukte (pSos CD23a+Linker und pSosCD23b+Linker) exprimierten den zytoplasmatischen Anteil von CD23a/b am N-terminalen Ende des humanen SOS und hatten folglich den N-terminalen Anteil als Andockstelle frei, entsprechend den Bedingungen in vivo. Eine flexible Verbindungsregion trennte die Fusionsproteine, um auf diese Weise die kurze Aminosäurekette deutlich „sichtbar“ werden zu lassen. Annähernd drei Millionen Klone wurden mittels der verschiedenen Konstrukte untersucht. Dabei konnte keine tatsächlich positive Interaktion gefunden werden. Stattdessen fand sich eine vergleichsweise hohe Zahl falsch-positiver Klone. Diese wiederum wurden in einem zweiten “Zwei-Hefen-Hybrid-System“ getestet. In Zukunft wird ein neues Konstrukt als Köder verwendet werden. Hierbei wurde ein Tyrosin-Rest im zytoplasmatischen Anteil von CD23a durch Glutamat ersetzt. Das System wurde bereits dazu verwendet, die Interaktion zwischen CD23 und p59fyn - einem Mitglied der Src-Familie von Proteinkinasen, welches mit CD23a assoziiert sein soll – zu testen. Jedoch konnte im CytoTrap “Zwei-Hefen-Hybrid-System“ keine Wechselwirkung nachgewiesen werden. Zusammenfassend zeigt das zentrale Ergebnis der Arbeit, dass Pax-5 der Schlüsselregulator ist, der die B-Zell-spezifische Expression von CD23a ermöglicht. Zusätzlich wurde ein “Zwei-Hefen-Hybrid-System“ etabliert, mit dem zytoplasmatische Interaktionspartner für die CD23 Isoformen gefunden werden können. / Two isoforms of human CD23 (CD23a and CD23b) have been described. They differ by only 6-7 residues in the N-terminal cytoplasmic tail. CD23a is restrictively expressed on B-cells while CD23b is inducible on B-cells, as well as monocytes, eosinophils, macrophages and a variety of other cell types, after IL-4 stimulation. The two isoforms seems to have different functions. CD23a appears to be the isoform associated with endocytosis of IgE immune complexes and mediating antigen presentation on B-cells. CD23b has a phagocytosis motif and seems to be involved in the phagocytosis of IgE-coated particles, cytokine release and the generation of superoxides. Previous studies indicate that the two isoforms connect to different signal transduction pathways. Comparing the cells that express only one or both CD23 isoforms suggests that CD23b is involved in upregulating cAMP and iNOS, whereas CD23a mediates an increase in intracellular calcium. In the main part of the study we investigated how the CD23a B-cell specific expression is regulated. Pax-5 is a B-cell restricted transcription factor with an essential role in early and late B-cell development. Putative Pax-5 binding sites have been predicted in the CD23a proximal promoter. Analyses of the CD23a promoter revealed three putative Pax-5 binding sites with more than 50% homology to the consensus sequence. One of these sites, named CD23-1 can compete a high affinity Pax-5 binding site or can directly bind Pax-5 protein in electrophoretic mobility shift assays. Introducing mutations into this site abrogates the binding. A different approach, in which overlapping peptides covering the length of the CD23a promoter were tested in competition assays against a high affinity binding site, also revealed CD23-1 as the only site that directly binds Pax-5 protein. Expression of Pax-5 in 293 cells resulted in a 7-fold activation of a CD23a core promoter construct. Co-transfection together with STAT6 showed that Pax-5 cooperates with this transcription factor in enhancing the level of transcription of a CD23a extended promoter construct. Most importantly, ectopic expression of Pax-5 in the monocytic cell line U-937 that regularly expresses only the CD23b isoform enabled a significant CD23a expression after stimulation with IL-4 and PMA. Our results suggest that Pax-5 is a key regulator of the B-cell restricted expression of the CD23a isoform. In the second part of the project, we used a yeast two-hybrid system (CytoTrapTM from Stratagene) in order to look for cytoplasmic interaction partners for the CD23 receptor. The system was established in order to reach a high efficiency of transformation and different bait vector constructs were made. The screening was performed using a human spleen library cloned in the target vector of the system. The first bait constructs used (pSosCD23a and pSosCD23b) expressed the very short (22 amino acids) cytoplasmic tails of the isoforms at the C-terminal end of the fusion protein (human SOS). Improved bait constructs, (pSosCD23a+Linker and pSos CD23b+Linker) expressed the cytoplasmic tail of CD23a/b at the N-terminal side of the human SOS and had in consequence the N-terminal part free as a bait, as it occurs in vivo. A flexible linker region separated the fusion proteins in order to make the small amino acid bait chain more obvious. Approximately three million library clones were screened with these various constructs. No “true positive” interaction was detected. A relatively high number of “false positive” clones were obtained and checked in another two-hybrid system. A new bait construct, in which the tyrosine residue in the cytoplasmic tail of CD23a was replaced by a glutamic acid residue will be used for future screening. The system was also used in order to test the interaction between CD23 and p59fyn, a member of the Src family of protein kinases that was mentioned to associate with CD23a. No interaction was detected by using the CytoTrap two-hybrid system. In conclusion, the key result of the study demonstrates that Pax-5 is a main regulator of the B-cell specific expression of the CD23a isoform. In addition, a two-hybrid system was established and employed in order to look for cytoplasmic interaction partners for CD23.

Orientador: Marco Eustáquio de Sá / Resumo: O tratamento químico de sementes é uma prática tradicional na agricultura, utilizado no controle de agentes fitopatogênicos presentes nas sementes e no solo. No tratamento biológico o uso de rizobactérias tem sido uma alternativa interessante, como a espécie Bacillus subtilis, resistente a condições adversas de calor e baixa umidade. Assim, objetivou-se avaliar a ação de dois produtos biológicos comerciais feito à base de Bacillus subtilis FMT001 e Serenade®, e um produto químico Vitavax Thiram® 200 SC, aplicados via tratamento de sementes, como promotores de crescimento de plantas e protetores das sementes, visando verificar o desempenho germinativo das sementes e os efeitos da inoculação de doses sob diferentes espaços de tempo de exposição aos tratamentos, em caupi (Vigna unguiculata) e em feijoeiro comum (Phaseolus vulgaris). O experimento foi conduzido no LAS da UNESP. utilizando cinco lotes de sementes de feijão, quatro lotes de Vigna unguiculata e um de Phaseolus vulgaris. As sementes foram submetidas aos tratamentos biológico e químico, foram estabelecidos seis tratamentos e quatro repetições, com quatro doses de inoculantes à base de Bacillus subtilis: FMT001 com doses 100 ml, 200 ml, 400 ml e 200 ml produto Serenade®, e 100 ml VitavaxThiram® 200 SC, para 50 kg-1 sementes. Foram avaliados condutividade elétrica, teste de germinação, comprimento de raiz e plântulas, envelhecimento acelerado, teste de frio, sob os períodos de 00, 24, 48, 72 e 96 horas após a inoculação... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The seed chemical treatment is a traditional practice in agriculture, used for controlling pathogenic agents present in the seeds and soil. In the biological treatment using rhizobacteria it has been an interesting alternative as a Bacillus subtilis species, resistant to adverse conditions of heat and low humidity. The objective of this study was to evaluate the action of two commercial biological products based on Bacillus subtilis FMT001 and Serenade® and a chemical product Vitavax Thiram® 200 SC, applied through seed treatment, as plant growth promoters and seed protectors, to verify the germination performance of the seeds and the effects of the inoculation of doses under different time periods of exposure to the treatments, in Vigna unguiculata cowpea and common bean Phaseolus vulgaris. The test was conducted in the LAS of UNESP. using five lots of bean seeds four lots of Vigna unguiculata and one of Phaseolus vulgaris. The seeds were submitted to biological and chemical treatments. Six treatments and four replications were established with four doses of Bacillus. subtilis inoculants: FMT001 with doses of 100 ml, 200 ml, 400 ml and 200 ml Serenade® product, e 100 ml VitavaxThiram® 200 SC, for 50 kg-1 seeds. Electrical conductivity were evaluated, the test germination, seedling root length and, accelerated aging, cold test, in periods of 00, 24, 48, 72 and 96 hours after inoculation. Additional chemical and biological treatments, particularly the chemical, are generally s... (Complete abstract click electronic access below) / Doutor

La tuberculosis es una enfermedad que sigue siendo una amenaza para la salud pública a escala mundial, con su tendencia a desarrollar enfermedad crónica, discapacitante y fatal; es por ello que la estrategia de control de tuberculosis requiere de la intervención de diferentes actores sociales incluyendo a los promotores de salud. Por lo cual el estudio plantea la necesidad de comprobar el nivel de conocimiento del promotor de salud y las características de su intervención con pacientes de tuberculosis pulmonar en centros de salud de Lima Metropolitana. El estudio es de enfoque cuantitativo, de nivel aplicativo, método descriptivo. Se utilizó como técnica la entrevista y observación, a través de referencias del profesional de enfermería y los instrumentos fueron un cuestionario y lista de chequeo respectivamente a 25 promotores de salud. Cuyos resultados nos indican un nivel de conocimiento medio (56%), destacando en aspectos conceptuales de la enfermedad con un nivel alto (52%); acerca de las características de intervención, intervienen en sus actividades un 80 %, destacando en el área de promoción con un 76%. Concluyendo que los promotores de salud sobre la atención que brindan a los pacientes de tuberculosis pulmonar presentan un nivel de conocimiento medio, y cumplen con el rol de intervención impartido por la norma técnica, guía y manuales en su totalidad, destacando en áreas de prevención y promoción. / Tesis

Corynebacterium glutamicum is a Gram-positive non-sporulating soil bacterium which is used in biotechnology as a producer of amino acids, nucleotides, biofuels and alcohols. The aim of this thesis was to create a hybrid σ factor of RNA polymerase which would be able to recognize a matching hybrid promoter without effect on expression of the host genes. Based on the σD and σH amino acid sequence, two types of hybrid factors, σDH and σHD , were designed by the sequence combination of sigD and sigH. As an alternative approach, based on the in silico homology modeling, mutations of wild-type σH in the region recognizing the -35 promoter element of the σH -dependent promoter were introduced. Hybrid promoters were constructed by combining the -35 and -10 promoter regions that were derived from the σD - and σH - dependent promoters. Promoter activity was determined by using gfpuv reporter gene under the control of hybrid promoter. The expression of gfpuv in strains with hybrid sigma factors σDH / σHD and hybrid promoters was rather low compared to strains that carried wild-type σ factor and the respective promoter. The aim of the thesis was achieved by using one of the mutant σH factor (σmutH_6A ) with alterations in the region recognizing the -35 element of the σH -dependent promoter. This mutant σ...

Zugl.: Giessen, Univ., Diss., 2005