Der Typ I Plasminogenmangel ist eine seltene Multisystemerkrankung mit einer gestörten extravaskulären Fibrinolyse, die zur Ausbildung fibrinreicher Pseudomembranen auf Schleimhäuten führt. Kausale Therapien existieren bisher nicht, Fallberichte beschreiben jedoch eine Besserung der Symptomatik bei Patientinnen bei Einnahme oraler Kontrazeptiva. Östrogen wirkt im Körper über Rezeptoren durch Beeinflussung der Genexpression an bestimmten regulatorischen Elementen im Bereich der Promotoren (Estrogen responsive Elements (EREs)). Dies führte zu der Fragestellung, ob und durch welche Promotorelemente der Plasminogen Promotor durch Östrogen regulierbar und die Genexpression hierdurch modulierbar ist. Hierfür wurden verschiedene Promotorkonstrukte mit und ohne regulatorische Elemente kloniert und mittels Dual Luciferase Reporter Assay analysiert. In silico wurden 2 EREs (-11,5 kb und +4,2 kb relativ zur Transkriptionsstartstelle) identifiziert und anschließend ebenfalls in den Konstrukten getestet. Der kleinste „Plasminogenminimalpromotor“ war nicht durch Östrogen beeinflussbar. Proximale Promotorelemente wie die DNAse hypersensitive Region II mit einer beschriebenen Estrogen Responsive Unit sowie ein -2,4kb umfassendes Promotorkonstrukt wirkten unter Östrogenstimulation hemmend auf die Promotoraktivität. Ursächlich dafür sind wahrscheinlich weitere Interaktionen des Östrogen Rezeptors mit transkriptionsmodulierenden Proteinen, z.B. sind Interaktionen vermittelt über eine AP-3 Bindungsstelle denkbar. Die hemmenden Effekte konnten als Plasminogen-Gen-spezifisch und Leberzell-spezifisch demonstriert werden. Im Gegensatz dazu lösten beide vor die Minimalpromotoren klonierten EREs eine starke Stimulation aus, die sich auch in nichthepatischen Zelllinien - dort jedoch in geringerem Ausmaß ¬- zeigte. Somit sind diese EREs starke Enhancer, die eine Leberspezifität aufweisen. Die in der Summe komplexe Regulation der Östrogen-vermittelten Plasminogen Transkriptionskontrolle lässt auf eine Mitwirkung zusätzlicher Faktoren schließen. Um den in vitro Effekt der scheinbar widersprüchlichen Ergebnisse zu untersuchen, wurden humane Leberzellen einer Primärzellkultur mit Östrogen stimuliert und anschließend hinsichtlich der Plasminogen Expression untersucht. Diese Methode ließ sich jedoch aufgrund der Limitierung des Probenmaterials auf Leberbiopsien von Patienten mit gastrointestinalen Karzinomen mit Lebermetastasierung, damit einhergehend fehlender Östrogenrezeptorexpression, sowie fehlender Plasminogenexpression nicht etablieren, sodass hier der Nachweis des wirklichen Effektes des Östrogeneinflusses nicht gelang. In dieser Arbeit konnte gezeigt werden, dass sich der Plasminogen Promotor durch Östrogen regulieren lässt. Der genaue Mechanismus und der in vitro Effekt ließ sich jedoch nicht abschließend klären und bedarf weiterer Forschung. Eine vielversprechende Fortführung der Arbeit, besonders im Hinblick auf adäquate Therapieoptionen des Typ I-Plasminogenmangels, wäre die Etablierung eines geeigneten Zellmodells und die Erprobung weiterer Plasminogen modifizierender Substanzen.

No description available.

It was shown that 95 % of human multi-exon genes are alternatively spliced and the regulation of alternative splicing is extremely complex. Most pre-mRNA splicing events occur co- transcriptionally and there is increasing body of evidence, that chromatin modifications play an important role in the regulation of alternative splicing. Here we showed that inhibition of histone deacetylases (HDACs) modulates alternative splicing of ~700 genes via induction of histone H4 acetylation and increase of Pol II elongation rate along alternative region. We identified HDAC1 the catalytic activity of which is responsible for changes in alternative splicing. Then, we analyzed whether acetylhistone binding protein Brd2 regulates alternative splicing and showed that Brd2 occupies promoter regions of targeted genes and controls alternative splicing of ~300 genes. Later we showed that knockdown of histone acetyltransferase p300 promotes inclusion of the alternative fibronectin (FN1) EDB exon. p300 associates with CRE sites in the promoter via the CREB transcription factor. We created mini-gene reporters driven by an artificial promoter containing CRE sites. Both deletion and mutation of the CRE site affected EDB alternative splicing in the same manner as the p300 knockdown. Next we showed that p300 controls histone...

Background: The major histocompatibility complex (MHC) molecules play an important role in the immune response regulation and in the maintenance of the immune homeostasis. Regulation of their expression is therefore a key factor influencing the adaptive immune response. DNA methylation of gene regulatory regions is one of the mechanisms of gene expression control that affects the accessibility of DNA to transcription factors. Ageing is connected with changes in DNA methylation and increased predisposition to autoimmune diseases in older age could be associated with changes in MHC class II genes methylation. Aims: The aim of this diploma thesis is to analyze the methylation profile of DQA1 and DQB1 genes regulatory regions and to compare its differences between the generations and between individual alleles. The next aim is to compare DQA1 mRNA expression between the generations and between single alleles. Methods: DNA and RNA were isolated from blood of three age group donors. DNA was converted by the bisulfite treatment and regulatory regions of HLA class II genes were amplified and cloned into bacteria. Positive clones were sequenced and then analyzed. RNA was reverse transcribed and its expression level was determined by real-time PCR. Results: Statistically significant differences were found by...

Die Familie der NFAT-Transkriptionsfaktoren (NFATc1-c4) ist im Zuge einer Immunreaktion endscheidend an der transkriptionellen Regulation der Genexpression beteiligt. Wurden NFAT-Faktoren zunächst als T-zell-spezifische Aktivatoren von Zytokinpromotoren beschrieben, so hat sich inzwischen gezeigt, dass sie in einer Vielzahl von Geweben eine wichtige Rolle spielen. Als Beispiele seien die Herzklappenentwicklung, die Bildung von Blutgefässen, die Ausbildung neuronaler Axone oder die Osteoklastendifferenzierung genannt [10, 24]. In der hier vorliegenden Arbeit zeigen wir, dass die starke Expression der kurzen Isoform NFATc1/αA in Effektor-T-Lymphozyten durch die induzierbare Aktivität des Promoters P1 kontrolliert wird. Die P1 Aktivierung führt zum Splicing des Exon 1 zu 3 (α-Isoformen) und endet meist durch Benutzung der Polyadenylierungsstelle pA1 hinter Exon 9 (A-Isoformen). Der zweite, schwächerer Promoter P2 befindet sich vor dem zweiten Exon und ist für die konstitutive Synthese der β-Isoformen verantwortlich. Der Transkriptionstart am zweiten Exon geht meist mit der Benutzung einer zweiten, hinter dem 11. Exon gelegenen Polyadenylierungsstelle pA2 einher, die durch alternatives Splicing zur Synthese der Isoformen B und C führt. Insgesamt können so vom nfatc1-Lokus sechs verschiedene Isoformen (αA, αB, αC, βA, βB und βC) generiert werden. Die induzierbare Aktivität des P1-Promoters ist, im Gegensatz zum eher konstitutiv aktiven P2-Promoter, NFAT-abhängig und somit eine Form der Autoregulation. In ruhenden T-Lymphozyten sind einzig die Transkripte der NFATc1/β-Isoformen nachweisbar. Nach einer T-Zell-Aktivierung nimmt ihre Häufigkeit dann ab, während nun die α-Isoformen dominant werden. In dieser Arbeit wird gezeigt, dass es nach Induktion primärer Effektor-T-Helfer-Zellen oder in T-Zell-Linien zu einer 15-20-fachen Akkumulation der NFATc1/αA mRNA bzw. einer 2-5-fachen Zunahme der NFATc1/αB und C mRNAs kommt. Zur maximalen Induktion des P1-Promotors bedarf es zum einen eines anhaltenden Anstiegs der intrazellulären Kalziumkonzentration, die zur Aktivierung der Phosphatase Calcineurin und damit zur Kernlokalisation der NFAT-Faktoren führt. Zum anderen ist die Aktivierung der Proteinkinase C-Enzyme und der MAP-Kinasen notwendig, wie sie durch Phorbolester in der Zelle vermittelt wird. Dies lässt darauf schließen, dass für eine optimale Aktivierung des P1-Promotors sowohl Signale des T-Zell-Rezeptors als auch Signale von Korezeptoren - wie von CD28 - notwendig sind. Da die Induktion von NFATc1/αA in NFATc2/NFATc3 doppeldefizienten Mäusen normal erfolgt, kann man schlussfolgern, dass NFATc1 in Form einer Autoregulation die Aktivität des P1-Promoters und damit die Synthese der α-Isoformen kontrolliert. Die NFAT-vermittelte Aktivierung des P1-Promoters erfolgt über zwei tandemartig angeordnete NFAT-Bindungsstellen der Nukleotidsequenz TGGAAA, an die jeweils ein NFAT-Protein binden kann. Daneben enthält der Promoter konservierte Bindemotive für CREB-, AP-1, Sp-, NF-kB- und GATA-Faktoren, die wahrscheinlich an der komplexen Kontrolle dieses induzierbaren NFATc1-Promoters beteiligt sind. Zusammengefasst ergibt sich aus diesen Daten das folgende Modell. Die Transkription im nfatc1-Genlokus erfolgt in naiven und in ruhenden Effektor-T-Zellen konstitutiv und gesteuert durch den P2-Promotor. In Folge einer Aktivierung der Zelle verringert sich die Aktivität des P2-Promotors, während gleichzeitig der P1-Promotor induziert wird, der zusammen mit einer verstärkten Nutzung der pA1-Polyadenylierungssequenz für die massive Zunahme der NFATc1/αA-Isoform verantwortlich ist. Dies deutet auf eine besondere Bedeutung dieser kurzen Isoform in der Effektorphase der T-Zell-Aktivierung hin, insbesondere in Th1-Zellen, die NFATc1/αA in hohen Konzentrationen produzieren. / NFAT transcription factors play critical roles in gene transcription during immune responses. Besides regulation of lymphokine promoters in T lymphocytes, NFAT factors are also expressed in other cell types and regulate the activity of numerous genes that control the generation of cardiac septa and valves in embryonic heart, the formation of blood vessels, the outgrowth of neuronal axons and the differentiation of osteoclasts during bone formation [10, 24]. Here we show that the induction of NFATc/αA in effector T cells is controlled by a strong inducible promoter, P1. It results in splicing of exon 1 to exon 3 transcripts and, in concert with the activity of a poly A site downstream of exon 9, leads to the massive synthesis of NFATc/αA in effector Th1 cells. A second, weak promoter, P2, lies in front of exon 2 and directs the synthesis of longer NFAT β isoforms. Both P1 and P2 direct the synthesis of three different RNAs: αA, αB, αC and βA, βB, βC correspondingly. The B and C isoforms arise from alternative splicing and poly A addition at the distal site pA2. P1 but not P2 activity is autoregulated by NFAT factors which bind to two tandemly arranged NFAT sites within P1 and enhance its induction. In resting T cells, the NFATc1/β RNAs are the most prominent nfatc1 transcripts and their synthesis is reduced upon T-cell activation. However, following activation in primary effector T cells or in T-cell lines of human or murine origin, a 15–20-fold induction of NFATc1/αA RNA was detected, whereas only a 2–5-fold increase was observed for the NFATc1/αB or NFATc1/αC RNAs. Optimal induction of P1 promoter require involving of a persistent increase in free cytosolic Ca2+ induced by ionomycin, which stimulates the nuclear translocation and transcriptional activation of all NFATc factors and phorbol esters, which activate protein kinase C and other protein kinase pathways in T cells. This suggests that both TCR and co-receptor signals contribute to give full P1 nfatc1 induction. Because NFATc1/αA induction is unaffected in NFATc2+c3 double-deficient T cells, NFATc1 autoregulates its own synthesis by controlling P1 activity and NFATc1/αA induction. P1 promoter contains tandemly arranged NFAT core binding motif TGGAAA to witch bind monomeric NFATc1 proteins and numerous conservative binding sites of other transcriptional factors like CREB, Fos, ATF-2, Sp1, NF-kB and GATA suggesting complex multi-factor regulation of NFATc1 gene. We also highlight that initial phase of nfatc1 transcription in naive CD4+ T cells is controlled by the promoter P2 which is constitutively active in resting T cells. The activation of resting T cells results in a decrease of P2 and the induction of P1 activity and, under optimal conditions, in the predominant synthesis of NFATc1/αA in effector T cells. In addition to the high concentrations of poly A factors required for optimal pA1 function, the levels of transcription factors, in particular NFATs, must also increase for P1 induction. That could be explained by achievement of certain threshold levels for transcriptional activation. Finally, the altered transactivation potential of NFATc1/αA suggests a specific role for this NFATc1 protein in gene control, such as in Th1 effector cells where NFATc1/αA is synthesized at high concentrations.

A suspeita de indução de resistência bacteriana a antibióticos melhoradores de desempenho (AMD) e a pressão dos consumidores e dos mercados importadores para a abolição da prática criam a necessidade da descoberta de novas substâncias que funcionem como substitutos aos antibióticos, como os probióticos, prebióticos e simbióticos. Sendo assim, o objetivo deste trabalho foi avaliar os efeitos de probiótico, prebiótico e simbiótico sobre o desempenho zootécnico, os rendimentos de carcaça e de cortes e a morfologia intestinal de frangos de cortes criados de 1 a 43 dias de idade. Foram utilizados 1200 pintos de 1 dia, machos, da linhagem comercial Cobb 500®, distribuídos em delineamento inteiramente casualizado, com 5 dietas experimentais: Controle, Antibiótico (virginiamicina), Probiótico (Bacillus subtilis), Prebiótico (mananoligossacarídeo - parede celular de Saccharomyces cerevisae) e Simbiótico (Bacillus subtilis + mananoligossacarídeo - parede celular de Saccharomyces cerevisae) e 8 repetições de 30 aves cada. Em relação ao desempenho zootécnico, os aditivos testados provaram ser alternativas viáveis ao antibiótico melhorador de desempenho utilizado, uma vez que apresentaram resultados melhores que os do grupo controle e iguais aos obtidos com o uso do AMD. Da mesma maneira, para os rendimentos de carcaça e de cortes, o probiótico, o prebiótico e o simbiótico, mostraram poder ser usados como substitutos ao antibiótico, uma vez que promoveram rendimentos iguais aos do grupo alimentado com AMD. Por fim, para a morfometria intestinal, os dados obtidos mostram não haver uma relação direta entre a presença de aditivo e uma maior altura de vilosidade, menor profundidade de cripta ou maior relação vilo:cripta, uma vez que nem sempre os animais suplementados tiveram melhores resultados que o grupo controle negativo. Desta maneira concluí-se que os aditivos usados podem substituír o antimicrobiano testado, em suas respectivas doses, para as fases de criação consideradas, uma vez que fornecem resultados tão bons quanto aos obtidos com o AMD. / The suspect of the resistance\'s indution of antibiotics (ATB) and the pressure of the consumers and of the importing markets for the abolishment of this kind of practice have criated the necessity of discovering new substances that work like substitutes to antibiotics, just like probiotics, prebiotics and symbiotcs. In this manner, the objective of this study it was evaluate the effects of probiotic, prebiotic and symbiotic on performance, carcass and parts yield and intestinal morphology of broilers from 1 to 43 days of age. There were used 1,200 one-day-old chicks, male, Cobb 500® strain, distributed on a completely randomized design, with 5 experimental diets: Control, Antibiotic (virginiamicin), Probiotic (Bacillus subtilis), Prebiotic (Saccharomyces cerevisae yeast cell) and Symbiotic (Bacillus subtilis + Saccharomyces cerevisae yeast cell) and 8 replicates with 30 chicks each one. Concernig on the performance, the tested additives have been proved to be viable alternatives to used antibiotic, once that they have showed better results than the control group, and similars to the ones that were obtained with the use of antibiotic (ATB). Equally, for the carcass and parts yield, probiotic, prebiotic and symbiotic have showed they can be used like substitutes to antibiotics, once they have promoted equall yields to the group fed antibiotic. At last, for the intestinal morphology, the data haven\'t showed relation between the additive\'s presence and a higher vilo high, a smaller cript depth and a higher vilo:cript ratio, once not always animals fed additives have had better results than the control group. This way, the conclusion is that the substitution of the antimicrobian by the additives, in its respective doses, can be adopted once promotes good results as the antibiotic.

Neste trabalho, estudamos duas malformações craniofaciais mendelianas, decorrentes de um distúrbio do desenvolvimento dos primeiro e segundo arcos faríngeos: a síndrome de Treacher Collins (STC) e a síndrome Aurículo-condilar (SAC). A identificação de genes e de mecanismos moleculares associados a essas condições, além de contribuir para a compreensão do desenvolvimento das estruturas derivadas desses arcos faríngeos, é fundamental para o desenvolvimento do diagnóstico molecular, uma ferramenta importante para diagnóstico diferencial e aconselhamento genético. Contribuímos para uma melhor caracterização clínica da SAC com a descrição de uma nova família com 11 afetados. Após excluirmos quatro genes/regiões candidatas para síndromes de 1º e 2º arcos faríngeos, realizamos estudos de ligação usando marcadores polimórficos ao longo do genoma. Mapeamos o primeiro lócus associado à SAC, 1p21.1-q23.3 (lod score=3.0), e nossos dados sugerem que há heterogeneidade genética para essa patologia. Com relação ao estudo da STC, realizamos uma extensa revisão da nomenclatura das mutações patogênicas descritas na literatura, além de investigar mecanismos mutacionais atípicos na STC, corroborando a hipótese de que mutações nos exons que sofrem splicing alternativo são capazes de gerar o fenótipo da síndrome. Demos continuidade à caracterização do espectro de mutações no gene TCOF1 e investigamos a correlação genótipo-fenótipo numa amostra de 58 pacientes com STC. A análise dos dados de polimorfismos da região codificadora do gene permitiu que fizéssemos inferências sobre o regime de seleção ao qual o TCOF1 está submetido, e os resultados sugeriram que o gene TCOF1 está sob seleção purificadora, que atua sobre mutações fracamente deletérias. Também inferimos a fase para um conjunto de polimorfismos da região codificadora, verificando se havia associação de algum haplótipo à gravidade do quadro clínico ou à predisposição para a doença. Dada a observação de ausência de correlação haplótipo/genótipo-fenótipo, testamos a hipótese de que variações nos níveis de expressão do alelo normal poderiam ser responsáveis pela variabilidade clínica observada nos pacientes portadores da STC. Para tanto, iniciamos o estudo funcional das regiões regulatórias do gene TCOF1 , inclusive, de regiões distantes do promotor mínimo, preditas como enhancers. Identificamos polimorfismos em sua região promotora, sendo um deles capaz de diminuir os níveis de transcrição e de afetar a ligação do fator de transcrição YY1 ao promotor do TCOF1 . Caracterizamos a ação de YY1 como repressora in vitro. Testamos também a hipótese de haploinsuficiência como mecanismo associado à STC. Quantificamos os níveis de transcritos do TCOF1 em indivíduos afetados e normais e observamos uma diferença significativa. Nosso trabalho corrobora a hipótese de haploinsuficiência e mostra pela primeira vez que pacientes têm degradação de transcritos. Também investigamos a possibilidade de os níveis de transcritos estarem correlacionados à variabilidade fenotípica. Comparamos os dados de expressão de cada paciente à freqüência de nove sinais clínicos principais da STC, mas nenhuma correlação foi observada. Investigamos o padrão de metilação da ilha CpG do gene TCOF1 em pacientes e em controles, com o intuito de verificar se diferentes níveis de metilação inter-individual estariam associados à grande variação na expressão gênica. Demonstramos que a metilação da ilha CpG não é o mecanismo regulatório por trás dessa ampla variação de expressão do TCOF1 / In the present study, we investigate two craniofacial mendelian disorders, resulting from abnormalities in the development of the first and second pharyngeal arches: the Treacher Collins Syndrome (TCS ) and the auriculo condylar syndrome (ACS). The identification of genes and molecular mechanisms associated to these conditions, in addition to contributing to the understanding of the development of structures derived from these pharyngeal arches, is fundamental for the development of molecular diagnostics, an important tool for differential diagnosis and genetic counseling. We contributed to a better clinical characterization of ACS with a description of a new family with 11 affected individuals. After excluding four candidate genes/regions for syndromes of the 1st and 2nd pharyngeal arches, we carried out linkage studies using polymorphic markers throughout the genome. We mapped the first locus associated to ACS, 1p21.1-q23.3 (lod score=3.0), and our data suggest genetic heterogeneity exists for this pathology. With respect to the study of TCS , we carried out an extensive review of the nomenclature for the pathogenic mutations described in the literature, in addition to investigating atypical mutation mechanisms in TCS , corroborating the hypothesis that mutations in the exons that undergo alternative splicing can result in the TCS phenotype. We continued the characterization of the mutation spectrum for TCOF1 and we investigated the genotype-phenotype correlation in a sample of 58 patients with TCS . The analysis of polymorphisms in the coding region allowed us to make inferences about the selective regime experienced by TCOF1 , and our results suggested that TCOF1 is under purifying selection, which acts upon weakly deleterious mutations. We also inferred the phase for a set of polymorphisms in the coding region, testing whether there was association between any haplotype and the severity of the phenotype or the susceptibility to the disease. Given the observation of no correlation between haplotype/genotype and phenotype, we tested the hypothesis that variation in the levels of expression of the normal allele could be responsible for the clinical variability observed in TCS patients. To do this, we started a functional study of the TCOF1 regulatory regions, including regions distant from the minimal promoter, predicted to be enhancers. We identified polymorphisms in the promoter region, one of which reduced the levels of transcription and affected the binding of the YY1 transcription factor to the TCOF1 promoter. Using an in vitro assay we characterized YY1 as a repressor. We also tested the hypothesis of haploinsufficiency as a mechanism associated to TCS . We quantified the levels of TCOF1 transcripts in normal and affected individuals and found a significant difference. Our study corroborates the haploinsufficiency hypothesis and for the first time shows that patients have transcript degradation. We also investigated the possibility that transcripts levels are correlated to phenotypic variability. We compared the expression data for each patient with the frequency of nine clinical signs of TCS , but no correlation was found. We investigated the pattern of methylation on the CpG island of TCOF1 in patients and controls, in order to test whether different levels of methylation among individuals were associated to the great variation in gene expression. We demonstrated that methylation of the CpG island is not the regulatory mechanisms underlying the broad variation in TCOF1 expression.

Insetos representam a maioria dos seres vivos na Terra. O sucesso biológico desses organismos está, em parte, relacionado às associações com microrganismos que influenciam aspectos de sua bioecologia, incluindo as relações com o primeiro e terceiro níveis tróficos. Assim como os insetos, plantas também apresentam associações com microrganismos que podem interferir na sua nutrição e relação com patógenos e herbívoros. Esse trabalho buscou analisar a capacidade de simbiontes associados a insetos de atuar como endofíticos de planta e influenciar, assim, o potencial biótico da planta e a sua capacidade de defesa contra a herbivoria, utilizando o modelo milho - Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae). Quatro simbiontes de insetos foram selecionados. Três deles (IIL-Sfm05, IILSfc-sus01 e IIL-Sfb05) associados ao trato digestivo de S. frugiperda e outro (IIL-ASP45) de Acromyrmex coronatus (IIL-ASP45). Adicionalmente, um endofítico (IILzm-Idp03) isolado do próprio milho foi adicionado como controle positivo. Os ensaios foram conduzidos após cultivo de sementes inoculadas com os diferentes microrganismos via bio-priming, aplicação no solo e aplicação foliar. Dos cinco isolados testados, três (IILSfc-sus01, IIL-Sfb05 e IILzm-Idp03) induziram maior crescimento em plantas de milho; porém, a forma de disponibilização dos microrganismos influenciou a resposta da planta. O isolado IILzm-Idp03 induziu o maior crescimento da planta pela sua disponibilização via bio-priming; IILzm-Idp03 e IIL-Sfb05 via inoculação do solo; e o isolado IILSfc-sus01 via inoculação foliar. Testes biológicos com S. frugiperda em plantas de milho inoculadas ou não com os microrganismos selecionados indicaram que dois deles (isolados IIL-Sfb05 e IIL-ASP45) resultaram em relações patogênicas, chegando à induzir a completa mortalidade de insetos alimentados em tecidos foliares de plantas inoculadas com esses microrganismos. Testes de preferência alimentar demonstraram que as lagartas não foram influenciadas pela alimentação com folhas de plantas inoculadas com as bactérias via bio-priming. Estudos comparativos dos índices nutricionais de plantas tratadas com as diferentes bactérias, indicaram que aquelas tratadas com IIL-ASP45 resultaram no menor crescimento e desenvolvimento larval de S. frugiperda, corroborado pelo menor consumo e índices nutricionais alcançados nessa fonte de alimento. A alimentação em plantas tratadas com IIL-Sfb05 causou 100% de mortalidade larval, não sendo possível a avaliação dos índices nutricionais. Os resultados desse trabalho demonstram o potencial de manipulação da planta hospedeira por bactérias associadas ao herbívoro, bem como o potencial biocida de microrganismos associados a insetos. / Insects represent most of the living things on Earth. The biological success of these organisms is in part related to associations with microorganisms that influence aspects of their bioecology, including relationships with the first and third trophic levels. Like insects, plants also have associations with microorganisms that may interfere in their nutrition and interactions to pathogens and herbivores. This work sought to assess the potential of symbionts associated with insects to act as plant endophytes and thus to influence plant growth and its utilization by the herbivory using the maize - Spodoptera frugiperda (JE Smith, 1797) (Lepidoptera: Noctuidae) model system. Four insect symbionts were selected. Three of them (IIL-Sfm05, IILSfc-sus01 and IIL-Sfb05) associated with the digestive tract of S. frugiperda and another (IIL-ASP45) with the cuticle of Acromyrmex coronatus (IIL-ASP45). Additionally, an endophyte (IILzm-Idp03) from maize was added as a positive control. Experiments were conducted after inoculation of the different microorganisms via bio-priming, soil application and foliar application. Of the five isolates tested, three (IILSfc-sus01, IIL-Sfb05 and IILzm-Idp03) induced higher growth in maize plants; however, the inoculation of the seeds with the microorganisms influenced the plant response. The isolate IILzm-Idp03 induced the highest plant growth when inoculated via bio-priming; IILzm-Idp03 and IIL-Sfb05 via soil inoculation; and IILSfc-sus01 via foliar inoculation. Biological tests with S. frugiperda on maize plants inoculated or not with the selected microorganisms indicated that two of them (IIL-Sfb05 and IIL-ASP45 isolates) were highly pathogenic, inducing very high larval mortality. Our analysis indicated no changes in larval feeding preference. Comparative analysis of larval food consumption and utilization indicated that plants inoculated with IIL-ASP45 resulted in the lowest growth and larval development of S. frugiperda, corroborated by the lower consumption and nutritional indexes reached in this food source. Feeding in plants inoculated with IIL-Sfb05 caused 100% larval mortality, and it was not possible to evaluate the nutritional indexes. Our data demonstrate the potential for manipulation of the host plant by bacteria associated with the herbivore, as well as the biocidal potential of insect - associated microorganisms.

A citricultura brasileira é a maior produtora e exportadora de citros e tem sido afetada por doenças que causam sérios prejuízos a produção e a qualidade dos frutos. No entanto, a cultura apresenta grandes problemas, entre eles, os fatores fitossanitários, que vem dizimando milhares de plantas e afetando a produtividade e a competitividade do setor. Atualmente, o huanglongbing (HLB), associado às bactérias de floema Candidatus Liberibacter spp., é considerado uma das mais destrutivas doenças de citros. A inexistência de cultivares de laranja doce resistentes ao HLB torna a transformação genética de citros uma ferramenta importante no controle desta doença. Para se defender do ataque de pragas e patógenos as plantas desenvolveram, durante o processo evolutivo, uma série de mecanismos de defesa, no qual pode-se incluir a produção de peptídeos com atividade antimicrobiana. As defensinas vegetais são peptídeos pequenos relacionadas à patogênese (PR), que possuem atividade antimicrobiana associada aos mecanismos de defesa das plantas. Assim, o objetivo deste trabalho foi a obtenção de plantas transgênicas de laranja doce (Citrus sinensis L.) cvs. \'Hamlin\', \'Natal\', \'Valência\' e \'Pera\', via Agrobacterium tumefaciens, superexpressando o gene codificador de defensina (def), isolado de Citrus sinensis cv. \'Valência\', dirigido pelo promotor com expressão preferencial no floema AtSUC2 (transportador de sacarose, clonado de Arabidopsis thaliana) ou pelo promotor constitutivo CaMV 35S (clonado do vírus do mosaico da couve-flor). Os explantes utilizados na transformação genética foram segmentos de epicótilo obtidos de plantas germinadas in vitro. A identificação das plantas transgênicas foi realizada por meio da análise da PCR, utilizando-se primers para a detecção do fragmento do gene de seleção nptII. As plantas PCR+ foram aclimatizadas e transferidas para casa-de-vegetação. A análise de Southern blot confirmou a integração do transgene em 36 plantas. Foram obtidas 7 plantas transgênicas da cultivar \'Hamlin\', 9 da cultivar \'Natal\', 1 da cultivar \'Pera\' e 9 da cultivar \'Valência\' contendo a construção gênica pC35S/def, e 3 plantas transgênicas da cultivar \'Hamlin\', 6 da cultivar \'Natal\' e 1 da cultivar \'Valência\' contendo a construção gênica pcAtSUC2/def. Os resultados obtidos neste trabalho serão importantes para futura avaliação e estudo visando o controle de Candidatus Liberibacter spp.. / The Brazilian citrus industry is the world\'s largest producer and exporter of citrus, however, it has been affected by diseases that cause serious production losses and damages to fruit quality. However, the culture faces problems, namely phytosanitary issues that have been damaging thousands of plants, affecting yield and competitiveness of the sector. Currently, Huanglongbing (HLB), associated with phloem bacteria Candidatus Liberibacter spp., is considered one of the most destructive citrus diseases. The lack of sweet orange cultivars resistant to HLB makes genetic transformation an important tool in the disease control. To defend from pest and pathogen attack, plants developed a series of defense mechanisms during the evolutionary process, which may include the production of peptides with antimicrobial activity. Plant defensins are small peptides related to pathogenesis (PR) which have antimicrobial activities, associated with plant defense mechanisms . The objective of this study was to obtain transgenic plants of sweet orange (Citrus sinensis L.) cultivars \'Hamlin\', \'Natal\', \'Valência\' and \'Pera\' with Agrobacterium tumefaciens overexpressing the defensin gene (def), isolated from Citrus sinensis cv. \'Valência\', controlled by the promoter with preferential expression in the phloem AtSUC2, (sucrose transporter, cloned from Arabidopsis thaliana) or by the constitutive promoter CaMV 35S (cloned from the mosaic virus of cauliflower). The explants used in genetic transformation were epicotyl segments obtained from germinated plants in vitro. The identification of transgenic plants was accomplished by PCR analysis using primers for the detection of nptII gene fragment. The PCR+ plants were acclimatized and transferred to greenhouse. The analysis of Southern blot confirmed the transgene integration in 36 plants. Seven transgenic plants were obtained for the cultivar \'Hamlin\', nine for \'Natal\', one for \'Pera\' and nine for \'Valência\' containing the gene construct pC35S/def and three transgenic plants for \'Hamlin\', six for \'Natal\' and one for \'Valência\' containing the gene construct pcAtSUC2/def. The results obtained in this work are important for future evaluation of the plants for resistance to Candidatus Liberibacter spp..

As rizobacterias promotoras do crescimento de plantas (RBCP) colonizam as raízes de plantas e induzem um aumento no crescimento vegetal por diversos mecanismos. A rizobacteria Bacillus subtilis é capaz de atuar como agente de controle de doenças de várias plantas cultivadas, como também promotor de crescimento vegetal. Diante disso, este trabalho teve como objetivos, nos quatro capítulos apresentados, de avaliar o crescimento micelial de patógenos, com a capacidade dos isolados de B. subtilis em inibir esses patógenos; avaliar a capacidade dos isolados de B. subtilis, inoculados em solo adubado com fosfato natural e sem adubação, em disponibilizar P e no crescimento da soja e feijão caupi em condições de casa de vegetação; avaliar a eficiência da inoculação de B. subtilis como promotor de crescimento vegetal na cultura do milho, em casa de vegetação; e avaliar a eficiência da inoculação de B. subtilis no desempenho agronômico da soja em condições de campo em duas localidades. Foram testados sete isolados de B. subtilis contra os patógenos Fusarium subglutinans, Curvularia lunata e Bipolaris spp., avaliando a capacidade de controle biológico in vitro. Na avaliação da capacidade de inibição do crescimento dos patógenos utilizando quatro métodos os isolados de B. subilis UFTBs 03, UFTBs 05, UFTBs 06, UFTBs 07 foram eficazes. Os isolados UFTBs 01, UFTBs 03, UFTBs 04, UFTBs 05, UFTBs 06 e UFTBs 07 foram capazes de inibir o crescimento micelial dos patógenos testados por metabólitos termoestáveis, sendo a antibiose seu principal mecanismo de ação. Quanto à eficiência da inoculação de B. subtilis em soja e feijão caupi com adubação de fosfato natural e sem adubação, em casa de vegetação, utilizando sete isolados e um MIX, observou-se que a maioria dos tratamentos onde recebeu a inoculação de B. subtilis proporcionou o crescimento da cultura da soja e do feijão caupi, também a maioria do isolados testados proporcionaram um maior teor de P disponível no solo e na parte aérea das plantas, tanto em solo suplementado com fosfato natural como também em solo sem adubação, em ambas as culturas. Em casa de vegetação testando inoculante de B. subitilis, composto por três cepas (UFTBs 01, UFTBs 02, UFTBs 03), em milho, mostrou que sementes de milho inoculadas com B. subtilis resultaram em plantas com maior acúmulo de biomassa, em estádio inicial de crescimento. No experimento de campo, onde foi testado inoculante de B. subtilis, composto por três cepas (UFTBs 01, UFTBs 02, UFTBs 03), na cultura da soja em duas localidades na safra 2013/1014, a inoculação com B. subtilis proporcionou aumento da biomassa e produtividade da soja nas duas regiões onde foram avaliadas. Os resultados do presente trabalho comprovam a eficiência de B. subtilis nativos, isolados de solos do Tocantins, no controle biológico de fitopatogênos, bem como a capacidade como promotores de crescimento de feijão caupi, milho e soja, e aumento de produtividade da cultura da soja. / The rhizobacteria promote plant growth (RBCP) colonize roots of plants and induce an increase in plant crecimento by different mechanisms. The rizobacteria Bacillus subtilis is able to act as a control agent of several cultivated plant diseases, as well as plant growth promoter. Thus, this study aimed to the four chapters presented, to evaluate the mycelial growth of pathogens such as the ability of isolates of B. subtilis to inhibit these pathogens; evaluate the effect of isolates of B. subtilis inoculated in soil fertilized with rock phosphate and without fertilization, as its ability to provide P, the growth of soybean and cowpea at home conditions of vegetation; evaluate the efficiency of B. subtilis inoculation as plant growth promoter in corn in a greenhouse; and evaluate the efficiency of inoculation of B. subtilis the agronomic performance of soybeans under field conditions in two locations. They tested seven isolates of B. subtilis pathogens Fusarium subglutinans, Curvularia lunata and Bipolaris spp., assessing the biological control capacity in vitro. In the evaluation of growth inhibition ability of the pathogens using four methods isolates of B. subilis UFTBs 03, UFTBs 05, UFTBs 06, UFTBs 07 were effective. Isolated UFTBs 01, UFTBs 03, UFTBs 04, UFTBs 05, UFTBs 06 and 07 UFTBs were able to inhibit the mycelial growth of the pathogens tested by thermostable metabolites, antibiose being its main mechanism of action. As the efficiency of inoculation of B. subtilis in soybean and cowpea with natural phosphate fertilizer and no fertilization in a greenhouse, using seven isolates and MIX, it was observed that most of the treatments which received inoculation of B. subtilis provided the growth of soybean and cowpea, also most of the isolates tested provided a higher P content available in soil and shoots of plants, both in soil supplemented with rock phosphate as well as in soil fertilization in both cultures. In greenhouse testing inoculant B. subitilis, composed of three strains (UFTBs 01, UFTBs 02, UFTBs 03) in corn, showed that maize seed inoculated with B. subtilis resulted in plants with higher biomass accumulation in stadium initial growth. In the field trial, where he was tested inoculant B. subtilis, composed of three strains (UFTBs 01, UFTBs 02, UFTBs 03) in soybean at two sites in the harvest 2013/1014, inoculation with B. subtilis gave rise biomass and soybean yield in the two regions where they were evaluated. The results of this study demonstrated the efficacy of B. subtilis native isolated from the Tocantins soil, the biological control of plant pathogens, as well as the capacity and bean growth promoters cowpea, corn and soybeans, and increased the soybean yield.