Gene transfer techniques such as retroviral transduction have many applications such as cell marking, cell reprogramming, and therapeutics. Transgene expression, however, is often variable and maintaining long-term expression is problematic in progenitor cell types. To better control transgene expression, research has focused on the optimized use of cis-regulatory elements, such as promoters, enhancers and insulators. In addition to controlling gene expression, these regulatory elements modulate the nuclear organization of the transgene. The integration site also exerts significant effects on steady state and temporal transgene expression via the neighbouring chromatin environment. The first part of this thesis describes the co-operation of modified β-globin intronic elements in providing high-level expression and favorable nuclear localization. I demonstrate that these elements are compatible with efficient lentivirus transduction for globin gene therapy purposes. In the second chapter, I examine high-expressing EGFP retroviral transgenes and show that such steady state expression may exhibit rapid transcriptional fluctuations, which is modulated by different transcriptional dynamics at different integration sites. Finally, in the last chapter, I evaluate the use of a 3’D4Z4 insulator element in maintaining long-term EGFP transgene expression in ES cells, and discover integration-site specific temporal dynamics in retroviral vector expression. Overall, my results demonstrate that using multiple regulatory elements and insulating these elements from different types of genomic loci optimize transgene expression and dynamics in progenitor cells.

Gene transfer techniques such as retroviral transduction have many applications such as cell marking, cell reprogramming, and therapeutics. Transgene expression, however, is often variable and maintaining long-term expression is problematic in progenitor cell types. To better control transgene expression, research has focused on the optimized use of cis-regulatory elements, such as promoters, enhancers and insulators. In addition to controlling gene expression, these regulatory elements modulate the nuclear organization of the transgene. The integration site also exerts significant effects on steady state and temporal transgene expression via the neighbouring chromatin environment. The first part of this thesis describes the co-operation of modified β-globin intronic elements in providing high-level expression and favorable nuclear localization. I demonstrate that these elements are compatible with efficient lentivirus transduction for globin gene therapy purposes. In the second chapter, I examine high-expressing EGFP retroviral transgenes and show that such steady state expression may exhibit rapid transcriptional fluctuations, which is modulated by different transcriptional dynamics at different integration sites. Finally, in the last chapter, I evaluate the use of a 3’D4Z4 insulator element in maintaining long-term EGFP transgene expression in ES cells, and discover integration-site specific temporal dynamics in retroviral vector expression. Overall, my results demonstrate that using multiple regulatory elements and insulating these elements from different types of genomic loci optimize transgene expression and dynamics in progenitor cells.

The aim of this study was to analyze number of incorporated T-DNA harbouring pmi and nptII transgenes and expression of the pmi gene in tomato transformants using Southern blot and Northern blot methods. The presence of a functional PMI protein was assesed using PMI-assay with Chlorophenol red dye. Mannose selection of tomato seeds was established and segregation patterns in T1 progeny were studied.

Dissertação(mestrado)- Universidade Federal do Rio Grande, Programa de Pós-Graduação em Aqüicultura, Instituto de Oceanografia, 2009. / Submitted by Cristiane Silva (cristiane_gomides@hotmail.com) on 2012-08-10T13:50:16Z No. of bitstreams: 1 Rafael.pdf: 548290 bytes, checksum: 93681ab1550a68d8844eefffd0bf0b49 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-09-18T02:09:25Z (GMT) No. of bitstreams: 1 Rafael.pdf: 548290 bytes, checksum: 93681ab1550a68d8844eefffd0bf0b49 (MD5) / Made available in DSpace on 2012-09-18T02:09:25Z (GMT). No. of bitstreams: 1 Rafael.pdf: 548290 bytes, checksum: 93681ab1550a68d8844eefffd0bf0b49 (MD5) Previous issue date: 2009 / O hormônio do crescimento (GH) é produzido e secretado pela adeno-hipófise tendo como efeito principal promover o crescimento somático em vertebrados. No presente estudo foi analisada a morfometria do crescimento, estrutura do tecido muscular e expressão de fatores miogênicos em indivíduos machos e fêmeas da linhagem F0104 de zebrafish transgênico para o GH. Os índices morfométricos sugerem que o excesso de GH circulante está promovendo uma antecipação na idade de maturação sexual. Também, foi observado um menor fator de condição nos transgênicos de ambos os sexos (P<0,05) e uma alteração nos padrões morfométricos ao longo do tempo evidente nos machos transgênicos. As análises multivariadas demonstraram dois fenótipos distintos de machos transgênicos. O primeiro assemelha-se às fêmeas transgênicas (tamanho grande e baixo fator de condição), e o segundo aos machos não transgênicos (menor tamanho e baixo fator de condição). Este padrão de crescimento heterogêneo nos machos transgênicos pode ser explicado pela alteração do perfil de secreção do GH associado à variabilidade genética individual na resposta ao excesso de GH circulante. As análises histológicas demostraram que os transgênicos apresentam uma hipertrofia muscular acentuada quando comparados com os não transgênicos, sendo as fêmeas transgênicas mais hipertróficas do que os machos transgênicos. A expressão dos genes relacionados com o crescimento muscular mostrou que a hipertrofia muscular observada nos transgênicos é independente do fator de crescimento tipo insulina I (IGF-I). Adicionalmente, nos machos transgênicos foi observada uma indução significativa na expressão da miogenina, indicando que esta proteína pode estar mediando, pelo menos em parte, o crescimento hipertrófico neste grupo. A expressão gênica também mostrou uma indução da a-actina somente em machos, independentemente da transgenia. Entretanto, não foi observada alteração no teor de proteínas totais de músculo. Dentro do contexto dos resultados obtidos no presente estudo, ficou evidente que o excesso de GH nos peixes transgênicos da linhagem F0104 provavelmente esteja promovendo uma maturação sexual precoce, uma hipertrofia muscular independente de IGF-I, e um crescimento heterogêneo em machos transgênicos devido à alteração do padrão de secreção do hormônio como efeito da expressão constitutiva do transgene. Este modelo é uma ferramenta interessante para o estudo de peixes com crescimento limitado. / Growth hormone (GH) is produced and released by the adenohypohysis being its main role to promote vertebrate somatic growth. The present study analyzed growth morphometrics, muscular tissue structure and myogenic factors expression in males and females from F0104 transgenic zebrafish lineage for GH. Morphometric indexes suggest that systemic GH excess is promoting an early sexual maturation. It was also observed a minor conditional factor in both sexes of transgenic group (P<0.05), and an evident time-course male altering in morphometric patterns. The first is similar to transgenic females (big size and low condiction factor) and the second to non-transgenic males (smaller size and low condiction factor). This heterogeneous male growth pattern may be explained by the altering of the GH secretion profile associated to individual genetic variability in answer to systemic GH excess. Histological analysis demonstrated that transgenic presented an enhanced muscle hypertrophy when compared to non-transgenic, being transgenic females more hypertrophic than transgenic males. Gene expression related to muscle growth revealed that transgenic hypertrophy observed is independent from insulin-like growth factor I (IGF-I). In addition, transgenic males had a significantly myogenin gene expression induction indicating that, at least partially, this protein may be mediating hypertrophic growth in this group. It was also shown an induction in males of the a-actin gene independently from transgenesis. However, there were no diferences in total protein content from the muscle. According the data presented in this study, it was evident that GH excess in F0104 transgenic fish lineage is probably promoting an early sexual maturation, a muscle hypertrophy independent from IGFI, and a heterogeneous transgenic males growth due to hormone secretion pattern altering as an effect of the transgene constitutive expression. This model is an interesting tool for the study of fishes with limited growth.

RNA interference (RNAi) is an important mechanism regulating gene expression. In plants, RNAi is triggered by double-stranded RNA (dsRNA) which is processed into small RNAs (sRNAs), usually 21-24 nt long. The sRNAs are loaded into Argonaut (AGO) protein and recognize the target based on sequence complementarity. When the target is mRNA, they can slice it or block translation leading to posttranscriptional gene silencing (PTGS). When the target is DNA, they can induce DNA methylation and chromatin changes, which when present in the promoter can lead to transcriptional gene silencing (TGS). The individual components of RNAi are well described, but less is known about the impact of different types of dsRNA precursors on the dynamics of RNAi. To study these aspects of RNAi, we used tobacco BY-2 cell line expressing GFP reporter and inducible silencers. The silencers used different ways of triggering the dsRNA formation by transcripts from antisense (AS), unterminated sense (UT) and inverted repeat (IR) GFP sequence to initiate PTGS. Additionally, one IR silencer based on the CaMV 35S promoter initiated TGS. This allowed us to study RNAi from the beginning throughout the steady state level and till the recovery phase, all in the highly homogeneous system. Using this system, we described several features...

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Malaria ist eine Krankheit, die durch den Protozoen Plasmodium verursacht und von Anopheles Moskitos durch infektiöse Stiche übertragen wird. Diese ̈Übertragung kann durch verschiedene Interventionsstrategien blockiert werden. Eine relativ neue Strategie, die bisher nur im Labor getestet wurde, ist der Einsatz genetisch veränderter Moskitos, die den Parasiten nicht auf einen neuen menschlichen Wirt übertragen können. Ein Ansatz ist die Entwicklung von Moskitos, die mit murinen Antikoepern ausgestattet sind, die gegen relevante Oberflächenproteine des Parasiten, dem Circumsporozoite Protein (CSP) gerichtet sind. Es ist jedoch nach wie vor unklar, welches Entwicklungsstadium angegriffen werden soll und welche Antikörper für diesen Ansatz effizient sind. Hier zeige ich, dass in Stechmücken, die mit einem humanen Anti-CSP-Antikörper ausgestattet sind, die Sporogonie der Oozysten in Abhängigkeit von der Parasiten-dichte blockiert wird und somit die Sporozoitenlast in den Mücken signifikant verringern. Insbesondere Antikörper, die sich an die ’Repeat Region’ des CSP binden, können die Sporozoitenlast in der Stechmücke verringern. Des Weitern, zeigen diese Stechmücken kaum Defekte in der Entwicklung und im Überleben. Diese Ergebnisse bestätigen die zuvor beschriebene Bedeutung von CSP während der Sporogonie und unterstreichen die Effizienz von humanen, ’Repeat Region’ bindenden Anti-CSP-Antikörpern bei der Beeinträchtigung der Parasitenentwicklung in dem Vektor. Darüber hinaus ist in Stechmücken, die mit humanen Anti-CSP Antikörpern ausgestattet sind, die Entwicklung von Sporozoiten teils limitiert und teils komplett verhindert. Dies macht sie zu einem vielversprechenden Instrument für Maßnahmen zum Malaria Kontrolle. In dieser Arbeit habe ich weitere Einblicke in den Mechanismus , durch den Anti-CSP-Antikörper die Parasitenentwicklung in der Mücke stören, und gezeigt, dass Oozysten ein effizientes Ziel für diesen Ansatz sind. / Malaria is a disease caused by the protozoan parasite Plasmodium and transmitted by Anopheles mosquitoes trough infectious bites, these transmission events can potentially be blocked by different intervention strategies. A relatively new strategy which has been only tested in the laboratory is the use of genetically modified mosquitoes unable to transmit the parasite to a new human host. In the past, murine derived antibodies directed against relevant parasite surface proteins were used with limited success. It remains unclear which developmental stage is targeted, and which antibodies are useful. Here, I show that in mosquitoes equipped with a human derived anti-CSP repeat binding antibody, oocyst sporogony is blocked in a parasite density dependent manner. Only repeat binding antibodies could decrease sporozoite loads in the mosquito. These results confirm the previously described importance of CSP during sporogony and highlight the efficiency of human derived repeat binding anti-CSP antibodies in interfering with parasite development even in a different host. Additionally, mosquitoes equipped with human derived anti-CSP antibodies show little (in high density infections) to no (in low density infections) sporogonic development, making them a promising tool for malaria transmission blocking interventions. I provided additional insights into the mechanism by which anti-CSP antibodies interfere with parasite development in the mosquito showing that oocysts are an efficient target for this approach. Therefore, the mosquitoes used here are potentially resistant in a more natural setting. Additionally, I provided a new tool allowing a faster screening of antibodies in a mosquito context by injection of single chain Fabs into the mosquito hemolymph. Taken together, this approach could one day give rise to alternative strategies in tackling malaria transmissions.

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