Estudo da associação dos genes GGR, GGPPS2 e GGPPS6 em Arabidopsis thaliana L. (Brassicaceae) a compostos alelopáticos

Dissertação (mestrado)—Universidade de Brasília, Departamento de Botânica, Programa de Pós-Graduação em Botânica, 2016.

Estudo da participação de microRNAs na regulação da resposta imune inata de macrófagos murinos à infecção por Paracoccidioides brasiliensis

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2016.

Identification of differentially expressed genes in the heart precursor cells of the chick embryo

Genetic evidence has implicated several genes as being critical for heart development. However, the inducers of these genes as well as their targets and pathways they are involved with, remain largely unknown. Previous studies in the avian embryo showed that at HH4 Cerberus (cCer) transcripts are detected in the anterior endomesoderm including the heart precursor cells and later in the left lateral plate mesoderm. We have identified a promoter element of chick cCer able to drive EGFP expression in a population of cells that consistently exit from the anterior primitive streak region, from as early as stage HH3+, and that later will populate the heart. Using this promoter element as a tool allowed us to identify novel genes previously not known to potentially play a role in heart development. In order to identify and study genes expressed and involved in the correct development and differentiation of the vertebrate heart precursor cell (HPC) lineages, a differential screening using Affymetrix GeneChip system technologies was performed. Remarkably, this screening led to the identification of more than 700 transcripts differentially expressed in the heart forming regions (HFR). Bioinformatic tools allowed us to filter the large amount of data generated from this approach and to select a few transcripts for in vivo validation. Whole-mount in situ hybridization and sectioning of selected genes showed heart and vascular expression patterns for these transcripts during early chick development. We have developed an effective strategy to specifically identify genes that are differentially expressed in the HPC lineages. Within this set we have identified several genes that are expressed in the heart, blood and vascular lineages, which are likely to play a role in their development. These genes are potential candidates for future functional studies on early embryonic patterning.

Improving safety and establishing episomal maintenance of Adeno-associated viral vectors

Adeno-associated viral (AAV) vectors are among the most widely used gene transfer systems in basic and pre-clinical research and have been employed in more than 160 clinical trials. AAV vectors are commonly produced in producer cell lines like HEK293 by co-transfection with a so-called vector plasmid and one (in this work) or two so-called helper plasmids. The vector plasmid contains the transgene cassette of interest (TEC) flanked by AAV’s inverted terminal repeats (ITRs) which serve as packaging signals, whereas the helper plasmid provides the required AAV and helper virus functions in trans. A pivotal aspect of AAV vectorology is the manufacturing of AAV vectors free from impurities arising during the production process. These impurities include AAV vector preparations that contain capsids containing prokaryotic sequences, e.g. antibiotic resistance genes originating from the producer plasmids. In the first part of the thesis we aimed at improving the safety of AAV vectors. As we found that encapsidated prokaryotic sequences (using the ampicillin resistance gene as indicator) cannot be re-moved by standard purification methods we investigated whether the producer plasmids could be replaced by Minicircles (MCs). MCs are circular DNA constructs which contain no functional or coding prokaryotic sequences; they only consist of the TEC and a short sequence required for production and purification. MC counterparts of a vector plasmid encoding for enhanced green fluorescent (eGFP) protein and a helper plasmid encoding for AAV serotype 2 (AAV2) and helper Adenovirus (Ad) genes were designed and produced by PlasmidFactory (Bielefeld, Germany). Using all four possible combinations of plasmid and MCs, single-stranded AAV2 vectors (ssAAV) and self-complementary AAV vectors (scAAV) were produced and characterized for vector quantity, quality and functionality. The analyses showed that plasmids can be replaced by MCs without decreasing the efficiency of vector production and vector quality. MC-derived scAAV vector preparations even exceeded plasmid-derived preparations, as they displayed up to 30-fold improved transduction efficiencies. Using MCs as tools, we found that the vector plasmid is the main source of encapsidated prokaryotic sequences. Remarkably, we found that plasmid-derived scAAV vector preparations contained a much higher relative amount of prokaryotic sequences (up to 26.1 %, relative to TEC) compared to ssAAV vector preparations (up to 2.9 %). By replacing both plasmids by MCs the amount of functional prokaryotic sequences could be decreased to below the limit of quantification. Additional analyses for DNA impurities other than prokaryotic sequences showed that scAAV vectors generally contained a higher amount of non-vector DNA (e.g. adenoviral sequences) than ssAAV vectors. For both, ssAAV and scAAV vector preparations, MC-derived vectors tended to contain lower amounts of foreign DNA. None of the vectors tested could be shown to induce immunogenicity. In summary we could demonstrate that the quality of AAV vector preparations could be significantly improved by replacing producer plasmids by MCs. Upon transduction of a target tissue, AAV vector genomes predominantly remain in an episomal state, as duplex DNA circles or concatemers. These episomal forms mediate long-term transgene expression in terminally differentiated cells, but are lost in proliferating cells due to cell division. Therefore, in the second part of the thesis, in cooperation with Claudia Hagedorn and Hans J. Lipps (University Witten/Herdecke) an AAV vector genome was equipped with an autonomous replication element (Scaffold/matrix attachment region (S/MAR)). AAV-S/MAR encoding for eGFP and a blasticidin resistance gene and a control vector with the same TEC but lacking the S/MAR element (AAV-ΔS/MAR) were produced and transduced into highly proliferative HeLa cells. Antibiotic pressure was employed to select for cells stably maintaining the vector genome. AAV-S/MAR transduced cells yielded a higher number of colonies than AAV-ΔS/MAR-transduced cells. Colonies derived from each vector transduction were picked and cultured further. They remained eGFP-positive (up to 70 days, maximum cultivation period) even in the absence of antibiotic selection pressure. Interestingly, the mitotic stability of both AAV-S/MAR and control vector AAV-ΔS/MAR was found to be a result of episomal maintenance of the vector genome. This finding indicates that, under specific conditions such as the mild selection pressure we employed, “common” AAV vectors persist episomally. Thus, the S/MAR element increases the establishment frequency of stable episomes, but is not a prerequisite.

Rôle du gène de fusion MLL-ENL dans le développement et le maintien du phénotype leucémique

Les translocations chromosomiques du gène MLL sont connues pour mener au développement de leucémies aiguës. La translocation avec un de ses partenaires de fusion les plus communs, ENL, peut engendrer des leucémies aiguës de plusieurs types différents pour cette même translocation. Une fois la leucémogenèse initiée par la fusion MLL-ENL, son rôle quant au maintien du phénotype leucémique n’est pas encore bien connu à ce jour. Pour mieux comprendre l’importance de MLL-ENL après la leucémogenèse, des cellules souches/progénitrices de sang de cordon ombilical humain purifiées ont ainsi été transduites par un virus exprimant le gène de fusion MLL-ENL bordé par des sites LoxP ainsi que le marqueur eGFP. Ces cellules infectées ont ensuite été injectées dans notre modèle de souris immunodéficientes irradiées et placées sous observation pendant 24 semaines pour voir le développement de leucémies aiguës. Elles ont alors été sacrifiées et les cellules la moelle osseuse et de la rate ont ensuite été analysées par cytométrie en flux pour déterminer si la xénogreffe a engendré une leucémie dans notre modèle ainsi que le phénotype de celle-ci. Les souris injectées par les cellules infectées par le MLL-ENL ont généré uniquement des leucémies lymphoïdes aiguës de type B. Les cellules de ces leucémies primaires isolées ont été par la suite infectées par un lentivirus exprimant la cre-recombinase et le marqueur BFP afin d’exciser le gène MLL-ENL des cellules leucémiques grâce aux sites LoxP. Les cellules ont ensuite été triées pour le marqueur BFP et injectées dans des souris secondaires pour de voir si les cellules leucémiques souches pouvaient toujours régénérer la leucémie. Les conséquences de l’absence de MLL-ENL dans le maintien du phénotype leucémique n’ont cependant pas pu être vérifiées à cause d’une erreur dans la séquence de la cre-recombinase, mais nous avons observé la régénération des leucémies secondaires.