Ao longo da vida reprodutiva dos machos adultos, espermatozoides são formados pelas células-tronco espermatogoniais (SSCs, do inglês spermatogonial stem cells) por um processo conhecido como espermatogênese. O cultivo in vitro de SSCs abriu novas possibilidades para a transgenia, no entanto os protocolos requerem a adição de fatores de crescimento, o que encarece a manutenção dessas células por um tempo prolongado, fazendo da criopreservação de SSCs uma alternativa para esse problema. O fenótipo molecular de células-tronco espermatogoniais tem sido objeto de estudo nas últimas décadas. Uma das moléculas mais estudadas como marcador na caracterização de espermatogônias é a alfa-6 integrina. Baseado nestas informações, a seguinte hipótese foi proposta: SSCs que apresentam maior expressão de alfa-6 integrina são mais eficientes para modificações genéticas e colonização de túbulos seminíferos. Para testar essa hipótese, as SSCs murinas foram dissociadas de testículos de camundongos com 8 a 11 dias de idade. Essas células foram cultivadas in vitro, caracterizadas quanto sua morfologia, congeladas e incubadas com anticorpo anti-alfa-6 integrina para a purificação das SSCs por separação celular ativada por fluorescência (FACS). Células testiculares foram geneticamente modificadas com a inserção do gene marcador LacZ e o transplante através dos ductos eferentes foi padronizado. As Células germinativas testiculares foram dissociadas e cultivadas in vitro, porém a viabilidade foi aquém da desejada, inviabilizando as etapas de transformação, transplante e posterior avaliação histológica. Usando o marcador molecular alfa-6 integrina foi possível separar populações de células germinativas testiculares por FACS e a expressão gênica de ITGα6, GFRα1, Oct-4 e Thy-1 foi detectada por RT-PCR quantitativo. Em conclusão, não foi possível comprovar a eficiência de transdução e colonização em túbulos seminíferos por células-tronco espermatogoniais selecionadas com alfa-6 integrina. / Throughout the reproductive life of adult males, spermatozoa are formed from spermatogonial stem cells (SSCs) by a process known as spermatogenesis. The in vitro culture of SSCs created new possibilities for transgenesis, however the protocols require addition of growth factors, which increases the maintenance costs of these cells for a prolonged time, making of the cryopreservation of SSCs an alternative for this problem. The molecular phenotype of spermatogonial stem cells have been target of studies in recent decades. One of the most studied marker molecule in the characterization of spermatogonia is integrin alpha-6. Based on these informations, the following hypothesis was proposed: SSCs that show increased expression of integrin alpha-6 are more efficient for genetic modification and colonization of seminiferous tubules. In order to test this hypotesis, murine SSCs were dissociated from testes of 8 to 11 days old mice. These cells were in vitro cultured, characterized based on its morphology, frozen and incubated with anti-integrin alpha-6 antibody for the purification of SSCs by activated cell sorting fluorescence (FACS). Testicular cells were genetically modified with the insertion of the marker gene LacZ and transplantation through the efferent ducts was standardized. The testicular germ cells were dissociated and in vitro cultured, however viability was below expected, precluding the steps of transformation, transplantation and subsequent histological evaluation. Using molecular marker integrin alpha-6 it was possible to separate testicular germ cell populations by FACS and gene expression of ITGα6, GFRα1, Oct-4 and Thy-1was detected by quantitative RT-PCR. In conclusion, it was not possible to prove the efficiency of transduction and colonization in the seminiferous tubules by spermatogonial stem cells selected with alpha 6 integrin.

Les avancées récentes dans la technique de transgénèse utilisant l’approche AAV ont permis de générer de nouveaux modèles animaux. Depuis quelques années, le développement des modèles de la maladie de Parkinson (MP) a amélioré la compréhension des mécanismes physiopathologiques de ce trouble dégénératif. Cependant, aucun modèle mammifère ne reproduit à ce jour la neurodégénérescence liée à l’âge associée à la pathologie synucléine et la symptomatologie motrice et non motrice. L’objectif de mon travail de thèse fut de développer de nouvelles stratégies de transgénèse chez le rat en utilisant ces vecteurs viraux pour la modélisation de la MP. Le challenge est de parvenir à une infection virale la plus précoce possible afin de transduire un maximum de neurones dopaminergiques. Pour cela, différentes voies d’approche ont été testées pour améliorer le processus de transgénèse : 1) l’injection de vecteurs viraux dans le rete testis de jeunes mâles pour transduire les cellules de la lignée germinale, 2) l’injection dans les embryons pré-implantatoires, 3) l’injection in utero intracérébroventriculaire et enfin 4) l’injection intracardiaque au premier jour de vie des animaux. Parmi elles, les injections in utero et intracardiaques ont permis d’aboutir à une transduction importante d’un grand nombre de structures cérébrales avec un tropisme neuronal. Ces techniques ouvrent la voie à la génération de nouveaux modèles animaux de maladies à composante génétique et à la thérapie génique. / Recent advances in the transgenesis technique using the AAV approach have led to the generation of new animal models. In recent years, the development of models of Parkinson's disease (PD) has improved understanding of the pathophysiological mechanisms of this degenerative pathology. However, no mammalian model recapitulates the required age-dependant parkinsonian degeneration, the α-synuclein inclusion pathology and motor and non-motor symptoms. The objective of my Ph.D work was to develop new transgenesis strategies in rats using these viral vectors for modeling PD. The challenge is to achieve a viral infection as early as possible in order to transduce as many dopaminergic neurons as possible. To this end, different strategies have been tested to improve transgenesis efficacy : i) injection in rete testis in young male to transduce germinal cells, ii) injection into early stage embryos, iii) in utero intracerebroventricular injection and iv) intracardiac injection in one day-old animals. Among them, in utero and intracardiac injections led to neuronal transgene expression in most regions of the brain. These techniques pave the way for the generation of new animal models of genetic diseases and offer unique opportunities for gene therapy.

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Le développement de Caenorhabditis elegans est finement régulé et aboutit au nombre constant de 959 cellules par individu. Une partie de ces régulations repose sur les microARN, ARN simple brin d’environ 22 nucléotides. Ils peuvent diminuer l'expression des gènes en ciblant des séquences homologues dans les régions 3' non transcrites (3'UTR) des ARN messagers. Pour déterminer l'impact de cette régulation, nous utilisons une méthode permettant de visualiser in vivo une différence d'expression induite post-transcriptionnellement. Cette méthode se base sur l'utilisation de deux protéines fluorescentes : la GFP (verte) et la mCherry (rouge) exprimées sous le contrôle du promoteur du gène d'intérêt. La mCherry est suivie par un 3'UTR contrôle et reflète l'activité du promoteur. La GFP est suivie par le 3'UTR du gène d'intérêt et subit les mêmes régulations que le gène endogène. La différence d'expression entre les deux protéines devrait donc refléter la régulation s'opérant sur le 3'UTR du gène. La transparence de C. elegans permet de localiser les protéines rapportrices par microscopie en fluorescence à tous les stades du développement dans l'organisme entier. Initialement, le 3'UTR du gène unc-54 (myosine de type II) a été choisi comme contrôle. Le rapporteur bicolore a révélé une régulation s’opérant sur unc-54 3’UTR jusqu’ici considérée comme permissif. La régulation observée s’opère dans les muscles et les neurones ADF. La caractérisation de cette régulation a permis de mettre en évidence le rôle potentiel de mir-1820. Le profil d’expression de mir-1820 a pu être établi grâce à une fusion avec la GFP et correspond au profil des régulations observées sur unc-54 3’UTR. / Caenorhabditis elegans development is very tightly regulated, leading to the same number of cells in each individual. Part of this regulation network relies on small single strand RNAs (miRNAs), which can target homologous sequences in the 3’ untranslated regions (3’UTR) of messenger RNAs. We want to investigate the contribution of miRNAs during neurons differentiation. In order to study the miRNA contribution to gene regulation we use double fluorescent reporters that allow us to visualize the posttranscriptional contribution to regulation throughout development. The GFP and the mCherry are expressed under the control of the gene promoter, but followed by either the 3’UTR of interest, or a control 3’UTR. We first chose as a control 3’UTR the unc-54 3’UTR (myosin class II). The gene unc-54 is expressed through all larval stages and in the adult worms. The two colors reporter system showed that unc-54 3’UTR undergoes a regulation in the ADF pair of neurons and partially in the body wall muscle. The characterization of this regulation pointed out a potential role for mir-1820. The GFP was cloned between mir1820 5’ and 3’ sequences and the construction displayed an expression profile overlapping with the regulation pattern observed on unc-54 3’UTR.

Le virus de l’hépatite C (VHC) est une des principales causes d’hépatite chronique. La protéine F du VHC est codée par un cadre de lecture alternatif du gène de la capside, Core. La protéine F a été découverte après que l’on ait associé Core à plusieurs des fonctions pathogènes du VHC. Nous proposons donc que certaines fonctions biologiques et pathogènes attribuées à la protéine Core résultent de l’activité de la protéine F. Nous avons choisi de développer trois lignées de poissons zébrés (Danio rerio) qui expriment différentes versions de la protéine F afin d’étudier les effets de la protéine F et leur incidence dans la pathogenèse du VHC. Deux versions de la séquence codant pour la protéine F (AF11 et AUG26) et une version mutante du gène core (CoremutI) ont été introduites sur les vecteurs d’un système d’expression répressible spécifique au foie. Ces vecteurs ont été co-injectés dans des embryons unicellulaires de poissons zébrés pour générer les poissons fondateurs des lignées transgéniques. 19, 21 et 36 poissons ont été choisis comme fondateurs pour les lignées AF11, AUG26 et CoremutI respectivement. De ce nombre, 9, 11 et 11 poissons ont atteint la maturité, dans l’ordre pour les mêmes lignées, et seront croisés pour donner naissance à des lignées transgéniques stables. Les résultats de ces expériences nous permettront de mieux cerner les propriétés biologiques de la protéine F et de définir son rôle dans la pathogenèse du VHC. / Hepatitis C virus (HCV) is a major cause of liver steatosis, fibrosis and hepatocellular carcinoma. HCV F protein is expressed from an alternative reading frame within the Core sequence. F protein was discovered after many of the pathogenic determinants of HCV had been associated with the effects of Core. Hence, we propose that a part of the functions attributed to Core result from the activity of the F protein. We produced and selected 19, 21 and 36 transgenic zebrafish (Danio rerio) to give rise to 3 independent lines expressing different versions of the F protein. Of these founders, 9, 11 and 11 were raised to maturity and will be bred to generate stable transgenic lines. Characterizing the phenotype of these transgenic fish will help determine the precise role of the F protein in the pathogenesis of hepatitis C.

La protéine core du virus de l’hépatite C (VHC) serait responsable des principaux effets pathogènes du VHC, dont le développement de fibrose, stéatose, cirrhose et carcinome hépatocellulaire. Un cadre de lecture alternatif existe dans le gène de core, permettant la synthèse d’une autre protéine appelée ARFP (pour alternatate reading frame protein) ou protéine F (pour frameshift), dont le rôle reste encore mal compris. La présence de la protéine F lors de l’étude des fonctions biologiques de core ne pouvant être exclue, il est possible que certains rôles attribués à core reflètent en réalité l’activité de la protéine F. Afin de déterminer les fonctions biologiques de la protéine F dans les hépatocytes et son influence dans la pathogenèse associée au VHC, nous avons généré des lignées transgéniques de poissons zébrés (Danio rerio) dans lesquelles l’expression de deux versions de la protéine F (AF11opti et AUG26opti) a été ciblée au foie par l’utilisation du promoteur de la liver fatty acid binding protein (L-FABP). Le phénotype des poissons transgéniques de génération F2 a été analysé au niveau morphologique, histologique et microscopique afin de rechercher des signes de pathologie hépatique. Nos résultats ont démontré l’implication de la protéine F dans le développement de stéatose hépatique chez les deux lignées transgéniques, mais aucun signe de fibrose ou d’oncogenèse n’a été détecté. L’identification des mécanismes cellulaires et moléculaires responsables de l’accumulation lipidique induite par la protéine F pourrait permettre de mieux comprendre son rôle dans la pathogenèse du VHC, et mener au développement de nouvelles stratégies antivirales. / Hepatitis C virus (HCV) core protein is thought to be responsible for the major pathogenic effects of HCV, including the development of fibrosis, steatosis, cirrhosis, and hepatocellular carcinoma. An alternate translational open reading frame exists in the core gene that allows the synthesis of another protein called ARFP (alternate reading frame protein) or F protein (frameshift), the role of which remains poorly understood. Since we cannot exclude the presence of F protein in most studies of core biological functions, it is possible that the roles attributed to core reflect the activity of ARFP. To determine the biological functions of F protein in hepatocytes and their influence on HCV-associated pathogenesis, we generated transgenic lines of zebrafish (Danio rerio) in which the liver fatty acid binding protein (L-FABP) promoter was used to direct liver-specific expression of two forms of ARFP (AF11opti and AUG26opti). The phenotype of F2 transgenic zebrafish was analyzed for morphological, histological and microscopic signs of liver-associated pathology. Our results demonstrated the implication of the HCV F protein in the development of hepatic steatosis in transgenic zebrafish liver but not fibrosis or oncogenesis. Identification of the cellular and molecular mechanisms underlying F protein-induced lipid accumulation will lead to a better understanding of the role of ARFP in HCV-associated pathology, which could lead to the development of novel antiviral strategies.

A baixa eficiência e a dificuldade de reprodução de resultados da técnica de transferência gênica mediada por espermatozoides (TGME) têm como possível explicação à ativação de endonucleases espermáticas. Assim, a inibição desta enzima poderia evitar a fragmentação de DNA (exógeno e genômico), possibilitando assim, o uso de maiores concentrações de DNA exógeno, aumentando a eficiência e garantindo a reprodutibilidade da técnica. O ácido aurintricarboxílico (ATA) é um inibidor geral de endonucleases (HALLICK et al., 1977), inclusive das endonucleases espermáticas (MAIONE et al., 1997; MAGNANO et al., 1998). Deste modo, o presente estudo objetivou avaliar a inibição das endonucleases espermáticas, pelo ácido aurintricarboxílico (ATA). Para isso, três experimentos foram realizados: 1) avaliar a inibição das endonucleases espermáticas pela adição do ácido aurintricarboxílico, após incubação com DNA exógeno; 2) verificar a eficiência do ATA na inibição de fragmentação de DNA genômico e 3) detectar o aumento nos índices de internalização após o uso de ATA. Para o primeiro experimento, um ensaio de digestão plasmidial com os plasmídeos PCX-EGFP e pmGENIE3 e três concentrações de ATA (10µM, 25µM e 50µM) foram testados. As digestões dos vetores plasmidiais ocorreram pela incubação dos plasmídeos PCX-EGFP e pmGENIE3, com e sem a presença de ATA, com extratos espermáticos. As incubações ocorreram durante 1 hora à 37ºC e os produtos foram analisados por eletroforese (2 horas, 100mV) em gel de agarose 0,7%. Os resultados foram avaliados em escala de cruzes, no qual 1 foi considerado digestão total dos plasmídeos e 3, a não digestão. Os resultados foram analisados em nível de significância de 5%. Os resultados demonstraram diferenças nas digestões dos dois vetores plasmidiais, sendo o pmGENIE3 mais susceptível à degradação pelo extrato espermático, demonstrando ausência de bandas em algumas replicatas (mediana=1). O PCX-EGFP apresentou inibição parcial da degradação já com 10µM de ATA. Já o pmGENIE só apresentou inibição da degradação com 25 ou 50µM de ATA. Assim a concentração utilizada nos experimentos consecutivos foi a de 50µM. Para o experimento 2, espermatozoides de camundongos da linhagem Bl-6/DBA (F1) foram incubados com duas concentrações (500 ou 1000ng) do plasmídeo PCX-EGFP, com ou sem pré-incubação com ATA. As incubações ocorreram durante 5 horas, em ar com 5% de CO2 à 37ºC. Assim, os espermatozoides foram submetidos ao teste de susceptibilidade à denaturação ácida e ao ensaio de cometa alcalino para verificar possível fragilidade da cromatina. Os dados demonstraram que o uso do ATA em espermatozoides murinos leva à fragilidade do genoma, independente de serem incubados com DNA exógeno e a concentração do mesmo. Além disso, foi possível verificar que pode existir um limiar de concentração ótima para que as endonucleases causem fragmentação do DNA cromossomal, o qual foi de 500ng. O uso de concentrações maiores, como 1000ng, pode ter agido como fator protetor ao DNA genômico, podendo este DNA exógeno ter sido o alvo primário das endonucleases, já que quando as amostras foram incubadas com essa concentração de plasmídeo, não houve altos índices de fragmentação no DNA endógeno. No experimento 3, espermatozoides de camundongos foram incubados com 500 ou 1000ng de PCX-EGFP/106 células, sendo ou não pré-incubados com ATA. O DNA genômico das células espermáticas foi extraído pelo método de fenol clorofórmio, diluído para concentração de 1ng/µl e submetidos à quantificação de DNA plasmidial pela técnica de quantificação absoluta em tempo real (qPCR). Os resultados demonstraram que o ATA não melhorou a eficiência de incorporação, já que tanto na concentração de 500 quanto na de 1000ng de DNA exógeno, a porcentagem foi menor (0,001% para os dois grupos) em relação aos grupos nos quais este não foi utilizado. Os grupos em que o ATA não foi utilizado não apresentaram diferença entre si demonstrando que a quantidade de 500ng de DNA exógeno foi suficiente na internalização deste ao espermatozoide, sendo a porcentagem de incorporação maior do que 1000ng (0,20% contra 0,10%). Contudo, o uso do inibidor de endonucleases, ao invés de aumentar os índices de incorporação apresentou resultados opostos, indicando que seu uso não trouxe melhorias para a técnica de TGME. / The low efficiency and low repeatability of sperm-mediated gene transfer (SMGT) could be due to the activation of sperm endonucleases. The inhibition of this enzyme would avoid genomic DNA fragmentation enabling the use of higher concentrations of exogenous DNA, increasing the efficiency and ensuring the reproducibility of this technique. Aurintricarboxilic acid (ATA) is a general inhibitor of endonucleases (HALLICK et al., 1977), including sperm endonucleases (MAIONE et al., 1997; MAGNANO et al., 1998). This study aimed to evaluate the inhibition of sperm endonucleases using the aurintricarboxilic acid (ATA). For that, three experiments were set: 1) evaluate the inhibition of sperm endonucleases by adding aurintricarboxilic acid after incubation with exogenous DNA, 2) study the inhibition efficiency of ATA in genomic DNA fragmentation and 3) detect exogenous DNA internalization after the use of ATA. For the first experiment, a plasmid digestion assay with pmGENIE3 and PCX-EGFP and three concentrations of ATA (10µM, 25µM and 50µM) were tested. The digestion of plasmid vector occurred by incubation of PCX-EGFP and pmGENIE3 with and without the presence of ATA with sperm extracts. Incubations took place for 1 hour at 37°C and the products were analyzed by electrophoresis (2 hours, 100mV) in 0,7% agarose gel. The results were evaluated on a cross scale whereas 1 was considered a total plasmid digestion and 3 no digestion. The results were analyzed with a significance level of 5%. The results show differences in the digestion of the two plasmid vectors, being pmGENIE3 more susceptible to degradation by sperm extract, demonstrating absence of bands in some replicates (median = 1). The PCX-EGFP showed a parcial inhibition of the degradation using 10µM ATA. PmGENIE3 presented inhibiting of degradation only using 25 or 50µM of ATA. Thus, the concentration used in the consecutives experiments was 50µM. For experiment 2, sperm from Bl-6/DBA (F1) mice strain were incubated with two concentrations (500 or 1000ng) of the PCX-EGFP plasmid, with and without pre-incubation with ATA. Incubation took place for 5 hours, with 5% CO2 in air, at 37°C. Sperm samples were subjected to acid denaturation susceptibility test and alkaline comet assay to check for possible chromatin fragility. The data showed that the use of ATA in murine sperm leads to a fragility of their genome, independently of the incubation with exogenous DNA and its concentration. Result showed that there might be a threshold concentration for chromosomal DNA fragmentation caused by endonucleases, which was 500ng of plasmid. The use of higher concentrations, as 1000ng, may be a protective factor for genomic DNA integrity, since exogenous DNA seems to be the primary target of endonucleases, showed by, lower DNA fragmentation levels. In experiment 3, sperm were incubated with 500 or 1000ng PCX-EGFP/106 cells, pre-incubated or not with ATA. Genomic DNA was extracted by phenol chloroform method, diluted to concentrations of 1ng/µl and subjected to quantification of plasmid DNA insertions by absolute quantification in real-time PCR (qPCR). The results showed that ATA did not improve the efficiency of DNA internalization, whereas both concentration of 500 and 1000ng presented a lower percentage of exogenous DNA integration (0.001% in both groups) compared with the groups in which ATA was not used. The groups without ATA did not differ indicating that the amount of 500ng of DNA was able to integrate exogenous DNA to sperm, and have higher percentage of incorporation compared to 1000ng (0,20% versus 0,10%). Thereby, the use of an endonuclease inhibitor instead of increasing integration indexes showed opposite results, indicating that its use did not bring improvements to the SMGT technique.

Non-invasive imaging techniques, such as micro-CT or optical imaging, provide valuable information about tumour microstructure, size, volume and growth dynamics. Although histology is an approach capable of describing several of these characteristics, the invasiveness of this analysis remains a disadvantage. The main aim of this work is the methodological development of non-invasive imaging of the dynamics of tumour growth and progression. The preparation of a dual-reporter lentiviral vector enables non-invasive study of tumour growth and dissemination of metastasis. The same dual reporter will also be a part of a second vector designed as a construct for targeting mouse embryonic stem cells with aim to produce corresponding transgenic reporter mouse line. This reporter mouse line can be beneficial for future projects by providing a novel approach for studying the dynamics of tumour growth under various genetic conditions. In addition to optical imaging, this project will also include the use of micro-CT technology which, as a non-invasive approach, has the potential to provide information about the microstructure of tumour tissue in 3D that histology is not able to report.

The mechanism to maintain stem cell properties and to exit into differentiation pathways is a pivotal question in stem cell research. Spermatogonia are the adult stem cells of the male germ line, which are used in biomedical research as a source of undifferentiated cells. The communication between germ line stem cells and specialized somatic cells (Sertoli cells and Leydig cells) plays important roles in stem cell maintenance, germ cell proliferation, and differentiation. With regard to the biology of stem cells and spermatogenesis, the medaka (Oryzias latipes) is used as a teleost model organism, and it is also used to assess the effects of endocrine disruptors on reproductive phenomena. However, the lack of suitable molecular markers hampers the detection, isolation and analysis of different testis cells including gonial stem cells and Leydig cells. Therefore, oct4, sox2 and cyp11b were chosen to create transgenic reporter lines for the labeling of stem cells and Leydig cells, respectively. The present study had the aim to examine the temporal and spatial expression of the respective genes during embryonic development and in adult gonads of the medaka, and to describe the application of these transgenic lines in stem cell biology and reproductive biology. The mCherry expression in transgenic fish of the line FSI-Tg(sox2-mCherry)17 marks embryonic stem cells, Leydig cells and interstitial cells in adult testis. Faithful EGFP and DsRed expression in transgenic reporters strains for oct4 and cyp11b mimics the endogenous expression of oct4/pou2 and cyp11b-protein, respectively. The reporter gene expression in the strains FSI-Tg(oct4-EGFP)9 and FSI-Tg(oct4-EGFP)A allows the visualization of oct4 positive cells during embryonic development, PGCs, early germ cells and adult gonial cells. The Leydig cells express brightly green or red fluorescence in the medaka strains FSI-Tg(cyp11b-EGFP)20 and FSI-Tg(cyp11b-DsRed)1434, respectively, allowing the easy identification of Leydig cells in adult testis. The oct4-EGFP reporter labels medaka embryonic and spermatogonial stem cells, in which the spermatogonial stem cells at the ends of the testicular lobules show brightly green fluorescence. The transgenic expression in stem cells is also shown in the flow plot of primary testis cells. The spermatogonia are the largest cells and have the strongest fluorescence, which decreased upon differentiation. Therefore, the oct4-EGFP reporter strains will provide an opportunity to detect and to isolate the EGFP expressing cells for transplantation. These strains will also facilitate further experiments on the effects of drugs or hypoxia on these cells, because the strongest EGFP expressing cells can be easily detected in transgenic lines. Labeling of Leydig cells in cyp11b reporter lines opens a new area to study the seasonal variation of spermatogenesis. The medaka is a seasonal breeder in its natural habitat and the simulation of seasonal changes allows the simultaneous quantitative analysis of oct4-EGFP and cyp11b-DsRed expressing cells under such conditions.