No description available.

In eukaryotic cells, the genome is packed into chromatin, a hierarchically organized complex composed of DNA and histone and nonhistone proteins. In this thesis we have addressed the role of chromatin in cellular response to DNA damage (DDR) using various methodologies encompassing functional genomics and proteomics. First, we analyzed histone post-translational modifications (PTM) in the context of specific kind of DNA lesions (ICL-Interstrand Crosslinks) in Fanconi anemia using quantitative proteomics methodology, SILAC (Stable Isotope Labeling of Amino acids during Cell Culture). Using mass spectrometry (MS), we have successfully identified and quantified a number of histone PTM marks in histone H3 and H4, mainly acetylations and methylations,which have shown dependence upon functional FA-pathway. As a next step, we applied a functional genomics approach to study DDR in FA cells. In this analysis we first monitored the expression profile of histone modifying enzymes related to histone acetylations and methylations. Our results suggest some correlations between histone PTMs and gene expression of histone modifying enzymes, although conclusive evidence warrants further investigations. Next, we analyzed the total transcriptome after DNA damage induction in FA mutant and wild type cells. We also included in this analysis IR irradiation, in an attempt to dissociate more generic DDR from more specific changes that are associated with the role of FA pathway to the DNA ICLs. By performing a factorial interaction analysis, we were able to isolate the part of transcriptional response to DNA damage that was requiring functional FA pathway, as well as the genes that were sensitized to DNA damage by the inactivation of FA pathway. In the final part of the thesis, we attempted to solve one of the limitations that we encountered in the histone PTM analysis. The current approaches used to study histone PTMs from particular loci involves classical chromatin immunoprecipitation, which due to involvement of formaldehyde crosslinking render the protein part mostly unavailable for MS-based proteomics. We have proposed a novel methodology, which is based upon the biotin tagging of histones proximal to a protein of interest and subsequent purification of nucleosomes carrying the tagged histone. This methodology does not involve any crosslinking, enabling us to purify histones from specific loci, and subject them to large scale MS-based histone PTM analysis. A time dimension can also be added to our approach, as we can follow the modification status of particular fraction of histones once they get biotinylated. Another advantage is the use of alternate variant histones, which allows us to study the PTM profile of different functional states of chromatin. This methodology certainly has an edge on current techniques to study histone PTMs pattern associated with a particular protein of interest or with particular chromatin state.

This thesis was performed within the research project “TrophinOak”, which addresses the impact of multitrophic interactions on the pedunculate oak (Quercus robur) clone DF159. In this frame, the present work focuses on the genetic and physiological mechanisms ruling the interaction of the mycorrhiza helper bacterium (MHB) Streptomyces sp. AcH 505 with microcuttings of DF159 either alone or in presence of the ectomycorrhizal fungus Piloderma croceum or the fungal leaf pathogen oak powdery mildew Microsphaera alphitoides. The work consists of 3 chapters. Chapter 1 characterises the growth of AcH 505 and P. croceum in a soil-based culture system used within the TrophinOak project. Besides the establishment and evaluation of quantification methods of these microorganisms by quantitative real-time PCR, the impact of the soil microbial community and the oak on the bacterium-fungus interaction was investigated, and AcH 505 and P. croceum were visualized by scanning electron microscopy. It was observed that the presence of the soil microorganisms and the oak both affect the bacterium-fungus interaction, and that P. croceum enhances the growth of AcH 505. Chapter 2 presents a study with the oak, AcH 505 and the EM fungus P. croceum, enabling to disentangle the direct effect of the MHB on the oak from the indirect one via the EM symbiosis. The used approach was transcriptomic based on RNA sequencing. It was shown that i) differential gene expression occurred between root and the distant leaf tissues (local vs. systemic effects), different developmental stages and treatments, suggesting that oak specifically coordinates its gene expression patterns, and ii) that genes related to plant growth, defence and DNA modification were dominant among the differential expressed genes, suggesting that these processes play essential roles in both symbiotic interactions investigated. Chapter 3 represents a second transcriptome study, addressing how AcH 505 suppresses powdery mildew infection in oak by analysing RNA Sequencing data from singly- and coinoculated oaks. This study combined the systemic impact of the root associated bacterium with local effects of the leaf pathogen, thereby linking belowground and aboveground interactions. Systemic defence response is induced by the bacterium and further enhanced upon pathogen challenge, suggesting that on the leaf level, some bacterial effectors are recognized as harmful for the plant.

For the first time the transcriptional reprogramming of distinct root cortex cells during the arbuscular mycorrhizal (AM) symbiosis was investigated by combining Laser Capture Mirodissection and Affymetrix GeneChip® Medicago genome array hybridization. The establishment of cryosections facilitated the isolation of high quality RNA in sufficient amounts from three different cortical cell types. The transcript profiles of arbuscule-containing cells (arb cells), non-arbuscule-containing cells (nac cells) of Rhizophagus irregularis inoculated Medicago truncatula roots and cortex cells of non-inoculated roots (cor) were successfully explored. The data gave new insights in the symbiosis-related cellular reorganization processes and indicated that already nac cells seem to be prepared for the upcoming fungal colonization. The mycorrhizal- and phosphate-dependent transcription of a GRAS TF family member (MtGras8) was detected in arb cells and mycorrhizal roots. MtGRAS shares a high sequence similarity to a GRAS TF suggested to be involved in the fungal colonization processes (MtRAM1). The function of MtGras8 was unraveled upon RNA interference- (RNAi-) mediated gene silencing. An AM symbiosis-dependent expression of a RNAi construct (MtPt4pro::gras8-RNAi) revealed a successful gene silencing of MtGras8 leading to a reduced arbuscule abundance and a higher proportion of deformed arbuscules in root with reduced transcript levels. Accordingly, MtGras8 might control the arbuscule development and life-time. The targeting of MtGras8 by the phosphate-dependent regulated miRNA5204* was discovered previously (Devers et al., 2011). Since miRNA5204* is known to be affected by phosphate, the posttranscriptional regulation might represent a link between phosphate signaling and arbuscule development. In this work, the posttranscriptional regulation was confirmed by mis-expression of miRNA5204* in M. truncatula roots. The miRNA-mediated gene silencing affects the MtGras8 transcript abundance only in the first two weeks of the AM symbiosis and the mis-expression lines seem to mimic the phenotype of MtGras8-RNAi lines. Additionally, MtGRAS8 seems to form heterodimers with NSP2 and RAM1, which are known to be key regulators of the fungal colonization process (Hirsch et al., 2009; Gobbato et al., 2012). These data indicate that MtGras8 and miRNA5204* are linked to the sym pathway and regulate the arbuscule development in phosphate-dependent manner. / Die Leguminose Medicago truncatula (gehört zur Gattung des Schneckenklees) ist in der Lage sowohl eine Symbiose mit stickstofffixierenden Bakterien (Rhizobien), als auch mit Mykorrhiza-Pilzen einzugehen. Der Mykorrhiza-Pilz Rhizophagus irregularis dringt in die Wurzelrindenzellen ein und bildet Strukturen aus, die als Arbuskeln bezeichnet werden. Diese ermöglichen den Transfer von Nährstoffen, wie Phosphat in die Wurzelzellen. Die Pflanze liefert hingegen bis zu 20 % ihrer Photosyntheseprodukte an den Pilz. Da die Lebenszeit der Arbuskeln nur wenige Tage beträgt, können Wurzelrindenzellen mehrere Arbuskeln nacheinander beherbergen. Somit können neben arbuskelhaltigen, auch nicht-arbuskelhaltige Zellen in kolonisierten Wurzeln auftreten. Die nicht-arbuskelhaltigen Zellen beeinträchtigen die Sensitivität von Genregulationsanalysen, wenn die Genregulation während der Mykorrhiza-Symbiose anhand von ganzen kolonisierten Wurzeln untersucht wird. In dieser Arbeit konnte eine Zelltyp-spezifische Analyse der Genregulation von arbuskelhaltigen und nicht-arbuskelhaltigen Zellen durchgeführt, und eine Erhöhung der Sensitivität erreicht werden. Mittels Laser Capture Microdissection wurden Zellen aus Gefrierschnitten von Wurzeln isoliert. Aus den so gewonnen Zellen konnte RNA von ausreichender Qualität und Quantität extrahiert werden, um das Transkriptom der beiden Zelltypen mittels Mikroarrayhybridisierung zu untersuchen. Transkriptionsfaktoren (TFs) spielen wahrscheinlich eine Schlüsselrolle in der Umprogrammierung von Wurzelzellen während der Mykorrhiza-Symbiose. Daher wurde die Genregulation von TF-Genen in den zwei Zelltypen gezielt untersucht. Anhand von quantitativer RT-PCR und Promoter-Reporter-Fusionen wurde die differentielle Expression von mehreren TF-Transkripten in den verschiedenen Zelltypen bestätigt. Die Charakterisierung eines potentiellen GRAS TF (MtGRAS8) konnte eine stark Symbiose- und Phosphat-abhängige Induktion von Transkripten bestätigt werden. Mutanten mit verringerter MtGras8 Transkriptmenge wiesen eine verringerte Arbuskelzahl und deformierte Arbuskeln auf. MtGras8 scheint daher an der Arbuskelentwicklung beteiligt zu sein. Vorherige Experimente zeigten, dass MtGras8 Transkripte, von der Phosphat-regulierten MikroRNA5204* geschnitten werden (Devers et al., 2011). Dies konnte durch Überexpression der MikroRNA5204* in vivo bestätigt werden. Weiterhin konnten Protein-Protein-Interaktionen von MtGras8 mit bekannten GRAS TFs (NSP1, NSP2, RAM1) nachgewiesen und daraus eine Verbindung zu bekannten Symbiose-induzierten Signalkaskaden geschlossen werden. In dieser Arbeit wurde erstmals die Umprogrammierung von nicht-arbuskelhaltigen Zellen untersucht und neue Regulationselemente für die Kontrolle der Arbuskelentwicklung, wie MtGRAS8 und MikroRNA5204*, charakterisiert.

The recent advances in molecular and computational biology have made possible the study of complicated transcriptional regulatory networks that control a wide range of biological processes and phenotypic traits. In this thesis, several approaches were combined including next generation sequencing, gene expression profiling, chromatin and RNA immunoprecipitation, bioinformatics and genome editing methods in order to characterize the biological significance of the ZBED6 and ZC3H11A genes. A mutation in the binding site of ZBED6, located in an intron of IGF2, disrupts the binding and leads to 3-fold upregulation of IGF2 mRNA in pig muscle tissues. The first part of the thesis presents a detailed functional characterization of ZBED6. Transient silencing of ZBED6 expression in mouse myoblasts led to increased Igf2 expression (~2-fold). ChIP-seq analysis of ZBED6 and histone modifications showed that ZBED6 preferentially binds active promoters and modulates their transcriptional activities (paper I). In the follow-up studies using CRISPR/Cas9 we showed that either the deletion of ZBED6 or its binding site in Igf2 (Igf2ΔGGCT) led to more than 30-fold up-regulation of Igf2 expression in myoblasts. Differentiation of these genetically engineered cells resulted in hypertrophic myotubes. Transcriptome analysis revealed ~30% overlap between the differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with significant enrichment of muscle-specific genes. ZBED6-overexpression in myoblasts led to cell cycle arrest, reduced cell viability, reduced mitochondrial activities and impaired the differentiation of myoblasts (paper II). Further studies on cancer cells showed that ZBED6 influences the growth of colorectal cancer cells with dramatic changes in the transcription of hundreds of cancer-related genes (paper III). The phenotypic characterization of Zbed6-/- and Igf2pA/mG mouse models showed that the ZBED6-Igf2 axis has a major effect on regulating muscle growth and the growth of internal organs. Transcriptome analysis demonstrated a massive up-regulation of Igf2 expression (~30-fold) in adult tissues, but not in fetal tissues, of transgenic mice (paper IV). In the second part of the thesis we investigated the cellular function of Zc3h11a, the gene harboring ZBED6 in one of its first introns. The function of the ZC3H11A protein is so far poorly characterized. We show that ZC3H11A is a novel stress-induced protein that is required for efficient mRNA export from the nucleus. The inactivation of ZC3H11A inhibited the growth of multiple viruses including HIV, influenza, HSV and adenoviruses (paper V).

No description available.

O inseticida spinosad tem sido um dos mais utilizados para o controle de Spodoptera frugiperda (J. E. Smith) no Brasil, devido à sua eficácia e ao seu mecanismo de ação único (modulador alostérico de receptores nicotínicos da acetilcolina). Para fornecer subsídios a um programa de manejo da resistência, foram realizados estudos para compreender as bases genéticas e moleculares da resistência de S. frugiperda a este inseticida. Inicialmente, foi selecionada uma linhagem de S. frugiperda resistente a spinosad (Spin-res) em laboratório por meio da técnica \"F2 screen\". A razão de resistência, baseada na CL50, foi de aproximadamente 890 vezes. A partir de cruzamentos recíprocos entre a linhagem suscetível (Sus) e Spin-res, constatou-se que o padrão de herança da resistência de S. frugiperda a spinosad é autossômica e incompletamente recessiva. Retrocruzamentos da progênie F1 de cruzamentos recíprocos com a linhagem Spin-res confirmaram a hipótese de herança poligênica da resistência, com número mínimo de segregações independentes variando de 1,86 a 2,45. Além disso, observou-se um elevado custo adaptativo associado à resistência de S. frugiperda a spinosad, baseado nos parâmetros da tabela de vida e fertilidade. A partir dos dados de seqüenciamento de quatro bibliotecas de cDNA de lagartas de quarto ínstar das linhagens Sus e Spin-res (expostas ou não a spinosad), utilizando a plataforma HiScan1000&reg; (Illumina&copy;), foi realizada a comparação do perfil de transcrição e expressão diferencial de genes entre as linhagens Sus e Spin-R. O transcritoma foi montado utilizando a estratégia de novo contendo cerca de 19 milhões de leituras single-end com qualidades de score acima de 30, gerando 42406 transcritos com o N50 de 598 pb. A busca por similaridade no banco de dados não-redundante (nr) do NCBI, possibilitou a anotação funcional de 24980 (59%) transcritos, alinhando-se a Bombyx mori L., Helicoverpa armigera (Hübner) e Spodoptera spp. com 22,5; 3,81 e 3,6% das sequências respectivamente. Foram identificados 2903 transcritos apresentando expressão diferencial (P <= 0,05, t-test; fold-change > 2) entre as linhagens Spin-res e Sus. Dentre os transcritos relacionados a enzimas do complexo metabólico, 23 P450 monooxigenases, 13 glutathiona S-transferases, uma carboxilesterase e uma esterase foram superexpressas na linhagem Spin-res. Além disso, foi observada a superexpressão de 15 genes relacionados à produção energética na linhagem Spin-res, o que pode estar relacionada ao elevado custo adaptativo associado à resistência. Análises de PCR quantitativo em tempo real confirmaram que os padrões de expressão foram consistentes com os resultados de RNA-seq. Bioensaios com os sinergistas PBO e DEM mostraram pouco envolvimento de enzimas P450 e nenhum envolvimento de glutationa S-transferases na resistência de S. frugiperda a spinosad. O sequenciamento da subunidade &alpha;6 do receptor nicotínico de acetilcolina de ambas linhagens demonstrou a existência de uma mutação sinônima entre as duas linhagens (G567A), indicando que a subunidade &alpha;6 não é a única relacionada à resistência de S. frugiperda a spinosad. / Spinosad has been one of the most used insecticides to manage Spodoptera frugiperda (J. E. Smith) in Brazil, due to its efficacy and unique mode of action (nicotinic acetylcholine receptor allosteric modulator). To support an insect resistance management program (IRM), we selected and characterized in laboratory a spinosad-resistant strain (Spin-res) of S. frugiperda using the F2 screen method. The resistance ratio, based on LC50, was &asymp; 890-fold. Based on reciprocal crosses between susceptible (Sus) and Spin-res, the inheritance of spinosad resistance in S. frugiperda was autosomal incompletely recessive. Backcrosses between the F1 from reciprocal crosses and the parental Spin-res revealed a polygenic resistance, with an estimation of at least 1.86 to 2.45 genes related to spinosad resistance. Furthermore, it was observed a strong fitness cost associated to spinosad-resistance in Spin-res strain, based on the life table and fertility parameters. The characterization of the transcriptional profile and the differential gene expression comparison between susceptible and spinosad-resistant strains of Spodoptera frugiperda were obtained from the sequencing of cDNA libraries from fourth instar larvae of Sus and Spin-res strains (exposed or not to spinosad) using a HiScan1000&reg; platform (Illumina&copy;). The transcriptome was de novo assembled using nearly 19 million single-end reads with quality score over 30, yielding 42,406 transcripts with a N50 of 598 bp. Based on similarity search in the non-redundant (nr) nucleotide database, 24,980 (59%) transcripts were annotated. Most of the transcripts aligned to Bombyx mori L., Helicoverpa armigera (Hübner) and Spodoptera spp., with 22.5%, 3.81, and 3.6, respectively. We identified 2,032 differentially expressed transcripts (P <= 0.05, t-test; fold-change > 2) between the susceptible and spinosad-resistant strains. Among metabolic enzyme transcripts, 23 P450 monooxigenases, 13 glutathione S-transferases, one carboxylesterase and one esterase were up-regulated in the spinosad-resistant strain. In addition, it was observed 15 genes superexpressed in spinosad-resistant strain related to energy production, which can be related to the high fitness cost associated with resistance. Quantitative real-time PCR analysis showed that patterns of gene expression were consistent with RNA-seq results. Synergistic bioassays using PBO and DEM showed little involvement of P450s in spinosad-resistance and lack of involvement regarding the glutathione Stransferases. Furthermore, we sequenced and compared the subunit &alpha;6 from the nicotinic acetylcholine receptor of S. frugiperda Spin-res and Sus strains. Only one synonymous mutation within the two strains (G567A) was found, showing that the &alpha;6 is not the only subunit involved in S. frugiperda resistance to spinosad.

La fièvre jaune est une pathologie virale humaine causée par un flavivirus, le virus de la fièvre jaune et transmise par des vecteurs arthropodes. Les formes sévères, parfois mortelles, sont caractérisées par une atteinte systémique aigüe qui affecte le foie. Bien que la vaccination existe depuis près de 80 ans, des recensements réguliers d'épidémies sont encore faits. Les vaccins à base d'une souche vivante atténuée YF 17D présentent d'excellents taux de séroconversion et sont notamment caractérisés par une forte diminution de l'hépatotropisme. Néanmoins les mécanismes associés à la pathogénèse hépatique sont encore mal compris et pourraient être une aide aux développements vaccinaux contre d'autres flavivirus ou virus hépatiques. L'étude développée ici s'est inscrite dans la problématique de la représentativité des modèles cellulaires hépatiques utilisés. Afin de répondre aux pertes métaboliques et immunitaires reportées dans plusieurs modèles, nous nous sommes orientés vers des modèles organotypiques associant plusieurs populations cellulaires hépatiques et un microenvironnement caractéristique. Les modulations induites par les souches vaccinales ou sauvages du virus de la fièvre jaune ont été évaluées par une approche transcriptomique globale utilisant la technologie RNASeq et des méthodes d'analyse définies. Nos résultats montrent une plus forte permissivité des modèles cellulaires à la souche atténuée YF 17D par rapport à la souche sauvage YF Asibi. Cette observation est associée pour la souche atténuée à l'établissement précoce d'une réponse antivirale complète impliquant une détection rapide des formes réplicatives du virus, la mise en place des réponses aux IFNs de type I et de type III, la clairance virale et un contrôle des métabolismes cellulaires et hépatiques. De son côté la souche sauvage présente un délai important dans l'établissement de ces réponses amenant à de potentiels mécanismes alternatifs de la clairance virale et de dérégulations métaboliques. Ces données mettent en exergue les interactions étroites qui existent entre les systèmes immunitaires et métaboliques au niveau du foie. Nous suggérons que la forte réponse antivirale induite par la souche atténuée pourrait contribuer à la rupture de la tolérance hépatique et à l'efficacité in vivo de la souche vaccinale. En outre, la cinétique des réponses immunitaires, en combinaison avec la charge virale, peuvent déterminer l'équilibre entre la récupération et l'immunopathologie après l'infection par le virus sauvage / Yellow fever is a human disease caused by a flavivirus, the yellow fever virus, transmitted by arthropod vectors. Severe forms, sometimes fatal, are characterized by acute systemic disease that affects the liver. Despite an effective vaccine being available for nearly 80 years, epizootic circulation occurs and results in periodic outbreaks in endemic regions and among travelers. Vaccines based on a live attenuated strain YF 17D exhibit excellent seroconversion rate and are characterized by a strong decrease in hepatotropism. However the mechanisms involved in liver pathogenesis are poorly understood and could be helpful for future vaccine development against other flaviviruses or hepatitis viruses.There is a need to develop liver cellular model better reflecting the in vivo liver microenvironment. In this work, we used new 3D models combining several liver cell populations to evaluate immune and metabolic responses induced by yellow fever viruses. Modulations induced by both vaccine and wild-type strains were evaluated by a global transcriptomic approach using RNA-Seq technology and well-defined analysis methods. Our results show a greater permissivity of cellular models to YF 17D strain compared to the wild type YF Asibi. In addition, YF 17D infection leads to an early establishment of a complete antiviral response involving rapid detection of replicating forms of the virus, development of a strong type I and type III IFN responses, initiation of viral clearance and modulation of cellular and liver metabolism. Wild-type strain presents a significant delay in the establishment of these responses leading to potential alternative mechanisms for viral clearance and metabolic dysregulation. These data highlight the close interactions between the immune and metabolic systems in the liver.We suggest that the strong antiviral response induced by attenuated strain could contribute to the breakdown of liver tolerance and in vivo efficacy of the vaccine strain. In addition, the kinetics of immune responses, in combination with viral load, can determine the balance between the recovery and immunopathology after infection with wild type virus

O inseticida spinosad tem sido um dos mais utilizados para o controle de Spodoptera frugiperda (J. E. Smith) no Brasil, devido à sua eficácia e ao seu mecanismo de ação único (modulador alostérico de receptores nicotínicos da acetilcolina). Para fornecer subsídios a um programa de manejo da resistência, foram realizados estudos para compreender as bases genéticas e moleculares da resistência de S. frugiperda a este inseticida. Inicialmente, foi selecionada uma linhagem de S. frugiperda resistente a spinosad (Spin-res) em laboratório por meio da técnica \"F2 screen\". A razão de resistência, baseada na CL50, foi de aproximadamente 890 vezes. A partir de cruzamentos recíprocos entre a linhagem suscetível (Sus) e Spin-res, constatou-se que o padrão de herança da resistência de S. frugiperda a spinosad é autossômica e incompletamente recessiva. Retrocruzamentos da progênie F1 de cruzamentos recíprocos com a linhagem Spin-res confirmaram a hipótese de herança poligênica da resistência, com número mínimo de segregações independentes variando de 1,86 a 2,45. Além disso, observou-se um elevado custo adaptativo associado à resistência de S. frugiperda a spinosad, baseado nos parâmetros da tabela de vida e fertilidade. A partir dos dados de seqüenciamento de quatro bibliotecas de cDNA de lagartas de quarto ínstar das linhagens Sus e Spin-res (expostas ou não a spinosad), utilizando a plataforma HiScan1000&reg; (Illumina&copy;), foi realizada a comparação do perfil de transcrição e expressão diferencial de genes entre as linhagens Sus e Spin-R. O transcritoma foi montado utilizando a estratégia de novo contendo cerca de 19 milhões de leituras single-end com qualidades de score acima de 30, gerando 42406 transcritos com o N50 de 598 pb. A busca por similaridade no banco de dados não-redundante (nr) do NCBI, possibilitou a anotação funcional de 24980 (59%) transcritos, alinhando-se a Bombyx mori L., Helicoverpa armigera (Hübner) e Spodoptera spp. com 22,5; 3,81 e 3,6% das sequências respectivamente. Foram identificados 2903 transcritos apresentando expressão diferencial (P <= 0,05, t-test; fold-change > 2) entre as linhagens Spin-res e Sus. Dentre os transcritos relacionados a enzimas do complexo metabólico, 23 P450 monooxigenases, 13 glutathiona S-transferases, uma carboxilesterase e uma esterase foram superexpressas na linhagem Spin-res. Além disso, foi observada a superexpressão de 15 genes relacionados à produção energética na linhagem Spin-res, o que pode estar relacionada ao elevado custo adaptativo associado à resistência. Análises de PCR quantitativo em tempo real confirmaram que os padrões de expressão foram consistentes com os resultados de RNA-seq. Bioensaios com os sinergistas PBO e DEM mostraram pouco envolvimento de enzimas P450 e nenhum envolvimento de glutationa S-transferases na resistência de S. frugiperda a spinosad. O sequenciamento da subunidade &alpha;6 do receptor nicotínico de acetilcolina de ambas linhagens demonstrou a existência de uma mutação sinônima entre as duas linhagens (G567A), indicando que a subunidade &alpha;6 não é a única relacionada à resistência de S. frugiperda a spinosad. / Spinosad has been one of the most used insecticides to manage Spodoptera frugiperda (J. E. Smith) in Brazil, due to its efficacy and unique mode of action (nicotinic acetylcholine receptor allosteric modulator). To support an insect resistance management program (IRM), we selected and characterized in laboratory a spinosad-resistant strain (Spin-res) of S. frugiperda using the F2 screen method. The resistance ratio, based on LC50, was &asymp; 890-fold. Based on reciprocal crosses between susceptible (Sus) and Spin-res, the inheritance of spinosad resistance in S. frugiperda was autosomal incompletely recessive. Backcrosses between the F1 from reciprocal crosses and the parental Spin-res revealed a polygenic resistance, with an estimation of at least 1.86 to 2.45 genes related to spinosad resistance. Furthermore, it was observed a strong fitness cost associated to spinosad-resistance in Spin-res strain, based on the life table and fertility parameters. The characterization of the transcriptional profile and the differential gene expression comparison between susceptible and spinosad-resistant strains of Spodoptera frugiperda were obtained from the sequencing of cDNA libraries from fourth instar larvae of Sus and Spin-res strains (exposed or not to spinosad) using a HiScan1000&reg; platform (Illumina&copy;). The transcriptome was de novo assembled using nearly 19 million single-end reads with quality score over 30, yielding 42,406 transcripts with a N50 of 598 bp. Based on similarity search in the non-redundant (nr) nucleotide database, 24,980 (59%) transcripts were annotated. Most of the transcripts aligned to Bombyx mori L., Helicoverpa armigera (Hübner) and Spodoptera spp., with 22.5%, 3.81, and 3.6, respectively. We identified 2,032 differentially expressed transcripts (P <= 0.05, t-test; fold-change > 2) between the susceptible and spinosad-resistant strains. Among metabolic enzyme transcripts, 23 P450 monooxigenases, 13 glutathione S-transferases, one carboxylesterase and one esterase were up-regulated in the spinosad-resistant strain. In addition, it was observed 15 genes superexpressed in spinosad-resistant strain related to energy production, which can be related to the high fitness cost associated with resistance. Quantitative real-time PCR analysis showed that patterns of gene expression were consistent with RNA-seq results. Synergistic bioassays using PBO and DEM showed little involvement of P450s in spinosad-resistance and lack of involvement regarding the glutathione Stransferases. Furthermore, we sequenced and compared the subunit &alpha;6 from the nicotinic acetylcholine receptor of S. frugiperda Spin-res and Sus strains. Only one synonymous mutation within the two strains (G567A) was found, showing that the &alpha;6 is not the only subunit involved in S. frugiperda resistance to spinosad.

Einführung: Eine entscheidende Ursache für das Aufkommen der den modernen Menschen charakterisierenden kognitiven Funktionen ist in der beachtlichen Vergrößerung des menschlichen Neocortex innerhalb der letzten 5-7 Millionen Jahre zu finden. Die Identifizierung der dieser Entwicklung zu Grunde liegenden genomischen Veränderungen ist letztlich nicht nur bedeutsam für die Beantwortung der Frage, welche evolutionären Anpassungen den Menschen kennzeichnen, sondern auch für ein besseres Verständnis einer möglicherweise besonderen Anfälligkeit gegenüber neurologischen und psychiatrischen Erkrankungen. Kürzlich konnten 15 menschenspezifische Gene, deren Expression sich vorzugsweise in neuronalen Vorläuferzellen (NPCs) des menschlichen fetalen Neokortexes nachweisen lässt, identifiziert werden (Florio et al., 2018). Drei davon (FAM72B, C und D) sind vor 3,4 – 1 Millionen Jahren im menschlichen Genom durch Genduplikationen entstanden und gehören zur Family of sequence similarity 72 (FAM72). Zielsetzung und Ansätze: Konkret wurde betrachtet, ob FAM72D durch die spezifischen Substitutionen von Aminosäuren eine sich von der Funktion des anzestralen Gens FAM72A unterscheidende Rolle in der neokortikalen Entwicklung einnimmt. Untersucht wurden deshalb die Effekte von FAM72A und D auf die Proliferationskapazität und Genexpression von NPCs nach der ektopen Expression von FAM72A oder D während der embryonalen Entwicklung des Neocortex der Maus. Methoden: Die in utero Elektroporation (IUE) embryonaler Mäusegehirne erfolgte zur Expression eines rot oder grün fluoreszierenden Proteins (RFP oder GFP) entweder gemeinsam mit einem leeren DNA pCAGGS Vektor als Kontrollbedingung oder aber einem pCAGGS-FAM72A oder pCAGGS-FAM72D Plasmid. Die in der zweiten Ergebnissektion (Results II) präsentierten IUE wurden dabei im dorsolateralen Neokortex zum Höhepunkt der Neurogenese am 14. Entwicklungstag (E 14.5) durchgeführt, im Unterschied zu den Experimenten in der dritten Sektion (Results III), die im medialen Neokortex am 18. Entwicklungstag (E 18.5) während der Spätphase der embryonalen Neurogenese realisiert wurden. Die Proliferation der NPCs wurde durch Immunfluoreszenzanalysen zweier Marker (Ki67 und phosphoryliertes Histon 3) bestimmt. Zudem wurde die Häufigkeit wichtiger Subtypen von NPCs ebenfalls durch Immunfluoreszenzanalysen eines Markers für basale intermediäre Vorläuferzellen (bIPs → Tbr2) sowie für basale und apikale radiale Gliazellen (aRGs, bRGs → Sox2) ermittelt. Die Gliogenese wurde durch Olig2 Immunfluoreszenz quantifiziert. Weitere Experimente wurden durchgeführt, um die Fähigkeit der NPCs, den Zellzyklus nach der IUE von FAM72D erneut einzuleiten, zu untersuchen. Zu diesem Zweck wurde schwangeren Mäusen 24 h nach der IUE das Thymidin-Analogon 5-Ethinyl-2'-desoxyuridin (EdU) intraperitoneal injiziert. Damit wurden alle Zellen markiert, die sich zu diesem Zeitpunkt in der S-Phase des Zellzyklus befanden und damit den Zellzyklus nach der IUE fortsetzten. Nach weiteren 24 h (48 h post-IUE) erfolgte die Auswertung: alle Ki67- und EdU-doppelt positiven Zellen wurden als solche betrachtet, die den Zellzyklus nach IUE fortführten (EdU+) und nach weiteren 24 h noch immer proliferierten (Ki67+). Zur Durchführung der Transkriptomanalyse wurden Mäuse am 13. Entwicklungstag mit pCAGGS-GFP und entweder dem leeren DNA-Vektor (pCAGGS, Kontrolle) oder einem die Expression von FAM72A (pCAGGS-FAM72A) oder FAM72D (pCAGGS-FAM72D) ermöglichenden Vektor elektroporiert. Anschließend wurden die elektroporierten dorsolateralen neokortikalen Bereiche am 14. Entwicklungstag mikroskopisch seziert und in einzelne Zellen dissoziiert. Die Isolation der elektroporierten (GFP+) Zellen erfolgte aus den Einzelzellsuspensionen durch Fluoreszenz-aktivierte Zellsortierung (FACS). Im Anschluss wurden die isolierten Zellen für die RNA-Sequenzierung vorbereitet. Die primäre Datenanalyse der Ergebnisse der RNA-Sequenzierung wurde entsprechend etablierter Protokolle durchgeführt (Florio et al., 2015). Ergebnisse: Die Analyse der Immunfluoreszenzquanitfizierungen (Results II und III) ergab keine signifikanten Veränderungen der proliferativen Parameter oder der Häufigkeit der NPCs in der ventrikulären Zone (VZ) oder subventrikulären Zone (SVZ) des sich entwickelnden Mausneokortex nach der ektopen Expression von FAM72A oder FAM72D im Vergleich zur Kontrollbedingung. Die Transkriptomanalyse (Results IV) zeigte jedoch 88 signifikant hoch- und 52 herunterregulierte Gene in Folge der FAM72A sowie 91 signifikant hoch- und 67 herunterregulierte Gene nach der FAM72D Expression im Vergleich zur Kontrolle. Es wurde festgestellt, dass nur zwei dieser differentiell exprimierten Gene in Folge der ektopen Expression sowohl von FAM72A als auch FAM72D hochreguliert wurden und ein Expressionslevel > 1 fpkm aufwiesen: Syde1 und Shisa5. Darüber hinaus wurden sechs Gene mit > 1 fpkm identifiziert, die spezifisch nach der Expression von FAM72D hochreguliert waren: Tapbp, Mtfp1, Slitrk5, Parp9, Cnp, Rbm43. Darüber hinaus zeigte die Genontologie-Analyse (Gen Ontology) eine signifikante Anreicherung von Angiogenese-assoziierten Genen (z. B. Vegfc) im Datensatz der artifiziell FAM72A exprimierenden Zellen. Interessanterweise konnte beobachtet werden, dass unter den im Vergleich zur Kontrolle differentiell exprimierten Genen mehr Gene mit typischer Expression in NPCs in Folge von FAM72D als FAM72A Expression hochreguliert und mehr NPC typische Gene nach FAM72A Expression herunterreguliert wurden. Im Falle der Gene, deren Expression eher in Neuronen zu finden ist, zeigte sich ein entgegengesetztes Bild (Results IV). Diese Befunde lassen den vorsichtigen Schluss zu, dass FAM72D stärker als FAM72A die Aufrechterhaltung von NPC-Eigenschaften positiv beeinflussen kann. Schlussfolgerungen: In einer früheren Studie erhöhte der Knockdown von Fam72a in NPCs erwachsener Mäuse die Neurogenese (Benayoun et al., 2014). Dies legt in Verbindung mit den vorliegenden Ergebnissen nahe, dass FAM72A und FAM72D nicht hinreichend, möglicherweise jedoch notwendig sind, um die Aufrechterhaltung des Vorläuferzellcharakters von NPCs zu fördern (Results II, III). Aus diesem Grund sollte das in dieser Studie verfolgte Gain of Function Design durch einen Loss of Function Ansatz ergänzt werden. Als Modellsystem bieten sich hierfür insbesondere Hirnorganoide aus Stammzellen des Schimpansen oder Menschen an. Da alle der kürzlich identifizierten menschenspezifischen Gene in den gleichen NPCs exprimiert werden, sollte auch die potenzielle synergistische Wirkung auf die NPC-Proliferation der FAM72 und der zwölf anderen humanspezifischen Gene wie etwa ARHGAP11B analysiert werden. Neben anderen möglichen Mechanismen, die auf Grundlage der Genexpressionsanalyse im Diskussionsteil dieser Arbeit (Results IV und Discussion) erörtert wurden, könnte die Hochregulierung von Slitrk5 in Folge der ektopen Expression des humanspezifischen FAM72D besonders relevant sein. Es ist bekannt, dass Slitrk5 am Recycling des TrKB-Rezeptors beteiligt ist (Song et al., 2015), der wiederum grundlegende Aspekte der Gehirnentwicklung beeinflusst. Ebenfalls konnte bereits gezeigt werden, dass FAM72A die Funktion des TrKB Rezeptors hemmt (Nehar et al., 2009). Somit ist denkbar, dass FAM72D im menschlichen Neokortex die Wiederherstellung der TrKB-Rezeptorfunktion indirekt über Slitrk5 verbessert und dadurch wesentliche Parameter wie das Überleben von Vorläuferzellen und die Neurogenese beim Menschen verlängern oder verstärken könnte. Diese Studie stellt damit die erste funktionelle Charakterisierung der evolutionär hochinteressanten, die FAM72 Gene beinhaltende Region des menschlichen Genoms während der Entwicklung in utero dar. Daraus ergeben sich zahlreiche Ansatzpunkte für zukünftige Untersuchungen, die in ihrer Gesamtheit ein umfassendes Verständnis der Evolution des menschlichen Gehirns ermöglichen werden.:1 INTRODUCTION 11 1.1 WHAT MADE US HUMAN? 11 1.2 THE NEOCORTEX 12 1.2.1 Origin and structure 12 1.2.2 Neurogenesis in the developing neocortex 14 1.2.3 How to increase the neuronal output 18 1.3 EVOLUTION AND GENE DUPLICATION 19 1.3.1 Gene duplication and evolutionary novelty 19 1.3.2 Mechanisms of replication 21 1.3.3 Fates of duplicated genes 22 1.3.4 Which genes tend to duplicate? 24 1.3.5 Human adaptation and gene duplication 24 1.4 HUMAN-SPECIFIC SIGNATURES OF NEOCORTICAL EXPANSION 25 1.5 IDENTIFICATION OF HUMAN-SPECIFIC GENES EXPRESSED IN THE DEVELOPING NEOCORTEX.. 25 1.6 FAMILY WITH SEQUENCE SIMILARITY 72 (FAM72) 26 1.6.1 Evolutionary origin 26 1.6.2 Subcellular localization 27 1.6.3 Cell cycle regulation 28 1.6.4 NPC maintenance 28 2 AIMS & APPROACHES 30 3 RESULTS I 31 3.1 FROM GENES TO PROTEINS: 1 FAMILY – 4 PARALOGUES 31 3.2 FAM72 MRNA EXPRESSION LEVELS IN THE DEVELOPING MOUSE AND HUMAN NEOCORTEX 32 3.3 COMPUTATIONAL ANALYSES 34 3.3.1 Proportion of cysteines 34 3.3.2 Transmembrane domain 34 3.4 AMPLIFICATION, SUBCLONING AND MUTAGENESIS 36 3.4.1 Amplification from human cDNA 36 3.4.2 Verification of the pCAGGs vectors 36 4 RESULTS II 38 4.1 ECTOPIC EXPRESSION OF FAM72A AND FAM72D IN THE MOUSE DORSOLATERAL NEOCORTEX AT MID-NEUROGENESIS 38 4.2 NPC PROLIFERATION 39 4.2.1 Assessment of NPC proliferation using Ki67 immunofluorescence 39 4.2.2 Cell cycle reentry 41 4.2.3 Assessment of mitosis using PH3 immunofluorescence 43 4.2.4 Conclusion 45 4.3 NPC ABUNDANCE 46 4.3.1 Assessment of NPC abundance using Tbr2 and Sox2 immunofluorescence 46 4.3.2 Conclusion 49 5 RESULTS III 50 5.1 ECTOPIC EXPRESSION OF FAM72A and FAM72D IN THE MOUSE MEDIAL CORTEX AT LATE-NEUROGENESIS 50 5.2 NPC PROLIFERATION 51 5.2.1 Assessment of the NPC proliferation using Ki67 immunofluorescence 51 5.3 NPC ABUNDANCE 52 5.3.1 Assessment of NPC abundance using Tbr2 and Sox2 immunofluorescence 52 5.4 GLIOGENESIS 53 5.4.1 Assessment of gliogenesis using Olig2 immunofluorescence 53 5.5 CONCLUSION 54 6 RESULTS IV 55 6.1 DIFFERENCES IN GENE EXPRESSION UPON ANCESTRAL FAM72A AND HUMAN-SPECIFIC FAM72D EXPRESSION AT MID-NEUROGENESIS 55 6.1.1 Rationale and experimental setup 55 6.2 DIFFERENTIALLY EXPRESSED GENES UPON ECTOPIC FAM72A AND FAM72D EXPRESSION IN THE DEVELOPING MOUSE DORSOLATERAL NEOCORTEX 56 6.3 UPREGULATED GENES UPON THE ECTOPIC FAM72A OR FAM72D EXPRESSION 57 6.3.1 Upregulated genes upon the ectopic FAM72A and FAM72D expression 57 6.3.2 Upregulated genes upon the ectopic FAM72A or D expression – cut off: fpkm >1 58 6.3.3 Upregulated genes upon the ectopic FAM72A and D expression – cut off: fpkm >1 59 6.4 UPREGULATED GENES SPECIFICALLY UPON THE ECTOPIC FAM72D EXPRESSION – CUT OFF: FPKM >1 60 6.4.1 Tapbp (TAP binding protein, Tapasin) 60 6.4.2 Mtfp1 (mitochondrial fission protein 1, Mtp18) 61 6.4.3 Slitrk5 (Slit and Ntrk-like protein 5) 61 6.4.4 Parp9 (Poly(ADP-ribose) polymerase 9) 63 6.4.5 Cnp (2',3'-Cyclic-nucleotide 3'-phosphodiesterase) 63 6.4.6 Rbm43 (RNA binding motif protein 43) 65 6.5 DOWNREGULATED GENES UPON THE ECTOPIC FAM72A OR FAM72D EXPRESSION 66 6.5.1 Downregulated genes upon the ectopic FAM72A or D expression – cut off: fpkm >1 66 6.5.2 Downregulated genes upon ectopic FAM72A expression – cut off: fpkm >1 66 6.5.3 Downregulated genes upon ectopic FAM72D expression – cut off: fpkm >1 67 6.6 GENES PREVIOUSLY SHOWN TO BE DIFFERENTIALLY EXPRESSED UPON FORCED FAM72A EXPRESSION 69 6.6.1 Cell cycle regulators 69 6.6.2 Tumor suppressor genes 69 6.6.3 PROTEINS PREVIOUSLY OBSERVED TO INTERACT WITH FAM72A 70 6.7 EFFECT OF ECTOPIC FAM72A AND FAM72D EXPRESSION ON GENES IMPLICATED IN NEURAL LINEAGE FATE DECISION 70 6.7.1 Upregulated and NPC-enriched genes 71 6.7.2 Downregulated and NPC-enriched genes 73 6.7.3 Upregulated and neuron-enriched genes 74 6.7.4 Downregulated and neuron-enriched genes 75 6.8 GO ENRICHMENT ANALYSIS 75 6.9 CONCLUSION 75 7 DISCUSSION 78 7.1 WHAT MAKES US HUMAN? 78 7.2 IN UTERO ELECTROPORATION OF A HUMAN-SPECIFIC GENE IN THE DEVELOPING MOUSE NEOCORTEX 81 7.2.1 Opportunities and limitations of the approach 81 7.3 THE FAMILY OF SEQUENCE SIMILARITY 72 AND HUMAN UNIQUENESS 83 7.3.1 Cell cycle regulation and NPC maintenance 83 7.3.2 Cell death 84 7.3.3 Neurogenic period 85 7.3.4 TrkB signaling 85 7.3.5 Mitochondria 86 7.3.6 Angiogenesis 88 7.3.7 An evolutionary immunological adaptation in the brain? 89 7.3.8 FAM72 and SRGAP2 90 7.3.9 FAM72, Neanderthals, and lncRNAs 91 7.4 FUTURE DIRECTIONS 92 7.4.1 Loss of function 92 7.4.2 Gain of function 92 8 SUMMARY / ZUSAMMENFASSUNG 95 8.1 SUMMARY 95 8.2 ZUSAMMENFASSUNG 98 9 MATERIALS AND METHODS 101 9.1 CHART OF ALL EXPERIMENTS 101 9.2 COMPUTATIONAL ANALYSIS 101 9.2.1 Reference sequences and multiple sequence alignments 101 9.2.2 Transmembrane domain prediction 102 9.3 AMPLIFICATION, SUBCLONING, MUTAGENESIS 102 9.3.1 Amplification from human brain cDNA 102 9.3.2 Subcloning 103 9.2.3 Mutagenesis 103 9.4 PLASMID VERIFICATION 104 9.4.1 Transfection of Cos7 cells 104 9.4.2 Immunoblots 104 9.4.3 In situ hybridization (ISH) 105 9.5 MICE 105 9.6 IN UTERO ELECTROPORATION 105 9.7 FIXATION AND CRYOSECTIONS 106 9.8 IMMUNOFLUORESCENCE AND ANTIBODIES 106 9.9 EDU DETECTION 107 9.10 IMAGE ACQUISITION 108 9.11 STATISTICS 108 9.12 MICRODISSECTION AND SINGLE CELL SUSPENSION 108 9.13 FACS 109 9.14 RNA SEQUENCING 109 9.15 TRANSCRIPTOME ANALYSIS 110 10 REFERENCES 111 11 APPENDIX 145 11.1 CONFERENCE PRESENTATION 145 V. ACKNOWLEDGMENTS 146 / Introduction: The higher cognitive functions that characterize modern humans can be attributed to the cerebral neocortex and its remarkable expansion in size during the last 5 – 7 million years of human evolution. The identification of the underlying genomic changes will be not only of importance to better understand the unique complexity of the human brain, but also its susceptibility to neurological and psychiatric diseases. Recently, 15 human-specific genes preferentially expressed in neural progenitor cells (NPCs) of the human fetal neocortex were identified (Florio et al., 2018). Three of them, FAM72B, C and D belong to the Family of sequence similarity 72 (FAM72) and occurred in the human genome by gene duplication 3.4 – 1 mya. Aims & Approaches: Specifically, it was asked whether FAM72D plays a diverse role compared to the ancestral FAM72A (Results II, III, IV) due to the specific sets of amino acid substitutions it acquired (Results I). Effects of FAM72A and FAM72D on the proliferative capacity and gene expressions of embryonic mouse NPCs were analyzed upon ectopic expression either of FAM72A or FAM72D during embryonic mouse neocortical development. Methods: In utero electroporation (IUE) of embryonic mouse brains was performed to drive the expression of a red or green fluorescent protein (RFP or GFP) either plus empty DNA vector (pCAGGS; control), pCAGGS-FAM72A or pCAGGS-FAM72D plasmids in the dorsolateral neocortex at mid-neurogenesis (embryonic day 13.5, E13.5; Results II) or in the medial neocortex at late-neurogenesis (E15.5; Results III). NPC proliferation was evaluated by immunofluorescence of Ki67 (immunohistochemistry, IHC), a cell proliferation marker, and phosphorylated Histone H3 (PH3), a marker of cell mitosis. Moreover, the abundance of NPCs using immunofluorescence of basal intermediate progenitor (Tbr2) and apical and basal radial glia (Sox2) markers, and the gliogenesis by Olig2 immunofluorescence was analyzed. Additional experiments were carried out to study the capacity of NPCs to reenter the cell cycle upon IUE of FAM72D. To this end, pregnant mice were intraperitoneally injected with the thymidine analog 5-Ethynyl-2´-deoxyuridine (EdU) 24 h post-IUE, to label all cells undergoing S-phase of the cell cycle (i.e., all cells that reentered the cell cycle after IUE) in the developing mouse brains. Embryonic brains were collected 24 h after EdU injection and co-stained with Ki67. Ki67 and EdU double positive cells were considered as cells that reentered the cell cycle. To execute the transcriptome analysis E13.5 mice were electroporated with pCAGGS-GFP either plus an empty DNA vector (pCAGGS, control), a vector driving expression of FAM72A (pCAGGS-FAM72A) or FAM72D (pCAGGS-FAM72D). Subsequently, the electroporated dorsolateral neocortical areas were microdissected at E14.5 and dissociated into single cells. The electroporated (GFP+) cells were isolated from the single cell suspensions by the fluorescence-activated cell sorting (FACS). The isolated cells were processed for RNA sequencing. Data analysis was performed as previously reported (Florio et al., 2015). Results: By immunohistochemistry, no significant changes in any of the proliferative parameters or in the abundance of progenitors in the ventricular zone (VZ) and subventricular zone (SVZ) of the developing mouse neocortex upon ectopic expression of FAM72D compared to FAM72A and control samples were detected (Results II, III). However, the transcriptome analysis (Results IV) showed 88 significantly up- and 52 down-regulated genes upon FAM72A and 91 significantly up- and 67 downregulated genes upon FAM72D expression compared to the control. Only two of these differentially expressed genes were found to be upregulated upon FAM72A and FAM72D with an expression >1 fpkm: Syde1 and Shisa5. Besides, six genes specifically upregulated upon ectopic expression of FAM72D exhibiting fpkm > 1 were identified and characterized using the existing literature: Tapbp, Mtfp1, Slitrk5, Parp9, Cnp, Rbm43. Beyond that, gene ontology analysis showed significant enrichment of angiogenesis-related genes (e.g., Vegfc) upon FAM72A expression. Interestingly, there were more genes found to be enriched in NPCs that were upregulated compared to control upon FAM72D than FAM72A expression, but more NPC enriched genes downregulated upon FAM72A compared to FAM72D expression. In the case of differentially expressed neuron-enriched genes, the data was were inverse, which slightly supports the idea that FAM72D rather than FAM72A could positively affect the maintenance of NPC characteristics. Conclusions: In a previous study knockdown of Fam72a in adult mouse NPCs increased neurogenesis (Benayoun et al., 2014). This suggests, in conjunction with the present results, that FAM72A and FAM72D are not sufficient, but may be required, to promote NPC maintenance (Results II, III). This is why the gain of function experiments conducted in this study should be complemented by a loss of function approach in the developing mouse neocortex, in chimpanzee or human-derived brain organoids. Because of their expression in the NPCs of the developing human neocortex, it might be productive to analyze the potential synergistic effect on NPC proliferation of the FAM72s and the 12 other human-specific genes such as ARHGAP11B. Among other mechanisms discussed based on the gene expression analysis in this thesis (Results IV and Discussion), the upregulation of Slitrk5 upon ectopic expression of the human-specific FAM72D could be particularly remarkable. Slitrk5 is known to be involved in the recycling of the TrKB receptor (Song et al., 2015), which affects fundamental aspects of brain development. While FAM72A was found to inhibit the TrKB receptor (Nehar et al., 2009), the occurrence of FAM72D could indirectly rescue the TrKB receptor function via Slitrk5 and thereby prolonging or enhancing essential features such as precursor cell survival and neurogenesis in humans. Therefore, this study provides the first functional characterization of the evolutionary highly interesting region in the human genome comprising the FAM72 genes during embryonic neocortical development in vivo and offers numerous starting points for further investigations, that will collectively facilitate a comprehensive understanding of the genomic adaptations underlying the astonishing evolution of the human brain.:1 INTRODUCTION 11 1.1 WHAT MADE US HUMAN? 11 1.2 THE NEOCORTEX 12 1.2.1 Origin and structure 12 1.2.2 Neurogenesis in the developing neocortex 14 1.2.3 How to increase the neuronal output 18 1.3 EVOLUTION AND GENE DUPLICATION 19 1.3.1 Gene duplication and evolutionary novelty 19 1.3.2 Mechanisms of replication 21 1.3.3 Fates of duplicated genes 22 1.3.4 Which genes tend to duplicate? 24 1.3.5 Human adaptation and gene duplication 24 1.4 HUMAN-SPECIFIC SIGNATURES OF NEOCORTICAL EXPANSION 25 1.5 IDENTIFICATION OF HUMAN-SPECIFIC GENES EXPRESSED IN THE DEVELOPING NEOCORTEX.. 25 1.6 FAMILY WITH SEQUENCE SIMILARITY 72 (FAM72) 26 1.6.1 Evolutionary origin 26 1.6.2 Subcellular localization 27 1.6.3 Cell cycle regulation 28 1.6.4 NPC maintenance 28 2 AIMS & APPROACHES 30 3 RESULTS I 31 3.1 FROM GENES TO PROTEINS: 1 FAMILY – 4 PARALOGUES 31 3.2 FAM72 MRNA EXPRESSION LEVELS IN THE DEVELOPING MOUSE AND HUMAN NEOCORTEX 32 3.3 COMPUTATIONAL ANALYSES 34 3.3.1 Proportion of cysteines 34 3.3.2 Transmembrane domain 34 3.4 AMPLIFICATION, SUBCLONING AND MUTAGENESIS 36 3.4.1 Amplification from human cDNA 36 3.4.2 Verification of the pCAGGs vectors 36 4 RESULTS II 38 4.1 ECTOPIC EXPRESSION OF FAM72A AND FAM72D IN THE MOUSE DORSOLATERAL NEOCORTEX AT MID-NEUROGENESIS 38 4.2 NPC PROLIFERATION 39 4.2.1 Assessment of NPC proliferation using Ki67 immunofluorescence 39 4.2.2 Cell cycle reentry 41 4.2.3 Assessment of mitosis using PH3 immunofluorescence 43 4.2.4 Conclusion 45 4.3 NPC ABUNDANCE 46 4.3.1 Assessment of NPC abundance using Tbr2 and Sox2 immunofluorescence 46 4.3.2 Conclusion 49 5 RESULTS III 50 5.1 ECTOPIC EXPRESSION OF FAM72A and FAM72D IN THE MOUSE MEDIAL CORTEX AT LATE-NEUROGENESIS 50 5.2 NPC PROLIFERATION 51 5.2.1 Assessment of the NPC proliferation using Ki67 immunofluorescence 51 5.3 NPC ABUNDANCE 52 5.3.1 Assessment of NPC abundance using Tbr2 and Sox2 immunofluorescence 52 5.4 GLIOGENESIS 53 5.4.1 Assessment of gliogenesis using Olig2 immunofluorescence 53 5.5 CONCLUSION 54 6 RESULTS IV 55 6.1 DIFFERENCES IN GENE EXPRESSION UPON ANCESTRAL FAM72A AND HUMAN-SPECIFIC FAM72D EXPRESSION AT MID-NEUROGENESIS 55 6.1.1 Rationale and experimental setup 55 6.2 DIFFERENTIALLY EXPRESSED GENES UPON ECTOPIC FAM72A AND FAM72D EXPRESSION IN THE DEVELOPING MOUSE DORSOLATERAL NEOCORTEX 56 6.3 UPREGULATED GENES UPON THE ECTOPIC FAM72A OR FAM72D EXPRESSION 57 6.3.1 Upregulated genes upon the ectopic FAM72A and FAM72D expression 57 6.3.2 Upregulated genes upon the ectopic FAM72A or D expression – cut off: fpkm >1 58 6.3.3 Upregulated genes upon the ectopic FAM72A and D expression – cut off: fpkm >1 59 6.4 UPREGULATED GENES SPECIFICALLY UPON THE ECTOPIC FAM72D EXPRESSION – CUT OFF: FPKM >1 60 6.4.1 Tapbp (TAP binding protein, Tapasin) 60 6.4.2 Mtfp1 (mitochondrial fission protein 1, Mtp18) 61 6.4.3 Slitrk5 (Slit and Ntrk-like protein 5) 61 6.4.4 Parp9 (Poly(ADP-ribose) polymerase 9) 63 6.4.5 Cnp (2',3'-Cyclic-nucleotide 3'-phosphodiesterase) 63 6.4.6 Rbm43 (RNA binding motif protein 43) 65 6.5 DOWNREGULATED GENES UPON THE ECTOPIC FAM72A OR FAM72D EXPRESSION 66 6.5.1 Downregulated genes upon the ectopic FAM72A or D expression – cut off: fpkm >1 66 6.5.2 Downregulated genes upon ectopic FAM72A expression – cut off: fpkm >1 66 6.5.3 Downregulated genes upon ectopic FAM72D expression – cut off: fpkm >1 67 6.6 GENES PREVIOUSLY SHOWN TO BE DIFFERENTIALLY EXPRESSED UPON FORCED FAM72A EXPRESSION 69 6.6.1 Cell cycle regulators 69 6.6.2 Tumor suppressor genes 69 6.6.3 PROTEINS PREVIOUSLY OBSERVED TO INTERACT WITH FAM72A 70 6.7 EFFECT OF ECTOPIC FAM72A AND FAM72D EXPRESSION ON GENES IMPLICATED IN NEURAL LINEAGE FATE DECISION 70 6.7.1 Upregulated and NPC-enriched genes 71 6.7.2 Downregulated and NPC-enriched genes 73 6.7.3 Upregulated and neuron-enriched genes 74 6.7.4 Downregulated and neuron-enriched genes 75 6.8 GO ENRICHMENT ANALYSIS 75 6.9 CONCLUSION 75 7 DISCUSSION 78 7.1 WHAT MAKES US HUMAN? 78 7.2 IN UTERO ELECTROPORATION OF A HUMAN-SPECIFIC GENE IN THE DEVELOPING MOUSE NEOCORTEX 81 7.2.1 Opportunities and limitations of the approach 81 7.3 THE FAMILY OF SEQUENCE SIMILARITY 72 AND HUMAN UNIQUENESS 83 7.3.1 Cell cycle regulation and NPC maintenance 83 7.3.2 Cell death 84 7.3.3 Neurogenic period 85 7.3.4 TrkB signaling 85 7.3.5 Mitochondria 86 7.3.6 Angiogenesis 88 7.3.7 An evolutionary immunological adaptation in the brain? 89 7.3.8 FAM72 and SRGAP2 90 7.3.9 FAM72, Neanderthals, and lncRNAs 91 7.4 FUTURE DIRECTIONS 92 7.4.1 Loss of function 92 7.4.2 Gain of function 92 8 SUMMARY / ZUSAMMENFASSUNG 95 8.1 SUMMARY 95 8.2 ZUSAMMENFASSUNG 98 9 MATERIALS AND METHODS 101 9.1 CHART OF ALL EXPERIMENTS 101 9.2 COMPUTATIONAL ANALYSIS 101 9.2.1 Reference sequences and multiple sequence alignments 101 9.2.2 Transmembrane domain prediction 102 9.3 AMPLIFICATION, SUBCLONING, MUTAGENESIS 102 9.3.1 Amplification from human brain cDNA 102 9.3.2 Subcloning 103 9.2.3 Mutagenesis 103 9.4 PLASMID VERIFICATION 104 9.4.1 Transfection of Cos7 cells 104 9.4.2 Immunoblots 104 9.4.3 In situ hybridization (ISH) 105 9.5 MICE 105 9.6 IN UTERO ELECTROPORATION 105 9.7 FIXATION AND CRYOSECTIONS 106 9.8 IMMUNOFLUORESCENCE AND ANTIBODIES 106 9.9 EDU DETECTION 107 9.10 IMAGE ACQUISITION 108 9.11 STATISTICS 108 9.12 MICRODISSECTION AND SINGLE CELL SUSPENSION 108 9.13 FACS 109 9.14 RNA SEQUENCING 109 9.15 TRANSCRIPTOME ANALYSIS 110 10 REFERENCES 111 11 APPENDIX 145 11.1 CONFERENCE PRESENTATION 145 V. ACKNOWLEDGMENTS 146