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Sox2 plays a crucial role in the biology of neural stem cells (NSC) by regulating differentiation and keeping NSC in a slowly proliferative state which allows to maintain over time a pool of stem cells ready to differentiate in more rapidly dividing progenitor cells. Sox2 exerts these functions by transcriptionally activating and repressing target genes. At the present time, it is still not clear how these 2 opposite functions are regulated. Here, I analyse whether SUMOylation of Sox2 plays a role in the regulation of Sox2 activity and, ultimately, in the regulation of NSC biology. Several mutant forms of Sox2 were designed and cloned, including a non-SUMOylatable mutant construct, where the SUMO acceptor lysine was mutated to arginine (Sox2_K247R), and a SUMO-fusion construct where SUMO2 was fused at the carboxyl terminal of Sox2_K247R to mimic constitutive SUMOylation (Sox2_K247R-SUMO2). Comparison between the activity of these constructs and the activity of wt Sox2 revealed that over-expression of Sox2_K247R-SUMO2 causes a reduction in the protein transcriptional activation ability in vitro. When analysed in vivo, constitutively SUMOylated Sox2 repressed a set of target genes which were also repressed by over-expressed wt Sox2. These genes were enriched for cell-cycle regulators. Conversely, few genes were differentially regulated by over-expression of wt Sox and over-expression of Sox2_K247R and the majority of these few genes was up-regulated, indicating a potential loss-of-function in Sox2 ability to transcriptionally repress some of its target genes in absence of SUMOylation. Moreover, hNSC over-expressing Sox2_K247R lost the ability of proliferating slowly, while hNSC over-expressing SUMOylated Sox2 proliferated at a slower rate, similar to hNSC over-expressing wt Sox2. This evidence further promotes the hypothesis that SUMOylation of Sox2 is involved in the regulation of neural stem cells proliferation through the repression of target proliferation genes. Biochemical analysis of Sox2 SUMOylation was also performed and confirmed that, as reported in literature, SUMOylation of Sox2 is challenging to detect, supposedly because a very small portion of Sox2 is SUMOylated at any given time in the cell as reported for other SUMO targets. Taken together, the evidences reported in this study suggest a model according to which SUMOylation enhances Sox2 transcriptional repression activity, leading to the repression of proliferation genes and therefore to a slow-cycling state of neural stem cells.

Human γ-herpesviruses 4 and 8 (also known as EBV and KSHV) are oncogenic viruses known primarily for their associations with Hodgkin Lymphoma and Kaposi Sarcoma respectively. Upon infection of target cells, γ-herpesviruses establish either a latent or a lytic viral cycle. Latency is characterized by expression of just a few viral oncogenes and microRNAs, whereas lytic replication leads to expression of virtually all of the virus encoded genes and release of infectious particles, leading to apoptosis of the host cell. The molecular mechanisms regulating the latent-to-lytic switch are poorly characterized, and a deeper understanding of it could lead to identification of specific targets for therapeutic intervention. Here I describe two novel molecular mechanisms leading to viral lytic replication. Firstly, I describe the requirement for viral microRNA miR-K10 for maintenance of steady state latency. I found that miR-K10 targets the transcription factor β-catenin, which can be triggered by stimulation of the Wnt pathway. Induction of β-cateninmediated transcription results in viral replication and virally-induced apoptosis in both EBV and KSHV. Because β-catenin activity is also induced by growth factors, we speculate that γ-herpesviruses have evolved to spread to neighbouring cells when the extracellular environment is rich in growth stimuli. I secondly describe the interplay of γ-herpesviruses with senescence, showing that senescent cells can be infected by KSHV, which there establishes a lytic but not a latent program. One of the major characteristics of cellular senescence is loss of telomerase activity. I show that inhibition of telomerase activity in non-senescent cells leads to viral replication of both KSHV and EBV. Thus, γ-herpesviruses do not establish latency in senescent cells, which would not perpetrate the viral genome. Given that both of these mechanisms can be pharmacologically targeted, this work suggests new avenues for the design of novel therapeutic strategies to treat γ-herpesvirus-associated malignancies.

Transition metal-catalysed cross coupling chemistry is a valuable tool for synthetic organic chemistry, enabling the preparation of compounds of great interest. The catalytic metal of choice is usually palladium, which generally offer better performances in term of catalytic activity and easy handling. On the other hand, the use of nickel in this class of reactions is gaining attention, as it would provide more economically and environmentally sustainable processes. Deprotonative cross couplings are a subgroup of these reactions, in which the nucleophile is generated in situ by direct deprotonation of a (relatively) acidic C–H bond, for example those of an enolizable ketone or an imine. The reaction products often represent intermediates towards more complex molecular architectures, by virtue of the well-known carbonyl chemistry. The development of a Pd-catalysed methodology for the prototypical deprotonative coupling, the a-arylation of ketones, is reported in this thesis. It requires significantly lower catalyst loadings compared to previous reports, and displays good tolerance towards functionalised substrates. A related protocol for the intramolecular a-arylation of imines towards indoles was subsequently disclosed: as it requires low catalyst loadings and displays good scalability and simple setup, this methodology is a promising hit for industrial applications. The parallel development of nickel-catalysed protocols afforded an efficient method for the a-arylation of ketones, using chloroarenes as electrophile for the first time in the literature. The method was further optimised for the synthesis of an intermediate towards a commercial medicinally active compound. Building up on these findings, the first nickel-catalysed protocol for the deprotonative arylation of benzylaminederived imines was also developed. Last, the first aqueous palladium-catalysed protocol for the a-arylation of ketones was investigated. The method proved flexible, showing excellent functional group tolerance: compounds containing base-sensitive functional groups, halogenated small-molecule drugs, and Boc-protected amino acids were all suitable substrates.

Brazil is the world's sixth largest cocoa producer with 270,000 tonnes of cocoa produced per year. In a world with an increasing demand for chocolate, but with agriculture threatened by climate change, the chocolate industry is worried about a possible shortage of cocoa. Furthermore, growing cocoa is a main cause of deforestation. However, in the state of Bahia, Northeast Brazil, cocoa is grown in traditional agroforests called 'cabrucas' which maintain a forest cover. Cocoa, an understorey crop, is planted under the shade of native Atlantic Forest trees and exotic fruit trees introduced by the farmers. These cabrucas have high conservation value but very low cocoa yield. In my thesis I investigate the factors limiting cocoa yield and how to increase yield in cabrucas. I explore the role of shade trees and the nutrient dynamics in litterfall. Finally, I explore the risk that climate change could represent for cocoa production in the future by looking at the effects of an unexpected drought caused by an El Niño Southern Oscillation (ENSO) event between November 2015 and May 2016. My study is based on data collected in permanent transects in 32 randomly chosen cabruca farms in Barro Preto a municipality of Bahia over a period of three years. I also established littertraps in 10 cocoa farms where I intensively studied nutrient dynamics and cocoa yield per tree over 12 months. My results showed that unproductive trees, low cocoa tree density, high shade cover and high cocoa mortality due to drought were the main factors limiting cocoa yield in cabrucas. Surprisingly, adding fertilisers to the cocoa trees did not increase yield. This suggests that there is no nutrient deficiency in cabrucas. In the farms, I found 69 species of shade trees for an average density of 125 ± 32 trees per hectare. Half of the species of shade trees were Atlantic Forest species of conservation value. The litterfall experiment showed the shade tree species and the quantity of litterfall produced, can affect the number of cocoa pods per tree. In cabrucas, a higher number of cocoa pods was found on cocoa trees under shade trees than under no shade. Finally, I showed that the exceptionally severe ENSO-related drought caused 80% loss in yield and 11% cocoa tree mortality in Barro Preto. Climate models predict an increased frequency of strong ENSO events in the future. Farmers in Bahia are not prepared to face regular drought events. The 2015/16 drought affected the dynamics of cocoa production in Brazil: it accelerated the decrease of extensive wildlife-friendly cocoa production in Bahia whereas it increased the development of cocoa production in intensive low shade plantations in the state of Pará. This suggests that climate change could be a threat to traditional cocoa agroforests in Bahia. Developing wildlife-friendly certification schemes and Payment for Ecosystem Services to internalise the value of forest conservation and to encourage farmers to maintain their shade trees could save cabruca systems from going extinct.

Memory T cell recruitment to the non-lymphoid tissue of the intestine (the gut lamina propria) requires the interactions between integrin a4B7 and its ligand Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1), which is exclusively expressed in the gut microvasculature. T cell homing to small intestine also requires the expression of chemokine receptor CCR9 on T cells, whose ligand - the gut chemokine CCL25 - is abundantly expressed in the small intestine. The a4B7 integrin and chemokine receptor CCR9 are known as the "homing" molecules whose expression is "imprinted" on T cells by intestinal dendritic cells during antigen presentation. In the first part of this thesis, data from in vitro studies showed that other than retinoic acid, soluble tissue-derived factors such as the gut chemokine CCL25 and T cell growth factor IL-2 also contributed to the induction of gut-homing receptor acquisition. Moreover, the expression of a4B7 integrin and CCR9 appeared to be modulated differently. The present study suggests that under the influence of tissue-derived factors, T cell acquisition of a4B7 integrin is the "default" response at steady state, and that only fully activated T cells are able to express high levels of CCR9. In the second part of this thesis, T cell gut homing was investigated in the context of disease, i.e. inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC). IBD is characterized by excessive lymphocyte infiltration to the intestine. By quantitatively analyzing peripheral blood and intestinal lymphocyte subsets, proportional changes in the circulating gut-homing T cells (a4B7+) was found to be associated with CD. In addition, my data suggest that downregulation of T cell receptor zeta chain (TcRC) correlates to disease severity in both CD and UC. Measuring TcRC expression may thus provide an additional tool for the monitoring and management of IBD.

T cell migration to sites of inflammation is an essential step of an effective immune response. T cell recruitment is enhanced by the recognition of their cognate antigen presented by the endothelium and their retention in the tissue is controlled by resident antigen presenting cells. Vav1 is a guanidine exchange factor for RhoGTPases that is activated through TCR signalling and is important for the cytoskeletal re-arrangements occurring during T cell activation and migration. In this study we have investigated the role of Vav1 in the recruitment and retention of antigen-specific T cells to sites of inflammation. HY-specific Ab-restricted WT and Vav1-/- T cells were generated and fully characterised for specificity and expression of migration-related markers. Vav1-/- T cells showed defects in adhesion, migration in response to ICAM-1, CXCL12 and CXCL10 and increased migratory speed in in vitro assays. Despite displaying defective motility in vitro, both constitutive migration and recruitment of Vav1-/- T cells to antigenic sites occurred normally. However, retention of Vav-1-/- T cells into antigenic tissue was profoundly impaired. This may be due to the inability by Vav-1-/- T cells to respond to ‘stop’ signals delivered by co-engagement of TCR and CD28. In contrast to recent reports on wild type T cells, Vav1-/- T cell migration and retention to sites of inflammation was not affected by engagement of the co-stimulatory molecule CD28, suggesting that Vav-1 recruitment during CD28-mediated signalling is instrumental for Vav-1-mediated regulation of T cell traffic