Le neuroblastome (NB) est une tumeur pédiatrique du système nerveux sympathique. Des mutations activatrices du gène ALK (Anaplastic Lymphoma Kinase) ont été identifiées dans 8 % des formes sporadiques et dans des formes familiales de NB. Le gène ALK code pour un récepteur tyrosine kinase appartenant à la famille des récepteurs à l’insuline, principalement exprimé dans le système nerveux central et périphérique. Le récepteur ALK représente une cible thérapeutique pertinente dans ce cancer. Des mutations de novo du gène ALK ont également été rapportées dans une forme syndromique associant NB congénital et encéphalopathie sévère avec dysmorphie du tronc cérébral, suggérant un rôle développemental du gène ALK en plus de son implication dans l’oncogenèse.Dans ce contexte, mon projet de thèse avait pour but de déterminer le rôle du récepteur ALK muté dans l’oncogenèse du NB et le développement, principalement à l’aide de modèles murins originaux obtenus au laboratoire. J’ai ainsi largement caractérisé deux lignées de souris KI (Knock-In) Alk pour les deux mutations les plus fréquemment observées dans le NB: F1174L et R1275Q chez l’homme, correspondant à F1178L et R1279Q chez la souris.Une analyse détaillée de ces deux lignées de souris n’a pas révélé de phénotype majeur chez les souris KI AlkR1279Q hétérozygotes et homozygotes ainsi que chez les hétérozygotes KI AlkF1178L. Par contre, nous avons documenté une forte létalité post-natale des animaux KI AlkF1178L homozygotes et montré que ces nouveaux-nés présentent des troubles majeurs d’alimentation. Les homozygotes KI AlkF1178L phénocopient donc partiellement les patients encéphalopathes. La différence d’effet observé entre les animaux hétérozygotes et homozygotes suggère fortement qu’il existe un seuil d’activation du récepteur Alk compatible avec la survie.Nous avons ensuite exploré le rôle du récepteur ALK muté dans le système nerveux sympathique des souris KI Alkmut. Cette analyse a montré que l’activation du récepteur induit un excès de prolifération des neurones sympathiques de E14.5 à la naissance. Néanmoins, nous n’avons pas observé de NB chez ces animaux. En croisant ces souris avec la lignée TH-MYCN, nous avons documenté une coopération des mutations Alk avec l’oncogène MYCN pour le développement de NB. La comparaison des profils transcriptomiques des tumeurs murines MYCN et MYCN/Alkmut a révélé que l’expression de l’oncogène Ret (codant également un récepteur à activité tyrosine kinase) était fortement induite par l’activation du récepteur Alk. Le traitement des souris par un inhibiteur de l’activité kinase du récepteur Ret a montré une diminution de la taille des tumeurs suggérant que le gène Ret joue un rôle majeur dans l’oncogenèse induite par le récepteur Alk muté. Par ailleurs, l’induction de l’expression du gène RET par le récepteur ALK muté dans les NB a été confirmée dans des lignées et des tumeurs humaines.Afin de déterminer le mécanisme par lequel l’activation du récepteur ALK aboutit à la régulation de l’expression du gène RET des expériences ont été effectuées sur des lignées humaines de NB dans lesquelles le récepteur ALK peut être activé ou inactivé. Ce travail a montré que l’expression du gène RET est dépendante de l’axe ALK-ERK-ETV5. En effet, la modulation de l’activité du récepteur ALK affecte l’expression des gènes ETV5 et RET. Cet effet est dépendant de l’activation de la voie MEK/ERK. Par ailleurs, ETV5 active l’expression du gène RET. Afin de confirmer le rôle de Ret dans l’oncogenèse dépendante du récepteur Alk, nous avons croisé des souris portant une mutation activatrice de Ret avec les souris TH-MYCN. Nous avons ainsi mis en évidence que le récepteur Ret activé coopère avec l’oncogène MYCN dans le développement de tumeurs et que ces tumeurs sont des NB présentant des caractéristiques très semblables à celles des tumeurs MYCN/Alkmut. Le gène Ret apparaît donc comme une cible essentielle du récepteur Alk muté dans l’oncogenèse du NB. / Neuroblastoma (NB) is a pediatric tumor arising from the sympathetic nervous system. Activating mutations of the ALK gene have been observed in around 8 % of sporadic neuroblastoma as well as in familial cases. The ALK gene encodes a tyrosine kinase receptor of the insulin receptor super-family. It is mainly expressed in the central and peripheral nervous system. The ALK receptor represents a therapeutic target in this cancer. De novo ALK mutations have also been reported in a syndrome associating congenital NB and severe encephalopathy with abnormal shape of the brainstem, suggesting a developmental role for the ALK gene in addition to its implication in oncogenesis.In this context, my PhD project was to determine the role of the mutated ALK receptor in NB oncogenesis and in development, mainly with original mouse models obtained in the laboratory. I extensively characterized two knock-in (KI) Alk mouse lines with the two mutations that are most frequently observed in NB: F1174L and R1275Q in human and F1178L and R1279Q in mouse.A detailed analysis of these two mouse lines showed that the KI AlkR179Q heterozygous and homozygous mice as well as the KI AlkF1178L heterozygous mice do not show striking clinical signs. On the contrary, we documented a high postnatal lethality for KI AlkF1178L homozygous mice and showed that these pups presented with a dramatic reduced milk intake. Thus, the KI AlkF1178L homozygous mice partially phenocopy the human patients with encephalopathy. The difference of phenotype between the heterozygous and the homozygous KI AlkF1178L mice highly suggest a threshold of activity of the Alk receptor compatible with survival.We then explored the role of the mutated ALK receptor in the sympathetic nervous system of the KI Alkmut mice. This analysis showed that the activation of the receptor induces an excess of proliferation in sympathetic neurons from E14.5 to birth. However, we could not observe NB in these animals. We next bread these mice with the transgenic TH-MYCN line. We documented cooperation between Alk mutations and the MYCN oncogene to induce NB. Comparison of transcriptomic profiles of MYCN vs MYCN/Alkmut tumors revealed that the expression of the Ret oncogene (encoding a tyrosine kinase receptor) was strongly induced by the activation of the Alk receptor. Besides, the induction of the expression of the RET gene by the mutated ALK receptor in NB was confirmed in human cell lines and tumors.In order to determine the mechanism by which the activation of the ALK receptor regulates RET gene expression, experiments were done on human NB cell lines in which the ALK receptor can be activated or inactivated. This work showed that RET gene expression is dependent of the ALK-ERK-ETV5 axis. Indeed, the modulation of the ALK receptor activity affects gene expression of ETV5 and RET. This effect is dependent of the activation of the MEK/ERK pathway. Besides, ETV5 increases RET gene expression. In order to confirm the role of the Ret receptor in oncogenesis driven by the mutated Alk receptor, we bread mice bearing an activating mutation of the Ret gene with the TH-MYCN mice. We showed that the activated Ret receptor cooperates with the MYCN oncogene in tumor formation and that these tumors are NB presenting with characteristics very close to MYCN/Alkmut tumors. Thus, the Ret gene appears to be an essential target of the mutated Alk receptor in NB oncogenesis.

The Neuregulin 1 (NRG1)-ErbB4 signalling pathway is implicated in critical processes for the development and function of neuronal circuits. Post mortem studies have reported that elevated expression of NRG1 type 1 isoform is associated with schizophrenia. Importantly previous behavioural studies in mice that overexpress the NRG1 type 1 isoform (NRG1^tg-type-I) have suggested a schizophrenia endophenotype including impairment in the hippocampus-dependent spatial working memory, prepulse inhibition (PPI) of the startle reflex and alterations in the gamma band rhythmogenesis This study aims to reveal the cellular targets of the NRG1-ErbB4 signalling pathway and putative alterations in the function of the hippocampal network in NRG1^tg-type-I mice. Immunocytochemical analysis showed that the NRG1 receptor ErbB4 is predominantly localized in interneurons comprising parvalbumin positive (PV) and cholecystokinin (CCK) expressing cells. Comparison of the density of ErbB4-positive cells between the hippocampus of wild type (WT) and NRG1^tg-type-I mice suggested that NRG1 over-expression resulted in decreased number of ErbB4 immunopositive hippocampal interneurons. This is consistent with the proposed role of the NRG1-ErbB4 signalling in the migration of GABAergic cells during neurodevelopment and with the NRG1-mediated internalisation of the ErbB4 receptors. CCK- positive cells are a major target of NRG1-ErbB4 signalling, and therefore the NMDA receptor and AMPA receptor components of glutamatergic transmission were analysed in this population of cells by performing whole cell recordings of evoked and miniature excitatory post synaptic currents. Glutamatergic neurotransmission in CCK-positive cells was found to be compromised in the hippocampus of NRG1^tg-type-I mice. This change was attributed to hypofunction of NMDA receptors but not AMPA receptors post-synaptically. Next, the inhibitory output of CCK-positive cells to pyramidal cells was examined. Analysis of the optogenetically elicited inhibitory post synaptic currents (IPSCs) did not reveal any changes in the properties of the GABAergic synapse formed by these cells due to NRG1 over-expression Finally, the effects of this NMDA receptor hypofunction in the recurrent inhibition were analysed by performing whole cell recordings during the gamma relevant optogenetic entrainment of the hippocampal network. It was found that the disynaptic inhibition, a key synaptic interaction for the generation of gamma oscillations, depends on the NMDA receptors and was altered in the hippocampus of NRG1^tg-type-I mice. Together these data point out a key modulatory role of the NRG1-ErbB4 signalling in the neurodevelopment of cortical microcircuits and a link between ErbB4 and NMDA receptor function with a possible association to schizophrenia pathogenesis.

Glucocorticoids, key hormonal regulators of the stress response, powerfully influence inflammation and metabolism. Reducing excessive glucocorticoid exposure is beneficial in treating metabolic and cognitive disorders, but manipulating systemic endogenous glucocorticoids risks compromising their beneficial effects. The enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activates glucocorticoids in target tissues and thus inhibition of this enzyme presents a clinical opportunity to reduce tissue-specific glucocorticoid action. Active glucocorticoids also exert potent angiostatic effects by binding the glucocorticoid receptor (GR), and 11β-HSD1 inhibitors have proven beneficial in models of myocardial infarction by promoting angiogenesis. The possibility that 11β-HSD1 inhibitors may increase pathological angiogenesis, such as that seen in solid tumours, remains unaddressed. This project tested the hypothesis that 11β-HSD1 inhibition promotes tumour growth as a result of increased angiogenesis, using murine models of squamous cell carcinoma (SCC) and pancreatic ductal adenocarcinoma (PDAC). Murine SCC or PDAC cells were injected (1x106 cells/flank) into WT female mice fed either standard diet, or diet containing the 11β-HSD1 inhibitor UE2316 (175 mg/kg, N=6/group), or into 11β-HSD1 knockout (Del1) mice fed standard diet. Developing tumours were measured by callipers over several weeks, before animals were culled and tissues collected. SCC tumours grew more rapidly in UE2316-treated mice to reach a significantly (P < 0.01) larger final volume (0.158 ± 0.037 cm3) than in control mice (0.051 ± 0.007 cm3). PDA tumours were unaffected by 11β-HSD1 inhibition or deletion. Immunofluorescent co-staining of tumour sections for CD31/α-smooth muscle actin revealed no differences in vessel density, and RT-qPCR showed no difference in angiogenic factor expression, after 11β-HSD1 inhibition/deletion in either tumour type. GR and 11β-HSD1 RNA expression were greater in SCC vs PDAC tumours (P < 0.001), as was 11β-HSD1 activity (P < 0.0001). In studies using the aortic ring assay of ex vivo angiogenesis, 11β-HSD1 deletion, but not inhibition with UE2316, was shown to prevent glucocorticoid-mediated angiostasis. The growth/viability of tumour cell lines was not affected by UE2316 or corticosterone, as assessed by live cell imaging using the Incucyte imaging system. RNA-sequencing of SCC tumours revealed that multiple factors involved in the innate immune/inflammatory response were reduced in UE2316-treated tumours, and that extracellular matrix regulation was also altered by UE2316. Imaging of tumour sections using Second Harmonic Generation microscopy confirmed that UE2316 altered Type I collagen deposition in SCC (P < 0.001) but not PDAC. 11β-HSD1 inhibition can increase tumour growth, possibly via suppression of inflammatory/immune cell signalling and alteration of the extracellular matrix, and tumours with higher GR and 11β-HSD1 content, such as SCC, may be more at risk. Interestingly this investigation found no evidence of increased angiogenesis in vivo or ex vivo after UE2316 treatment, suggesting that 11β-HSD1 inhibition does not promote angiogenesis in all ischaemic environments. Future work must focus on the effects of 11β-HSD1 inhibition on the immune and extracellular matrix component of the tumour microenvironment. While promotion of pathological angiogenesis does not appear to pose a major threat, 11β-HSD1 inhibitors may still interact with the immune and inflammatory environment in tumours to the detriment of health.

Disrupted in schizophrenia 1 (DISC1), located on chromosome 1, was first identified due to its disruption by a chromosomal translocation, t(1;11)(q42;q14). This translocation co-segregates with psychiatric illness in the Scottish family within which it was discovered. DISC1 is a component of the mitochondrial trafficking machinery and regulates trafficking of mitochondria in neurons, possibly implicating defective mitochondrial trafficking as a contributory factor in psychiatric illness. The product of another candidate gene for psychiatric illness, Glycogen synthase kinase 3β (GSK3β), is known to interact directly with DISC1 and has also been reported to be involved in mitochondrial trafficking. The interaction of these proteins has not been investigated in this process. The work in this thesis centres around a novel mouse model of the t(1:11) translocation. I use time-lapse imaging of live cells to show that hippocampal neurons cultured from this mouse model exhibit altered axonal mitochondrial trafficking, including reduced mitochondrial pausing. I also demonstrate that the DISC1 interactor GSK3β is a component of the mitochondrial trafficking machinery and investigate effects of the t(1:11) event upon this multi-protein complex. Finally, I demonstrate altered mitochondrial motility responses to overexpression of GSK3β in mutant neurons. Defective mitochondrial trafficking, particularly reduced pausing, could result in an altered distribution of mitochondria within neurons, leading to an impaired ability to respond to cellular conditions, such as the requirement to power synaptic vesicle release or the ion pumps that restore membrane potential following action potential generation. This could ultimately affect neuron viability, leading to brain dysfunction. My data therefore support a proposed disease mechanism whereby defective mitochondrial trafficking contributes to susceptibility to psychiatric illness in carriers of the t(1:11) translocation, and may be relevant to psychiatric illness in general.

In the brain, axons are wrapped by myelin sheaths which ensure fast saltatory conduction of impulses and provide metabolic support. In multiple sclerosis (MS), the myelin sheaths are lost which leaves the axon denuded. This not only results in slower conduction of action potentials, but if prolonged, can also lead to axon death due to the loss of metabolic support. This neurodegeneration is the main cause of permanent disability in multiple sclerosis patients. The axon death and disability which stem from it could be prevented by restoring the myelin wrap before axon damage has occurred. This remyelination process is carried out by oligodendrocyte precursor cells which are present throughout life. To remyelinate, OPCs migrate to the area of damage and differentiate into myelinating oligodendrocytes which ensheathe axons with new myelin. In multiple sclerosis, this process occurs but is insufficient to overcome the damage. Therefore, central to the therapeutic efforts in multiple sclerosis is the aim to improve endogenous remyelination. Enhancing recruitment of oligodendrocyte precursor cells (OPCs) to the areas of damage is a clinically unexplored target. To investigate the therapeutic potential of OPC recruitment modulators, I have looked at 2 different targets involved in migration NDST1/HS and Sema3A/NP1. The first target, heparan sulfate (HS) is a proteoglycan which is important to OPC migration, investigated by Pascale Durbec's group in France. In a demyelinating mouse model, its key synthesising enzyme, NDST1, is upregulated by oligodendroglia in a belt around the lesion to aid OPC recruitment. Loss of NDST1 in oligodendrocytes was found to impair remyelination and reduce OPC migration in mice. In collaboration with them, I investigated the relevance of this molecule in post-mortem MS human tissue. I found that in human as well as mouse, NDST1 was primarily expressed by oligodendroglia. The protein level and the proportion of oligodendroglia expressing NDST1 was increased in MS compared to control indicating NDST1 upregulation as a disease response in human. We also found that low numbers of NDST1+ oligodendroglia correlate with bigger sizes of lesions and chronic lesion types that fail to repair, highlighting its importance in repair. Moreover, high numbers of NDST1+ cells in a patient correlated with increased remyelination potential. This indicates that in human, intra-patient variation in NDST1 level may explain differences in potential for endogenous repair. Secondly, I looked at Sema3A, a chemorepulsive molecule which is upregulated in demyelinated injury rodent models aswell as multiple sclerosis lesions, particularly in OPC-depopulated chronic active lesions. Research has consistently found that the level of Sema3A negatively correlates to remyelination because Sema3A hinders OPC migration. This has highlighted Sema3A as a potential target to improve OPC recruitment in MS however the size and shape of the molecule make it hard to design therapeutics against it. Therefore, I looked at its druggable receptor, Neuropilin 1 (NP1), to see whether inhibition of NP1 had the same positive effect on OPC recruitment and remyelination as lowering the level of Sema3A. NP1 is a tyrosine kinase receptor for both Sema3A and vascular endothelial growth factor (VEGF) and is found in many cell types. To check if NP1 inhibition is beneficial, I assessed remyelination in a mouse where the Sema3A binding site of NP1 has been mutated to prevent Sema3A binding and exerting its effect. This is a proxy for a (currently unavailable) ideal NP1 inhibitor of the Sema3A site only. Contrary to my expectations, OPC recruitment and remyelination in the mutant mice were not improved. However, the NP1 mutation resulted in an altered immune response. To exclude the possibility that no improvement in the OPC recruitment and remyelination of those mice was seen because it was negated by the altered immune response, I explored a cell specific mutant mouse in which NP1 was deleted in oligodendroglia only. In this mutant as well, I did not see improvement of OPC recruitment and remyelination. I therefore propose that Neuropilin 1 is not imperative for Sema3As action in remyelination and is not suitable as a therapeutic target in multiple sclerosis. Loss of the whole NP1, but not loss of the Sema3A site also resulted in biggermyelinated and unmyelinated axons as well as a different myelin thickness post remyelination. This showed that VEGF and the VEGF site on NP1 in oligodendroglia have a previously unknown but important role in determining axon size and myelin thickness which should be further investigated. To further elucidate those results in a simple system, I looked at how Sema3A, NP1-Sema3A inhibitors, VEGF and NP1-VEGF inhibitor affect OPC behaviour. I confirmed Sema3As chemorepulsive effect but also showed that at different concentrations it can improve proliferation and survival of OPCs. Inhibiting the Sema3A site and the VEGF site of NP1 by specific blocking antibodies also affects OPC proliferation and maturation. This suggested that NP1s ligands are involved in more than just OPC migration. In summary, this work supports the relevance of the mouse findings that NDST1 is upregulated in demyelination and important for repair for human illustrating that it might be a suitable therapeutic target to investigate. However, despite the importance of Sema3A in MS models, its only reported receptor, NP1, is not essential for Sema3As action. Therefore, it is an unsuitable therapeutic target. The fact that NP1 is an inappropriate drug target for MS is further demonstrated by the involvement of its ligands in multiple OPC behaviours both in positive and negative aspects.

Adaptive phenotypic plasticity, the ability of a genotype to give rise to different phenotypes in different environments, evolves to allow organisms to fine-tune their life-history traits according to the varying conditions they encounter during their lives. Reproductive investment - the manner in which organisms divide their resources between survival and reproduction - is well studied in evolutionary ecology because it is a key determinant of fitness. However, whilst plasticity in reproductive effort is well understood for free-living multicellular taxa (such as insects, birds, and mammals), the application of evolutionary theory for plasticity and life history strategies to unicellular parasites and pathogens is lacking. In this thesis, I use empirical and theoretical approaches to uncover how differential resource allocation to non-replicating, sexual stages (gametocytes) versus asexually replicating stages can be harnessed by the rodent malaria parasite Plasmodium chabaudi to maximise its fitness across the often very variable conditions it encounters during infections. Differential allocation between those stages is equivalent to the fundamental life-history trade-off between survival and reproduction because gametocytes are responsible for between-host transmission (i.e. reproduction of the infection) whereas asexual parasites mediate host exploitation and within-host survival. A suite of within-host models reveal that malaria parasites could gain considerable fitness benefits in the face of low levels of drug treatment if they reduce their investment into gametocyte production ("reproductive restraint"), thereby assuring the continuity of the infection and capitalising on opportunities for future transmission. In contrast, high levels of drug treatment typically select parasites to commit all of their resources to gametocyte production ("terminal investment"), to escape a host that does not offer much opportunity for future transmission. My experiments reveal that P. chabaudi increases both its reproductive investment and its asexual replication rate in anaemic hosts (i.e. host that have a low density of red blood cells), suggesting that parasites profit from host anaemia and can afford high investment in gametocytes ("affluent investment"). I also uncover plasticity in a number of traits that underpin asexual replication rate, including invasion preference for different ages of red blood cells, but it is plasticity in the number of progeny (merozoites) per infected cell that is the main contributor to asexual replication rate. My experiments also reveal genetic variance in plasticity of the life-history traits investigated, which has profound implications for their evolution. Furthermore, plastic modification of these traits is associated with minimal costs or constraints, so that parasites can rapidly match life-history traits appropriately to the within-host environment. Severe anaemia is one of the deadliest symptoms of malaria, so observing that virulence and infectiousness increases in anaemic hosts has also fundamental clinical implications. Finally, the empirical and theoretical observations of affluent investment, reproductive restraint and terminal investment match theoretical predictions of how organisms should behave in varying environments, confirming P. chabaudi as a useful model system to test life-history theory.

Haematopoietic stem cells (HSCs) are capable of self-renewing and multi-lineage reconstitution of the haematopoietic system of irradiated recipient mice. In the mouse embryo, HSCs originate in a step-wise manner from the haematogenic endothelium. The first HSC precursor has been detected at E9.5 in the dorsal aorta, while HSCs emerge in the aorta-gonad-mesonephros (AGM) region around E11. To date, the molecular mechanisms regulating these events are poorly characterized. Through the activating role of Angiopoietin1 (Ang1) on Tie2 receptor, the Ang-Tie signalling pathway plays a critical role in HSC maintenance in the adult bone marrow niche. Tie2 ligand Angiopoietin2 (Ang2) is described as being a Tie2 inhibitor, however its role is unknown. The aim of this thesis was to characterise the role of Ang-Tie signalling pathway in HSC formation in the mouse embryo. First, I used an ex vivo aggregate system to culture with angiopoietins cells derived from the AGM region at stages of development preceding HSC formation (E9.5-E11). Ang2- treated cells were able to reconstitute the peripheral blood of recipient mice to a higher extent compared to control, indicating a role for Ang2 in promoting HSC maturation. Then, I characterized the expression pattern of Ang-Tie molecules in the AGM region. Ang2-expressing cells were identified as perivascular and sub-aortic mesenchymal cells located in the ventral side of the aorta and in proximity of intra-aortic haematopoietic clusters. Finally, I performed an RNA-seq analysis with the aim of unravelling the molecular mechanisms involved in Ang2-mediated HSC maturation. Pre-HSC-I were cultured in presence or absence of Ang2 and their transcriptional profiles were compared, revealing a number of genes and pathways up-regulated or down-regulated in presence of Ang2, which might indicate a role for Ang2 in increasing cell proliferation, favouring cell migration, and regulation of other signalling pathways involved in HSC development. All together, these data support Ang2 as a novel regulator for HSC formation.

The difference between the number of patients needing transplantation for chronic liver disease and the number of organ donors is growing, drawing attention to the urgent requirement for novel therapies. Chronic liver injury is commonly caused by viral hepatitis, alcohol consumption, obesity and metabolic disorders. Prolonged liver injury leads to fibrosis, hepatic scarring and eventually cirrhosis. This project is based on previous studies demonstrating the therapeutic effects of bone marrow-derived macrophages (BMDM) in a murine model of liver fibrosis. BMDM facilitated fibrosis regression and improved liver regeneration. Pro-resolution macrophages exhibited increased expression of MMPs, growth factors and phagocytosis-related genes. However, macrophages derived from bone marrow are inherently heterogeneous and difficult to genetically manipulate. To overcome this limitation, our laboratory has established a protocol whereby pure populations of macrophages can be produced in significant numbers from murine embryonic stem cells (ESC) in vitro, providing an essentially limitless source of macrophages. The first goal of this project was to compare macrophages derived from ESCs (ESDM) with classical BMDM. ESDM displayed characteristic macrophage morphology, could be activated and responded to different cytokines in vitro, and were functionally phagocytic. However, they displayed some differences in their gene expression profile, and were found to be less phagocytic than BMDM. We then assessed whether ESDM could be used in the treatment of a murine model of hepatic injury induced by carbon tetrachloride administration. ESDM therapy helped in the regression of liver fibrosis, down-regulated the number of fibrogenic myofibroblasts, and activated liver progenitor cells. However, a higher number of ESDM compared to BMDMs were required to exert that effect. To assess whether ESDM may be similar to yolk sac derived tissue-resident macrophages, rather than monocyte-derived, we compared their behaviour in a Kupffer cell repopulation assay. Macrophages were depleted using liposomal clodronate treatment then animals were transplanted with either ESDM or BMDM. We demonstrated that ESDM were more efficient than BMDM at repopulating the Kupffer cell compartment and reversing the effects of liposomal clodronate treatment in mice. It is well known that macrophages are very difficult to genetically modify. So our strategy was to genetically modify ESC and then differentiate them to macrophages that carry the modification. By genetically modifying ESCs, we attempted to produce pro-fibrolytic ESDM that over-express MMP12 which is a member of the matrix metalloproteinase family of genes that mainly degrades elastin, an extracellular matrix component. We initially employed a Tet-On 3G expression system to create an ESC line where MMP12 could be expressed in an inducible manner in differentiated macrophages. However, although this inducible strategy functioned in undifferentiated ESCs we could not induce the expression of MMP12 in differentiated macrophages. In an attempt to overcome possible gene-silencing issues, we designed and constructed an expression strategy such that Mmp12 was expressed specifically in macrophages. The ESC line was built such that Mmp12 expression would be driven by the promoter of macrophage colony stimulating factor-1 receptor gene (Csf-1r or c-fms). Using the CRISPR/Cas9 strategy, we successfully targeted the Mmp12 cDNA to the Csf-1r locus but ESDM that were differentiated from targeted ESC lines did not express Mmp12. Thus, despite having adopted two independent strategies, we have failed to generate genetically modified macrophages. As a first step to translate the therapeutic effects of macrophages into the clinical setting, we optimized a feeder- and serum-free protocol to efficiently generate macrophages from human induced pluripotent stem cells.

Duchenne Muscular Dystrophy (DMD) is an X-linked recessive neuromuscular disease characterized by progressive muscle-wasting and loss of mobility. One-third of patients with DMD are also affected by cognitive impairments such as a lower than average IQ and impaired working memory, comorbid with neuropsychiatric disorders such as anxiety and autism-related behaviours. DMD is caused by mutations in the DMD gene resulting in the deletion of the full-length dystrophin protein (Dp427) and, dependent on mutation, other dystrophin isoforms. These isoforms are predominantly found in the brain and deletion may impact on cognition. The most commonly used animal model to study DMD is the mdx mouse which completely lacks Dp427 but no other DMD isoforms. Although the muscle phenotype is well-established, behavioural characterization of the mdx mouse model has been inconclusive. In this thesis I investigated the hippocampal and amygdala cellular and behavioural phenotypes of the mdx mouse. I show that post-natal neural stem-like cell division in the SGZ is altered in the absence of Dp427 resulting in enhanced symmetric division. I show in vitro that primary mdx cultures are fewer and smaller than wild-type, consistent with an increase in symmetrical self-renewal whereas secondary cultures are fewer and larger, consistent with a shift in symmetric division producing transit-amplifying type 2a daughter cells. I next characterized the mdx mouse model using a battery of behavioural tests. Data presented here show that mdx mice do not exhibit an anxious phenotype, do not display autism-related behaviours, and do not display impairments in and spatial learning or memory. However, associative learning, as measured in the fear conditioning paradigm is enhanced in mdx mice. Lastly, I attempted to generate three different brain-specific dystrophin knock-out mouse models to examine role of other dystrophin isoforms. While none of the models were able to deplete dystrophin from brain, given the inverse relationship between Cre-mediated efficiency and the genetic distance of the loxP sites in the fDMDH mouse employed, I do provide important insight into the presence and absence of the muscle-specific enhancers in constructs commonly used to generate brain-specific mouse models. Taken together, this thesis provides converging evidence to indicate that loss of Dp427 impacts on fear associative learning and stem-cell like division in the SGZ but likely does not underlie the non-progressive cognitive impairments affecting one-third of all DMD patients.

Patients with Axenfeld-Rieger Syndrome (ARS) present various dental abnormalities. ARS is genetically associated with mutations in the PITX2 gene, which encodes one of the earliest transcription factors to initiate tooth development. Thus, Pitx2 has long been considered as an upstream regulator of the transcriptional hierarchy in tooth development. However, it is unclear how its mutant forms cause ARS dental anomalies. In this report, we outline the transcriptional mechanism that is defective in ARS. We demonstrate that during normal tooth development Pitx2 activates Amelogenin (Amel) expression, whose product is required for enamel formation, and that this regulation is perturbed by missense PITX2 mutations found in ARS patients. We further show that Pitx2-mediated Amel activation is enhanced and controlled by co-factors and target genes of Pitx2. These co-factors include cooperative transcription factors such as Dlx2 and FoxJ1; chromatin-associated remodeler factor Hmgn2; and Wnt signaling components such as Lef-1, β-catenin and Dact2. We also unveil a novel Pitx2 target gene Irx1 that functions in dental epithelium differentiation. Consistent with a physiological significance to these modulations, we show that FoxJ1, Dact2, Irx1 knockout mice and K14-Hmgn2 transgenic mice display various types of amelogenesis defects including enamel hypoplasia - consistent with the human ARS phenotype. Collectively, these findings define transcriptional mechanisms and multi-level regulations involved in normal tooth development and shed light on the molecular underpinnings of the enamel defect observed in ARS patients who carry PITX2 mutations. Moreover, our findings validate the etiology of the enamel defect in novel mouse models of enamel hypoplasia. The impact of this study on current understanding of the dental epithelium development and the translational value lie in the gene network we identified. By manipulating components of the network, pluripotent dental cells can be reprogrammed and serve as new source for tooth regeneration. Our findings brought insights of novel gene therapy approach that can alleviate the dental problems of patients with ARS and other developmental anomalies.