La maladie de Parkinson (MP) est marquée par la présence d'une inflammation, pouvant être bénéfique ou délétère à la neurodégénérescence dopaminergique (DAgique). Nous avons adressé la nature des interactions pathologiques entre neurones DAgiques, cellules gliales et leucocytes infiltrant, nécessaire à la mise en place de cette inflammation. Dans un modèle MPTP murin de la MP, les objectifs de ma thèse ont été 1) d'identifier des signaux inflammatoires neuronaux et gliaux, par une approche transcriptomique associée à de la microdissection laser et 2) de déterminer leurs rôles dans la neuroinflammation ainsi que leurs effets sur la perte DAgique. Nous avons retenu parmi les candidats identifiés: les axes CXCL16-CXCR6 et CCL2-CCR2. Nous reportons dans le modèle MPTP, une expression microgliale de CXCL16 ainsi qu'une infiltration de population lymphocytaire CXCR6. Bien que la déplétion de CXCR6 permette de réduire cette infiltration, aucun effet n'est observé sur la perte DAgique. Nous décrivons une infiltration de monocyte CCR2 en concomitance avec une expression astrocytaire précoce de CCL2 dans le modèle MPTP murin, ainsi qu'une expression plus prolongée de CCL2 chez le primate non-humain MPTP, suggérant une relevance de l'axe CCL2-CCR2 dans la MP. En effet nous montrons que des souris surexprimant CCL2, intoxiquées au MPTP, ont non seulement une augmentation accrue de l'infiltrat monocytaire CCR2, mais également de la lésion DAgique. De manière inattendue, nous montrons que la neurotoxicité accrue observée chez des souris CX3CR1-/- MPTP passe indirectement par la voie CCL2-CCR2. Ainsi, nos données supportent l'hypothèse d'une neurotoxicité des monocytes CCR2 dans la MP. / Parkinson's disease (PD) presents signs of neuroinflammation, which can be beneficial or deleterious for dopaminergic (DA) neurodegeneration. We have analyzed the characteristics of such pathological interactions between DA neurons, glial cells and infiltrating immune cells. Using the neurotoxic MPTP mouse model of PD and focusing on chemokines, my thesis objectives were: 1) to identify using laser-microdissection and RNA profiling, the neuronal and glial inflammatory signals in the affected Substantia Nigra (SN) and 2) to assess the role of promising identified chemokine candidates during DA neurodegeneration. We have focused on the lymphocytic CXCL16-CXCR6 and the monocytic CCL2-CCR2 axes. We have found early microglial CXCL16 induction and parallel SN infiltration of CXCR6 lymphocyte subpopulations. CXCR6-deletion reduced infiltration of specific lymphocyte subpopulations, but did not affect the known deleterious infiltration of CD4 T-lymphocytes. For the CCL2-CCR2 axis, we found evidence for limited SN infiltration of CCR2 monocytes, which was preceded by transient astrocytic CCL2 induction in MPTP mice, but a prolonged CCL2 induction in MPTP monkeys, suggesting a potential relevance for human PD. While CCR2-gene deletion did not affect loss of DA neurons, astrocytic CCL2 overexpression increased MPTP induced DA neural loss, revealing the principally neurotoxic nature of infiltrating CCR2 monocytes in a PD-like environment. Unexpectedly, we also found that the known increased DA loss in CX3CR1-KO mice was mediated indirectly via over-induction of the CCL2-CCR2 axis. Combined, our results suggest a potential deleterious role of the CCL2-CCR2 axis in actual human PD.

Autism spectrum disorder (ASD) now affects 1 in 68 children in the United States. Disorders within this spectrum share hallmark deficits in verbal and nonverbal communication, repetitive behavior, and social interaction. The cause of ASD is still unknown. Even though hundreds of genetic abnormalities have been identified in ASD, these markers account for less than 1% of all ASD cases. Researchers continue to search for pathological markers common to all or most cases of ASD. The research presented in this dissertation used a novel combination of state-of-the-art methods to investigate brain pathology in ASD. Postmortem anterior cingulate cortex (ACC) from ASD and typically developing brain donors was obtained from 2 national brain banks. The ACC was chosen for study because of its documented role in influencing behaviors characteristically disrupted in ASD. An initial study revealed elevated glial fibrillary acidic protein (GFAP) in ACC white matter from ASD brain donors compared to typically developing control donors. Laser capture microdissection was then employed to isolate specific cell populations from the ACC from ASD and control brain donors. Captured cells were used to interrogate potential gene expression abnormalities that may underlie biological mechanisms that contribute behavioral abnormalities of ASD. The expression of 4 genes associated with synaptic function, NTRK2, GRM8, SLC1A1, and GRIP1, were found to be significantly lower in ACC pyramidal neurons from ASD donors when compared to control donors. These expression abnormalities were not observed in ACC glia. Given robust evidence of neuronal and glial pathology in the ACC in ASD, a novel method for whole transcriptome analysis in single cell populations was developed to permit an unbiased analysis of brain cellular pathology in ASD. A list of genes that were differentially expressed, comparing ASD to control donors, was produced for both white matter and pyramidal neuron samples. By examining the ASD brain at the level of its most basic component, the cell, methods were developed that should allow future research to identify common cellular-based pathology of the ASD brain. Such research will increase the likelihood of future development of novel pharmacotherapy for ASD patients.

The airway epithelium has many roles in innate immunity including detection of pathogens and transmitting danger signals to other cell types. However, its role as a primary defender against infection is not well recognized. We have investigated methods of augmenting antiviral immunity by application of agents that stimulate viral killing, either in the extracellular space or within the cytoplasm. A recently described property of airway epithelial cells is direct oxidative killing of bacteria through the coordination of Duox and lactoperoxidase enzymes. We have exploited this property by supplementing airway cells with the lactoperoxidase substrate iodide to prevent viral infection. A second method for enhancing antiviral defenses is to supply small interfering RNAs (siRNAs) targeting essential viral genes. We have optimized antiviral siRNAs targeting respiratory syncytial virus by designing them to specifically target positive sense viral RNAs. Finally, we have initiated a project to discover host defense genes that are expressed in either the submucosal glands surface epithelium of human airway. This information will enable a better characterization of the roles for these structures in host defense pathways, and may identify other targets for augmentation of antiviral immunity.

<p>Mutation of the p53 gene appears to be an early event in skin cancer development. The present study is based on morphology and represents a cellular and genetic investigation of p53 alterations in normal human skin and basal cell cancer.</p><p>Using double immunofluorescent labelling, we have demonstrated an increase in thymine dimers and p53 protein expression in the same keratinocytes following ultraviolet radiation. Large inter-individual differences in the kinetics of thymine dimer repair and subsequent epidermal p53 response were evident in both sunscreen-protected and non-protected skin. The formation of thymine dimers and the epidermal p53 response were partially blocked by topical sunscreen. We have optimized a method to analyze the p53 gene in single cells from frozen tissue sections. In chronically sun-exposed skin there exist clusters of p53 immunoreactive keratinocytes (p53 clones) in addition to scattered p53 immunoreactive cells. Laser assisted microdissection was used to retrieve single keratinocytes from immunostained tissue sections, single cells were amplified and the p53 gene was sequenced. We have shown that p53 mutations are prevalent in normal skin. Furthermore, we detected an epidermal p53 clone which had prevailed despite two months of total protection from ultraviolet light. Loss of heterozygosity in the PTCH and p53 loci as well as in the sequenced p53 gene was determined in basal cell cancer from sporadic cases and in patients with Gorlin syndrome. Allelic loss in the PTCH region was prominent in both sporadic and hereditary tumors, while loss of heterozygosity in the p53 locus was rare in both groups. p53 mutations found in the hereditary tumors differed from the typical mutations found in sporadic cases. In addition, we found genetically linked subclones with partially different p53 and/or PTCH genotypes in individual tumors. Our data show that both genes are important in the development of basal cell cancer.</p>

<p>The aim of this thesis was to gain further insight into the biology of normal human skin and basal cell carcinoma (BCC). Morphology in combination with microdissection was used as primary tool for sampling.</p><p>Using the X-chromosome inactivation assay, we found normal human skin to consist of a mosaic of cells, with either the maternal or the paternal X-chromosome inactivated. We believe that each tile is made up of several epidermal proliferative units with identical X-chromosome inactivation patterns. Using the same method, we found BCC to be a monoclonal neoplasm imbedded in polyclonal stroma. However, one tumor displayed clear evidence of being composed of two intermingled monoclonal tumors.</p><p>To better enable molecular analysis of defined cells from tissue sections, we investigated a zinc-based fixative as alternative to neutral-buffered formalin. Zinc-based fixative preserves good quality of genomic DNA, with only slight impairment of morphology. In addition, it partly abrogates the need for antigen retrieval.</p><p>The patched gene is involved in BCC development. We analyzed the distribution of a coding polymorphism (Pro/Leu) at codon 1315 in populations with different skin types. We found a reduced Pro/Pro genotype frequency in populations with lighter pigmentation. This in combination with genotype analyses of patients with multiple BCCs, showed that failure to lose the Pro allele during a shift towards lighter pigmented skin may be associated with an increased risk of developing BCC.</p><p>We compared the expression profile of BCC cells with putative progenitor cells in the basal layer of epidermis. In addition to discovering several unknown genes, we found the Wnt signaling pathway to upregulated. Furthermore, differentiation markers were downregulated together with proteins important for scavenging of oxygen radicals.</p><p>In conclusion, the combination of morphology, microdissection and subsequent molecular applications provided valid information deepening our understanding of normal skin and BCC.</p>

Mutation of the p53 gene appears to be an early event in skin cancer development. The present study is based on morphology and represents a cellular and genetic investigation of p53 alterations in normal human skin and basal cell cancer. Using double immunofluorescent labelling, we have demonstrated an increase in thymine dimers and p53 protein expression in the same keratinocytes following ultraviolet radiation. Large inter-individual differences in the kinetics of thymine dimer repair and subsequent epidermal p53 response were evident in both sunscreen-protected and non-protected skin. The formation of thymine dimers and the epidermal p53 response were partially blocked by topical sunscreen. We have optimized a method to analyze the p53 gene in single cells from frozen tissue sections. In chronically sun-exposed skin there exist clusters of p53 immunoreactive keratinocytes (p53 clones) in addition to scattered p53 immunoreactive cells. Laser assisted microdissection was used to retrieve single keratinocytes from immunostained tissue sections, single cells were amplified and the p53 gene was sequenced. We have shown that p53 mutations are prevalent in normal skin. Furthermore, we detected an epidermal p53 clone which had prevailed despite two months of total protection from ultraviolet light. Loss of heterozygosity in the PTCH and p53 loci as well as in the sequenced p53 gene was determined in basal cell cancer from sporadic cases and in patients with Gorlin syndrome. Allelic loss in the PTCH region was prominent in both sporadic and hereditary tumors, while loss of heterozygosity in the p53 locus was rare in both groups. p53 mutations found in the hereditary tumors differed from the typical mutations found in sporadic cases. In addition, we found genetically linked subclones with partially different p53 and/or PTCH genotypes in individual tumors. Our data show that both genes are important in the development of basal cell cancer.

The aim of this thesis was to gain further insight into the biology of normal human skin and basal cell carcinoma (BCC). Morphology in combination with microdissection was used as primary tool for sampling. Using the X-chromosome inactivation assay, we found normal human skin to consist of a mosaic of cells, with either the maternal or the paternal X-chromosome inactivated. We believe that each tile is made up of several epidermal proliferative units with identical X-chromosome inactivation patterns. Using the same method, we found BCC to be a monoclonal neoplasm imbedded in polyclonal stroma. However, one tumor displayed clear evidence of being composed of two intermingled monoclonal tumors. To better enable molecular analysis of defined cells from tissue sections, we investigated a zinc-based fixative as alternative to neutral-buffered formalin. Zinc-based fixative preserves good quality of genomic DNA, with only slight impairment of morphology. In addition, it partly abrogates the need for antigen retrieval. The patched gene is involved in BCC development. We analyzed the distribution of a coding polymorphism (Pro/Leu) at codon 1315 in populations with different skin types. We found a reduced Pro/Pro genotype frequency in populations with lighter pigmentation. This in combination with genotype analyses of patients with multiple BCCs, showed that failure to lose the Pro allele during a shift towards lighter pigmented skin may be associated with an increased risk of developing BCC. We compared the expression profile of BCC cells with putative progenitor cells in the basal layer of epidermis. In addition to discovering several unknown genes, we found the Wnt signaling pathway to upregulated. Furthermore, differentiation markers were downregulated together with proteins important for scavenging of oxygen radicals. In conclusion, the combination of morphology, microdissection and subsequent molecular applications provided valid information deepening our understanding of normal skin and BCC.

Prion diseases are fatal and incurable neurodegenerative diseases that share many pathological similarities to other neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease. One of the earliest pathological signs commonly detected in all of these diseases is the dysfunction followed by loss of neuronal synapses, spines and eventually dendrites that collectively contribute to disruption of normal brain function. These pathologies tend to progressively accumulate within the brain tissue such that extensive damage typically precedes clinical symptom manifestation and ultimate death of neurons. Clearly, understanding the molecular processes responsible for these pathologies could uncover critical pathway(s) that are responsible for propagating this brain damage and could therefore be exploited for therapy development. However, molecular mechanisms implicated in this early pathology remain unidentified. To address this gap in knowledge, this thesis describes a transcriptional approach coupled with specific isolation of neuronal-enriched tissue which was used to help delineate cellular pathways involved in prion-induced neurodegeneration. Profiling cell bodies of CA1 hippocampal neurons known to be affected during early prion disease revealed temporal alteration in both gene and microRNA (gene regulators) expression throughout disease. On a gene expression level, changes in transcript expression during preclinical disease were reminiscent of an activity-dependent neuroprotective gene signature previously described in the literature. These neuroprotective genes were induced during preclinical disease, diminished as disease progressed and were abolished at clinical disease. In support of this process, upregulation of the phosphorylated form of the neuroprotective transcription factor CREB was detected during preclinical disease in these neurons. Furthermore, several genes known to be induced by CREB were also upregulated at preclinical disease in prion-infected mice. Interestingly, expression of numerous deregulated microRNAs paralleled the neuroprotective gene signature of which several are known to remodel neuronal spines and dendrites. To determine whether other preclinically induced microRNAs were also capable of remodeling neuronal structures, gain-of-function studies were performed in primary mouse hippocampal neurons for the uncharacterized miR-26a-5p. Neurons over-expressing miR-26a-5p had enhanced spine density and dendrite arborization, similar to other preclinically-induced microRNAs. Together, these data suggests that CA1 hippocampal neurons induce a neuroprotective transcriptional signature that is evident early in the course of disease within CA1 hippocampal neurons and is abolished by clinical disease. Reestablishment of key molecules that can induce this neuroprotective signature at a time when these genes begin to dissipate could prolong prion disease onset and delay clinical symptom manifestation. / October 2015

Résumé: L’expression des gènes modifiés contribue à l’initiation et la progression du cancer. Malheureusement, les recherches actuelles s’attardent essentiellement à l’expression globale des gènes sans tenir compte des différentes isoformes des ARNm résultant de l’épissage alternatif, codant potentiellement pour des protéines de fonctions différentes. En effet, la haute similitude et la complexité des isoformes ARNm rendent la discrimination des isoformes laborieuse, raisons pour lesquelles l’épissage alternatif est historiquement étudié gène par gène. C’est la raison pour laquelle nous avons développé une méthodologie à haut débit pour la quantification des isoformes d’ARNm. Basé sur cette technique, cette Thèse présente une série d’évidences qui appuie l’hypoThèse selon laquelle certaines isoformes d’ARNm sont nécessaires à la tumorigénèse. Précédemment, des études bioinformatiques à grande échelle ainsi que des validations expérimentales à petite échelle ont révélé la présence d’isoformes spécifiques à certains types de cancers. Toutefois, ces conclusions ont été tirées à partir de tumeurs dont le contenu cellulaire est hétérogène. Or, il est bien connu que les niveaux des isoformes d’ARNm diffèrent largement en fonction du type cellulaire et il est possible que les différences observées dans des tumeurs entières soient des artéfacts résultant de la complexité cellulaire des tissus comparés. En nous basant sur le cancer de l’ovaire comme modèle, nos préparations homogènes de différents types cellulaires nous ont permis de révéler la présence d’isoformes associées au cancer indépendamment de l’hétérogénéité des tumeurs. Étonnamment, les changements les plus intéressants ne se retrouvent pas dans les cellules cancéreuses à proprement dites, mais dans les cellules "normales" en bordure de la tumeur. Notre analyse des facteurs de régulation de ces isoformes nous a permis d’affirmer que ces changements ne sont pas dûs à un dérèglement anarchique des cellules cancéreuses, mais découlent plutôt d’un programme destiné à avantager les cellules cancéreuses. Pour évaluer la fonction des isoformes d’ARNm, nous avons généralisé des outils moléculaires permettant de reprogrammer spécifiquement l’expression d’isoformes de gènes cible dans des lignées cellulaires cancéreuses. Ainsi, le criblage systématique d'isoformes d’ARNm de gènes cibles associés au cancer a permis de révéler leur caractère pro-cancer dans des essais in vitro. L'ensemble de ces travaux démontrent la contribution des isoformes d’épissage dans le développement des tumeurs solides. // Abstract: Cancer cells modify their gene expression pattern to promote tumor growth. Unfortunately, current research focuses almost exclusively on global gene expression changes, without taking into account the fact that the majority of genes produce multiple alternative splicing variants, which potentially code for proteins of different functions In fact, the main reason splicing isoforms are still studied on a gene by gene basis is because the high sequence similarity and complexity of mRNA isoforms make it very difficult to detect and quantify them. To overcome this difficulty, we developed a high-throughput methodology to accurately quantify splicing isoforms. This methodology has been applied in this thesis to generate evidence supporting a causal role for splicing isoforrns in the tumorigenesis of solid tumors. A few years ago, genome-wide bioinformatics predictions, and some experimental validations in human tissues, suggested the existence of cancer-specific splicing isoforms. However, these conclusions may be simply explained by the cellular heterogeneity of tumor. In fact, it is well established that alternative splicing is a cell type-specific process. To address this, we microdissected cancer tissues and demonstrated that some splicing isoforms are truly cancer-associated and independent of tumor cell type content. Surprisingly, the most interesting changes were found in the "normal" stromal cells surrounding the tumor and not within the cancer cells. Our analysis of splicing-factor expression changes using the same tissue set allowed us to confirm that these splicing shifts were not a random process, but rather part of a defined splicing program promoting tumor development. To evaluate the functional contribution of splicing isoforms, we developed molecular tools that modulate splicing of endogenous targets in cancer cell lines. We used these tools to decipher the pro-cancer properties of cancer-associated splicing isoforms using in vitro assays. Overall, this work demonstrated the contribution of splicing isoforms in solid tumor development.

Small intestinal neuroendocrine tumors (SI-NETs) originate from serotonin-producing enterochromaffin (EC) cells in the intestinal mucosa. Somatostatin analogs (SSAs) are mainly used to control hormonal secretion and tumor growth. However, the molecular mechanisms leading to the control of SI-NETs are unknown. Although microRNAs (miRNAs) are post transcriptional regulators deeply studied in many cancers, are not well-defined in SI-NETs. We adopted a two-pronged strategy to investigate SSAs and miRNAs: first, to provide novel insights into how SSAs control NET cells, and second, to identify an exclusive SI-NET miRNA expression, and investigate the biological functions of miRNA targets. To accomplish the first aim, we treated CNDT2.5 cells with octreotide for 16 months. Affymetrix microarray was performed to study gene variation of CNDT2.5 cells in the presence or absence of octreotide. The study revealed that octreotide induces six genes, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15. To accomplish the second aim, SI-NET tissue specimens were used to run genome-wide Affymetrix miRNA arrays. The expression of five miRNAs (miR-96, -182, -183, -196a and -200a) was significantly upregulated in laser capture microdissected (LCM) tumor cells versus LCM normal EC cells, whereas the expression of four miRNAs (miR-31, -129-5p, -133a and -215) was significantly downregulated in LCM tumor cells. We also detected nine tissue miRNAs in serum samples, showing that the expression of five miRNAs is significantly increased in SSA treated patients versus untreated patients. Conversely, SSAs do not change miRNA expression of four low expressed miRNAs. Silencing miR-196a expression was used to investigate functional activities in NET cells. This experimental approach showed that four miR-196a target genes, HOXA9, HOXB7, LRP4 and RSPO2, are significantly upregulated in silenced miR-196a NET cells. In conclusion, ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15 genes might regulate cell growth and differentiation in NET cells, and play a role in an innovative octreotide signaling pathway. The global SI-NET miRNA profiling revealed that nine selected miRNAs might be involved in tumorigenesis, and play a potential role as novel markers for follow-up. Indeed, silencing miR-196a demonstrated that HOXA9, HOXB7, LRP4 and RSPO2 genes are upregulated at both transcriptional and translational levels.