The gut-brain axis describes a paradigm wherein the trillions of microorganisms inhabiting the gastrointestinal tract engage in bidirectional communication with the host central nervous system. Adaptive immunity represents an important intermediate in this dynamic crosstalk; previous work in our lab has demonstrated that T-lymphocytes, a main class of immune effector cells, contribute to neurodevelopmental processes and behavioral outcomes across the lifespan. Parallels between the phenotype of T-cell deficient and germ free mice led us to hypothesize that bidirectional T-cell-microbe communication is critical for normal neurodevelopment, and that T-cell deficiency impacts the neural circuitry underpinning behavior via disruption of the gut-brain axis. The main objective of this thesis was to elucidate the mechanisms by which T-cells mediate developmental gut-brain signalling. The first installation examined the gut microbiome, gut metabolome, and neurochemical profile in wild-type and T-cell deficient mice from adolescence to adulthood, demonstrating that absence of T-cells impacts the developmental trajectory of functional microbiome output and levels of neuroactive molecules in the brain. Experiment two investigated the impact of T-cell deficiency on gut-brain communication through the lens of host gene expression in the parenchyma and the intestine. T-cell deficient mice showed significant changes in genes related to intestinal immunity and barrier function, in addition to decreases in microglia-related genes in the prefrontal cortex during early life. The final experiment transitioned into a wild-type model to measure the co-evolution of T-cell subsets in mucosal and central immune compartments with composition and diversity of the microbiota. We demonstrated a parallel diversification of the gut microbiome and the functional T-cell repertoire, whereby emergence and proliferation of specific T-cell subsets is linked to compositional shifts in dominant microbial communities across development. Together, our results demonstrate the importance of T-cells for normal development of the holo-organism, with implications for the developmental wiring of functional brain circuitry. / Thesis / Doctor of Philosophy (PhD) / Modern medicine has increasingly placed emphasis on the mind-body connection. This has been exemplified by a series of recent discoveries surrounding the importance of the gut microbiome in maintaining our physical and mental health. One of the key channels through which the microbiome communicates with the host is through the immune system, an equally complex network of cells and proteins that protect the body against invading pathogens. Indeed, these systems evolve alongside each other and engage in constant crosstalk throughout the lifespan, with downstream impacts on the developing brain. This thesis sought to further explore the role of T-cells, a key component of the adaptive immune system, in coordinating gut-microbiome-brain interactions across development. The first experiment examined the microbiome as well as small molecules in the gut and brain of normal mice and mice lacking T-cells. The second experiment built on this work to examine how T-cells influence the expression of different genes in the gut and brain. Finally, the third experiment mapped different populations of T-cells and microbiome composition from the first week of life to adulthood, to better understand how they interact at different stages of development. This work will offer insight into how T-cells talk to the microbiome and how they transmit signals from the gut to the brain, with implications for understanding neurodevelopmental disorders and how they arise.

Spinal muscular atrophy (SMA) is a devastating disorder caused by deletions and mutations in the survival of motor neuron (SMN1) gene and is marked by motor neuron loss and muscle weakness. While its genetic basis is clear, the underlying molecular mechanisms remain elusive. Decreased levels of the survival of motor neuron (SMN) protein, encoded by the SMN1 gene, are implicated in SMA pathology. Despite splicing has been under the spotlight as a major mechanism impaired in SMA, recent evidence suggests that SMN deficiency also disrupts protein translation in vivo in a mouse model of severe SMA, complicating SMA's molecular landscape. This thesis examines the impact of SMN protein loss on translation in SMA mouse models across tissues, post-natal and pre-natal disease stages, focusing on both mild (Chapter 1) and severe forms of SMA (Chapter 2) respectively. To tackle this question, in this thesis, I took advantage of multiple cutting-edge and sequencing-based techniques (ribosome profiling and RNA-seq) coupled with biochemical and molecular biology-based assay (polysome profiling, co-sedimentation profiles, qPCR, and western blotting), which applied to study in molecular detail the translational defects in the brain, spinal cord and liver at asymptomatic, pre-symptomatic and early symptomatic stages of SMA. Polysome profiling in control mice (Smn2B/+) reveals a gradual increase in SMN association with ribosomes/polysomes during postnatal development, indicating dynamic SMN function in protein translation during post-natal development. In SMA condition, where SMN protein levels drop, this binding reveals a tissue-specific decrease in the spinal cord and liver. Through ribosome profiling, numerous alterations in translation were identified at the pre-symptomatic stage of the disease, suggesting that translational defects are features of the early stages of SMA. Importantly these alterations are independent of transcriptional and splicing changes. Although no gene was found to be in common, I found that genes altered in at least 2 tissues are involved in the same processes. The dysregulated mRNAs exhibit rare codons at the beginning of coding sequences in all three tissues, as shown in the case of the severe model of SMA. 4 From these common processes I have identified specific mRNA targets that play key roles in the organization of the extracellular matrix I validated the presence of translational changes in Col1a1, Col1a2, and Spp1 highlighted effects of SMN deficiency on translational regulation, in the absence of transcriptional alterations. Validation studies in both mice and SMA patient-derived fibroblasts further underscored the potential of translational dysregulation and drop in Col1a1 protein expression during SMA progression. Finally, prenatal studies have revealed distinct translational changes in embryonic tissues from Taiwanese mice. Despite no alterations in global translation, a drop in SMN association with ribosomes/polysomes and tissue-specific differences in ribosome occupancy were observed. Also, in this case, dysregulated mRNAs exhibit rare codons at the beginning of coding sequences. These findings shed light on the unique molecular landscape of prenatal development in the context of SMN deficiency. In summary, this study provides insights into translation dysregulation in SMA pathology, emphasizing tissue-specific effects and developmental stage-dependent alterations. By elucidating the complex relationship between SMN protein function and translational dynamics, it lays the groundwork for targeted therapeutic strategies and biomarkers to improve SMA management. Ongoing investigations into prenatal development and translation dynamics are crucial for a comprehensive understanding of SMA pathogenesis and effective treatment development.

Mosquito-borne viruses (MBV) are the cause of significant health and economic concerns worldwide. However, the mechanisms by which these viruses are transmitted to humans remain elusive. This is due to the scarcity of animal models recapitulating mosquitoes to human MBV transmission. While transgenic mice defective in their immune responses are permissive to MBV infection, the partial immunodeficiency of these models and the lack of human skin preclude an accurate understanding of the molecular and cellular mechanisms driving effective MBV transmission. The goal of this project is to develop the first mouse model co-engrafted with a functional human immune system and syngeneic induced pluripotent stem cells (iPSC)-derived skin organoids. This novel mouse model will illuminate critical mechanisms and host-pathogen interactions driving MBV transmission and provide a novel technology to model skin-related diseases. / 2026-10-31T00:00:00Z

La réaction du greffon contre l’hôte (GvHD) est une complication majeure de la transplantation de cellules souches hématopoïétiques (HSCT). Les traitements de prophylaxie contre le développement de la GvHD reposent essentiellement sur l’utilisation d’agents immunosuppresseurs, ce qui contribue à ralentir la reconstitution immunitaire post-greffe et à prolonger la durée de l’état immunosupprimé des patients. Le développement de prophylaxie pour la GvHD à base d’agents immunomodulateurs est ainsi privilégié. À l’aide d’un modèle murin xénogénique chez les souris NOD/scid-IL2rγ-/- (NSG), on a étudié le potentiel immunomodulateur des immunoglobulines intraveineuses (IVIG) dans la prévention de la GvHD, ainsi que leurs effets sur la qualité et la cinétique de la reconstitution immunitaire. On a déterminé qu’un traitement hebdomadaire d’IVIG peut effectivement réduire l’incidence de la GvHD, ainsi que la mortalité qui y est reliée, avec une efficacité similaire à celle obtenue avec la cyclosporine A, un immunosuppresseur couramment utilisé dans la prophylaxie de la GvHD. Par ailleurs, on a déterminé que le mécanisme d’action des IVIG dans la réduction de la GvHD est distinct de celui des immunosuppresseurs. De plus, on a démontré que les IVIG induisent l’expansion et l’activation des cellules NK présentes au sein du greffon, lesquelles sont nécessaires pour l’obtention de l’effet protecteur des IVIG contre le développement de la GvHD, et sont dépendantes de la présence de lymphocytes T activés. Grâce à un modèle murin humanisé, on a également démontré que le traitement hebdomadaire d’IVIG induit un délai transitoire de la reconstitution humorale, ce qui n’affecte toutefois pas la qualité globale de la reconstitution immunitaire. Ces résultats mettent cependant en doute la pertinence de l’utilisation des IVIG dans les protocoles cliniques de prophylaxie de la GvHD, puisque les immunosuppresseurs seront toujours utilisés, et qu’on a démontré que les IVIG ont besoin de lymphocytes T activés afin de prévenir efficacement le développement de la GvHD. / Graft-versus-Host Disease (GvHD) is a major complication following hematopoietic stem cell transplantation (HSCT). Prophylactic treatments for the prevention of GvHD rely mostly on the use of immunosuppressors, which contribute to inhibit the patient’s immune reconstitution and prolong their immunosuppressed state. Development of immunomodulator-based prophylactic treatments is therefore preferred. Using NOD/scid-IL2rγ-/- mice, we developed a xenogeneic mouse model to assess the immunomodulatory potential of intravenous immunoglobulins (IVIG) for the prevention of GvHD, along with assessing their effect on the kinetics and the quality of the immune reconstitution in mice. We determined that weekly IVIG treatments reduced the incidence of GvHD and its related mortality. The effectiveness of IVIG for the prevention of GvHD was similar to that of cyclosporine A, an immunosuppressive drug routinely used for the prophylactic treatment of GvHD. Furthermore, we demonstrated that IVIG has a mechanism of action that is different from that of immunosuppressors. IVIG induce the expansion and activation of NK cells from the graft, which is mandatory for the preventive effect of IVIG on GvHD development. Furthermore, this IVIG-induced expansion and activation of NK cells require the presence of activated T lymphocytes. Using our humanized mouse model, we have also demonstrated that weekly IVIG treatments cause a transient delay of the humoral reconstitution, but do not affect the overall quality of the immune reconstitution. We have demonstrated that activated T lymphocytes are mandatory for the effective expansion and activation of NK cells, which in turn are essential to the IVIG-induced prevention of GvHD, and immunosuppressors will always be part of the prophylactic regimen of GvHD, therefore shining a doubt on the usefulness of adding IVIG to the prophylactic treatments for the prevention of GvHD.

<p>Over three million infections were reported in the United States of America in 2019. These infections were caused by either antibiotic-resistant pathogens or <i>Clostridioides difficile</i> and resulted in more than 50,000 deaths. Unfortunately, antibacterial agents are rapidly losing their ability to treat infections and the process of discovering new antibiotics is too slow to cope up with bacterial evolution. Repurposing FDA-approved drugs of well-studied safety, pharmacology and pharmacokinetics represents a faster alternative method of antibacterial drug discovery. Repurposing is more successful and less depleting method of drug discovery than classical de novo method in regard to both cost and time. In the following studies, two major pathogens are targeted, vancomycin-resistant <i>Enterococcus</i> (VRE) and <i>C. difficile</i>. Both bacteria are more prevalent in healthcare settings were more vulnerable population of elderly and immunocompromised individuals reside. In addition, healthcare settings are usually associated with higher frequency of receiving antibiotics which in turn, compromises the integrity of normal microbiota responsible for protection against invading pathogens. Furthermore, hospital stays are associated with exposure to bacterial shedding from other patients. Our aim was to identify FDA-approved drugs with novel ability to eradicate these two bacterial pathogens in the gastrointestinal tract (GIT). Notably, the GIT is considered the actual site of infection in case of <i>C. difficile while it is only a transition site for VRE where the bacteria colonize before causing true infections in other tissues. Studies against both bacteria started with an <i>in vitro</i> screening of FDA-approved drugs and clinical molecules to identify potential candidates for further investigation.</i></p><p><i>For VRE, two drugs where identified with potent inhibitory activity and favorable pharmacokinetic profiles, auranofin and ebselen. Auranofin was approved in the 1960s for the treatment of rheumatoid arthritis due to its anti-inflammatory activity. Auranofin was found to exert potent bacteriostatic activity against both vancomycin-sensitive and vancomycin-resistant <i>Enterococcus</i> strains (minimum inhibitory concentration against 90% of the strains, MIC90 = 1 µg/mL). In addition, bacteria could not develop resistant mutants against auranofin upon prolonged exposure. On the other hand, ebselen is an organoselenium compounds currently in clinical trials for several indications. Similarly, ebselen was found to be a potent inhibitor of VRE growth (MIC90 = 2 µg/mL). In addition, ebselen successfully inhibited bacterial biofilm formation and eradicated mature biofilms. In a mouse model of VRE colonization, both drugs inhibited bacterial shedding and reduced bacterial counts in the GIT of the colonized animals.</i></p><p><i>For <i>C. difficile</i>, auranofin was also found to exert potent inhibitory activity against bacterial growth (MIC90 = 2 µg/mL), toxin production and spore formation. Additionally, it was beneficial in protecting colon cells against <i>C. difficile</i> toxin-induced inflammation. Further, auranofin was found to not promote growth of VRE as seen with the current anticlostridial agents. In addition to auranofin, two more antiprotozoal drugs were found to potently inhibit <i>C. difficile</i> growth, ronidazole and secnidazole. Both drugs are 5-nitroimidazoles approved for human (secnidazole) or veterinary (ronidazole) applications. Secnidazole and ronidazole halted <i>C. difficile</i> growth at very low concentrations (MIC90 = 0.5 and 0.125 µg/mL, respectively). Furthermore, both drugs were superior to metronidazole in bacterial killing and had favorable activities against protective gut microbiota. In addition, they demonstrated efficient protection to mice in a <i>C. difficile</i> infection model. </i></p><p><i>Overall, several drugs were presented to possess favorable activities against <i>C. difficile</i> or VRE. These drugs merit more evaluation as potential candidates for the treatment of infection caused by either bacteria. </i></p><div><i><br></i></div>

These studies were aimed at investigating the potential application of nanoparticles engineered from oil-in-water microemulsion precursors for enhancing immune responses to HIV-1 Tat and Gag p24 proteins. Both of the HIV-1 proteins have been reported to be critical in the virus life cycle and are being evaluated in clinical trials as vaccine candidates. Anionic nanoparticles were prepared using emulsifying wax as the oil phase and Brij 78 and sodium dodecyl sulfate as the surfactants. The resulting nanoparticles were coated with Tat and were demonstrated to produce superior immune responses after administration to BALB/c mice compared to Tat adjuvanted with Alum. Similarly, cationic nanoparticles were prepared using emulsifying wax and Brij 78 and cetyl trimethyl ammonium bromide as the surfactants. The cationic nanoparticles were investigated for delivery of immunostimulatory adjuvants, namely three Toll-like receptor ligands, for obtaining synergistic enhancements in immune responses to a model antigen, Ovalbumin (OVA). In vitro and in vivo studies were carried out to elucidate possible mechanisms by which nanoparticles may result in enhancements in immune responses. In vitro studies were carried out to evaluate the uptake of nanoparticles into dendritic cells and to assess the release of pro-inflammatory cytokines from dendritic cells in the presence of nanoparicles. In vivo studies were carried out using a MHC class I restricted transgenic mouse model to investigate the potential for nanoparticles coated with OVA to enhance presentation of the protein to CD8+ T cells compared to OVA alone. Finally, the preparation of nanoparticles with a low amount of surface chelated nickel for high affinity binding to histidine-tagged (his-tag) proteins was investigated. It was hypothesized that this strengthened interaction of his-tag protein to the nickel chelated nanoparticles (Ni-NPs) would result in a greater uptake of antigen in vivo; therefore, enhanced immune responses compared to protein bound to anionic nanoparticles. In vivo evaluation of his-tag HIV-1 Gag p24 bound to Ni-NPs resulted in enhanced immune responses compared to protein either adjuvanted with Alum or coated on the surface of nanoparticles.

La candidose oropharyngée (COP) constitue l’infection fongique opportuniste la plus fréquente chez les patients infectés au VIH-1. Malgré la profonde immunosuppression causée par le VIH-1, l’infection à Candida albicans demeure confinée au niveau de la muqueuse buccale sans dissémination aux organes profonds. La souris transgénique (Tg) CD4C/HIVMut exprimant le génome tronqué du VIH-1 présente, suite à l’inoculation orale de C. albicans, une COP chronique reproduisant fidèlement l’infection chez les patients séropositifs. Cette souris Tg a donc été utilisée afin de déterminer si les macrophages contribuent au confinement de C. albicans à la muqueuse buccale. Cette étude a permis de démontrer que i) les macrophages sont recrutés aux muqueuses buccale et gastrique en réponse au champignon malgré l’expression du transgène, ii) les macrophages de ces souris Tg présentent une polarisation vers un phénotype d’activation alternative et iii) la production de monoxyde d’azote par les macrophages des souris Tg n’est pas requise pour limiter la prolifération de Candida à la muqueuse buccale et pour restreindre sa dissémination aux organes profonds. Les macrophages ne semblent donc pas directement responsables de l’établissement de l’infection chronique à Candida chez la souris Tg CD4C/HIVMut. / Oropharyngeal candidiasis (OPC) is the most frequent opportunistic fungal infection among HIV-infected patients. Despite the profound immunosuppression caused by HIV-1, Candida albicans infection is limited to the oral epithelium and rarely disseminates to deep organs. The CD4C/HIVMut transgenic (Tg) mice, which expresses the truncated HIV-1 genome, developed a chronic OPC after oral inoculation with C. albicans that closely reproduces infection in seropositive patients. Here, we used this Tg mouse to investigate the contribution of macrophages in limiting candidiasis to the oral mucosa. This study shows that i) macrophages are recruited to the oral and gastric mucosa in response to C. albicans despite transgene expression, ii) the macrophages of this Tg mouse exhibited a polarization toward an alternatively activated phenotype and iii) nitric oxide production by these macrophages is dispensable for limiting chronic oral carriage and for preventing systemic dissemination of the fungi in these Tg mice. Overall, these result indicate that macrophage do not directly determine the susceptibility to chronic carriage of Candida in these CD4C/HIVMut Tg mice.

The ras genes, including Harvey ras (H-ras) and Kirsten ras (K-ras), were among the first oncogenes discovered, and are the most commonly mutated oncogenes in human cancer. The H-ras and K-ras proteins are 85% identical and share considerable functional overlap. However, there is increasing evidence for functional differences between the two proteins that may impart different properties to tumors arising from mutations in these two genes. To study the functional differences between H-ras and K-ras in an in vivo setting, we used two different transgenic mouse tumor models, MMTV-H-ras and MMTV-K-ras mice. The MMTV-H-ras mice were originally developed in Dr. Leder's lab and have been well characterized with regard to tumor properties. We created a similar line of transgenic mice expressing mutant K-ras (G12V) under the control of the MMTV promoter. Female mice of both lines develop primarily mammary tumors. We compared differences between the H-ras and K-ras lines with regard to age of tumor onset, rate of tumor growth, and rates of tumor proliferation and apoptosis. The tumors were also characterized by microarray analysis to look for genes that are differentially expressed in the two tumor types. Finally, the response of tumors to two common chemotherapeutic agents, doxorubicin and taxol, was also measured. We found that tumors in the MMTV-H-ras and MMTV-K-ras mice were similar with respect to several tumor properties, including age of onset, histopathology, and proliferation and apoptotic indices. While tumors from mice of these two genotypes clustered separately in an unsupervised analysis of gene expression profiles, the differentially expressed genes did not fall within any well-defined signaling pathways. However, drug studies indicated differences in response to doxorubicin between the two isoforms, with H-ras tumors responding better than K-ras tumors. In conclusion, our studies point to specific differences between H-ras and K-ras that may represent novel signaling pathways not currently known to be regulated by Ras. In spite of the few differences in properties of tumors arising from H-ras and K-ras mutation, there might be differences in response to chemotherapeutic agents that could have clinical significance.

p53 is an important tumor suppressor gene which is mutated in ~50% of all human cancers. Some of the p53 mutants appear to have acquired novel functions beyond merely losing wild-type functions. To investigate these gain-of-function effects in vivo, we interbred MMTV-v-Ha-ras transgenic mice to either p53-/- knock-out mice or p53R172H/+ knock-in mice to generate mice of three different genotypes: MMTV-ras, MMTV-ras/p53-/-, and MMTV-ras/p53R172H/R172H. Male mice of each of these genotypes were characterized with regard to age of salivary tumor onset and the tumors were characterized with regard to mean growth rates, proliferation fraction, apoptotic levels, and tumor histopathology, as well as responses to doxorubicin treatment. Microarray analysis was also performed to profile gene expression.The MMTV-ras/p53-/- and MMTV-ras/p53R172H/R172H mice display similar properties in age of tumor onset, tumor growth rates, and tumor histopathology, as well as response to doxorubicin. However, a subset of genes show differential expression between the two groups of tumor , and do not appear to be regulated by wild-type p53. At the same time, the MMTV-ras/p53R172H/R172H and MMTV-ras/p53+/+ tumors share similar expression levels of a group of genes that are differentially expressed in the MMTV-ras/p53-/- tumors. Thus, the gain-of-function effects may be caused in part by perturbed regulation of genes not normally regulated by wild-type p53, in addition to imbalances in the regulation of normal p53 target genes.

La Neuropathie à Axones Géants (NAG) est une maladie neurodégénérative rare et fatale caractérisée par une détérioration du système nerveux central et périphérique, impliquant les fonctions motrices et sensorielles. La détérioration massive du système nerveux est accompagnée d'une désorganisation générale des Filaments Intermédiaires ce qui la différencie de nombreuses maladies neurodégénératives où seuls les neurofilaments(NFs) sont affectés. La protéine déficiente, la gigaxonine, est la sous-unité d'une ubiquitine ligase E3, responsable de la reconnaissance spécifique des substrats MAP1B, MAP1S et TBCB, seuls connus à ce jour.Dans le but d'étudier le rôle de la gigaxonine sur la survie neuronale, la désorganisation du cytosquelette et d'avoir un modèle animal suffisamment fort pour envisager des tests thérapeutiques, j'ai caractérisé un modèle murin de NAG. Pour ce faire, j'ai réalisé une étude comportementale des fonctions motrices et sensorielles ainsi qu'une étude histopathologique. Les souris NAG (129/SvJ) développent un phénotype moteur modéré dès 60 semaines alors que les souris NAG (C57BL/6) présentent un phénotype sensoriel dès 60 semaines. Les données histopathologiques ne présentent pas de mort neuronale mais les NFs sont sévèrement altérés. Les NFs sont plus abondant, leur diamètre est augmenté et leur orientation hétérogène, comme c'est observé chez les patients NAG.Nos résultats montrent que l'absence de gigaxonine induit un phénotype moteur et sensoriel modéré mais par contre reproduit la désorganisation massive des NFs observée chez les patients. Ce modèle va nous permettred'étudier le rôle de la gigaxonine, une ligase E3, sur l'organisation des NFs et ainsi comprendre les processus pathologiques impliqués dans d'autres maladies neurodégénératives caractérisée par une accumulation des NFs et un dysfonctionnement du système ubiquitine-protéasome comme les maladies d'Azheimer, de Parkinson etd'huntington ou la sclérose latérale amyotrophique. / Giant Axonal Neuropathy (GAN) is a rare and fatale neurodegenerative disorder characterized by a deterioration of the peripheral and central nervous system. The broad deterioration of the nervous system is accompanied with a general disorganization of the Intermediate Filaments which makes it different from other neurodegenerative disorders wherein only neurofilaments (NFs) are affected. The defective protein, gigaxonin, is the substrate adaptator of an E3 ubiquitin ligase, in charge of the specific recognition of MAP1B, MAP1S and TBCB. In order to study the role of gigaxonin on neuronal survival, the cytoskeleton disorganization and to have a relevant GAN animal model to evaluate efficacy of GAN treatments, I have characterized a GAN mouse model. I did a motor and sensory behavioural study and an histopathologic study. The GAN mice (129/SvJ) shown mild motordeficits starting at 60 weeks of age while sensory deficits were evidenced in C57BL/6 GAN mice. No apparent neurodegeneration was evidenced in GAN mice, but dysregulation of NFs was massive. NFs were more abundant, they shown the abnormal increased diameter and misorientation that are characteristics of the human pathology. Our results show that gigaxonin depletion induces mild motor and sensory deficits but recapitulates the severe NFs dysregulation seen in patients. Our model will allow us to study the role of the gigaxonin-E3 ligase in organizing NFs and understand the pathological processes engaged in other neurodegenerative disorders characterized by accumulation of NFs and dysfunction of the Ubiquitin Proteasome System, such as Amyotrophic Lateral Sclerosis, Huntington's, Alzheimer's and Parkinson's diseases.