O Vírus Respiratório Sincicial Humano (HRSV) é um dos patógenos mais importantes do trato respiratório. Analisamos as interações das proteínas virais nucleoproteína (N), fosfoproteína (P) e matriz (M) em células HEK-293T. N interage com as proteínas celulares Hsp70, PRMT5 e WDR77; P com Hsp70 e Tropomiosina; e M com Nucleofosmina e Tropomiosina. Cada gene celular foi co-expresso em bactérias com um gene viral possibilitando a co-precipitação das proteínas. Analisamos a interação entre Hsp70 e N ou P, confirmando sua ocorrência em bactérias. Com um conjunto de proteínas mutantes, definimos que as interações são através dos amino terminais de N e P, ou do carboxi terminal de P, e do domínio amino terminal de Hsp70. Superexpressão de Hsp70 por transfecção provocou efeito de estímulo sobre a replicação de HRSV. Imunizações em camundongos com vacinas de DNA para N e P mostraram a indução de resposta celular e humoral. Ensaios de desafio resultaram em redução da carga viral após imunização com N, indicando potencial para sua aplicação em formulação vacinal. / Human respiratory syncytial virus (HRSV) is one of the most important pathogens of the respiratory tract. We analyzed the interactions of viral nucleoprotein (N), phosphoprotein (P) and matrix (M) in HEK-293T. N interacts with the cellular proteins Hsp70, PRMT5 and WDR77; P interacts with Hsp70 and Tropomyosin; and M with Nucleophosmin and Tropomyosin. Each cellular gene was co-expressed with a viral gene in bacteria allowing co-precipitation of proteins. Hsp70 co-expression with N or P proteins confirmed that these interactions also occur in bacteria. Using a set of mutants we found that the N and P amino terminus, P carboxy terminus, and Hsp70 amino terminal domain participate in the interactions. The overexpression of Hsp70 by transfection resulted in stimulation of HRSV replication. Mice immunization with N and P showed that DNA vaccines were capable of inducing humoral and cellular response. In challenge assays it was possible to detect significant virus titer reduction in animals immunized with N, indicating its potential for a vaccine formulation.

Les cellules dendritiques (DC) sont une charnière entre immunité innée et adaptative. Les DC du sous-type CD8a+ excellent à la présentation croisée d’antigènes exogènes pour activer les lymphocytes T (LT) CD8+. Mon projet de thèse consistait à analyser la contribution de gènes sélectivement exprimés par les DC CD8a+ à leur spécialisation fonctionnelle. Nous avons d’abord analysé le rôle du récepteur de chimiokine XCR1 que les DC CD8a;+ expriment spécifiquement. XCR1 reconnait XCL1, une chimiokine qui a un pouvoir attractant sur les DC CD8a+ in vitro, et qui est exprimée par les cellules NK, les lymphocytes innés de type 1 et les LT CD8+ activés. Nous avons montré que XCR1 était indispensable à l’hôte pour résister à une infection par le cytomégalovirus murin, en permettant l’activation optimale des cellules NK. L’axe XCL1-XCR1 est donc un moyen privilégié de communication entre les cellules NK et les DC CD8a+. Ensuite, nous avons utilisé un modèle murin ciblant spécifiquement les DC CD8a+ pour montrer leur rôle majeur dans la réactivation des LT CD8+ mémoires au cours d’infections secondaires. Enfin, nous avons tenté d’analyser si une autre molécule préférentiellement exprimée par les DC CD8a+, la GTPase Rab7b, était impliquée dans la présentation croisée des antigènes. Dans d’autres cellules, Rab7b interviendrait dans l’échange de matériel entre le réseau trans-Golgien et les endosomes tardifs. Dans les DC CD8a+, cette activité pourrait favoriser le trafic intracellulaire des antigènes vers les endosomes où s’effectue la présentation croisée. Mon travail de thèse a permis de mieux comprendre comment les DC CD8a+ activent les lymphocytes effecteurs innés et adaptatifs. / Dendritic cells (DC) link innate and adaptive immunity. The CD8α+ DC subtype is particularly efficient in activating CD8+ T cells through cross-presentation of exogenous antigens. The aim of my PhD project was to analyze the role of genes specifically expressed by CD8a+ DC in the functional specialization of these cells. First we investigated the role of the chemokine receptor XCR1 in the biology of CD8a+ DC. In mice, XCR1 is a powerful chemo-attractive molecule for CD8a+ DC in vitro. It recognizes XCL1, a chemokine expressed by NK cells, type 1 innate lymphoid cells, and activated CD8+ T cells. We have shown that XCR1 is crucial for the resistance to murine cytomegalovirus (MCMV) infection, allowing optimal activation of NK cells. Thus, the XCL1-XCR1 axis is a privileged way of communication between NK cells and CD8a+ DC. We then used a mouse model which specifically targets CD8a+ DC, to show their major role in the reactivation of memory CD8+ T cells upon diverse secondary infections. Finally, we tried to investigate the potential role in antigen cross-presentation of another molecule selectively expressed by CD8a+ DC, the Rab7b GTPase. ,. Indeed, in another cell type, Rab7b could be involve in material exchange between the trans-golgi network and late endosomes. In CD8a+ DC, this process could favor the intracellular trafficking of endocytosed antigens towards the endosomes where cross-presentation occurs. In summary, my PhD work advanced our understanding of how CD8a+ DC activate innate and adaptive effector lymphocytes

The wide scale use of ZnO nanoparticles (NPs) in the world consumer market has resulted in likelihood of exposure to human beings. The present study was aimed to assess the in vitro and in vivo interactions of ZnO NPs in the mammalian system and to elucidate the possible mechanism of their toxicity. Our in vitro results using human epidermal cells (A431), primary human epidermal keratinocytes and human liver cells (HepG2) demonstrated that cells exposed to ZnO NPs exhibit a decrease in cell viability which was independent of NP dissolution. ZnO NPs also induced oxidative DNA damage as evidenced by an increase in the Fpg sensitive sites. The reactive oxygen species triggered a decrease in mitochondrial membrane potential and an increase in the ratio of Bax/Bcl2 leading to apoptosis through the intrinsic pathway. In addition, ZnO NPs induced phosphorylation of JNK, P38 and P53ser15. The results from our in vivo studies using a mouse model showed that ZnO NPs induce lipid peroxidation, oxidative DNA damage and apoptosis in liver which further confirmed our in vitro findings. The data from the present study provide valuable insights into the cellular interactions of ZnO NPs and the underlying molecular mechanism of their toxicity. The results also stress the need for a comprehensive environmental health and safety assessment of engineered nanomaterials to ensure safer nanotechnology based products.

Les infections nosocomiales liées aux dispositifs médicaux, et plus particulièrement ceux de la perfusion, sont un problème majeur dans le milieu hospitalier. Ces infections sont liées à la présence de biofilm. Pour lutter contre le biofilm, les mesures préventives en hygiène ne sont pas suffisantes. Les recherches se dirigent vers la modification des surfaces des matériaux des dispositifs médicaux: ajout de substances biocides, développement de surfaces antiadhésives par voie chimique ou topographique. L’objectif de cette thèse est de créer des polymères nanostructurés pouvant entrer dans la composition de dispositifs médicaux de la perfusion et de tester leur impact sur l’adhésion bactérienne et le développement du biofilm. Dans un premier temps, la technique de nanostructuration choisie repose sur la réplication d’un moule nanostructuré en alumine nanoporeuse qui se caractérise par des nanopores auto-organisés en nid d’abeille. Après avoir mis en place une station d’anodisation permettant la nanostructuration de ce moule, la reproductibilité du procédé de fabrication a été validée (diamètre des pores : 51 ± 6 nm, profondeur: 97 ± 9 nm, espace interpores: 102 ± 6 nm). Ensuite, les travaux de réplication ont été effectués avec le polymère ABS (acrylonitrile-butadiène-styrène). Plusieurs méthodes de réplication ont été testées à partir de dépôt de solutions de polymères ou de fonte du matériau sur le moule d’alumine. La méthode sélectionnée sur des critères de reproductibilité et de facilité de transposition industrielle donne des nanostructures de type nanopicots (diamètres des picots : 56 ± 7 nm, distances interpicots : 101 ± 16 nm, longueurs : 73 ± 33 nm). Les surfaces développées sont ensuite caractérisées (MEB, DSC analyse calorimétrique différentielle, spectrométrie Infra Rouge, angle de contact). La fabrication des nanostructures ne semble pas dégrader le matériau ABS et la modification topographique rend la surface plus hydrophile. Une étude de stabilité montre que les nanostructures résistent à plusieurs modes de stérilisation (oxyde d’éthylène, plasma H2O2 et rayon Beta) et sont conservés dans le temps, ce qui les rend applicables à la surface d’un dispositif médical. La seconde étape du travail consiste à évaluer l’adhésion bactérienne sur les surfaces témoins et nanostructurées. Différents tests de culture de biofilm ont été réalisés avec S. epidermidis en conditions statique ou dynamique. Après un temps de 3 à 48h, les bactéries sont décrochées de la surface puis dénombrées sur gélose. Il n’y a pas de différence significative d’adhésion bactérienne entre les deux types de surface. L’observation en microscopie électronique à balayage et confocale à 24h semble confirmer ce résultat. Des tests réalisés avec d’autres souches bactériennes (S. aureus, K. pneumoniae, P. aeruginosa) en condition statique montrent également que l’adhésion est également identique sur les deux surfaces. Par conséquent, nous pouvons conclure que nos surfaces ABS développées avec ces nanopicots spécifiques n’ont pas un effet anti-adhésion sur les bactéries testées. Des recherches récentes mettent en évidence que l’espacement entre les nanopciots est un facteur critique sur l’adhésion bactérienne. L’étape suivante de notre travail consiste à tester de nouvelles nanostructures réalisées avec un moule AAO ayant une distance interpore plus grande. / Medical device-related infections are a public health concern and an economic burden. The role of biofilms in medical device-related infections is clearly established. Preventive hygiene measures are not often sufficient to prevent biofilms formation. One promising way of preventing device-related infections is the development of medical devices with surfaces or materials that reduce either microbial viability using biocidal substances or microbial adhesion with topographical modifications.Developing nanostructured polymeric surfaces, which could have applications in medical devices, and testing their impact on bacterial adhesion and biofilm development were the main goals of this thesis. First of all, the polymer was replicated on an aluminum anodized oxide nanostructured mold (AAO), characterized by highly ordered nanopores. An anodization station was made in order to create molds. Then, the reproducibility of the process fabrication was validated (pore diameter: 51 ± 6 nm, deepness 97 ± 9 nm, interpore espace: 102 ± 6 nm). Several replication techniques with ABS were tested including polymers solutions and melted polymers. The selected method was the one with the most reproducible results pillar diameter: 56 ± 7 nm, interpillar distance: 101 ± 16 nm, length: 73 ± 33 nm) and the most representative of industrial injection processes. The created surfaces were then characterized (MEB, DSC, ATR-FTIR, wettability). The fabrication process does not seem to degrade the ABS material and the topographical change increases the hydrophilicity of the surface. A stability study showed that the nanopillars were resistant to several sterilization processes (ethylene oxide, H2O2 plasma, Beta irradiation) and were maintained through time, which is an important element for applications in medical-devices.The second step of our work consisted of assessing bacterial adhesion on control and nanostructured ABS samples. Several biofilm tests were made with S. epidermis in static and dynamic conditions. Between 3 and 48 hours of culture, bacteria were removed from the surfaces and then viable plate counting was performed. No significant differences were observed between the samples. Microscopic observations (MEB, CSLM) seemed to confirm this result. Other bacteria with different morphologies were tested (S. aureus, K. pneumoniae, P. aeruginosa): bacterial adhesion was similar for the two surfaces. Therefore, we can conclude that our developed ABS surfaces with these specific nanopillars do not have an anti-adhesion effect on the tested bacteria. Recent researches showed that spacing between nanopillars is a critical factor on bacterial adhesion. The following step of our work would be to test new nanostructures using AAO molds with bigger interpore distance.

The broad aim of the work described in this thesis was to use the arbuscular mycorrhizal (AM) defective rmc tomato to explore the development and function of different types of fungus-plant interfaces (phenotypes) and to characterize the cellular modifications preceding colonization of rmc by a range of different AM fungi. Three main patterns of colonization with rmc have been described: 1) Pen- phenotype in which the AM fungus is restricted to the root surface with several attempts to penetrate the epidermal cells without success; 2) Coiphenotype where AM fungi penetrate the epidermis but cannot develop cortical colonization; and 3) Myc+ phenotype (with G. intraradices WFVAM23), where the AM fungus penetrates the cortex and forms a “normal” colonization after a delayed penetration of the epidermal cells (Review of literature). Little is known about cellular interactions, nutrient transfer or the ability of the fungi to complete their life cycles in the different phenotypes. These aspects were the main foci of this work. In addition further fungal isolates were screened to asses their ability to colonize rmc. The first experiments involved compartmented pots to follow the fungal life cycle, production of external mycelium and spores in the different rmc phenotypes (Chapter 3). The results showed that in the Pen- and Coiphenotypes, AM fungi are unable to form spores to complete the life cycle. However, in the Coi-phenotype, the fungus remained alive up to week 18, suggesting that some C transfer occurred. The fungus forming the Myc+ phenotype, G. intraradices WFVAM23, was able to produce spores, although they were significantly smaller than those produced with the wild-type tomato. The results suggested that arbuscules are essential for completion of the fungal life cycle. Labeled 32P was used to determine whether arbuscules are also essential for P transfer (Chapter 4). A compartmented pot system was used in which only fungal hyphae but not roots could obtain 32P. 32P was found in the shoots of rmc inoculated with S. calospora (Coi- phenotype), indicating that interfaces other than arbuscules can be involved in transfer of P. A nurse pot system was used to obtain synchronized colonization to determine how long AM fungi stay alive during the interactions with rmc and to elucidate the cellular modifications preceding colonization of rmc by a range of different AM fungi (Chapter 5). The results showed that rmc did attract the AM fungi, that the plant nucleus moved to the middle of the plant cell only after fungal penetration of plant roots and that callose deposition in rmc was not involved in blocking the AM fungi. Fourteen AM fungi with different taxonomic affiliations and fourteen different G. intraradices isolates were screened to try to relate phylogeny of AM fungi with phenotypes in rmc (Chapter 6). There were a large number of interactions, depending on the inoculated AM fungi, and although there were some similarities in the rmc phenotypes within phylogenetic groups, there was no clear relationship between phylogeny and development of interactions with rmc. This study showed the following. 1) Arbuscules/arbusculate coils are necessary for the completion of the AM fungal cycle. However, intraradical hyphae also participate in transfer of both P and C as demonstrated with the Coi- phenotype. 2) rmc clearly attracted AM fungi and the fungi stay alive and induce plant cellular responses such as nuclear movement only after penetrating rmc roots. 3) Plant defense responses such as callose deposition are not involved in blocking AM fungi in rmc; and 4) there was no relationship between the phenotypes described in rmc and phylogeny of the Glomeromycota. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1292816 / Thesis(Ph.D.)-- School of Earth and Environmental Sciences, 2007.

Ce projet explore les mécanismes de la morphogenèse des glandes mammaires, comme modèle de progression du carcinome du sein. La morphogenèse de la glande mammaire résulte de la coordination de réponses cellulaires distinctes (prolifération, différenciation, motilité, invasivité, apoptose) régulées par de nombreuses voies, y compris Wnt, EGF, FGF, Notch, SHH, Myc et l'activation hormonale. Nous estimons qu'il est essentiel d'analyser individuellement l'impact de ces voies dans la modulation de la prolifération, de la différenciation, de la motilité, de l'invasivité, de l'apoptose, de la cohésion intercellulaire et de la polarité dans les cellules impliquées dans une migration morphogénétique cohérente.Nous avons développé des modèles en 3D améliorés pour analyser l'impact d'EMT-TF dans un environnement 3D. Notre système permet de surveiller simultanément les voies mentionnées au niveau cellulaire pendant trois semaines, une période ajustée pour tester les médicaments de chimiothérapie. Notre premier modèle décrit l'émergence primaire des cellules envahissantes de carcinome mammaire de l'épithélium mammaire. Les cellules sont traitées avec des médicaments définis ou seront transfectées avec diverses constructions (en cours de validation) améliorant ou réprimant des voies spécifiques telles que Slug, en plus des constructions permettant de suivre l'évolution des structures cellulaires par marquage GFP en vidéomicroscopie. / This project explores the mechanisms of mammary gland morphogenesis, as a model for breast carcinoma progression. Mammary gland morphogenesis results from the coordination of distinct cell responses (proliferation, differentiation, motility, invasiveness, apoptosis) integrated by numerous pathways, including Wnt, EGF, FGF, Notch, SHH, Myc and hormonal activation. For the purpose of this study, we feel it is critical to analyze individually the impact of theses pathways in modulating proliferation, differentiation, motility, invasiveness, apoptosis, intercellular cohesion, and polarity in cells involved in a coherent morphogenetic migration.We have designed improved 3D models to analyze the impact of EMT-TF in a 3D environment. Our system allows monitoring simultaneously the mentioned pathways at a cellular level for three weeks, a period adjusted to test chemotherapy drugs.Our first model describes the primary emergence of invading breast carcinoma cells from mammary epithelium. Cells are treated with defined drugs or will be transfected with various constructs (under validation) enhancing or repressing specific pathways such as Slug, in addition to constructs allowing the monitoring of cell structures by GFP labeling for video microscopy..

Le muscle squelettique a la capacité de se régénérer suite à une lésion grâce aux cellules satellites qui sont les cellules souches du muscle. Après dommage musculaire, les cellules satellites s’activent, prolifèrent, se différencient et fusionnent afin de reformer le muscle lésé. Cependant les cellules myogéniques ne sont pas les seules cellules impliquées dans la régénération musculaire. Des études précédentes réalisées au laboratoire ont montré chez la souris que les macrophages sont des cellules essentielles à la régénération musculaire. En effet, peu de temps après un dommage musculaire, les monocytes infiltrent le tissu lésé et se différencient en macrophages pro-inflammatoires Ly6Cpos (M1). Ces macrophages stimulent la prolifération des myoblastes et inhibent leur fusion. Puis les macrophages pro-inflammatoires changent de phénotype et deviennent des macrophages anti-inflammatoires Ly6Cneg (M2) qui stimulent la différenciation des myoblastes et les protègent de l’apoptose. Ainsi, en fonction de leur phénotype, les macrophages exercent des rôles trophiques séquentiels sur les myoblastes tout au long du processus de régénération musculaire. Dans la première partie de notre étude, nous montrons in vitro que les macrophages humains soutiennent les différentes étapes de la myogenèse. Les macrophages M1 sont fortement attirés par les myoblastes. De plus ils stimulent la prolifération des myoblastes et inhibent leur fusion. Les macrophages M2 attirent les myoblastes et stimulent leur différenciation permettant ainsi la formation de larges myotubes. En utilisant des anticorps bloquants spécifiques, nous avons identifié plusieurs molécules sécrétées par les macrophages régulant la myogenèse chez l’homme. Nos analyses in vivo chez l'homme confirment nos résultats obtenus in vitro. En effet, les macrophages M1 sont préférentiellement associés aux aires de régénération contenant des myoblastes non différenciés alors que les macrophages M2 sont associés aux aires de régénération contenant des myoblastes en différenciation. Dans un contexte de myopathie dégénérative, nous avons montré que les macrophages adoptent des phénotypes et des fonctions totalement différents des macrophages présents au cours de la régénération musculaire. Nous avons observé chez la souris et chez l’homme, que les macrophages exprimant des marqueurs M1 sont associés à la fibrose et qu’un traitement anti-inflammatoire réduit leur nombre dans le muscle dystrophique murin. Par isolement spécifique et cocultures ex vivo, nous avons montré qu'au cours de la régénération musculaire, les macrophages Ly6Cneg stimulent la production de collagène par les fibroblastes. A l'inverse au cours des myopathies dégénératives, ce sont les macrophages Ly6Cpos qui stimulent fortement l’établissement de la fibrose en agissant directement sur les fibroblastes. De plus, ces macrophages Ly6Cpos, qui régulent négativement les fibroblastes au cours de la régénération musculaire, stimulent la différenciation des fibroblastes et myofibroblastes dans les myopathies. De plus, ils les protègent de l'apoptose, participant ainsi à la persistance de ces cellules fibrosantes. Ainsi, nous avons confirmé chez l’homme in vitro et in vivo, le rôle de support séquentiel des macrophages au cours de la régénération musculaire. De plus, nous avons identifié différents effecteurs sécrétés par les macrophages M1 et M2 impliqués dans la régulation du processus myogénique chez l'adulte. Nous avons également montré que lors des myopathies dégénératives et au cours de la régénération musculaire, les macrophages adoptent un phénotype et des fonctions totalement différents, avec notamment un rôle profibrotique des macrophages pro-inflammatoires. / Skeletal muscle has the ability to regenerate after a chemical or physical injury thanks to satellite cells, the muscle stem cells. After damage, satellite cells proliferate, differentiate and fused to reform muscle. Myogenic cells are not the only on cells involved. Previous studies in the laboratory showed that, in mice, macrophages are crucial for skeletal muscle regeneration. Soon after an injury, macrophages infiltrate damaged muscle and differentiate into Ly6Cpos pro-inflammatory (M1) macrophages. They stimulate proliferation of myoblasts and inhibit their fusion. Then, pro-inflammatory macrophages skew towards a Ly6Cneg anti-inflammatory phenotype (M2). Anti-inflammatory macrophages stimulate differentiation of myoblasts and protect them from apoptosis. Thus, depending on their phenotype, macrophages exert sequential trophic roles on myoblasts throughout muscle regeneration. Here, we showed in vitro that human macrophages also support different steps of myogenesis. M1 macrophages are strongly attracted by mpcs. Moreover, they secrete molecules, which stimulate proliferation of mpcs and inhibit their fusion. M2 macrophages attract mpcs and stimulate differentiation of mpcs in order to form large myotubes. Using specific blocking antibodies, we identified molecules involved in the regulation of myogenesis by M1 and M2 macrophages in a human in vitro system. In vivo analysis of regenerating human muscle sections confirmed our results obtained in vitro. M1 macrophages are preferentially associated with proliferating myogenic cells while M2 macrophages are associated with differentiating myogenic cells. In degenerative myopathies, we showed that macrophages are completely different from those present during skeletal muscle regeneration. We observed in mouse and human that M1 marker-expressing macrophages are associated with fibrosis while anti-inflammatory treatment reduced this population, in association with an improvement of the dystrophic muscle. Isolated Ly6Cneg macrophages exhibit a mixed M1/M2 phenotype. In ex-vivo coculture experiments, we showed that Ly6Cpos macrophages strongly favor establishment of fibrosis by directly acting on fibroblasts while in regenerating muscle, these Ly6Cpos macrophages negatively regulate fibrosis. To resume, we confirm in human the supportive sequential roles of macrophages during skeletal muscle regeneration in vitro and in vivo. Moreover, we identified effectors secreted by M1 and M2 macrophages involved in the regulation of the myogenic process. We also highlight that during muscle regeneration and in degenerative myopathies, macrophages exhibit different phenotype associated with opposite functions, with a pro-fibrotic role for pro-inflammatory macrophages.

Retinal ganglion cells (RGCs) serve as a vital connection between the eye and the brain with damage to their axons resulting in loss of vision and/or blindness. Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, providing a valuable model of RGC development in vitro. The working hypothesis of these studies is that hPSC-derived RGCs are capable of extensive outgrowth and display target specificity and pathfinding abilities. Initial efforts focused on characterizing RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner expressing a compliment of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful to investigate and model the extensive axonal outgrowth necessary to reach post-synaptic targets. As such, additional efforts aimed to elucidate factors promoting axonal outgrowth. Results demonstrated significant enhancement of axonal outgrowth through modulation of both substrate composition and growth factor signaling. Furthermore, RGCs possessed guidance receptors that are essential in influencing outgrowth and pathfinding. Subsequently, to determine target specificity, aggregates of hPSC-derived RGCs were co-cultured with explants of mouse lateral geniculate nucleus (LGN), the primary post-synaptic target of RGCs. Axonal outgrowth was enhanced in the presence of LGN, and RGCs displayed recognition of appropriate targets, with the longest neurites projecting towards LGN explants compared to control explants or RGCs grown alone. Generated from the fusion of regionally-patterned organoids, assembloids model projections between distinct regions of the nervous system. Therefore, final efforts of these studies focused upon the generation of retinocortical assembloids in order to model the long-distance outgrowth characteristic of RGCs. RGCs displayed extensive axonal outgrowth into cortical organoids, with the ability to respond to environmental cues. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC development, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing outgrowth as well as modeling long-distance projections and pathfinding abilities.

Mesoporous silica nanoparticles (MSNPs) possess large surface areas and ample pore space that can be readily modified with specific functional groups for targeted binding of bioactive materials to be transported through cellular barriers. Engineered silica nanoparticles (ESNP) have been used extensively to deliver bio-active materials to target intracellular sites, including as non-viral vectors for nucleic acid (DNA/RNA) delivery such as for siRNA induced interference. The reverse process guided by the same principles is called “nanoharvesting”, where valuable biomolecules are carried out and separated from living and functioning organisms using nano-carriers. This dissertation focuses on ESNP design principles for both applications. To investigate the bioactive materials loading, the adsorption of antioxidant flavonoids was investigated on titania (TiO2) functionalized MSNPs (mean particle diameter ~170 nm). The amount of flavonoid adsorbed onto particle surface was a strong function of active group (TiO2) grafting and a 100-fold increase in the adsorption capacity was observed relative to nonporous particles with similar TiO2 coverage. Active flavonoid was released from the particle surface using citric acid-mediated ligand displacement. Afterwards, nanoharvesting of flavonoids from plant hairy roots is demonstrated using ESNP in which TiO2 and amine functional groups are used as specific binding sites and positive surface charge source, respectively. Isolation of therapeutics was confirmed by increased pharmacological activity of the particles. After nanoharvesting, roots are found to be viable and capable of therapeutic re-synthesis. In order to identify the underlying nanoparticle uptake mechanism, TiO2 content of the plant roots was quantified with exposure to nanoparticles. Temperature (4 or 23 °C) dependent particle recovery, in which time dependent release of ESNP from plant cells showed a similar trend, indicated an energy independent process (passive transport). To achieve the selective separation and nanoharvesting of higher value therapeutics, amine functionalized MSNPs were conjugated with specific functional oligopeptides using a hetero-bifunctional linker. Fluorescence spectroscopy was used to confirm and determine binding efficiency using fluorescently attached peptides. Binding of targeted compounds was confirmed by solution depletion using liquid chromatography–mass spectrometry. The conjugation strategy is generalizable and applicable to harvest the pharmaceuticals produced in plants by selecting a specific oligopeptide that mimic the appropriate binding sites. For related gene delivery applications, the thermodynamic interaction of amine functionalized MSNPs with double-stranded (ds) RNA was investigated by isothermal titration calorimetry (ITC). The heat of interaction was significantly different for particles with larger pore size (3.2 and 7.6 nm) compared to that of small pore particles (1.6 nm) and nonporous particles. Interaction of dsRNA also depended on molecular length, as longer RNA (282 base pair) was unable to load into 1.6 nm particles, consistent with previous confocal microscopy observations. Calculated thermodynamic parameters (enthalpy, entropy and free energy of interaction) are essential to design pore size dependent dsRNA loading, protection and delivery using MSNP carriers. While seemingly diverse, the highly tunable nature of ESNP and their interactions with cells are broadly applicable, and enable facile nano-harvesting and delivery based on a continuous uptake-expulsion mechanism.

<p>Skeletal muscle takes up nearly 40% of total body mass, is critical for daily function by</p> <p>providing balance, supports breathing, movement, and energy expenditure. Preserving</p> <p>skeletal muscle can also significantly improve one’s quality by maintaining balance, movement</p> <p>and improving metabolic health [1, 2]. This becomes more imperative with age, as skeletal muscle mass naturally declines, and further compounds decline in quality of life and health [1, 2]. Thus, it is critical to understand the physiology of skeletal muscle and the underlying cellular and</p> <p>molecular mechanisms that contribute to normal function. Using mouse models to further our</p> <p>understanding, this dissertation leverages single-cell RNA-sequencing (scRNA-seq) to dissect the</p> <p>cellular and molecular underpinnings of skeletal muscle injury and repair. Specifically, chapter 1</p> <p>provides an overview of skeletal muscle structure, muscle regeneration, and the current state of</p> <p>scRNA-seq literature in muscle regeneration. In chapter 2, I will discuss the large-scale scRNAseq of regenerating muscle which identified dynamic population of resident and infiltrating cells. In chapter 3, I will discuss the potential immunomodulatory role of MuSCs and leveraging scRNAseq data to understand the cellular mechanisms that govern successful muscle regeneration. Finally, in chapter 4 I will discuss the role of the transcription factor Sox11, which was identified by scRNA-seq and was specific to differentiating MuSCs. Thus, this dissertation spans the cellular and molecular components of muscle regeneration.</p>