Damage to the central nervous system (CNS) leads to the generation of an immune response which culminates with the encapsulation of the damaged area. The encapsulation, known as a glial scar, essentially breaks neural signal pathways and blocks signal transmissions to and from the CNS. The effect is the loss of motor and sensory control for the damaged individual. One method that has been used successfully to treat this problem is the use of a brain-machine interface (BMI) which can intercept signals from the brain and use these signals to control a machine. Although there are many types of BMI devices, implantable devices show the greatest promise with the ability to target specific areas of the CNS, with reduced noise levels and faster signal interception, and the fact that they can also be used to send signals to neurons. The largest problem that has plagued this type of BMI device is that the materials that have been used for their construction are not chemically resilient, elicit a negative biological response, or have difficulty functioning for extended periods of time in the harsh body environment. Many of these implantable devices experience catastrophic failure within weeks to months because of these negative factors. New materials must be examined to advance the future utilization of BMI devices to assist people with CNS damage or disease. We have proposed that two semiconductor materials, cubic silicon carbide (3C-SiC) and nanocrystalline diamond (NCD), which should provide solutions to the material biocompatibility problems experienced by implantable BMI devices. We have shown in this study that these two materials show chemical resilience to neuronal cellular processes, and we show evidence which indicates that these materials possess good biocompatibility with neural cell lines that, in the worst case, is comparable to celltreated polystyrene and, in most cases, even surpasses polystyrene. We have utilized 3C-SiC within an electrode device and activated the action potential of differentiated PC12 cells. This work details our initial efforts to modify the surfaces of these materials in order to improve cellular interaction and biocompatibility, and we examine our current and future work on improving our implantable BMI devices.

Les conditions du corps humain ne sont pas reproduites fidèlement par la culture cellulaire traditionnelle en 2D. Dans cette thèse, des cultures cellulaires 3D sont réalisées dans une plateforme microfluidique hautement intégrée. Des cellules mammifères adhérentes sont encapsulées dans des gouttes immobilisées dans un tableau de pièges capillaires à haute densité. Dans chaque goutte, les cellules se réorganisent pour former un unique microtissu 3D et fonctionnel appelé sphéroïde. L'utilisation d'un hydrogel permet d'alonger le temps de culture et de perfuser le tableau avec des solutions aqueuses, par exemple pour de l'immuno-cyto-chimie. Un unique sphéroïde, viable, peut aussi être extrait de cette puce microfluidique. Des données quantitatives sont extraites à haut débit au niveau de la population, du sphéroïde (dizaines de miliers de sphéroïdes) et au niveau cellulaire emph{in situ} (centaines de miliers de cellules) grâce à de l'imagerie de fluorescence et au dévelopement d'un code d'analyse d'image. Une première preuve de concept a été obtenue en démontrant la viabilité, la prolifération et la fonctionalité de sphéroïdes d'hépatocytes et en les corrélant à des paramètres morphologiques. Ensuite, des aggrégats de cellules souches mésenchymales ont été produits et les hétérogénéités spatiales dans l'expression de protéines impliquées dans leurs propriétés thérapeutiques ont été étudiées. Enfin, cette technologie a été encore dévelopée pour permettre d'appliquer des conditions biochimiques différentes dans chaque goutte. La production et la culture de sphéroïdes dans cette plateforme microfluidique peut mener à des dévelopements importants dans beaucoup de domaines tels que l'analyse de la toxicité des médicaments, le criblage de médicaments à haut débit, le traitement personnalisé du cancer, l'ingénierie tissulaire ou la modélisation de maladies. / Conventional 2D cell culture fails to reproduce emph{in vivo} conditions. In this PhD thesis, 3D cell culture is implemented into a highly integrated microfluidic platform. Adherent mammalian cells are encapsulated in droplets immobilized on a high density array of capillary traps called anchors. In each droplet, the cells reorganize into a single functional 3D microtissue called spheroid. The use of an hydrogel allows to extend the culturing time in microdroplets and to perfuse the array with aqueous solutions, for instance for immuno-cyto-chemistry. A single and viable spheroid can also be selectively retrieved from the microfluidic chip. High throughput and quantitative data is extracted at the population, spheroid (tens of thousands of spheroids) and cellular level emph{in situ} (hundreds of thousands of cells) thanks to fluorescent imaging and a custom image analysis software. As a first proof of concept, the viability, proliferation and functionality of hp sh s were demonstrated and correlated with morphological parameters. Drug toxicity experiments were also performed on this liver model. Then, human mesenchymal stem cell aggregates were produced and the spatial heterogeneities of the expression of proteins involved in their therapeutic properties were investigated. Finally, this technology was further developed to enable applying different biochemical conditions in each droplet. The production and culture of spheroids in this microfluidic platform could lead to major advances in many fields such as drug toxicity, high throughput drug screening, personalized cancer treatment, tissue engineering or disease modeling.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19048号 / 農博第2126号 / 新制||農||1032(附属図書館) / 学位論文||H27||N4930(農学部図書室) / 31999 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 植田 和光, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19759号 / 農博第2155号 / 新制||農||1039(附属図書館) / 学位論文||H28||N4975(農学部図書室) / 32795 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 宮川 恒, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Exercise has been considered a cornerstone of diabetes prevention and treatment for decades, but the benefits of resistance training are less clear. Nineteen non-diabetic subjects (10 metabolic syndrome, 9 sedentary controls) underwent 8 weeks of supervised resistance training. After training, strength and V̇ O2max increased by 10% in both groups. Percent body fat decreased in subjects with the metabolic syndrome. Additionally, lean body mass increased in both groups (p<0.05). Expression of glucose transporter protein-4 (GLUT4), the principle insulin-responsive glucose transporter, increased significantly in both groups. 5-adenosine monophosphateactivated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) expression increased in both groups, indicating increased protein synthesis and mitochondrial biogenesis. Markers of insulin resistance measured by a euglycemic hyperinsulinemic clamp did not improve in subjects with the metabolic syndrome but increased significantly in control subjects (13%). Resistance training upregulates intracellular signaling pathways that may be beneficial for ameliorating the metabolic syndrome.

This thesis will be organized into two chapters discussing the role of hypoxia in the human placenta. The goal of this thesis is to characterize pyruvate kinase M2, mammalian target of rapamycin, mitochondrial function, and cell invasion in hypoxic conditions in the trophoblast. Understanding the mechanisms of placental metabolism can lead to further treatments for placental diseases. Chapter one covers the background of intrauterine growth restriction, hypoxia, placental metabolism, and pyruvate kinase M2 (PKM2). Little is currently understood about the role of the mitochondria in placental diseases. Expression of PKM2, trophoblast cell invasion, and mitochondrial function is shown to be inhibited by hypoxia. PKM2 inhibition decreases trophoblast cell invasion and nuclear expression of PKM2, but increases mitochondrial function. Studying how hypoxia affects the placenta during placental diseases can help clarify the mechanisms by which these diseases occur. Chapter two further characterizes the background of intrauterine growth restriction and hypoxia. It also covers the background of mammalian target of rapamycin. The objective of this chapter was to assess activated mTOR in the trophoblast in hypoxia. Decreased placental and fetal weights, as well as trophoblast cell invasion were observed in hypoxia. A decrease in the activation of mTOR was also found in the hypoxic placenta. This study could provide insight into the physiological relevance of the pathways and could be targeted to help alleviate placental diseases.

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Thesis advisor: Abhishek Chatterjee / The study of protein structure and function has advanced significantly with the development of genetic code expansion (GCE) technology for the incorporation of noncanonical amino acids (ncAAs), revolutionizing synthetic biology by enabling the introduction of novel functionalities into proteins. Within eukaryotic systems, these advancements have paved the way for deeper investigations into complex protein functions critical to human biology and have spurred the development of innovative biotherapeutic solutions.The work described within this dissertation has aimed to further advance various applications of mammalian GCE. This includes the construction of next-generation homogenous antibody-drug conjugates (ADCs) both using a genetically encoded photocaged cysteine and with a dual incorporation system for the construction of a dual-drug conjugate. Multiple new platforms were developed for the incorporation of two or even three ncAAs within a single protein, utilizing a novel aaRS/tRNA pair and evolved hyper-efficient tRNAs. GCE-enabled precise protein modification was also utilized to spectroscopically study the conformational dynamics of dimeric EGFR. Additionally, platforms were established for the precise installation of post-translational modification (PTM) mimics within mammalian proteins, allowing for their programmed activation. Finally, an innovative strategy for the study of protein-protein interactions using genetically encoded photocrosslinkers was developed. Collectively, these efforts have contributed to the development of novel tools for studying protein function in mammalian cells and advancing the creation of new biotherapeutics through GCE technology. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.