Micro and nanofabrication technologies have been used extensively in many biomedical and biological applications. Integration of MEMS technology and biology (BioMEMS) enables precise control of the cellular microenvironments and offers high throughput systems. The focus of this research was to develop three dimensional (3-D) isotropic microstructures for comprehensive analysis on cell-substrate interactions. The aim was to investigate whether the normal and cancerous cells differentially respond to their underlying substrate and whether the differential response of the cells leads to a novel label-free technique to distinguish between normal and cancerous cells. Three different generations of 3-D isotropic microstructures comprised of curved surfaces were developed using a single-mask, single-etch step process. Our experimental model included HS68 normal human fibroblasts, MCF10A normal human breast epithelial cells and MDA-MB-231 metastatic human breast cancer cells. Primary findings on the first generation of silicon substrates demonstrated a distinct adhesion and growth behavior in HS68 and MDA-MB-231 cells. MDA-MB-231 cells deformed while the fibroblasts stretched and elongated their cytoskeleton on the curved surfaces. Unlike fibroblasts, MDA-MB-231 cells mainly trapped and localized inside the deep microchambers. Detailed investigations on cytoskeletal organization, adhesion pattern and morphology of the cells on the second generation of the silicon substrates demonstrated that cytoskeletal prestress and microtubules organization in HS68 cells, cell-cell junction and cell-substrate adhesion strength in MCF10A cells, and deformability of MDA-MB-231 cells (obtained by using AFM technique) affect their behavior inside the etched cavities. Treatment of MDA-MB-231 cells with experimental breast cancer drug, SAHA, on the second generation of substrates, significantly altered the cells morphology, cytoarchitecture and adhesion pattern inside the 3-D microstructures. Third generation of silicon substrates was developed for comprehensive analysis on behavior of MDA-MB-231 and MCF10A cells in a co-culture system in response to SAHA drug. Formation of colonies of both cell types was evident inside the cavities within a few hours after seeding the cells on the chips. SAHA selectively altered the morphology and cytoarchitecture in MDA-MB-231 cells. Most importantly, the majority of MDA-MB-231 cells stretched inside the etched cavities, while the adhesion pattern of MCF10A cells remained unaltered. In the last part of this dissertation, using AFM analysis, we showed that the growth medium composition has a pronounced effect on cell elasticity. Our findings demonstrated that the proposed isotropic silicon microstructures have potential applications in development of biosensor platforms for cell segregation as well as conducting fundamental biological studies. / Ph. D.

L'épilepsie du lobe temporal (ELT) est la forme la plus fréquente de l'épilepsie chez l'adulte. Elle se traduit par des crises spontanées et récurrentes, qui sont résistantes à tout traitement dans 90% des cas. Une agression initiale du cerveau (traumatisme crânien, méningite, convulsions fébriles etc.), est souvent à l'origine de la transformation d'un cerveau « sain » en cerveau épileptique. L'ensemble des processus responsables de cette transition s'appelle l'épileptogenèse. Pouvoir bloquer et/ou retarder l'épileptogenèse chez les patients à risque est une question de santé majeure. En plus des crises, l'ELT soulève d'autres questions. Elle est souvent associée à des déficits cognitifs, qui sont la conséquence de la réorganisation des circuits neuronaux. Ces déficits pourraient être traités de façon indépendante de l'épilepsie elle-même. Le projet de recherche de cette thèse s'inscrit dans ce cadre général. / Temporal Lobe Epilepsy (TLE) is the most common form of epilepsy in adults. It translates into spontaneous and recurrent seizures, which are resistant to any treatment in 90% of cases. An initial brain insult (head injury, meningitis, febrile seizures etc.), is often the cause of the transformation of a "healthy" brain into an epileptic one. The process responsible for this transition is called epileptogenesis. Blocking and/or delaying epileptogenesis in at-risk patients is a key issue for public health. In addition to the seizures, TLE raises other problems. It is often associated with cognitive deficits, which are the result of the reorganization of neuronal circuits. These deficits may be treated independently of epilepsy itself. The work presented here fits into this general framework.

Pubertal timing displays wide, normally distributed variation in a healthy population of sexually maturing adolescents. However, like many complex traits, factors contributing to the variation are not well understood. Epigenetic regulation may contribute to some of the population variation. The role that epigenetics, specifically DNA methylation and histone acetylation, may play in regulating pubertal timing was investigated in C57BL/6 female mice: investigating whether population variation in pubertal timing among inbred mice could be explained by environmental factors; whether perturbing the epigenome using a histone deacetylase inhibitor or methyl-donor would alter pubertal timing; and examining genome-wide methylation patterns in hypothalami of early versus late maturing mice. Results demonstrate that measurable micro-environmental factors have only negligible effects on pubertal timing; pubertal timing was significantly altered by administration of epigenetic modifying agents; differences in methylation patterns are subtle. This initial evidence supports the involvement of epigenetic mechanisms in regulating pubertal timing.

Pubertal timing displays wide, normally distributed variation in a healthy population of sexually maturing adolescents. However, like many complex traits, factors contributing to the variation are not well understood. Epigenetic regulation may contribute to some of the population variation. The role that epigenetics, specifically DNA methylation and histone acetylation, may play in regulating pubertal timing was investigated in C57BL/6 female mice: investigating whether population variation in pubertal timing among inbred mice could be explained by environmental factors; whether perturbing the epigenome using a histone deacetylase inhibitor or methyl-donor would alter pubertal timing; and examining genome-wide methylation patterns in hypothalami of early versus late maturing mice. Results demonstrate that measurable micro-environmental factors have only negligible effects on pubertal timing; pubertal timing was significantly altered by administration of epigenetic modifying agents; differences in methylation patterns are subtle. This initial evidence supports the involvement of epigenetic mechanisms in regulating pubertal timing.

L'enveloppe cellulaire des bactéries à Gram négatif constitue la première ligne de défense contre les antibiotiques. Sous l’effet, d’une part, de la faible perméabilité de la membrane externe qui s'oppose à la pénétration des agents antibactériens, d’autre part des pompes d'efflux qui favorisent leur expulsion, elle empêche nombre de composés potentiellement actifs in vitro d'atteindre leur cible, limitant l’effet antibactérien. Un enjeu important pour restaurer l’activité de ces molécules est de trouver une stratégie pour en augmenter la concentration intracellulaire. L'objectif de cette thèse est de développer des métallodrogues comme nouvelle stratégie de vectorisation de drogues dans les cellules. Cette stratégie repose sur l’association d'une drogue active in vitro, et d’un ligand auxiliaire ayant des propriétés perméabilisantes ou inhibitrices de pompe d’efflux, dans un complexe qui jouera le rôle de chaperone. Les agents antibactériens utilisés sont des inhibiteurs (dérivés d’acides hydroxamiques) de peptide déformylase (PDF) et de méthionine aminopeptidase (MetAP) développés au laboratoire. Tout d’abord, une étude globale de la stratégie de vectorisation a été réalisée (i) étude de stabilité de métallodrogues modèles : en utilisant un acide hydroxamique fluorescent, nous avons montré que, seules, des métallodrogues à Co(III), à la différence de celles à Cu(II) et Fe(III), satisfaisaient aux conditions de stabilité compatibles avec les conditions de tests biologiques. (ii) Etude de la libération de la drogue : nous avons établi par une étude RMN 1H et UV-vis qu’en milieu tampon pH = 7,4, la libération de la drogue se faisait par échange de ligand avec un thiol exogène. Récemment, une nouvelle série d’inhibiteurs de PDF a été synthétisée au laboratoire. Elle est basée sur un squelette hétérocyclique à 5 chaînons fonctionnalisé par une chaîne en C4, puis via un espaceur monocarboné, à un acide hydroxamique. Les meilleurs résultats ont été obtenus avec un oxadiazole (AT002 16 µg/ml sur E. coli en présence de perméabilisant PMBN). Au cours de cette thèse, pour améliorer la lipophilie, des groupements aromatiques ont été fixés sur cet hétérocycle. Les MICs sur la souche d’E. coli sauvage n’ont pas été améliorées mais en présence de PMBN, le dérivé présentant la meilleure activité est le composé AT015 (2 µg/ml sur E. coli en présence de PMBN) qui a donc été choisi pour concevoir des métallodrogues. La métallodrogue réunit autour d’un métal deux parties: (i) un ligand auxiliaire fonctionnalisé via un espaceur par un perméabilisant peptidique analogue de peptide antimicrobien ou par un modulateur de l’efflux (ii) un acide hydroxamique inhibiteur de PDF. Au cours de la SAR réalisée en faisant varier la drogue, le ligand auxiliaire et le métal, nous avons montré que les meilleures métallodrogues permettent d’améliorer l’activité de la drogue sur la souche d’E. coli sauvage d’un facteur 16. Un des ligands auxiliaires fonctionnalisé par un tétrapeptide présente, seul, une activité sur une souche d’E. aerogenes résistante aux fluoroquinolones. Sur ce cas, l’activité biologique a été reliée, par des expériences de mapping par fluorescence, à son accumulation intracellulaire, en utilisant un analogue fluorescent de ce composé. / The gram negative bacterias’ cell envelopes are the first line of defense against antibiotics. First thanks to the low permeability of the external membrane that prevents the penetration of the antibiotics, but also thanks to the efflux pumps that help expelling the antibiotics from the cell. These mechanisms prevent many compounds, potentially active in vitro, from reaching their targets, thus limiting the antimicrobial effect. To increase the molecules’ intracellular concentration is one of the means to restore their activity. This thesis’ objective is to develop metallodrugs as a new drug vectorization strategy in cells. We here associate an active drug in vitro and an auxiliary ligand with permeabilization or efflux pumps inhibition abilities in a complex playing the role of a chaperone. We used peptide deformylase (PDF) and methionine aminopeptidase (MetAP) inhibitors (derived from hydroxamic acids) developed at the laboratory as antimicrobial agents. I’ll begin with a global study of the vectorization strategy we’ve adopted (i) Stability study of the metallodrugs models: using a fluorescent hydroxamic acid, we showed that only Co(III) metallodrugs are in agreement with the stability conditions compatible with the biological tests, in opposition with the Cu(II) and Fe(III) ones. (ii) Drug release study: we showed in 1H NMR and UV-vis studies that in a buffer solution pH 7.4, a ligand exchange with an exogenous thiol is responsible for the drug release. Recently, a new series of PDF inhibitors was synthesized at the laboratory. It is composed of a 5 membered heterocyclic skeleton functionalized by a chain in C4 followed by an hydroxamic acid via a monocarbonated spacer. The best results were obtained with an oxadiazole (AT002 16 µg/ml with E. coli and PMBN as permeabilizing agent). During this thesis, to enhance lipophilicity, we attached aromatic groups on the heterocycle. CMIs on the E. coli strain have not been increased but the compounds displaying the best activity in presence of PMBN (AT015, 2 µg/ml with E. coli and PMBN) was chosen to conceive metallodrugs. The metallodrug is composed of a metal center and two other parts: (i) an auxiliary ligand functionalized via a spacer by a permeabilizing peptide, an antimicrobial peptide analogue, or by an efflux modulator. (ii) An hydroxamic acid PDF inhibitor. We showed that the best metallodrugs enhance the drug activity on the wild E.coli strain by a 16 factor, with the SAR we realized, changing the drug, the auxiliary ligand and the metal. One of the auxiliary ligands functionalized by a tetrapeptide show an activity on a fluoroquinolone-resistant E. aerogenes strain while alone. Utilizing a fluorescent analogous of this compound, we linked the biological activity to its intracellular accumulation with fluorescence mapping experiments.

Pas de résumé en français / The goal of this research was the synthesis and spectroscopic, structural andphysical-chemical characterization of cyclic 6- and 7-membered hydroxamicacids, a tetrahydroxamic calix[4]arene-based tetrapodal receptor, and their metalcomplexes with trivalent and tetravalent metal cations. They were characterizedby several techniques such as 1H and 13C NMR, IR, and mass spectroscopies,single crystal X-ray analysis, and potentiometry. Cyclic hydroxamic acids arefound in a few mix siderophores but their coordination properties were stillunknown. The structural features of metal complexes formed with Fe(III),Ga(III), Ce(IV), Zr(IV), Hf(IV), U(IV) and U(VI) have been investigated both inthe solid state and in solution. The synthesis and complexation studies of anoriginal calix[4]arene-based tetrapodal receptor is described. Reactionparameters for all key steps in the synthetic route have been optimized. Thesingle X-ray crystal analysis of benzyl-protected receptor was obtained.Complexation studies with zirconium(IV) and hafnium(IV) evidenced theformation of two metal two ligand complexes rather than 1:1 species, whichwere shown to interact in solution with a third alkali cation

<p>Predviđena istraživanja u ovoj doktorskoj disertaciji su usmerena u pravcu&nbsp;<br />modifikacije klasičnih metoda i postupaka za transformaciju karboksilne grupe&nbsp;<br />prirodnih i sintetičkih karboksilnih kiselina. Modifikacije su rađene novim,&nbsp;<br />savremenim, ekonomski i ekolo&scaron;ki opravdanim metodama rada u&nbsp;<br />mikrotalasnom reaktoru. Modifikovanim sintetskim postupcima u&nbsp;<br />mikrotalasnom reaktoru urađena je sinteza amida, hidroksamskih&nbsp; derivata,&nbsp;<br />kao i redukcija individualnih naftnih kiselina i&nbsp; sme&scaron;e&nbsp; prirodnih naftnih kiselina&nbsp;<br />&bdquo;Velebit&ldquo; do alkohola. Prirodne naftne kiseline kori&scaron;ćene u ovom radu su prvo&nbsp;<br />izolovane iz gasne frakcije (interval destilacije 168-290 &deg;C) vojvođanske nafte&nbsp;<br />&bdquo;Velebit&ldquo; a potom preči&scaron;ćene i razdvojene na uže frakcije na osnovu različite&nbsp;<br />kiselosti. Nakon toga, izvr&scaron;ena je njihova karakterizacija GC-MS-EI analizom&nbsp;<br />(čime je potvrđeno da dolazi do strukturne diferencijacije kiselina). U radu je&nbsp;<br />takođe ispitana biolo&scaron;ka aktivnost sintetizovanih derivata. Proučavan je uticaj&nbsp;<br />prirodnih naftnih kiselina &bdquo;Velebit&ldquo; i njenih derivata na rast&nbsp; pet sojeva&nbsp;<br />Pseudomonas sp., kao i uticaj odabranih sintetisanih jedinjenja na proliferaciju&nbsp;<br />četiri ćelijske linije humanih tumora pri čemu je kao kontrola služila jedna&nbsp;<br />zdrava humana ćelijska linija.</p> / <p>The investigation of this doctoral dissertation is directed toward the modification of the &nbsp;transformation of the carboxylic group of natural and synthetic carboxylic acids. The dissertation takes into consideration the classical methods and procedures of the reaction and modifies them using microwave reactor. The synthesis of amides, hydroxamic derivatives, as well as the reduction&nbsp; of individual petroleum acids and acid mixtures of natural oil &quot;Velebit&quot; to alcohol were achieved&nbsp; by the modifications made to the synthetic methods in the microwave reactor. The natural oil acids used within this study were first isolated from the gas fraction (distillation interval 168-290 &deg;C) of the Vojvodina&#39;s crude oil &quot;Velebit&quot;, and then purified and separated by the narrow fractions under the different acidity. After that, their characterisation was made by the GC-MS-EI analysis which confirmed that the structural differentiation of&nbsp;&nbsp; acids had been achieved. Also, the biological activity of the synthesized derivatives are analysed. The impact of natural petroleum acids &quot;Velebit&quot; and its derivatives on the growth of five strains of&nbsp; Pseudomonas&nbsp; sp. was studied, as well as the impact of selected synthesized compounds on the proliferation of four human tumor cell lines wherein one healthy human cell lines used as the control.</p>

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