Le glioblastome (GBM) est le cancer du cerveau le plus fréquent et grave chez l’adulte. Son pronostic sombre est en partie dû à la résistance de ces tumeurs aux chimiothérapies. L’une des principales causes de ces résistances est l’incapacité des chimiothérapies à pénétrer dans le cerveau depuis le sang à cause de la barrière hémato-encéphalique (BHE), une spécificité des vaisseaux sanguins cérébraux. Par l’injection de microbulles et l’envoi d’ultrasons dans le cerveau, il est possible d’ouvrir cette BHE pour permettre à des chimiothérapies d’entrer dans le cerveau. Nous avons montré quela chimiothérapie carboplatine gagnait en efficacité lorsqu’elle était injectée après ce procédé. Les GBM peuvent aussi montrer une résistance aux chimiothérapies par des mécanismes génétiques intrinsèques à la tumeur. Nous avons étudié l’expression et l’impact sur le pronostique des patients atteints de GBM des gènes de la famille des transporteurs ABC, dont le membre ABCA13 s’est avéré important. / Glioblastoma (GBM) is the most frequent and severe brain cancer for adults. Its dark prognosis in partly due to the resistance of these tumors to chemotherapies. One of the main causes of these resistances is the incapacity of chemotherapies to enter the brain from the blood circulation because of the bloodbrain barrier (BBB), a specificity of cerebral blood vessels. By the injection of microbubbles and the delivery of ultrasound into the brain, it is possible to open this BBB to allow chemotherapies to enter the brain. We have showed that the chemotherapeutic agent carboplatin gained efficacy when it was injectedafter this procedure. GBM can also exhibit resistance to chemotherapies by genetic mechanisms intrinsic to the tumor. We studied the expression and the impact on prognosis for GBM patients of the genes of the ABC transporters family, which member ABCA13 appeared important.

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Over the last six decades, the field of drug delivery has advanced considerably, from sustained oral release technology to pH-responsive polymers. Innovation in the space has progressed alongside the development of new categories of drugs, as well as improvements in electronics and material science which have enabled new modalities of external stimulation. Nevertheless, the traditional challenges of drug delivery persist, including the need to reduce off-target toxicity, minimize invasiveness of administration, and bypass biological barriers; these challenges are particularly apparent for drug delivery applications in difficult-to-reach areas of the body, such as tumors or areas beyond the blood-brain barrier. Furthermore, as therapeutics become more targeted, the need for corresponding delivery methods becomes even more vital to ensure treatment effectiveness with minimal side effects. In this dissertation, we aim to demonstrate a new strategy for on-demand and localized drug delivery which is easy to fabricate and delivers a large payload relative to device size, is responsive to external stimulation for triggered release, and can be integrated into a system for real-time actuation during a physiological process. In Aim 1, we developed a microfluidic fabrication technique for making biphasic microcapsules loaded with model drug. This method relied on microfluidic droplet methods, with sufficient interfacial tension between two on-chip phases to cause droplet formation. Typically, these systems rely on an aqueous-oil interface for sufficient interfacial tension; to fabricate a biocompatible microcapsule, we formed biphasic microcapsules composed of an aqueous-based inner and outer phase, without an oil intermediate phase, with aqueous two-phase system properties. Additionally, we incorporated on-chip photopolymerization, designing the microfluidic chip and light source to minimize refracted ultraviolet exposure. The resulting drug-loaded microcapsules were stable, with minimal background leakage. This fabrication technique can produce a high-throughput supply of monodisperse microcapsules, which can be modified for a variety of therapeutic payloads and easily injected in targeted region in the body. In Aim 2, we adapted these drug-loaded microcapsules for ultrasound-triggered release. Focused ultrasound (FUS) is a minimally-invasive method of stimulating release from a device, which can penetrate deep within the body and is compatible with a variety of materials; when applied at sufficient intensity and duration, it can induce heating, cavitation, or both. We tuned the applied ultrasound parameters to minimize temperature increases in surrounding tissue phantoms, while inducing step-like release profiles from the microcapsules over the course of multiple cycles of pulsed FUS. Under these applied conditions, we detected acoustic signatures consistent with inertial cavitation and visually observed structural breakdown of the microcapsules corresponding to cavitation-related effects. This release strategy is highly targeted, inducing drug release from microcapsules within a narrow focal area with minimal risk to surrounding tissue. Finally, in Aim 3, we performed in vitro demonstrations of drug-loaded actuators, as initial demonstrations towards a system of integrated sensors, actuators, and adaptive learning algorithms for closed-loop control over physiological processes involved in wound healing. We experimented with both the aforementioned microcapsules and with a liposome-loaded scaffold as drug-loaded actuators, and tested both actuators with three ultrasound transducers which offered a range of portability, intensity ranges, and imaging capacities. Next, we developed in vitro testing setups incorporating the actuators with either a cell monolayer or a three-dimensional cell construct, mimicking a wound site, and validated ultrasound-triggered drug-release with minimal cell damage. To demonstrate cell uptake of the released therapeutic agents, we modified the microcapsules’ payload, performed the in vitro release experiments, and then observed correlating cell response over the following week of culturing. These demonstrations have provided guidance towards a more integrated system, which will validate the impact of the localized actuators in stimulating enhancing wound healing rates. More broadly, the eventual integrated system, incorporating both sensors and the adaptive algorithm, will be able to sense and respond to physiological changes within a wound in real-time. This work explores how wireless, deep-tissue devices coupled with external control modalities will facilitate interventions with high spatiotemporal accuracy; when combined with sensing and regulating algorithms, it will empower real-time monitoring and interventions in physiological processes. Aim 1 focused on the fabrication of such implantable microcapsule devices and Aim 2 demonstrated a method for triggering the devices using an external control modality. In Aim 3, we investigated a use case for these microcapsules to promote rapid wound healing, alongside flexible electronics, sensors, and additional actuators. To provide additional context on implantable microdevices and biocompatibility, we provide a framework for designing medical microrobotics in Appendix I and an application of a thermally-responsive hydrogel coating in Appendix II. Overall, the sum of this work illustrates the potential impact of soft microdevices for localized and on-demand applications, towards a future of spatiotemporally-targeted biological interventions.

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Despite evidence for an association between atrial fibrillation (AF) and cognitive decline and dementia independent of stroke, pathways underlying this relationship remain unclear. Critically, elevated levels of inflammatory markers, common in AF, are associated with the breakdown of the blood-brain barrier (BBB) and may contribute to neuroinflammation and neurodegeneration. To investigate this potential contributing pathway, we estimated associations of inflammatory markers with cognitive decline and dementia in AF adults. We used data from two population-based cohorts and found that inflammatory markers were associated with cognitive decline but not dementia. Some associations were modified by sex and apolipoprotein E (APOE) genotype. These findings provide preliminary evidence for inflammatory-mediated BBB dysfunction as a potential contributing pathway linking AF to cognitive decline. Future work examining the role of BBB dysfunction in AF and cognition may benefit from the use of markers of central inflammation to increase sensitivity, while considering possible differences by sex, dementia subtype, and APOE genotype.

In Drosophila melanogaster oxidative stress (OS) decreases lifespan and motor function (Coulom & Birman, 2004; Hosamani, 2013) through degeneration of dopaminergic (DA) neurons (Brooks et al., 1999). The mitogen-activated protein kinases, P38, c-JUN-NH2 terminal kinase (JNK) and extracellular-signal related kinase (ERK) are activated in response to OS (Apel & Hirt, 2004). My thesis investigated the protective role of up-regulation of the antioxidants superoxide dismutase (Sod) and catalase (Cat) in the glia of Drosophila against oxidative stress induced by paraquat (PQ). Exposure to PQ killed ~20-80% of flies and impacted motor functions as measured in a negative geotaxis assay. Pan-glial expression of Sod2 using Repo-GAL4 did not reduce the lethality caused by PQ exposure. These flies displayed a marked reduction in locomotion even when not exposed to PQ. However, their motor functions were not affected by PQ exposure. Pan-glial expression of Cat was not sufficient to prevent the negative effects of PQ exposure (viability and locomotion). Pan-neuronal expression of Sod2 using Elav-GAL4 protected the locomotive ability but not the lethality caused by PQ exposure. Pan-neuronal up-regulation of Cat protected against both the lethality and motor defects caused by PQ exposure. Over-expression of Sod2 and Cat in all sub-perineurial glial (SPG) cells using NP2276-GAL4 protected the motor function from exposure to PQ. Up-regulation of Sod1 and Sod2 in the SPG cells that form the blood brain barrier (BBB) using Spg Moody-GAL4 protected the motor function but not the lethality caused by PQ exposure. Over-expression of Sod2 in the SPG cells that form the BBB protected DA neurons from the deleterious effects of PQ exposure. A cluster of DA neurons, the paired posterior lateral 1 (PPL1), was identified as important for motor function. In both the parental lines and in flies in which Sod2 was up-regulated at the SPG cells, phospho-JNK and phospho-ERK were detected after 1h, 6h and 24h of PQ exposure by Western blot. Phospho-P38 levels were markedly reduced after 24h exposure to PQ in the parental controls. During all time points of PQ exposure, phosphorylated form of P38 was detected when Sod2 was up-regulated at the BBB. In conclusion, up-regulation of Sod2 in the SPG cells forming the BBB protects DA neurons from PQ exposure, maintains the phosphorylation status of P38, which may ultimately translate into protection of the motor function. It is possible that increased Sod2 expression at the BBB sustains phospho-P38 levels which may play a role in increasing the tolerance of the flies to oxidative stress induced by PQ. / Thesis / Master of Science (MSc)

Bacterial meningitis occurs when blood-borne bacteria are able to penetrate highly specialized brain endothelial cells (BECs) and gain access to the meninges. Neisseria meningitidis (Nm) is a human-exclusive pathogen for which suitable in vitro models are severely lacking. Until recently, modeling BEC-Nm interactions has been almost exclusively limited to immortalized human cells that lack proper BEC phenotypes. Specifically, these in vitro models lack barrier properties, and continuous tight junctions. Alternatively, humanized mice have been used, but these must rely on known interactions and have limited translatability. This motivates the need to establish novel human-based in vitro BEC models that have barrier phenotypes to research Nm-BEC interactions. Recently, a human induced pluripotent stem cell (iPSC) model of BECs has been developed that possesses superior BEC phenotypes and closely mimics the in vivo blood vessels present at the blood-meningeal barrier. Here, iPSC-BECs were tested as a novel cellular model to study Nm-host pathogen interactions, with focus on host responses to Nm infection. Two wild type strains and three mutant strains of Nm were used to confirm that these followed similar phenotypes to previously described models. Importantly, the recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, at distinct time points of infection, and the secretion of IFN γ and RANTES by iPSC-BECs. Nm was directly observed to disrupt tight junction proteins ZO-1, Occludin, and Claudin-5 at late time points of infection, which became frayed and/or discontinuous upon infection. This destruction is preceded by, and might be dependent on, SNAI1 activation (a transcriptional repressor of tight junction proteins). In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability was observed at late infection time points. Notably, bacterial transmigration correlated with junctional disruption, indicating that the paracellular route contributes for bacterial crossing of BECs. Finally, RNA-Sequencing (RNA-Seq) of sorted, infected iPSC-BECs was established through the use of fluorescence-activated cell sorting (FACS) techniques following infection. This allowed the detection of expression data of Nm-responsive host genes not previously described thus far to play a role during meningitidis. In conclusion, here the utility of iPSC-BECs in vitro to study Nm infection could be demonstrated. This is the first BEC in vitro model to express all major BEC tight junctions and to display high barrier potential. Altogether, here this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes and suggests that the paracellular route contributes to Nm traversal of BECs. / Eine bakterielle Meningitis tritt auf, wenn durch Blut übertragene Bakterien hochspezialisierte Hirnendothelzellen (BEC) durchdringen und Zugang zu den Meningen erhalten. Neisseria meningitidis (Nm) ist ein human-exklusiver Erreger, für dessen Untersuchung es an geeigneten In-vitro-Modellen mangelt. Bis vor kurzem war die Modellierung von BEC-Nm-Wechselwirkungen fast ausschließlich auf immortalisierte humane Zellen beschränkt, denen wichtige BEC-Phänotypen fehlen. Besonders hervorzuheben sind das Fehlen physiologischer Barriereeigenschaften durch unkontinuierliche dichte Zell-Zell-Verbindungen. Als alternative Modellorganismen können humanisierte Mäuse verwendet werden, die sich jedoch auf bekannte Wirt-Erreger-Wechselwirkungen stützen und durch Speziesunterschiede eine eingeschränkte Übersetzbarkeit aufweisen. Dies begründet die Notwendigkeit, neuartige humane In-vitro-BEC-Modelle zu etablieren, die physiologische Barrierephänotypen aufweisen, um Nm-BEC-Wechselwirkungen zu untersuchen. Kürzlich wurde ein humanes Modell entwickelt, welches auf aus induziert pluripotenten Stammzellen (iPSCs) abgeleiteten humanen BECs basiert und sich durch einen physiologischen Blut-Hirn-Schranken-Phänotyp auszeichnet. Die iPSC-BECs wurden in dieser Arbeit als neuartiges zelluläres Modell getestet, um Nm-Wirt-Pathogen-Wechselwirkungen zu untersuchen, wobei der Schwerpunkt auf Wirtsreaktionen auf Nm-Infektionen lag. Zwei Wildtypstämme und drei Mutantenstämme von Nm wurden verwendet, um zu bestätigen, dass diese ähnlichen Phänotypen wie in zuvor beschriebenen Modellen folgten. Hervorzuheben ist, dass die Rekrutierung des kürzlich veröffentlichten Pilus-Adhäsin-Rezeptors CD147 unter Meningokokken-Mikrokolonien in iPSC-BECs verifiziert werden konnte. Es wurde auch beobachtet, dass Nm die Expression der entzündungsfördernden und neutrophilen spezifischen Chemokine IL6, CXCL1, CXCL2, CXCL8 und CCL20 zu bestimmten Zeitpunkten der Infektion sowie die Sekretion von IFN-γ und RANTES durch iPSC-BECs signifikant erhöht. Es wurde zudem beobachtet, dass Nm die Tight Junction-Proteine ZO-1, Occludin und Claudin-5 zu späten Zeitpunkten der Infektion zerstört, verursacht durch die Infektion wurde ein ausgefranster und/oder diskontinuierlicher Tight Junction-Phänotyp beobachtet. Dieser Zerstörung geht die SNAI1-Aktivierung (ein Transkriptionsrepressor für Tight Junction-Proteine) voraus und könnte von ihr abhängig sein. In Übereinstimmung mit dem Verlust der Tight Junctions wurde zu späten Infektionszeitpunkten ein starker Verlust des transendothelialen elektrischen Widerstands und eine Zunahme der Natriumfluoreszein-Permeabilität beobachtet. Bemerkenswerterweise korrelierte die bakterielle Transmigration mit dem Verlust der Tight Junctions, was darauf hinweist, dass der parazelluläre Weg zur bakteriellen Überwindung von BECs eine entscheidende Rolle spielt. Schließlich wurde die RNA-Sequencing (RNA-Seq) von sortierten, infizierten iPSC-BECs durch die Verwendung von fluoreszenzaktivierten Zellsortiertechniken (FACS) nach der Infektion durchgeführt. Dies ermöglichte erstmalig den Nachweis von Expressionsdaten von Nm-responsiven Wirtsgenen, welche bei der Meningitidis eine Rolle zu spielen scheinen. Zusammenfassend konnte im Rahmen der vorliegenden Arbeit der Nutzen von iPSC-BECs In-Vitro-Modellen zur Untersuchung von Nm-Infektionen gezeigt werden. Dies ist das erste BEC-In-vitro-Modell, das alle wichtigen BEC-Tight Junctions exprimiert und ein hohes Barrierepotential aufweist. Insgesamt liefert das eingesetzte Modell neue Einblicke in die Nm-Pathogenese, einschließlich der Beeinflussung der Barriereeigenschaften und der Tight Junction-Komplexe durch Nm, und gibt erste Hinweise darauf, dass die parazelluläre Route zum Nm-Übertritt von BEC-Barrieren eine entscheidende Rolle spielt.

5 2 ABSTRACT Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Consultant: PharmDr. Eva Šnejdrová Ph.D. Student: Mimoza Veliu Title of Thesis: Preparation of oxim reactivators loaded PLGA nanoparticles In theoretical part the main attention is paid to polymeric nanoparticles (NP) for brain targeting. The drug properties and methods of preparation of the nanoparticles with hydrophilic drugs are presented. The part is devoted to physico-chemical properties that affect the permeability of these substances across biological barriers to the target site. The polymers used for NP preparation for brain targeting are focused. The experimental part deals with the nanoparticle formulation. Nanoparticles were prepared by double emulsion method. The method of preparation was chosen based on the drug solubility. In the W/O/W system oil phase was formed by linear copolymers of poly(lactic-co-glycolic) acid (PLGA) and its derivatives branched on polyacrylic acid (A2) or tripentaerythritol (T3). Poloxamer 407 and dimethyldidodecylammonium bromide (DDAB) were chosen for NP stabilization. The effect of the polymers on particle size was studied. The effect the polymers used, and the formulation factors on the size, polydispersity and stability of NP were studied. The concentration...

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