Growing consumer awareness of the health benefits associated with fruits and vegetables and demand for easy to prepare products has prompted the development of a wide variety of minimally processed fruits and vegetables. Minimally processed fruits and vegetables are often peeled, cut, or diced which compromise the produces’ natural protective barriers, exposing a nutrient rich medium and providing an ideal environment for the growth of microorganisms, including foodborne pathogens. The germination conditions of sprout vegetables consisting of relatively high temperatures and humidity, low light and abundance of nutrients are also conducive to the proliferation of foodborne pathogens. Recent outbreaks and recalls indicate additional measures are needed to improve food safety and maintain the integrity of the food industry. The objective of this research was to evaluate the efficacy of Lactic Acid Bacteria (LAB) against E. coli O157:H7, L. monocytogenes, and Salmonella spp. on apple slices and alfalfa sprouts and it’s influence on product quality. Apple slices inoculated with E. coli O157:H7, L. monocytogenes, and Salmonella spp. (each at 104 CFU/g) were treated with Lb. plantarum alone and in combination with Pediococcus acidophilus and P. pentosaceus (LPP) (107 CFU/g) while alfalfa seeds were inoculated with L. monocytogenes and Salmonella spp. (each at 101 CFU/g and 103 CFU/g) and treated with LPP (107 CFU/g). The growth of the microorganisms on the apple slices was assessed during five and seven days of storage at 4◦C and 20◦C, respectively. Growth on alfalfa seeds was reported during five days of sprouting at 20◦C. Populations of LAB were maintained between 7.0 log CFU/g and 8.0 log CFU/g throughout storage and sprouting on the sliced apples and alfalfa seeds, respectively. Although LAB had no significant effect on pathogen populations on apple slices during storage at 4°C (p > 0.05), populations were significantly different at 20°C (p < 0.05). Populations of L. monocytogenes in the presence of Lb. plantarum and LPP were 1.84 log CFU/g and 2.84 log CFU/g less than the controls after five days of storage at 20°C (p < 0.05). Populations of E. coli O157:H7 in the presence of Lb. plantarum and LPP were 1.83 log CFU/g and 1.86 log CFU/g less than the control after one and three days of storage, respectively. Finally, populations of Salmonella spp. were 0.86 log CFU/g less than populations in the absence of LPP after three days of storage. LPP had a significant effect on the growth of L. monocytogenes and Salmonella spp. on alfalfa seeds (p < 0.05). After five days of sprouting, populations of L. monocytogenes at an initial concentration of 101 CFU/g and 103 CFU/g on seeds treated with LPP were approximately 4.5 log CFU/g and 1.0 log CFU/g less than the untreated seeds, respectively. Populations of Salmonella spp. at an initial concentration of 101 CFU/g and 103 CFU/g were 1.0 log CFU/g less than the control. Overall, on apple slices the combination of Lb. plantarum with P. acidophilum and P. pentosaceus demonstrated greater efficacy than Lb. plantarum alone and reduction of L. monocytogenes by Lb. plantarum and LPP was greater than Salmonella spp. and E. coli O157:H7 on apple slices and alfalfa seeds, alike. LAB had a minimal effect on the quality of the apple slices and alfalfa seeds. LAB could be an effective strategy in reducing pathogen populations at abusive temperatures and germination conditions without influencing the quality of minimally processed fruit and vegetables.

Background: Glucose homeostasis is tightly regulated by hormones secreted within the pancreatic islets of Langerhans. The most important are insulin and glucagon produced by beta and alpha cells respectively. Changes in beta cell mass and/or their functional deficit can lead to hyperglycemia, a major hallmark of both type 1 (T1D) and type 2 (T2D). Moreover, a dysregulation in glucagon secretion is thought to also play a major role in patients with diabetes, suggesting a failure in the counterregulatory mechanisms of glucose homeostasis in disease pathogenesis. Dysfunction at the alpha cell level in T1D manifests are blunt glucagon response to low glucose levels, which can cause severe hypoglycemic events in patients with T1D. Furthermore, exaggerated glucagon responses to glucose or amino acid intake significantly contributes to dysglycemia in both T1D and T2D patients. Most of our knowledge about glucagon and alpha cell physiology in the human setting was generated using in vivo systemic assessments or in vitro investigations of isolated human islets or dispersed single cells. Despite the increasing knowledge regarding alpha cells and glucagon biology, the underlying mechanisms of alpha cell dysfunction are still uncertain. Studies on alpha cell physiology were hindered by limited human tissue accessibility, technical methodologies and translational value of findings from rodents to humans. To fill the gap between the currently available in vivo and in vitro approaches and a more precise understanding of mechanisms of diabetes pathogenesis detailed investigation of islet cells within their native environment is needed. Aim The overall objective of this thesis was characterize alpha cell function in diabetes pathogenesis. To this end, the human pancreas slice preparation would to be adapted and advanced for the study of alpha cell physiology. These adjustments would be then used to investigate changes in alpha cell mass and function in T1D and T2D. Methods: Pancreas tissue slices were prepared from donor organs with and without T1D and from tissue donors after pancreatectomy at different stages of T2D. Immunofluorescent staining with subsequent 3D morphometry was used to quantify alpha cell volumes from 120μm thick tissue slices. Furthermore, human tissue slices were subjected to dynamic slice perifusion for the assessment of glucagon and insulin secretion kinetics in response to specific stimuli. Finally, functional and morphometrical analysis was performed on the same tissue slices to enable direct correlation of glucagon secretion and alpha cell volume in a subset of cases in the context of T1D. Results: Here we developed a semi-automatic 3D approach to quantify total endocrine cell volumes within a given volume of pancreas tissue. In addition, we established an in situ method for dynamic insulin release measurements from islets preserved in their native environment. We successfully modified this protocol to allow the measurement of glucagon release in slices from organ donors. After further optimizations, we were additionally able to also measure alpha cell function from surgical specimens after pancreatectomy. To gain insight into alpha cell pathophysiology in T1D we investigated alpha cell volume in donor organs with different disease duration and age at onset. Alpha cell volumes in slices of individuals with T1D did not show a dramatic change (neither increase nor decrease) in comparison to slices generated from non-diabetic (ND) pancreata. Furthermore, functional assessment of glucagon release using a specific stimulation protocol for alpha cells suggests preserved stimulatory capacity of these cells in slices from autoantibody positive donors. Interestingly, this is also the case in the so far studied slices from donors with different durations of diabetes. Nevertheless, normalization of secreted glucagon to the total alpha cell mass within the slice indicated reduced glucagon release in the here investigated two cases of T1D. In the context of T2D, 3D morphometrical analysis revealed that overall endocrine cell volume, including alpha cell volume, is maintained in our cohort of IGT and T2D individuals. Glucagon release can also be measured in tissue procured from patients undergoing pancreatectomy, given the presence of amino acids in the perifusion media and increased trypsin inhibitors. While we provided proof of concept using tissue from ND individuals, we are confident that the approach will give valuable insight in different states of diabetes. Conclusion: These results demonstrate that human pancreas tissue slices represent a complementary platform to study alpha cell pathophysiology in both major types of diabetes. We provide evidence that this approach can be used to study alpha cell pathophysiology in T1D and T2D. Our preliminary data indicates no defect in the stimulatory capacity in slices from Aab+ and T1D donors, however more cases need to be investigated given the heterogeneous nature of the disease. We anticipate that the here proposed protocol for measurement of glucagon release from tissue slices will help us to gain insight in the role of alpha cells in diabetes pathophysiology. / Hintergrund: Die Aufrechterhaltung der Glukosehomöostase wird durch die Hormonsekretion der Langerhans’schen Inseln im Pankreas reguliert. Die wichtigste Rolle hierbei spielen die Insulin-produzierenden Betazellen und die Glukagon-produzierenden Alphazellen. Der Verlust der Betazellmasse und/oder der Funktion kann zur Entwicklung einer Hyperglykämie führen, die ein Hauptmerkmal des Typ 1 (T1D) und Typ 2 (T2D) Diabetes ist. Darüber hinaus wird vermutet, dass auch der Mechanismus der Gegenregulierung durch die Sekretion von Glukagon eine wichtige Rolle in der Pathogenese des Diabetes spielt. Während eine fehlende Glukagonsekretion zu schweren hypoglykämischen Phasen bei Typ 1 Diabetikern führen kann, geht man zusätzlich davon aus, dass eine erhöhte Reaktivität von Alphazellen sowohl auf Glukose als auch Aminosäuren ebenfalls zum Verlust der Glukosehomöostase im T1D und T2D beitragen kann. Trotz der stetig wachsenden Erkenntnisse über die Physiologie der Alphazellen, die vor allem durch systemische Untersuchungen in vivo oder an isolierten Langerhans’schen Inseln und Einzelzellen in vitro durchgeführt wurden, sind die zugrundeliegenden Mechanismen für deren Fehlfunktion beim Menschen noch nicht eindeutig aufgeklärt. Dies beruht hauptsächlich auf nur bedingt vorhandenem humanem Gewebe, technischen Schwierigkeiten bei der Isolation der Zellen, sowie der nur bedingten Vergleichbarkeit zu Studien in Nagern. Um diese Wissenslücken zwischen in vivo und in vitro Studien schließen zu können, ist es notwendig detaillierte Untersuchungen der Zellen unter nahezu physiologischen Bedingungen und in der nativen Umgebung der Pankreas in situ durchzuführen, um Alphazell-spezifische Mechanismen in der Diabetespathogenese genauer beleuchten zu können. Ziele: Ziel dieser Dissertation war es, durch Anpassung und Weiterentwicklung der Technik zur Gewinnung von Gewebeschnitten des humanen Pankreas, die Funktion der Alphazellen sowohl unter physiologischen Bedingungen als auch in der Entwicklung des T1D und T2D genauer zu charakterisieren. Methoden: Zur Untersuchungen von Alphazellen in Gewebeschnitten in situ wurden Gewebestücke sowohl von Organspendern mit und ohne T1D, sowie von metabolisch charakterisierter Patienten in verschiedenen Stadien der T2D Pathogenese nach einer Pankreatektomie verwendet. Die aus dem Gewebe gewonnenen 120 μm dicken Schnitte wurden zum einen für immunhistochemischer Färbungen verwendet, die eine 3-dimensionale morphometrische Analyse der Alphazellmasse ermöglichen. Ferner wurden Schnitte zur Ermittlung der Kinetik von Glukagon- und Insulinsekretion nach Stimulation mittels Perifusion benutzt. Schließlich wurden sowohl die morphologischen als auch funktionellen Analysen auf denselben Gewebeschnitten durchgeführt, um die Funktion der Alphazellen mit deren Masse besser korrelieren zu können. Ergebnisse: Zusätzlich zur Mitentwicklung eines halbautomatisierten Verfahrens zur 3D Analyse von endokrinen Zellvolumina in Gewebeschnitten des Pankreas wurde die bereits vorhandene Methode zur Messung der Insulinsekretionskinetik weiterentwickelt. Außerdem erfolgte die Etablierung adäquater Protokolle zur Messung der Glukagonsekretion in humanen Gewebeschnitten, die im Kontext beider Diabetestypen verwendet wurden. Um einen besseren Einblick in die T1D Pathogenese zu erhalten, wurde das Alphazellvolumen- und die Funktion in Gewebeschnitten von Organspendern mit unterschiedlichem Diabetes Verlauf (Alter bei Diagnose, Dauer seit Diagnose) mit nicht-diabetischen, aber Autoantikörper-positiven Spendern und nicht-diabetische Kontrollen verglichen. In Bezug auf das Alphazellvolumen waren zwischen den einzelnen Gruppen keine Unterschiede zu erkennen, die auf Veränderungen in der Entwicklung und Manifestation des T1D hinweisen. Darüber hinaus ergab die funktionelle Analyse, dass die Glukagonsekretion in nicht-diabetischen, Autoantikörper-positiven Spendern erhalten bleibt. Dies konnte zusätzlich auch in den bisher untersuchten Geweben von Typ 1 Diabetikern nachgewiesen werden, obwohl die Volumen-normalisierte Sekretion auf eine geringere Glukagonausschüttung hindeutet. Im Hinblick auf die T2D Pathogenese konnte bei der 3D Morphometrie von Nichtdiabetikern, Patienten mit beeinträchtigter Glukosetoleranz und Typ 2 Diabetikern keinerlei Unterschiede in den endokrinen Zellvolumina festgestellt werden. Durch die Anpassung der Konditionen für die Perifusion von reseziertem Gewebe konnte bei Nichtdiabetikern die erfolgreiche Messung der Glukagonsekretion gezeigt werden und ermöglicht zukünftig auch die Untersuchung einer möglichen Alphazelldysfunktion in der Entwicklung eines T2D. Schlussfolgerung: Die Ergebnisse dieser Arbeit zeigen, dass die etablierte Plattform zur morphologischen und funktionellen Analyse humaner Pankreasgewebeschnitte in situ eine wichtige Rolle in der Untersuchung der Alphazellen in der Diabetes-Pathogenese spielt. Die bisher erhobenen Daten zur Untersuchung des T1D haben gezeigt, dass die Kapazität der Glukagonsekretion nicht signifikant verändert ist. Aufgrund des heterogenen Krankheitsverlaufs beider Diabetesformen ist es jedoch notwendig Gewebe von einer größeren Anzahl an Spendern/Patienten zu untersuchen, um einen besseren Einblick in die Rolle der Alphazellen in der Entstehung des Diabetes zu erhalten.

No description available.

The thesis presents algorithms based on a linear algebraic solution for the identification of the parameters of the FIR system using only higher order statistics when only the output of the system corrupted by additive Gaussian noise is observed. All the traditional parametric methods of estimating the parameters of the system have been based on the 2nd order statistics of the output of the system. These methods suffer from the deficiency that they do not preserve the phase response of the system and hence cannot identify non-minimum phase systems. To circumvent this problem, higher order statistics which preserve the phase characteristics of a process and hence are able to identify a non-minimum phase system and also are insensitive to additive Gaussian noise have been used in recent years. Existing algorithms for the identification of the FIR parameters based on the higher order cumulants use the autocorrelation sequence as well and give erroneous results in the presence of additive colored Gaussian noise. This problem can be overcome by obtaining algorithms which do not utilize the 2nd order statistics. An existing relationship between the 2nd order and any Ith order cumulants is generalized to a relationship between any two arbitrary k, Ith order cumulants. This new relationship is used to obtain new algorithms for FIR system identification which use only cumulants of order > 2 and with no other restriction than the Gaussian nature of the additive noise sequence. Simulation studies are presented to demonstrate the failure of the existing algorithms when the imposed constraints on the 2nd order statistics of the additive noise are violated while the proposed algorithms perform very well and give consistent results. Recently, a new algebraic approach for parameter estimation method denoted the Linear Combination of Slices (LCS) method was proposed and was based on expressing the FIR parameters as a linear combination of the cumulant slices. The rank deficient cumulant matrix S formed in the LCS method can be expressed as a product of matrices which have a certain structure. The orthogonality property of the subspace orthogonal to S and the range space of S has been exploited to obtain a new class of algorithms for the estimation of the parameters of a FIR system. Numerical simulation studies have been carried out to demonstrate the good behaviour of the proposed algorithms. Analytical expressions for the covariance of the estimates of the FIR parameters of the different algorithms presented in the thesis have been obtained and numerical comparison has been done for specific cases. Numerical examples to demonstrate the application of the proposed algorithms for channel equalization in data communication and as an initial solution to the cumulant matching nonlinear optimization methods have been presented.

No description available.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Martin Huličiak Supervisor: PharmDr. Lukáš Červený, Ph.D Title of diploma thesis: Study of interactions of antiviral drugs with intestinal drug efflux ABC transporters P-gp, MRP2 and BCRP are efflux transporters, members of the family of ATP binding cassette (ABC) transporters. These transporters are located on the apical membrane of the intestinal epithelium, where they may limit absorption of orally administered drugs. Study of drug interactions with/on intestinal efflux transporters is necessary to provide safe and effective treatment. The Caco-2 cell line is FDA recommended in vitro model of intestinal barrier and it is used for bidirectional testing of substrates and inhibitors of ABC transporters in preclinical research. However, this methodology has several shortcomings, so the need of introduction of new experimental models is increasing and the ex vivo method based on human or rat intestine is a promising option. Precision-cut intestinal slices (PCIS) represent a mini-model of the organ and contain all types of cells of the tissue. We used both in vitro model using Caco-2 cell monolayers for drug transport study and in our lab established ex vivo method of PCIS for accumulation study...

Aim: Isolated papillary muscles and enzymatically dissociated myocytes of guinea-pig hearts are routinely used for experimental cardiac research. The aim of our study is to investigate adult mammalian ventricular slices as an alternative preparation. Method: Vibratome cut ventricular slices (350 μm thick) were examined histologically and with 2-photon microscopy for fibre orientation. Intracellular action potentials were recorded with conventional glass microelectrodes, extracellular potentials were measured with tungsten platinum electrodes and multi-electrode arrays (MEA). Results: Dominant direction of fibre orientation was absent in vertical and horizontal transmural slices, but was longitudinal in tangential slices. Control action potential duration (APD90, 169.9 ± 4 ms) and drug effects on this parameter were similar to papillary muscles. The L-type Ca-channel blocker nifedipine shortened APD90 with a half maximal effective concentration (EC50) of 4.5 μM. The IKr blocker E4031 and neuroleptic drug risperidone prolonged APD90 with EC50 values of 31 nM and 0.67 μM, respectively. Mapping field potentials on multi-electrode arrays showed uniform spread of excitation with a mean conduction velocity of 0.47 m ⋅ s-1. Conclusion: Slices from adult mammalian hearts could become a useful routine model for electrophysiological and pharmacological research. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

La démyélinisation et la mort oligodendrocytaire sont bien connues dans la sclérose en plaques (SEP). Au cours de ces dernières années, plusieurs études ont également décrit ce type de lésion après un traumatisme crânien (TC), participant à l’aggravation des lésions de la substance blanche, responsables des dysfonctionnements cognitifs et moteurs. Malgré de nombreux efforts, aucune thérapie efficace n’est disponible à ce jour pour traiter les lésions de la substance blanche. Dans ce contexte, une stratégie thérapeutique prometteuse serait de freiner la neuro-inflammation et la démyélinisation, en plus de promouvoir la maturation des oligodendrocytes afin de favoriser la remyélinisation des axones et de limiter ainsi leur dégénérescence. Notre choix de stratégie porte sur la stimulation des processus de réparation endogène via la protéine neuroprotectrice et neurotrophique sAPPα, forme soluble de la protéine βAPP libérée par l’action des α-sécrétases (ADAM10). Dans ce contexte, l’objectif de mes travaux de thèse était d’étudier l’intérêt thérapeutique de l’étazolate, un activateur desα-sécrétases, sur les conséquences biochimiques, histologiques et fonctionnelles, dans différents modèles de TC et de SEP in vivo chez la souris, et ex vivo sur des tranches organotypiques de cervelet. Les résultats obtenus sur le modèle de TC par percussion mécanique chez la souris ont montré pour la première fois le potentiel anti-inflammatoire de l’étazolate, associé à la restauration du taux de la sAPPα. De plus, l’étazolate s’est également opposé aux troubles fonctionnels post-TC tels que l’hyperactivité locomotrice et le déficit cognitif à court et à long terme. Par la suite, j’ai développé un nouveau modèle ex vivo de TC par percussion mécanique sur des tranches organotypiques de cervelet. Nous avons montré pour la première fois que l’étazolate était neuroprotecteur dans le tissu cérébelleux, et qu’il s’opposait à la démyélinisation post-traumatique. Par ailleurs, les effets bénéfiques de l’étazolate sur les gaines de myéline ont été reproduits dans un modèle ex vivo de démyélinisation induite par la lysolécithine, modèle ex vivo de SEP. De façon intéressante nous avons montré que l’étazolate exerçait un effet remyélinisant en stimulant la différenciation des oligodendrocytes. Cet effet a été reproduit in vitro dans des cultures primaires mixtes de cellules gliales issues de souris PLP-eGFP, où la maturation morphologique des oligodendrocytes a été favorisée en présence d’étazolate. L’ensemble des effets bénéfiques exercés par l’étazolate a été inhibé en présence d’un inhibiteur pharmacologique spécifique d’ADAM10, le GI254023X, suggérant que l’effet remyélinisant de l’étazolate dépend, au moins en partie, d’ADAM10. Par la suite, l’effet remyélinisant de l’étazolate a été étudié dans un modèle in vivo de démyélinisation chronique induite par la cuprizone. Dans ce modèle, l’étazolate a été capable de promouvoir la remyélinisation en stimulant la différenciation des oligodendrocytes, confirmant nos résultats in vitro et ex vivo. L’ensemble de mon travail permet de considérer le potentiel thérapeutique de l’étazolate, en visant l’ADAM10 comme nouvelle cible thérapeutique neuroprotectrice et remyélinisante. Cela aura pour intérêt de limiter la neuro-inflammation, la démyélinisation, ainsi que de promouvoir la différenciation des oligodendrocytes et la remyélinisation, afin de favoriser la récupération fonctionnelle suite aux lésions de la substance blanche survenant après un TC ou la SEP chez l’homme. / Demyelination and oligodendrocyte cell death are well established in multiple sclerosis (MS) and are increasingly described after traumatic brain injury (TBI), participating in the aggravation of white matter injury responsible of motor and cognitive deficits. Despite many efforts in clinical research, no efficient therapy against white matter injury progression is available nowadays. Thus, promoting remyelination and counteracting neuroinflammation and demyelination are major therapeutic strategies in order to restore white matter integrity. Here, we studied the stimulation of endogenous repair mechanisms through the neuroprotective and neurotrophic protein sAPPα, the soluble form of βAPP protein released by the α-secretases (ADAM10). In this context, the aim of this work was to evaluate the therapeutic potential of etazolate, an α-secretase activator on short- and long-term biochemical, histological and functional outcome in different mouse models of TBI and MS in vivo, and ex vivo on organotypic cerebellar slices. The results obtained from the TBI mouse model by mechanical percussion showed for the first time the anti-inflammatory effect of etazolate associated to a restoration of sAPPα levels. The same treatment was able to attenuate functional deficits (hyperactivity, cognitive deficit). We also developed a new ex vivo model of TBI by mechanical percussion on organotypic cerebellar slices. We confirmed the neuroprotective effect of etazolate on cerebellar tissue reducing the lesion size. Interestingly, etazolate treatment attenuated post-traumatic ex vivo demyelination. Moreover, the beneficial effect of etazolate on myelin sheaths have been well reproduced after lysolecithin-induced demyelination, an ex vivo model of MS. Interestingly, etazolate was able to enhance remyelination promoting oligodendrocyte differentiation. This effect has been reproduced in the primary mixed glial culture from PLP-eGFP mice, enhancing oligodendrocyte morphological maturation. However, etazolate failed to promote its beneficial effects in the presence of GI254023X, a specific ADAM10 (α-secretase) inhibitor, suggesting that the mechanism of action of etazolate is partly through the activation of ADAM10. Furthermore, etazolate reproduced in vivo the oligodendrocyte differentiation, accompanied by an increase of the myelinated axons, observed by electron microscopy in a mouse model of cuprizone-induced chronic demyelination. Taken together, the findings of this work provide a first evidence for the therapeutic potential of etazolate, with ADAM10 as new therapeutic target in white matter repair. The interest of this approach is to attenuate neuroinflammation and demyelination and to enhance oligodendrocyte differentiation and thus remyelination, in order to promote functional recovery following white matter lesions arising after TBI or MS.

Les récepteurs de type Toll (TLR) sont des récepteurs cellulaires jouant un rôle pivot dans le déclenchement de la réponse immunitaire après une infection ou une blessure, c'est-à-dire une inflammation. L'activation de la signalisation TLR a été associée à l’épilepsie. Dans ce projet, j'utilisai trois modèles distincts pour étudier comment le déclenchement des TLR contribue à l'épileptogenèse. Il existe une corrélation entre les malformations corticales développementales telle la dysplasie corticale focale (FCD) et convulsions fébriles dans les enfants de bas âge. Récemment, une réponse neuro-inflammatoire fut identifiée dans les lésions FCD. Nous postulâmes que l'inflammation induite par le FCD peut augmenter la sensibilité aux crises (chapitre 2). Nous modélisâmes FCD en induisant une congélation-lésion corticale chez le rat néonatal. La lésion corticale déclencha des effecteurs en aval de TLR4, spécifiquement le précurseur de la cytokine Caspase-1, dans l'hippocampe ipsilatéral à la lésion. Les rats lésés développèrent des crises fébriles expérimentales nettement plus rapidement que les rats témoins. Le blocage de l'activité de la Caspase-1 prolongea significativement la latence des crises chez les rats lésés. Nos résultats impliquent l'inflammation médiée par la Caspase-1 en tant que déclencheur des crises fébriles chez les enfants avec FCD préexistante. Des études antérieures déterminèrent que l'activation systémique de la cascade TLR4 abaisse le seuil de crise. Nous étudiâmes si la pénétration des cellules immunitaires périphériques dans le cerveau pendant la stimulation TLR4 favorise l'activité ictal en stimulant la voie TLR4 dans les leucocytes prélevés sur la rate de rat (splénocytes). Ensuite, nous co-cultivâmes des splénocytes avec des coupes organotypiques dérivées du cerveau in vitro (chapitre 3). L'ajout de splénocytes stimulés par TLR4 donna lieu à une neuro-inflammation et à une excitation neuronale accrue. L’ajout de splénocytes non-stimulés n’eut aucun effet pro-inflammatoire ou pro-excitateur dans les coupes organotypiques. De plus, l'inhibition de la Caspase-1 dans des coupes organotypiques co-cultivées avec des splénocytes stimulés diminua la neuro-inflammation et l'hyperexcitabilité neuronale. Nos résultats suggèrent que l'infiltration de leucocytes activés par TLR4 dans le cerveau augmente la prédisposition aux crises via les mécanismes médiés par la Caspase-1. Précédemment, des rapports montrèrent que l'activation de la signalisation TLR3 facilite l'évolution des crises. L'introduction d'un agoniste synthétique TLR3 chez la souris in vivo et des coupes organotypiques hippocampiques in vitro produisirent des mécanismes anti-inflammatoires dépendants de la dose et du temps (chapitre 4). La stimulation TLR3 supprimait les crises d'hippocampe in vivo et réduisait l'excitabilité synaptique dans le réseau hippocampique à la fois in vivo et in vitro. Nous avons déterminé que les effets anticonvulsivants médiés par TLR3 étaient principalement provoqués par les cascades en aval IRF3 / IFN-β. Ainsi, nos données suggèrent que l'activation de TLR3 peut protéger le cerveau contre les crises par la production d'IFN-β. Nos résultats donnent un aperçu des nouveaux mécanismes cellulaires sous-jacents à la modulation inflammatoire de l'excitabilité neurale. Notre découverte des rôles de la Caspase-1 et de l'IFN-β dans l'influence du seuil de crise améliorera notre compréhension des fondements moléculaires de la génération de crises ce qui pourraient améliorer le traitement de l'épilepsie. / Toll-like receptors (TLRs) are cellular receptors that play a pivotal role in initiating immune response following infection or injury, i.e. inflammation. Nevertheless, activation of TLR signaling has been associated with seizure manifestation. In this research, I employed three distinct models to study how triggering TLRs contributes to ictogenesis. There is a correlation between developmental cortical malformations, e.g. focal cortical dysplasia (FCD), and fever-provoked, i.e. febrile, seizures in young children. Recently, neuroinflammation was reported in FCD lesions. Therefore, we posited that FCD-induced inflammation may increase seizure susceptibility (Chapter 2). To recapitulate FCD pathology, we induced a cortical freeze-lesion in neonatal rats. Lesioning the cortex triggered TLR4 downstream effectors, specifically the cytokine precursor Caspase-1, in the hippocampus ipsilateral to the lesion. Further, lesioned rats developed experimental febrile seizures markedly faster than sham control rats. Strikingly, blocking Caspase-1 activity prior to seizure induction significantly prolonged seizure latency in lesioned rats. Our results implicate Caspase-1-mediated inflammation as a main driver of febrile seizures in children with pre-existing brain malformations. In addition, previous reports determined that systemic activation of TLR4 cascade lowers seizure threshold. Hence, we developed an in vitro model to investigate whether penetration of peripheral immune cells into the brain during TLR4 stimulation promotes ictogenic activity (Chapter 3). First, we stimulated TLR4 pathway in leukocytes harvested from rat spleen, i.e. splenocytes. Thereafter, we co-cultured splenocytes with brain-derived organotypic slices in vitro. Adding TLR4-stimulated splenocytes gave rise to neuroinflammation and enhanced neuronal excitation, whereas adding unstimulated splenocytes failed to evoke pro-inflammatory or proexcitatory effects in organotypic slices. Moreover, Caspase-1 inhibition in organotypic slices cocultured with stimulated splenocytes diminished neuroinflammation and neuronal hyperexcitability. Our findings suggest that infiltration of TLR4-activated leukocytes into the brain elevate seizure predisposition via Caspase-1-mediated mechanisms. Beside TLR4 pathway, it was previously shown that activation of TLR3 signaling facilitates seizure evolution. In chapter 4, introducing a synthetic TLR3 agonist to mice in vivo and to hippocampal organotypic slices in vitro yielded anti-inflammatory mechanisms in a dose- and time-dependent manner. Also, we found that TLR3 stimulation suppressed hippocampal seizures in vivo and reduced synaptic excitability in the hippocampal network both in vivo and in vitro. Finally, we determined that TLR3-mediated anticonvulsive effects were chiefly driven by IRF3/IFN-β downstream cascades. Thus, our data suggests that TLR3 activation may protect the brain from seizures through production of IFN-β. Altogether, our findings provide insight into novel cellular mechanisms underlying inflammatory modulation of neural excitability. Furthermore, our discovery of the roles of Caspase-1 and IFN-β in influencing seizure threshold will improve our understanding of the molecular underpinnings of seizure generation, which may ultimately have therapeutic benefits for epilepsy treatment.

Aufgabe der elektrophysiologischen Studie zur Charakterisierung der neuronalen hypothalamischen Plastizität beim Haushuhn war es, den Einfluss des Alters sowie GABAerger Substanzen auf die Feuerrate und die Temperatursensitivität (thermischer Koeffizient: TC) von Hypothalamusneuronen mittels extrazellulärer Ableitungen in Hirnschnitten zu untersuchen. Im Vergleich zu adulten Vögeln und Säugetieren wurde bei juvenilen Hühnern eine hohe neuronale Kältesensitivität nachgewiesen, die offensichtlich eine spezifische Eigenschaft juveniler Vögel ist. Die Ontogenese der neuronalen hypothalamischen Thermosensitivität ist deutlich artspezifisch. Einige Neurone wiesen eine inherente Kältesensitivität auf. Eine mögliche zentrale Rolle kältesensitiver Neurone im Rahmen der Thermoregulation juveniler Hühner wurde postuliert. Muscimol und Baclofen hemmen signifikant die Feuerrate der Hypothalamusneurone, unabhängig von der jeweiligen Thermosensitivität. Demgegenüber bewirken Bicucullin und CGP35348 einem Anstieg der Feuerrate. Nur bei kältesensitiven Neuronen wurde der TC signifikant durch GABAB-Rezeptor-Liganden verändert (signifikant erhöht durch Baclofen und durch CGP35348 gehemmt). Der Effekt von Muscimol und Baclofen auf Feuerrate und TC wurde durch Co-Perfusion mit einer 10-fach höheren Konzentration der entsprechenden Antagonisten Bicucullin und CGP35348 aufgehoben. Der wesentliche GABAerge Einfluss auf thermosensitive und –insensitive Hypothalamusneurone ist mit dem bei Säugetieren nachgewiesenen vergleichbar. Der einzige Unterschied betrifft die GABAB-Rezeptor vermittelte Änderung des TC. Beim Hühnerküken betraf dies die kältesensitiven und beim Säugetier die wärmesensitiven Neurone. Der grundlegende Mechanismus der GABAergen Beeinflussung thermosensitiver und –insensitiver Neurone scheint einen älteren evolutionären Ursprung zu haben. Eine funktionelle Rolle GABAerger Substanzen im Rahmen der zentralen Kontrolle der Körpertemperatur beim Vogel ist möglich. / In the present electrophysiological studies, characterization of neuronal hypothalamic plasticity in the chicken aims to investigate the influence of age during development by extracellular recordings. High neuronal cold sensitivity has been found in juvenile chicken in contrast to adult mammals and birds. High hypothalamic cold sensitivity seems to be a specific characteristic feature in juvenile birds. Between species a species specificity of the early development of neuronal hypothalamic thermosensitivity could be clearly demonstrated. Existence of inherent nature to a certain degree suggests a possible thermoregulatory role of cold-sensitive neurons in chicken. The effects of the GABAergic substances on neuronal tonic activity (firing rate) and temperature sensitivity (temperature coefficient) in hypothalamic neurons have been examined. Muscimol and baclofen in equimolar concentrations significantly inhibited tonic activity, regardless of their type of thermosensitivity. In contrast bicuculline and CGP 35348 increased firing rate. Temperature coefficient was significantly changed by ligands of GABAB receptors, restricted to cold-sensitive neurons. The TC was significantly increased by baclofen and significantly decreased by CGP 35348. Effects of muscimol and baclofen on firing rate and TC were prevented by co-perfusion of appropriate antagonists bicuculline and CGP 35348, respectively in tenfold higher concentration. Thus the main effects of GABA in chicken are similar with that described in mammals. The only difference is in respect of the GABAB receptors mediated change restricted to cold-sensitive neurons in chicken but in mammals only seen in warm-sensitive neurons. However, the results indicate that the fundamental mechanism of GABAergic influence in chicken are conserved during evolution. The response of hypothalamic neurons to temperature changes suggest a possible functional role of GABAergic substances in the control of body temperature in birds.