Tissue slices can be made from organs, such as liver, kidney, brain, and heart, and from various species including humans, dogs, non-human primates, rats and mice. It has been demonstrated that human and rat liver slices are viable for up to 2 days, and liver slices have been extensively used as an in vitro method to study hepatic drug metabolism and toxicity in humans. The objective of this study was to determine the utility of canine hepatic slices as an in vitro model for studying drug metabolism and hepatotoxicity in dogs. Canine hepatic slices were incubated in media containing various drugs to determine the hepatotoxicity of the agents and the ability of the slices to metabolize the drugs. The toxicity of phenobarbital, primidone, lidocaine and carprofen to canine hepatic slices was assessed by determining changes in supernatant concentrations of potassium ions and adenosine triphosphate (ATP); histologic lesions were determined as necrosis, extent of vacuolation and severity of vacuolation. Xenobiotic drug metabolizing enzymatic activity was investigated by determining the metabolism of lidocaine to monoethylglycinexylidide (MEGX), and administration of phenobarbital plus primidone was used as a positive control for hepatotoxicity in dogs. The function of drug-metabolizing enzymes was demonstrated by the successful metabolism of lidocaine to MEGX. Carprofen, a drug which causes idiosyncratic hepatic disease in dogs, did not show any hepatotoxicity at concentrations of 10, 50 and 100 µg/ml using potassium ion levels, ATP concentrations and histology as indicators of hepatotoxicity. Slices incubated in media without drug showed no toxicity over 24 hours based on potassium ion and ATP supernatant concentrations while significant increases in histologic lesions were noted at 8, 12 and 24 hours. Canine hepatic slices were a useful model for examining drug metabolism and toxicity for up to 24 hours.

Tissue slices can be made from organs, such as liver, kidney, brain, and heart, and from various species including humans, dogs, non-human primates, rats and mice. It has been demonstrated that human and rat liver slices are viable for up to 2 days, and liver slices have been extensively used as an in vitro method to study hepatic drug metabolism and toxicity in humans. The objective of this study was to determine the utility of canine hepatic slices as an in vitro model for studying drug metabolism and hepatotoxicity in dogs. Canine hepatic slices were incubated in media containing various drugs to determine the hepatotoxicity of the agents and the ability of the slices to metabolize the drugs. The toxicity of phenobarbital, primidone, lidocaine and carprofen to canine hepatic slices was assessed by determining changes in supernatant concentrations of potassium ions and adenosine triphosphate (ATP); histologic lesions were determined as necrosis, extent of vacuolation and severity of vacuolation. Xenobiotic drug metabolizing enzymatic activity was investigated by determining the metabolism of lidocaine to monoethylglycinexylidide (MEGX), and administration of phenobarbital plus primidone was used as a positive control for hepatotoxicity in dogs. The function of drug-metabolizing enzymes was demonstrated by the successful metabolism of lidocaine to MEGX. Carprofen, a drug which causes idiosyncratic hepatic disease in dogs, did not show any hepatotoxicity at concentrations of 10, 50 and 100 µg/ml using potassium ion levels, ATP concentrations and histology as indicators of hepatotoxicity. Slices incubated in media without drug showed no toxicity over 24 hours based on potassium ion and ATP supernatant concentrations while significant increases in histologic lesions were noted at 8, 12 and 24 hours. Canine hepatic slices were a useful model for examining drug metabolism and toxicity for up to 24 hours.

La cardiomyopathie associée à la DMD est une atteinte pour laquelle il n’existe pas actuellement de traitement. Les cellules souches embryonnaires humaines (hES), par leurs propriétés d’autorenouvellement et de différenciation, sont envisagées comme outil thérapeutique. Pour une recherche sur l’implantation de cellules, il n’existait pas de modèle in vitro au long cours. Nous avons ainsi développé un modèle de culture organotypique de tranches de cœur dans lequel nous avons, plusieurs mois après transplantation, retrouvé les cellules hES greffées, différenciées en cardiomyocytes. Par ailleurs, nous avons optimisé la différenciation cardiaque pour améliorer le rendement en cardiomyocytes, en induisant la différenciation cardiaque et en cultivant les cellules hES en bioréacteur. Le modèle d’étude au long terme permettant le suivi des cellules greffées dérivées de cellules hES nous permettra de valider les premières étapes précédant les études in vivo dans des modèles pathologiques. / Cardiomyopathy associated with DMD is a frequent occurrence with no treatment. Human embryonic stem cells (hESC), because of their self-renew differentiation properties are the best candidates to cardiac cellular therapy. To investigate cells implantation, there was no long term in vitro model. Thus, we have developed an organotypic model of heart slices in which we have, several months after transplantation, found grafted hES cells with evidence of cardiac differentiation. In addition, we have optimised cardiac differentiation to improve cardiac yield, inducing cardiac differentiation and cultivating hES cells in bioreactors. During long term culture model allows the study of grafted hES cells, combines to hESC-derived cells in reasonable number and purity, will constitute validation of the first steps before in vivo studies in pathological models.

Faults are common-place and inevitable in complex applications. Hence, automated techniques are necessary to analyze failed executions and debug the application to locate the fault. For locating faults in programs, dynamic slices have been shown to be very effective in reducing the effort of debugging. The user needs to inspect only a small subset of program statements to get to the root cause of the fault. While prior work has primarily focussed on single-threaded programs, this dissertation shows how dynamic slicing can be used for fault location in multithreaded programs. This dissertation also shows that dynamic slices can be used to track down faults due to data races in multithreaded programs by incorporating additional data dependences that arise in the presence of many threads. In order to construct the dynamic slices, dependence traces are collected and processed. However, program runs generate traces in the order of Gigabytes in a few seconds. Hence, for multithreaded program runs that are long-running, the process of collecting and storing these traces poses a significant challenge. This dissertation proposes two techniques to overcome this challenge. Experiments indicate that the techniques combined can reduce the size of the traces by 3 orders of magnitude. For applications that are critical and for which down time is highly detrimental, techniques for surviving software failures and letting the execution continue are desired. This dissertation proposes one such technique to recover applications from a class of faults that are caused by the execution environment and prevent the fault in future runs. This technique has been successfully used to avoid faults in a variety of applications caused due to thread scheduling, heap overflow, and malformed user requests. Case studies indicate that, for most environment bugs, the point in the execution where the environment modification is necessary can be clearly pin-pointed by using the proposed system and the fault can be avoided in the first attempt. The case studies also show that the patches needed to prevent the different faults are simple and the overhead induced by the system during the normal run of the application is less than 10 \%, on average.

Development of a three dimensional (3D) HYBROT model with targeted in vivo like intact cellular circuitry in thick brain slices for multi-site stimulation and recording will provide a useful in vitro model to study neuronal dynamics at network level. In order to make this in vitro model feasible, we need to develop several associated technologies. These technologies include development of a thick organotypic brain slice culturing method, a three dimensional (3D) micro-fluidic multielectrode Neural Interface system (µNIS) and the associated electronic interfaces for stimulation and recording of/from tissue, development of targeted stimulation patterns for closed-loop interaction with a robotic body, and a deep-tissue non-invasive imaging system. To make progress towards this goal, I undertook two projects: (i) to develop a method to culture thick organotypic brain slices, and (ii) construct a multiphoton imaging system that allows long-term and deep-tissue imaging of two dimensional and three dimensional cultures. Organotypic brain slices preserve cytoarchitecture of the brain. Therefore, they make more a realistic reduced model for various network level investigations. However, current culturing methods are not successful for culturing thick brain slices due to limited supply of nutrients and oxygen to inner layers of the culture. We developed a forced-convection based perfusion method to culture viable 700µm thick brain slices. Multiphoton microscopy is ideal for imaging living 2D or 3D cultures at submicron resolution. We successfully fabricated a custom-designed high efficiency multiphoton microscope that has the desired flexibility to perform experiments using multiple technologies simultaneously. This microscope was used successfully for 3D and time-lapse imaging. Together these projects have contributed towards the progress of development of a 3D HYBROT. ----- 3D Hybrot: A hybrid system of a brain slice culture embodied with a robotic body.

The original Bio-Rad gene gun was unable to transfect acute or organotypic brain slices, as the amount of helium gas used, the distance for the gold-coated microcarriers to travel to target area were not optimised for fragile tissues, such as the brain. Typically, tissues were severely damaged by a helium shock wave and only a few cells were transfected. It was essential to improve gene gun accuracy by restricting the gold particles from being propelled superficially over a wide area. It was also necessary to increase the amount of DNA or dye delivery into intact tissues. Furthermore, for the gene gun to perform successfully on brain slices the helium gas pressure had to be lowered thereby reducing the degree of cell damage incurred during a biolistic delivery. Without knowing it at the time, the modified gene gun had worked particularly well on a variety of other fragile tissues, and not just the brain. However, the modified gun was not optimised for cultured cells as other transfection methods were available. A particularly notable point of this work was the successful labelling of individual Purkinje dendritic spines from live nerve cells in the cerebellum region of the brain. Biolistic images of Purkinje cells show that the distribution of dendritic spines are not random (O’Brien and Unwin, 2006). Spines were shown to grow in elaborate regular linear arrays, that trace short-pitch helical paths around the dendrites. It was apparent that the spines are arranged to maximize the probability that the dendritic arbour would interact with any afferent axon. This was an important discovery as there has been much debate as to how spines develop on a dendritic shaft. There are three general views to this question, each proposing a theory describing a model for spinogenesis. Classification of the three models in relation to our findings is described in chapter six of this thesis. The Investigation of spine morphology by biolistics was further optimized; gold particles were reduced from a micrometre to forty nanometres (O’Brien and Lummis, 2011), demonstrating that it is possible to use gold-coated DNA nanoparticles of this size to transfect tissue revealing exquisite structural detail. It was possible to observe boutons making synaptic contacts with the pyramidal nerve spines in the hippocampal region of the brain. The findings so far have shown spines from the pyramidal shaft are similar to the spines in the cerebellum, forming regular linear arrays. Recent studies had linked defects in the function of presynaptic boutons to the etiology of several neurodevelopment and neurodegenerative diseases, including autism and Alzheimer’s disease. Our discovery could help to understand why there are abnormalities in dendritic spines which are associated with pathological conditions characterized by cognitive decline, such as mental retardation, Alzheimer’s, stroke and schizophrenia (Yuste and Bonhoeffer, 2001). This thesis provides a synthesis of knowledge about biolistic technology. It is presented as a narrative from improving the gene gun transfection efficiency in brain slices to the development of nano-biolistics. The delivery of DNA and fluorescent dyes into living cells by biolistic delivery should enable a detailed map of the anatomical connections between individual cells and groups of cells to be constructed, providing a “wiring diagram” of connections. The implications of this are discussed in Chapter twelve. The original Bio-Rad gene gun was unable to transfect acute or organotypic brain slices, as the amount of helium gas used, the distance for the gold-coated microcarriers to travel to target area were not optimised for fragile tissues, such as the brain. Typically, tissues were severely damaged by a helium shock wave and only a few cells were transfected. It was essential to improve gene gun accuracy by restricting the gold particles from being propelled superficially over a wide area. It was also necessary to increase the amount of DNA or dye delivery into intact tissues. Furthermore, for the gene gun to perform successfully on brain slices the helium gas pressure had to be lowered thereby reducing the degree of cell damage incurred during a biolistic delivery. Without knowing it at the time, the modified gene gun had worked particularly well on a variety of other fragile tissues, and not just the brain. However, the modified gun was not optimised for cultured cells as other transfection methods were available. A particularly notable point of this work was the successful labelling of individual Purkinje dendritic spines from live nerve cells in the cerebellum region of the brain. Biolistic images of Purkinje cells show that the distribution of dendritic spines are not random (O’Brien and Unwin, 2006). Spines were shown to grow in elaborate regular linear arrays, that trace short-pitch helical paths around the dendrites. It was apparent that the spines are arranged to maximize the probability that the dendritic arbour would interact with any afferent axon. This was an important discovery as there has been much debate as to how spines develop on a dendritic shaft. There are three general views to this question, each proposing a theory describing a model for spinogenesis. Classification of the three models in relation to our findings is described in chapter six of this thesis. The Investigation of spine morphology by biolistics was further optimized; gold particles were reduced from a micrometre to forty nanometres (O’Brien and Lummis, 2011), demonstrating that it is possible to use gold-coated DNA nanoparticles of this size to transfect tissue revealing exquisite structural detail. It was possible to observe boutons making synaptic contacts with the pyramidal nerve spines in the hippocampal region of the brain. The findings so far have shown spines from the pyramidal shaft are similar to the spines in the cerebellum, forming regular linear arrays. Recent studies had linked defects in the function of presynaptic boutons to the etiology of several neurodevelopment and neurodegenerative diseases, including autism and Alzheimer’s disease. Our discovery could help to understand why there are abnormalities in dendritic spines which are associated with pathological conditions characterized by cognitive decline, such as mental retardation, Alzheimer’s, stroke and schizophrenia (Yuste and Bonhoeffer, 2001). This thesis provides a synthesis of knowledge about biolistic technology. It is presented as a narrative from improving the gene gun transfection efficiency in brain slices to the development of nano-biolistics. The delivery of DNA and fluorescent dyes into living cells by biolistic delivery should enable a detailed map of the anatomical connections between individual cells and groups of cells to be constructed, providing a “wiring diagram” of connections. The implications of this are discussed in Chapter twelve.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Patrícia Čečková Supervisor: PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Study of drug interactions with OATP family transporters using intestinal tissue slices An essential role in the action of orally administered drugs is their absorption through the intestinal barrier. It expresses a variety of transporters, including the OATP2B1 and OATP1A2 influx transporters, belonging to the SLC family. They are located on the apical membrane of enterocytes and allow the flow of endogenous and exogenous substances from the lumen of the intestines to the enterocyte. They affect not only the pharmacokinetics of drugs, but also their safety and efficacy. They represent sites of drug interactions with other drugs/food components that may altered drug efficacy or toxicity. Since FDA (The Food and Drug Administration) and EMA (European Medicines Agency) do not have intestinal OATP transporters included in their guidelines for preclinical studies, there is no single model of interaction study. The limitations of cell models and genetically modified organisms lead to the development of new methods such as the ex vivo method of precision cut intestinal slices (PCIS), which represents a tissue model...

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Yes / Trait inferences occur routinely and rapidly during social interaction, sometimes based on scant or fleeting information. In this research, participants (perceivers) made inferences of targets' big‐five traits after briefly watching or listening to an unfamiliar target (a third party) performing various mundane activities (telling a scripted joke or answering questions about him/herself or reading aloud a paragraph of promotional material). Across three studies, when perceivers judged targets to be either low or high in one or more dimensions of the big‐five traits, they tended to be correct, but they did not tend to be correct when they judged targets as average. Such inferences seemed to vary in effectiveness across different trait dimensions and depending on whether the target's behaviour was presented either in a video with audio, a silent video, or just in an audio track—perceivers generally were less often correct when they judged targets as average in each of the big‐five traits across various information channels (videos with audio, silent videos, and audios). Study 3 replicated these findings in a different culture. We conclude with discussion of the scope and the adaptive value of this trait inferential ability.