The transcription factor nuclear factor κB (NF-κB) regulates multiple immediate-early gene expressions involved in immune and inflammatory responses and cellular defenses. Ischemia-reperfusion induces many immediate- early gene expressions, but little is known about the NF-κB activation in myocardium during ischemia and reperfusion. This study demonstrated that ischemia alone rapidly induced NF-κB activation in the myocardium of isolated working rat hearts. Electrophoretic mobility shift assay showed that NF-κB binding activity significantly increased in the nucleus after 5 min of ischemia and remained elevated for up to 30 min. Western blot analysis suggested that the levels of inhibitory IκBα protein in the cytoplasm became markedly decreased at 4, 5, 7.5, and 10 min of ischemia but were gradually restored following 10 min of ischemia. Reduction of IκBα protein in the cytoplasm by ischemia resulted in NF-κB translocation to the nucleus. Northern blot hybridization showed that IκBα mRNA levels were not significantly elevated during myocardial ischemia. Pyrrolidine dithiocarbamate, an antioxidant, significantly inhibited the loss of IκBα protein from the cytoplasm and prevented NF-κB binding activity in the nucleus. Reperfusion following short periods of ischemia augmented NF-κB binding activity in the nucleus induced by ischemia. The results suggest that early activation of NF-κB induced by ischemia in the myocardium could be a signal mechanism for controlling and regulating immediate-early gene expression during ischemia-reperfusion.

Background: Increasingly frequent reports of vancomycin treatment failures for serious methicillin-resistant Staphylococcus aureus (MRSA) infections provide impetus for comparative in vitro studies to assess the activity of newer antimicrobial agents against a range of MRSA isolates. Methods: A sample of 168 MRSA derived from a long-term MRSA collection was subjected to susceptibility testing to telavancin, daptomycin, linezolid, tigecycline and vancomycin by broth micro-dilution. Data were reviewed for sporadic occurrence of isolates with reduced susceptibility. Analyses were performed to test for temporal trends toward decreasing susceptibility and to compare susceptibility of isolates from different infection sites. Results: No MRSA isolate from any time period was resistant to test antibiotics. For daptomycin, linezolid and tigecycline, there were no susceptibility differences between the pre- and postclinical availability periods. All newer agents here active against MRSA isolates with minimum inhibitory concentrations (MICs) of vancomycin >1 mg/l, but there were significant correlations in susceptibility among several pairs of antibiotics. Conclusions: Telavancin and other newer antistaphylococcal agents were fully active against MRSA from various infection sites including isolates with vancomycin MIC >1 mg/l.

The brain network is a multiscale hierarchical organization from neurons and local circuits to macroscopic brain areas. The precise synaptic transmission at each synapse is therefore crucial for neural communication and the generation of orchestrated behaviors. Activation of presynaptic voltage-gated calcium channels (CaV2) initiates synaptic vesicle release and plays a key role in neurotransmission. In this dissertation, I have aimed to uncover how CaV2 activity affects synaptic transmission, circuit function and behavioral outcomes using Caenorhabditis elegans as a model. The C. elegans genome encodes an ensemble of highly conserved neurotransmission machinery, providing an opportunity to study the molecular mechanisms of synaptic function in a powerful genetic system. I identified a novel gain of function CaV2α1 mutation that causes CaV2 channels to activate at a lower membrane potential and slow the inactivation. Cell-specific expression of these gain-of-function CaV2 channels is sufficient to hyper-activate neurons of interest, offering a way to study their roles in a given circuit. CaV2(gf) mutants display behavioral hyperactivity and an excitation-dominant synaptic transmission. Imbalanced excitation and inhibition of the nervous system have been associated with several neurological disorders, including Familial Hemiplegic Migraine type 1 (FHM1) which is caused by gain- of-function mutations in the human CaV2.1α1 gene. I showed that animals carrying C. elegans CaV2α1 transgenes with corresponding human FHM1 mutations recapitulate the hyperactive behavioral phenotype exhibited by CaV2(gf) mutants, strongly suggesting the molecular function of CaV2 channels is highly conserved from C. elegans to human. Through performing a genome-wide forward genetic screen looking for CaV2α(gf) suppressors, we isolated new alleles of genes that required for CaV2 trafficking, localization and function. These regulators include subunits of CaV2 channel complex, components of synaptic and dense core vesicle release machinery as well as predicted extracellular proteins. Taken together, this work advances the understanding of CaV2 malfunction at both cellular and circuit levels, and provides a genetically amenable model for neurological disorders associated with excitation-inhibition imbalance. Additionally, through identifying regulators of CaV2, this research provides new avenues for understanding the CaV2 channel mediated neurotransmission and potential pharmacological targets for the treatments of calcium channelopathies.

The prefrontal cortices, in particular lateral prefrontal cortex (LPFC) and anterior cingulate cortex (ACC), have been implicated in top-down control of attention switching and behavioral flexibility. These cortices and their networks are disrupted in autism, a condition in which diverse behaviors such as social communication and attention control are dysregulated. However, little is known about the typical development of these cortical areas or the ways in which this process is altered in neurodevelopmental disorders. In order to identify changes that could affect the local processing of signals transmitted by the short-range pathways connecting the ACC and LPFC I assessed developmental changes in the distinct cortical layers, which send and receive different pathways and have unique inhibitory microenvironments that dictate excitatory-inhibitory balance. Normative developmental trends were compared with those seen in individuals with autism to identify changes that may contribute to symptoms of attention dysfunction. Unbiased quantitative methods were used to study overall neuron density, the density of inhibitory neurons labeled by the calcium-binding proteins calbindin (CB), calretinin (CR), and parvalbumin (PV), and the density, size, and trajectory of myelinated axons in the individual cortical layers in children and adults with and without a diagnosis of autism. There was a reduction in neuron density and an increase in the density of myelinated axons in both areas during neurotypical development. Axons in layers 1-3 of LPFC were disorganized in autism, with increased variability in the trajectory of axons in children and a decrease in the proportion of thin axons in adults. These findings were most significant in layer 1, the ultimate feedback-receiving layer in the cortex. While there were no differences in neuron populations between cohorts in children, in adults with autism there was a significant reduction in the density of CR-expressing neurons in LPFC layers 2-6 and a significant increase in the density of PV-expressing neurons in ACC layers 5-6. In autism, these findings suggest that dysregulation of the normal development of axonal networks, seen in children, may induce compensatory developmental changes in cell and axon populations in adults that could be connected to attention dysregulation. / 2021-10-07T00:00:00Z

La créativité repose sur la capacité à générer des idées à la fois originales et adaptées aux contraintes de la tâche afin de résoudre des problèmes pour lesquels aucune solution optimale n'est connue. Toutefois, dans ce type de circonstances, les connaissances intuitives des individus ainsi que leurs stratégies habituelles de résolution de problème les conduisent à générer des solutions peu créatives aboutissant à un phénomène de fixation, alors même que d'autres classes de solutions plus originales mais moins aisément accessibles pourraient être explorées. D'après le modèle triadique de la créativité, ces effets de fixations résulteraient de l'activation rapide et spontanée d'un système 1 intuitif et heuristique, alors qu'il serait plus avantageux d'explorer d'autres solutions en utilisant les processus cognitifs d'un système 2 délibératif et analytique. Ce modèle suggère également que le processus d'inhibition cognitive appartenant à un troisième système serait la clef pour diminuer la prégnance de ces effets de fixation créée par le système 1, et augmenter l'exploration d'autres voies plus créatives appartenant au système 2. Ainsi, l'objectif général de cette thèse consistait à apporter des arguments expérimentaux en faveur de ce modèle dans une approche interdisciplinaire allant de la psychologie expérimentale du développement aux neurosciences cognitives. À travers une série de cinq études expérimentales réalisées chez les enfants, les adolescents et les adultes, nous avons démontré 1) que les effets de fixation se développent avec l'âge et sont modulables par l'introduction d'indices comme des exemples de solutions, 2) qu'il est possible de stimuler la créativité des adolescents et des adultes en changeant la représentation qu'ils ont du problème de créativité par l'intermédiaire d'un paradigme d'amorçage, 3) qu'être capable de proposer des solutions créatives en dehors de la fixation implique le processus d'inhibition cognitive et la capacité à détecter que les solutions initialement générées ne sont pas originales, 4) que cette capacité de détection de conflit se développement au cours de l'adolescence et 5) que résister aux effets de fixation implique une modulation de l'activité des réseaux cérébraux au niveau des cortex frontaux et pariétaux sous tendant le contrôle cognitif et les associations sémantiques. / Creativity defined as the ability to think of something original, and adaptive concerning task constraints is crucial during circumstances in which individuals must generate new solutions to solve an unknown problem. In such circumstances, people propose solutions that are built on the most common and accessible knowledge within a specific domain leading to a fixation effects whereas other classes of more creative solutions could be explore. According to our triple systems model of creativity, the difficulty to generate creative ideas results from a specific failure to inhibit intuitive responses leading to fixation effect generated automatically by the intuitive and heuristic System 1 and activate the deliberative and analytic system 2 to explore more creative solutions. This model posits that inhibitory control is a core process to overcoming fixation effects and generating original solutions in a creative task. Therefore, the aim of this thesis was to provide empirical evidences in support of the triple system model of creativity by using an interdisciplinary approach from the field of experimental developmental psychology to the field of cognitive neuroscience. In a series of five experimental studies in children, adolescents and adults, we have demonstrated that 1) fixation effects develop with age and changes with the introduction of external cues such as examples of solutions 2) changing the individuals' representation of the creative task using a priming procedure can stimulate creative ideas generation in adolescents and adults, 3) overcoming fixation to explore creative solutions involves inhibitory control and the ability to detect that initial responses that come quickly to mind are not original, 4) this conflict detection ability develops with age during adolescence and 5) overcoming fixation is related to modulations of brain networks activations within the frontal and the parietal cortex involve in cognitive control and semantic associations respectively.

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells , while the steroid 24,25- dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by l,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for the 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against a putative 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake (P2D3, each in Cells exposed to hormone alone again showed an increased accumulation of 32P from T=5-10 min, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for 32P uptake, occurred. Incubation of cells with 100 nM phorbol-13-myristate (PMA) increased 32P uptake to 143% of controls, while cells pretreated with 50 μM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity at T=1-3 min (P2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.

No description available.

Agtarap, Wright, Mlynski, Hammad, and Blackledge took an initial step in providing support for the predictive validity of a new conceptual analysis concerned with behavioral restraint, defined as active resistance against a behavioral impulse or urge. The current study was designed to partially replicate and extend findings from their study, employing a common film protocol and a procedure for inducing low- and high levels of fatigue. Analyses on key data indicated that the fatigue manipulation was ineffective. On the other hand, they supported the suggestion that behavioral restraint should be proportional to the strength of an urge being resisted so long as success is perceived as possible and worthwhile. Analyses also provided evidence of gender differences for this behavioral restraint task. Women showed relatively enhanced CV responses to my manipulation of urge magnitude, performed less well, rated the behavioral restraint challenge as harder, and rated success on the more difficult behavioral restraint task as more important. A broad indication is that men and women can differ in the strength of impulses they experience in response to stimulus presentations as well as in the importance they place on resisting the impulses.

Large aspiny neurons are the only non-GABAergic neurons in the striatum. After transient cerebral ischemia, large aspiny neurons survive while medium spiny neurons die. Previous studies have shown that differential changes in the intrinsic membrane properties and excitatory synaptic transmission play a role in this selective vulnerability. However, the role of inhibitory synaptic transmission in this selective vulnerability is still unknown. Since inhibitory tone is very important in the control of neuronal excitability, the present study is aimed at examining if there are any changes in inhibitory synaptic transmission in striatal neurons after ischemia and the possible mechanisms. We also examined if facilitation of inhibitory synaptic transmission by muscimol could attenuate ischemic neuronal injury in the striatum after ischemia. Results from this study will improve the understanding of the mechanisms underlying selective neuronal injury after transient cerebral ischemia. We hope this study could contribute to the translational studies for the stroke patients after cardiac arrest. / Indiana University-Purdue University Indianapolis (IUPUI) / In the striatum, large aspiny (LA) interneurons survive transient cerebral ischemia while medium spiny (MS) neurons die. Excitotoxicity is believed to be the major cause for neuronal death after ischemia. Since inhibitory tone plays an important role in the control of neuronal excitability, the present study is aimed at examining if there are any changes in inhibitory synaptic transmission in striatal neurons after ischemia and the possible mechanisms. Transient forebrain ischemia was induced in male Wistar rats using the four-vessel occlusion method. Inhibitory postsynaptic currents (IPSCs) were evoked intrastriatally and whole-cell voltage-clamp recording was performed on striatal slices. The expression of glutamate decarboxylase65 (GAD65) was analyzed using immunohistochemical studies and Western blotting. Muscimol (a specific GABAA receptor agonist) was injected intraperitoneally to the rats (1 mg/kg) to observe ischemic damage, evaluated by counting the survived cells in the striatum after hematoxylin & eosin (HE) staining. The amplitudes of evoked IPSCs were significantly increased in LA neurons while depressed in MS neurons after ischemia. This enhancement was due to the increase of presynaptic release. Muscimol (1 μM) presynaptically facilitated inhibitory synaptic transmission in LA neurons at 24 h after ischemia. The optical density of GAD65-positive terminals and the number of GAD65-positive puncta was significantly increased in the striatum at both 1 day and 3 days after ischemia. Consistently, data from western blotting suggested an increased expression of GAD65 in the striatum after ischemia. For the rats treated with muscimol, the number of survived cells in the striatum was greatly increased compared to the non-treatment group. The present study demonstrates an enhancement of inhibitory synaptic transmission in LA neurons after ischemia, which is contributed by two mechanisms. One is the increased presynaptic release of GABA mediated by presynaptic GABAA receptors. The other is the increased expression of GAD. Facilitation of inhibitory synaptic transmission by muscimol protects striatal neurons against ischemia. Therefore, the enhancement of inhibitory synaptic transmission might reduce excitotoxicity and contribute to the selective survival of LA neurons after ischemia.

The submitted thesis is devoted to the quantitative analysis of tyrosine kinase inhibitors, specifically imatinib and nilotinib, by liquid chromatography-mass spectrometry method. The main purpose of developing this new method of analysis at the Department of Clinical Biochemistry and Diagnostics at the University Hospital in Hradec Králové was measuring and monitoring serum or plasma concentration levels of these drugs in patients with chronic myeloid leukemia, less often in patients with gastrointestinal stromal tumour. The main task during the elaboration of the thesis was to fully optimize and validate the method. Previously, this method for the analysis of tyrosine kinase inhibitors was routinely performed here by high-performance liquid chromatography with spectrophotometric (UV) detection. As part of the modernization of laboratory technology, they started to use high-performance liquid chromatography with mass spectrometry at the workplace. The analytes with their internal standards were obtained by a liquid-liquid extraction process. Then, samples were separated on a C18 reverse phase column using isocratic elution. Subsequently, both analytes were detected by a triple quadrupole tandem mass spectrometer with ESI ion source in a positive mode. As a part of the method validation was to...