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Doctor of Philosophy / Department of Animal Sciences and Industry / Cassandra K. Jones / Porcine epidemic diarrhea virus (PEDV) is a heat-sensitive virus that devastated the United States swine industry. Because of its heat sensitivity, it was hypothesized that a pellet mill mimicking commercial thermal processing may mitigate PEDV infectivity. From the results, it was determined that a conditioning time of 30 sec or greater and temperatures above 54.4°C were effective point-in-time kill steps to inactive PEDV in a research setting. However, this does not prevent subsequent recontamination after pelleting as it is a point-in-time mitigation step. To further explore this, various mitigation additives were evaluated to prevent or mitigate PEDV post-pellet contamination in swine feed and ingredients. Various additives were examined across 3 experiments and included mitigation additives of medium chain fatty acids (MCFA), organic acids (OA), essential oils (OA), formaldehyde based products, and sodium bisulfate. From Exp. 1, formaldehyde, medium chain fatty acids (MCFA), essential oils (EO), and organic acid (OA) each decreased detectable PEDV RNA compared to the control (P<0.05). Additionally, PEDV stability over time was influenced by matrix as the meat and bone meal and spray-dried animal plasma resulted in a greater (P<0.05) quantity of detectable PEDV RNA over 42 days compared to that of the swine diet and blood meal. In Exp. 2, the 1% MCFA inclusion was equally effective at mitigating PEDV as a commercially available formaldehyde product in the complete swine diet. To further explore the effects of MCFA against PEDV, Exp. 3 was conducted to evaluate lower inclusion levels of MCFA and fat sources containing MCFA. It was noted that formaldehyde, 1% MCFA (1:1:1: of caproic, caprylic, and capric acids), 0.66% caproic, 0.66% caprylic, and 0.66% capric acids enhance the RNA degradation of PEDV in swine feed as determined by a bioassay. The MCFA were also evaluated against Salmonella Typhimurium, Generic Escherichia coli, Enterotoxigenic Escherichia coli, and Campylobacter coli. It was noted that the efficacy of the MCFA varied between each bacteria species with caproic and caprylic being the most effective. Commercial developmental products were also tested and determined that Product A and B provided the lowest MIC values across Salmonella Typhimurium, Generic Escherichia coli, and Enterotoxigenic Escherichia coli (P < 0.05). Product A and B were further tested in an animal disease trial utilizing a strain of enterotoxigenic Escherichia. coli O149:K91: K88. From d 7 to 14, chlortetracycline, 1:1:1 blend, and Product B, all improved G:F compared to the control (P<0.05). This also led to chlortetracycline and Product B having an improvement (P<0.05) over the control diet from d 0 to 14. A treatment × day interaction for the enterotoxigenic E. coli plate scores was observed (P < 0.05), which occurred because of the decrease (P<0.05) in plate scores for Product B from d 1 to d 14 and an increase (P<0.05) in chlortetracycline from d 7 to 14. A decrease (P<0.05) in plasma urea nitrogen and haptoglobin was observed as time increased from d -2 to 14. In summary MCFA have shown to be an effect interventional mitigation strategy against PEDV and various bacteria.

Early experience critically shapes cognitive, affective, and behavioral development, and experiences of early life stress (ELS) have been documented to negatively impact developmental trajectories. An increasing need exists for innovative intervention development to ameliorate negative impacts of ELS. Translational neuroscience approaches hold promise for addressing the needs of individuals who have experienced ELS through the development and testing of targeted intervention strategies grounded in neuroscientific knowledge. The current dissertation work employed a translational neuroscience approach to improve inhibitory control (IC) in a group of adolescents (N=20) exposed to a range of ELS via a brief, computerized training paradigm. Baseline analyses of the association between ELS and IC revealed a positive relationship (i.e., increased ELS, increased IC performance), counter to hypotheses. The IC training did not significantly improve IC in the training group compared to the control group over time, contrary to hypotheses and previous work demonstrating the effectiveness of this IC training in young adults. Transfer of training effects to a non-trained IC task and to real-world risk-taking behavior were limited. ELS demonstrated some moderation of neural effects of IC training. The use of neuroimaging in this dissertation work allowed for further investigation of neural mechanisms even in the absence of significant behavioral training, transfer, and moderation effects. Results are discussed in the context of the utility of such IC training approaches for adolescents with ELS exposure and of translational neuroscience approaches more broadly. Future directions for IC training, translational neuroscience intervention approaches, and the incorporation of neuroimaging in this work are explored. This dissertation includes unpublished co-authored material. / 10000-01-01

Neste estudo avaliamos atividades biológicas pré-clínicas de moléculas obtidas de Mangifera indica e de Valeriana glechomifolia. Mangiferina, isolada de M. indica, estimulou a proliferação celular e induziu um aumento significativo na secreção do fator de crescimento do nervo e do fator de necrose tumoral em células de glioblastoma humano U138-MG in vitro. Uma injeção sistêmica de mangiferina melhorou a consolidação da memória de longa duração (LTM) de reconhecimento de objetos (RO) e prejudicou a retenção da memória aversiva no teste da esquiva inibitória (EI) em ratos. A melhora da LTM no RO promovida pela administração sistêmica de mangiferina também foi observada com a administração intrahipocampal. Já o prejuízo da memória no teste da EI observado sistemicamente não ocorreu com a infusão no hipocampo ou amígdala. Camundongos atáxicos também apresentaram melhora na memória de RO após administração crônica de mangiferina, sem efeito na EI; um extrato comercializado de M. indica não afetou a memória no RO, mas facilitou a memória na EI. Os resultados indicam que mangiferina melhora a LTM no RO com envolvimento do hipocampo por meio de um mecanismo que pode envolver um aumento dos níveis da neurotrofina NGF e da citocina TNF-α. Valepotriatos, isolados de V. glechomifolia, demonstraram inibição da viabilidade de células tumorais U138-MG nas doses de 30 e 100μg/μl; o 8-Br- AMPc, um análogo do AMPc, atenuou à inibição dos valepotriatos na viabilidade celular, sugerindo que os valepotriatos interagem com a rota de sinalização celular do AMPc/PKA na inibição da viabilidade de células cancerosas. A administração sistêmica de valepotriatos, em camundongos 30 minutos antes dos testes, apresentou os seguintes resultados: durante a exploração no campo aberto, a dose 10mg/kg causou redução na locomoção e no comportamento exploratório e diminuição da ansiedade no teste do labirinto em cruz elevado. Não ocorreu diferença entre os tratamentos na memória de EI e na memória RO, exceto no grupo que recebeu 3mg/kg de valepotriatos que apresentou piora na LTM de RO. Os resultados indicam que os valepotriatos causaram atividades ansiolítica e sedativa sem déficits na memória de EI e RO em camundongos tratados com 10mg/kg. As atividades biológicas in vitro e in vivo encontradas nas moléculas estudadas (especialmente mangiferina e valepotriatos) geram interesse de investigações para utilizações terapêuticas na memória e no câncer. / We evaluated the biological activities of molecules obtained from Mangifera indica and Valeriana glechomifolia. Mangiferin, isolated from M. indica, stimulated cell proliferation and induced a significant increase in levels of nerve growth factor and tumor necrosis factor secreted in human glioblastoma cells U138-MG in vitro. A systemic injection of mangiferin improved long term memory (LTM) consolidation of object recognition (NOR) and impaired memory retention in aversive inhibitory avoidance test (IA) in rats. The improvement in NOR memory promoted by systemic administration of mangiferin was also observed with intrahippocampal administration. The memory impairment observed systemically in the IA did not occur with the infusion into the hippocampus or amygdala. Ataxic mice also showed improvement in NOR memory after chronic administration of mangiferin, with no effect on IA; a standardized extract of M. indica had not effect on memory in NOR, but facilitated the memory in IA. The results indicate that mangiferin improvement NOR memory involving the hippocampus through a mechanism that may involve increased levels of neurotrophins and cytokines. Valepotriates isolated from V. glechomifolia showed inhibition of the viability of U138-MG tumor cells at doses of 30 and 100μg/μl; 8-BrcAMP, an analogue of cAMP, reversed the inhibition of valepotriates on cell viability, suggesting that valepotriates interact with the cAMP/PKA signaling route in the inhibition of the viability of cancer cells. Systemic administration of valepotriates in mice, 30 minutes before tests, showed the following results: during the open-field, the dose of 10mg/kg caused a reduction in locomotion and exploratory behavior and decreased anxiety in the test of elevated plus maze. There was no difference between treatments in IA or NOR memories, except from the group receiving valepotriates at 3mg/kg, which worsened in the NOR. The results indicated that valepotriates at 10mg/kg caused anxiolytic and sedative activities without inducing memory deficits in IA and NOR. The biological activities in vitro and in vivo found with the studied molecules (notable mangiferin and valepotriates) support further research for potential therapeutic uses in cancer and in memory.

A ciência e tecnologia andam juntas. A ciência fornece o conhecimento para a geração de novas tecnologias que, porsua vez, disponibiliza novas ferramentas para a geração de novos conhecimentos. Uma educação científica deve vir acompanhada de uma educação tecnológica permitindo então que o aluno entenda não só o experimento, mas também como os resultados foram obtidos, como os equipamentos, técnicas e protocolos funcionam. Para uma educação científica efetiva o aluno deve ir além de expectador, se colocando no papel de pesquisador, a fim de construir seu próprio conhecimento. No entanto, para essa tarefa, é necessário o uso de equipamentos adequados ao experimento a ser realizado. Essa tese visa suprir a necessidade de equipamentos para experimentos na área de neurociência focados no estudo de memória e aprendizado, utilizando roedores e peixes como modelos animais na tarefa de esquiva inibitória passiva. Esses equipamentos visam uma maior acessibilidade do ponto de vista operacional e financeiro, sendo mais precisos e com isso fornecendo dados mais confiáveis e robustos do que os equipamentos disponíveis. Possibilitando aos alunos, da alfabetização à pós-graduação, a realização de experimentos comportamentais, tanto de maneira simples quanto complexas, interagindo em todas as etapas experimentais, resultando em aprendizagem e desenvolvimento cognitivo além de possibilitar novas descobertas científicas. / Science and technology walking together, science provides the knowledge to generate new technologies which in turn provides new tools for the generation of new knowledge. A science education must be accompanied by a technological education to enable students to understand not only the experiment, but also how the results were obtained, understanding how the equipment, techniques and protocols work. For effective science education the student must go beyond bystander, putting themselves in the researcher function in order to construct their own knowledge. However for this task, it is necessary to use suitable equipment to the experiment to be performed. This PhD Thesis aims to fill a need for equipment for experiments in neuroscience focused on the study of memory and learning, using rodents and fish as animal models in the passive inhibitory avoidance task. These devices achieve greater accessibility of the operational and financial point, being more accurate and thus providing more reliable and consistent data that current equipment. Enabling student’s literacy to post graduate conducting simple and complex behavioral experiments, interacting in all experimental steps, resulting in learning and cognitive development and enable new scientific discoveries.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Etela Kouklíková Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Study of the effect of cyclin-dependent kinase inhibitors on the expression of selected AKR and CBR enzymes in human cell lines Cyclin-dependent kinase inhibitors (CDKi) are considered as a suitable treatment especially in patients with wrong prognosis or advanced stage of cancer. It has only recently been discovered that CDKi are able to influence the activity of some enzymes from aldo-keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) superfamilies. AKR and SDR enzymes belong to a group of carbonyl reducing enzymes that are involved in the metabolism of endobiotics and xenobiotics. An important group of drugs that are metabolized by these enzymes to less efficient compounds are anthracyclines. The aim of this diploma thesis was to find out whether purvalanol A, roscovitin, dinaciclib, AZD5438 and R547 can affect the expression of the most important anthracycline reductases (AKR1A1, AKR1B10, AKR1C3, AKR7A2 and CBR1) in human HepG2 and HL-60 cell lines. Expression of anthracycline reductases in cells exposed to CDKi was evaluated at mRNA level by RT-qPCR and at protein level by Western blotting. The...

Inhibitory control, described as the ability to suppress one response in favor of a goal-directed response, is thought to play an important role in the development of emotional regulation as well as various forms of psychopathology, including ADHD. Up until very recently, inhibitory control has been researched within two completely separate fields of study: temperament and neuropsychology. In the temperament/personality literature, inhibitory control is a major component of the overarching temperament/personality factor of Effortful Control/Conscientiousness. In the field of neuropsychology, inhibitory control is considered one aspect of executive function. Further complicating the current understanding of inhibitory control is the complexity of the underlying neural networks implicated in inhibitory control. This study examined inhibitory control in temperament and executive function in children with and without ADHD, and it explored the relationship between inhibitory control and the superior frontal cortex (SFC) and orbital frontal cortex (OFC) volumes. In order to assess subareas of the OFC and SFC, an innovative parcellation method was used. Results suggested that the temperament and executive function measures of inhibitory control did form a single factor as long as they were measured within the same modality, parent-report. In contrast, the performance-based measure of inhibitory control was not correlated with any of the parent-report measures of inhibitory control and was, therefore, analyzed separately in relation to OFC and SFC volumes. Parent-rated inhibitory control was predicted by ADHD status only, but exploratory analyses suggested that left anterior SFC, right and left anterior medial OFC, and gender were related to parent-rated inhibitory control. In contrast, performance-based inhibitory control was predicted by gender and left SFC, specifically posterior left SFC. Taken together, these findings suggest a conceptual overlap between temperament and executive function that brings together two areas of the literature and has implications for the understanding of various forms of psychopathology characterized by deficits in inhibitory control. This study provides evidence for the role of the SFC and the OFC in inhibitory control, depending upon the measurement method, and contributes to the broader understanding of the neural mechanisms of inhibitory control in children.

O esquecimento intencional pode cumprir uma função estratégica no sistema cognitivo, que permite aos indivíduos não pensar sobre acontecimentos indesejados do passado, tais como eventos traumáticos, dolorosos e violentos, dos quais se prefere não recordar. Enquanto esquecer involuntariamente é uma falha da lembrança, por outro lado, esquecer intencionalmente parece ser uma função estratégica da memória. A presente dissertação teve por objetivo investigar os mecanismos neurocognitivos que contribuem para o esquecimento de memórias. A dissertação se organizou em dois estudos. O primeiro estudo consiste em uma revisão sistemática de artigos empíricos publicados nos últimos dez anos sobre a supressão de memórias indesejadas. O segundo estudo é um ensaio empírico, no qual foi executado um experimento adaptado do paradigma Think/No-Think com a utilização de marcadores eletrofisiológicos de eletroencefalograma. Participaram do experimento 22 sujeitos, alocados aleatoriamente em dois grupos com estratégias distintas de esquecimento: Supressão de memória e substituição de pensamentos. Durante toda a tarefa experimental, os participantes tiveram dados de EEG continuamente gravados. Os resultados decorrentes do ensaio empírico estão de acordo com os achados da literatura, indicando que a positividade parietal em torno de 400-800ms após a apresentação do estímulo é um marcador de lembrança consciente durante a recuperação de memória. Apenas na estratégia de supressão de memória houve uma redução da positividade centro-parietal durante o esquecimento, entre 450 e 700ms após apresentação do estímulo. Além disso, uma maior deflexão no componente N2 durante a supressão é um preditor de esquecimento induzido. Os achados indicam que é possível mapear o sistema neurocognitivo subjacente à supressão de memórias. / Intentional forgetting can be characterized as a strategic function of the cognitive system that allows us not to think about unwanted memories from our past, as for example emotional events or traumatic experiences that we would prefer not to remember. While forgetting involuntarily is a failure of recollection, on the other hand, forgetting intentionally seems to be a strategic function of memory. The aim of this dissertation was to investigate the neurocognitive mechanisms that contribute to forgetting memories. The dissertation was organized in two studies. The first study consists of a systematic review of empirical articles published in the last ten years on the suppression of unwanted memories. The second study is an empirical essay, in which an experiment adapted from the Think/No-Think paradigm was performed, with the use of electrophysiological markers of electroencephalogram. Twenty-two subjects participated in the experiment, randomly assigned to two groups with distinct strategies of forgetting: Memory suppression and thought substitution. Throughout the experimental task, participants had continuously recorded EEG data. The results of the empirical essay are in agreement with the literature findings, indicating that the parietal positivity around 400-800 ms after the presentation of the stimulus is a marker of conscious memory during memory recovery. Only direct memory suppression reduced centro-parietal positivity during forgetting, between 450 and 700 ms post-stimulus. Also, a greater deflection in the N2 component during suppression is an induced forgetting predictor. The findings indicate that it is possible to map the neurocognitive system underlying memory suppression.

Neste estudo avaliamos atividades biológicas pré-clínicas de moléculas obtidas de Mangifera indica e de Valeriana glechomifolia. Mangiferina, isolada de M. indica, estimulou a proliferação celular e induziu um aumento significativo na secreção do fator de crescimento do nervo e do fator de necrose tumoral em células de glioblastoma humano U138-MG in vitro. Uma injeção sistêmica de mangiferina melhorou a consolidação da memória de longa duração (LTM) de reconhecimento de objetos (RO) e prejudicou a retenção da memória aversiva no teste da esquiva inibitória (EI) em ratos. A melhora da LTM no RO promovida pela administração sistêmica de mangiferina também foi observada com a administração intrahipocampal. Já o prejuízo da memória no teste da EI observado sistemicamente não ocorreu com a infusão no hipocampo ou amígdala. Camundongos atáxicos também apresentaram melhora na memória de RO após administração crônica de mangiferina, sem efeito na EI; um extrato comercializado de M. indica não afetou a memória no RO, mas facilitou a memória na EI. Os resultados indicam que mangiferina melhora a LTM no RO com envolvimento do hipocampo por meio de um mecanismo que pode envolver um aumento dos níveis da neurotrofina NGF e da citocina TNF-α. Valepotriatos, isolados de V. glechomifolia, demonstraram inibição da viabilidade de células tumorais U138-MG nas doses de 30 e 100μg/μl; o 8-Br- AMPc, um análogo do AMPc, atenuou à inibição dos valepotriatos na viabilidade celular, sugerindo que os valepotriatos interagem com a rota de sinalização celular do AMPc/PKA na inibição da viabilidade de células cancerosas. A administração sistêmica de valepotriatos, em camundongos 30 minutos antes dos testes, apresentou os seguintes resultados: durante a exploração no campo aberto, a dose 10mg/kg causou redução na locomoção e no comportamento exploratório e diminuição da ansiedade no teste do labirinto em cruz elevado. Não ocorreu diferença entre os tratamentos na memória de EI e na memória RO, exceto no grupo que recebeu 3mg/kg de valepotriatos que apresentou piora na LTM de RO. Os resultados indicam que os valepotriatos causaram atividades ansiolítica e sedativa sem déficits na memória de EI e RO em camundongos tratados com 10mg/kg. As atividades biológicas in vitro e in vivo encontradas nas moléculas estudadas (especialmente mangiferina e valepotriatos) geram interesse de investigações para utilizações terapêuticas na memória e no câncer. / We evaluated the biological activities of molecules obtained from Mangifera indica and Valeriana glechomifolia. Mangiferin, isolated from M. indica, stimulated cell proliferation and induced a significant increase in levels of nerve growth factor and tumor necrosis factor secreted in human glioblastoma cells U138-MG in vitro. A systemic injection of mangiferin improved long term memory (LTM) consolidation of object recognition (NOR) and impaired memory retention in aversive inhibitory avoidance test (IA) in rats. The improvement in NOR memory promoted by systemic administration of mangiferin was also observed with intrahippocampal administration. The memory impairment observed systemically in the IA did not occur with the infusion into the hippocampus or amygdala. Ataxic mice also showed improvement in NOR memory after chronic administration of mangiferin, with no effect on IA; a standardized extract of M. indica had not effect on memory in NOR, but facilitated the memory in IA. The results indicate that mangiferin improvement NOR memory involving the hippocampus through a mechanism that may involve increased levels of neurotrophins and cytokines. Valepotriates isolated from V. glechomifolia showed inhibition of the viability of U138-MG tumor cells at doses of 30 and 100μg/μl; 8-BrcAMP, an analogue of cAMP, reversed the inhibition of valepotriates on cell viability, suggesting that valepotriates interact with the cAMP/PKA signaling route in the inhibition of the viability of cancer cells. Systemic administration of valepotriates in mice, 30 minutes before tests, showed the following results: during the open-field, the dose of 10mg/kg caused a reduction in locomotion and exploratory behavior and decreased anxiety in the test of elevated plus maze. There was no difference between treatments in IA or NOR memories, except from the group receiving valepotriates at 3mg/kg, which worsened in the NOR. The results indicated that valepotriates at 10mg/kg caused anxiolytic and sedative activities without inducing memory deficits in IA and NOR. The biological activities in vitro and in vivo found with the studied molecules (notable mangiferin and valepotriates) support further research for potential therapeutic uses in cancer and in memory.

Doctor of Philosophy / Diagnostic Medicine/Pathobiology / Michael D. Apley / Bovine respiratory disease (BRD) remains a major disease in beef production systems. The administration of antimicrobials for both the control and treatment of acute BRD is common. According to most published accounts, antimicrobial resistance among BRD pathogens is increasing; therefore, judicious antimicrobial usage is vital for continued efficacy. The introduction of a novel antimicrobial class has not occurred for well over a decade, therefore it is paramount to maximize efficacy of the antimicrobials currently available. The challenge is targeting the perfect scenario: maximizing clinical efficacy while minimizing antimicrobial resistance. The host-pathogen-drug interaction is very complex and despite current sophisticated technology, this interaction is still not well understood for many infectious diseases. This dissertation work sought to investigate the effects of the administration of a macrolide for both control and treatment of acute BRD on the prevalence of resistance among isolated Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. Whole genome sequencing of M. haemolytica was utilized to investigate the presence/absence of macrolide resistance genes and their relationship to the observed minimum inhibitory concentration. Cattle were sampled (plasma and pulmonary epithelial lining fluid) after administration of gamithromycin for drug concentration analysis. A non-linear mixed effects approach was used to fit a compartmental model to the resulting sparse pharmacokinetic data so that a complete time concentration curve could be simulated. From these curves, the CMAX and AUC were measured and used to calculate standard PKPD indices using the MIC values of the isolated bacteria. Clear associations between the use of gamithromycin for control and treatment of BRD and a statistically significantly increased likelihood of macrolide resistance were not found, possibly due to sample size limitations. The calculation of pharmacokinetic-pharmacodynamic indices found that a longer drug exposure was more closely associated with a successful treatment outcome, but there was not a statistically significant correlation. However, there were few clinical failures in this study giving further credence to the complexity of the in vivo system. There are many factors beyond pharmacokinetics/pharmacodynamics and MICs that contribute to the success of a treatment regimen for cattle suffering from BRD.