Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Klára Dohnalová Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Inhibitory effect of SPA70 on hPXR activation This work focuses on pregnane X receptor (PXR) and its antagonists. PXR is a ligand-activated nuclear receptor that plays a major role in detoxification of xenobiotics and protecting the organism from their toxic effects. Recent evidence also shows endogenous action of PXR in the metabolism of lipids, glucose and bile acids. However, PXR activation could be harmful, since induction of biotransformation enzymes by PXR agonists may result in reduced treatment efficacy, increased toxicity of drug metabolites and resistance to chemotherapeutic agents. Recent research has been intensively focused on PXR antagonists capable of abolishing these unfavourable effects. Recently discovered human PXR antagonist SPA70 has a promising potential for future usage. In this study, we investigated the inhibitory effect of SPA70 on activated PXR. To activate PXR we used agonists binding directly to PXR (rifampicin, hyperforin, SR12813) and also agonists activating PXR indirectly via cell signalling pathways (U0126, PD184352, PD0325901). Experiments were performed using luciferase...

In the process of pre-mRNA splicing introns are removed from pre-mRNA and exons are joined together. Current studies show, that about 95 % of genes, which contain more than two exons, can undergo alternative splicing. In this process some exons are included in or excluded from the final mRNA. Majority of pre-mRNA splicing take place co- transcriptionaly at this time RNA polymerase II is still attached to pre-mRNA. Alternative splicing is complex process that takes place in a close proximity of DNA and histones that might modulate alternative splicing decisions. Futher studies have validated fibronectin gene (FN1) and his alternative exons EDA and EDB (extra domain A and B) as suitably model for studying alternative splicing. Study using FN1 minigene reporter system, which is composed from EDA exon and two surrounding introns and exons, has proved that insertion of transcription enhancer SV40 infront of promotor, the level of EDA inclusion is decreased. So far, has not been prooved if this mechanism can function in real genome context and if distal transcription elements can influence alternative splicing. In this study, we have predicted transcription enhancer for FN1 gene by using The Ensemble Regulatory Build and FANTOM 5. The predicted transcription enhancer, is located 23,5 kbp upstream of TSS...

A velocidade de renovação das proteínas musculares define o crescimento muscular, e afetam a qualidade da carne, destacando-se a renovação de colágeno e taxa proteolítica que tem implicação no fenótipo de maciez. No entanto, existe pouca informação na literatura sobre mudanças no processo de remodelação de proteínas musculares em resposta à taxa de recuperação do ganho de peso, após um período de subnutrição em animais fisiologicamente maduros. Dessa forma, o uso de sequenciamento de RNA pode indicar mudanças no tecido muscular através de um perfil de expressão diferencial que explicam mudanças nos fenótipos associados ao crescimento muscular. Portanto, o objetivo deste estudo foi avaliar as mudanças no transcriptoma associado a fenótipos de carne de vacas adultas Nelore submetidas a diferentes taxas de recuperação de peso. Neste experimento foram utilizadas 28 vacas adultas de 5 a 16 anos, sendo que 23 animais, por apresentarem baixo escore corporal no inicio do experimento como resultado de perda de peso durante uma estação seca, foram aleatoriamente distribuídas em 3 grupos baseados em ganho diário de peso vivo: GB - Base (abatidos com baixo escore corporal, n=4); GL - Lento (0,6 kg/dia, n=9); GR - Rápido (1,2 kg/dia, n=10). Um quarto grupo (GM - Manutenção, n=5), foi formado de animais com alto escore corporal do início ao final do experimento. Os animais foram abatidos em quatro pontos definidos por dias (d) de confinamento (A1(0d) = GB; A2(51d) = GL e GR; A3(74d) = GR; A4(104d) = GL e GM), e as amostras do musculo Longissimus thoracis foram coletadas para obtenção do RNA total. Após a obtenção do mRNA e a preparação das bibliotecas de cDNA, o sequenciamento das amostras foi feito em um equipamento HiScanSQ. Vinte e quatro horas após o abate, foram determinados o pH e a cor instrumental (L*, a*, b*) medidos no músculo Longissimus thoracis. Dois bifes foram amostrados para a análise de força de cisalhamento às 24h e 21 dias. Na comparação entre fenótipos e genes, os contrastes utilizaram o grupo GB como referência. As análises estatísticas foram realizadas no programa R, usando ANOVA e teste de Tukey. Menores força de cisalhamento aos 21 dias (P<=0,05) foram encontradas para o grupo GL (A4) e GM (A4) comparadas ao GB. Valores de L*, a*, b* no músculo do GR (A3) também foram maiores em relação ao GB. Somente o valor de b* na gordura foi diferente (P<=0,05) em GR (A2 e A3) em relação ao GB. Foram identificados o total de 578 genes diferencialmente expressos (DE; FDR 5%) nos cinco contrastes avaliados. A análise funcional de genes DE entre contrastes identificou uma via KEEG para o grupo GL (\"Ribosome\") e cinco para o grupo GR (\"ECM receptor interaction\", \"Focal Adhesion\", \"Protein digestion and absorption\", \"PI3K-Akt signaling pathway\"). Para o enriquecimento GO, foram obtidas quatro categorias, sendo \"Semaphorin receptor activity\" para o grupo GL, enquanto \"Cellular response to amino acid stimulus\", \"Collagen trimer\" e \"Extracellular matrix structural constituent\" foram identificadas para GR. As vias em geral estão associadas a alterações no tecido conjuntivo do músculo. As diferenças encontradas entre fenótipos e genes indicam que as taxas de crescimento afetam de forma diferente os fenótipos associados à qualidade da carne. A realimentação em animais adultos, ao alterar o transcriptoma com possíveis impactos no tecido conjuntivo, parece mostrar um cenário de fibrose muscular no ganho rápido de peso, diferentemente do ganho lento, que mostrou ser uma alternativa para melhorar a qualidade da carne. Todavia, os impactos na estrutura muscular e proteica que possam explicar incremento em aspectos de textura da carne precisam ser determinados. / The speed of renewal of muscle proteins defines muscle growth, and affect meat quality, especially the renewal the of collagen turnover and proteolytic rate that has implication in the tenderness phenotype. There is little information in the literature about changes in the muscle protein remodeling process in response to the rate of recovery of weight gain after malnutrition in physiologically mature animals. In this way, the use of RNA sequencing may indicate changes in muscle tissue through a differential expression profile that explains changes in phenotypes associated with muscle growth. Therefore, the objective of this study was to evaluate changes in the transcriptome associated with meat phenotypes of Nellore adult cows submitted to different rates of weight recovery. In this experiment, 28 adult cows from 5 to 16 years old were used, being that twenty-three animals were distributed randomly in 3 groups based on daily gain of live weight, since they presented low body score at the beginning of the experiment as a result of weight loss during a dry season: GB - Base (slaughtered with low body score, n=4); GL - Slow (0.6kg/day, n=9); GR - High (1.2 kg/day, n=10). A fourth group (GM - Maintenance, n = 5) was formed from animals with high body score from the beginning to the end of the experiment. The animals were slaughtered at four points defined by confinement days(d) (A1(0d) = GB; A2(51d) = GL and GR; A3(74d) = GR; A4(104d) = GL and GM), and Longissimus thoracis muscle samples were collected to obtain the total RNA. After obtaining the mRNA and preparation of the libraries, the sequencing of the samples was done in a HiScanSQ equipment. Twenty-four hours after slaughter, the pH and instrumental color (L*, a*, b*) were determined in the Longissimus thoracis muscle. Two steaks were collected for shear force analysis at 24h and 21 days. In the comparison of the phenotypes and genes, contrasts were used the GB group as reference. Statistical analysis were performed in R software, using ANOVA and Tukey test. Lower shear forces at 21 days (P<=0.05) were found for the GL (A4) and GM (A4) groups compared to GB. Values of L*, a*, b* in the GR (A3) muscle were also higher in relation to GB. Only the value of b* in fat was different (P<=0.05) in GR (A2 and A3) in relation to GB. A total of 578 differentially expressed genes (DE; false discovery rate 5%) in the five contrasts evaluated. Functional analysis of DE genes between contrasts identified a KEEG pathway for the GL group (\"Ribosome\") and five for the GR group (\"ECM receptor interaction\", \"Focal Adhesion\", \"Protein digestion and absorption\", \"PI3K-Akt signaling pathway\"). For GO enrichment, four categories were obtained, being \"Semaphorin receptor activity\" for GL, while \"Cellular response to amino acid stimulus\", \"Collagen trimer\" and \"Extracellular matrix structural constituent\" were identified for GR. Pathways in general are associated with changes in connective tissue of muscle. Re-feeding in adult animals by altering the transcriptome with possible impacts on connective tissue, seems to show a scenario of muscle fibrosis in the fast gain of weight, unlike the slow gain, which proved to be an alternative to improve meat quality. However, the impacts on muscle and protein structure that may explain the increase in meat texture aspects need to be determined.

La distribution de la neige à l'échelle du paysage dans les zones alpines est une des variables les plus importantes contrôlant la structure et la fonction des écosystèmes de montagne. Des changements d'épaisseur neigeuse et de durée d'enneigement peuvent entraîner de grands changements dans les conditions édapho-climatiques, ainsi que dans la composition des communautés végétales et surtout sur les cycles biogéochimiques majeurs et par conséquence la structure et le fonctionnement de l'écosystème. Nous avons utilisé l'approche métatranscriptomique pour essayer de comprendre la diversité fonctionnelle réelle et les activités exprimées dans les sols alpins par les micro-organismes, en réponse à différentes contraintes environnementales. La transcriptomique, et par extension, la métatranscriptomique, peut être vue comme l'analyse quantitative complète de tous les gènes exprimés par un ou plusieurs organismes, ou par l'écosystème entier. L'utilisation de cette approche implique d'abord l'extraction des ARN une bonne qualité et avec un bon rendement, ensuite la conversion de ces ARN en cDNA en ciblant les fractions de ARNm. La capacité d'évaluer le metatranscriptome des communautés microbiennes complexes dans différentes conditions environnementales représente en soi une avancée significative dans notre capacité de relier la structure et les fonctions des communautés avec les génotypes d'ADN (les séquences) et avec la correspondance phénotype. Dans cette étude, nous présentons l'utilisation pour la première fois de l'approche métatranscriptomique concernant les activités des communautés microbiennes des eucaryotes des sols alpins sous deux conditions d'enneigement très contrasté nommés LSM (lately snowmelt) et ESM (early snowmelt), qui sont caractérisés par des gradients climatiques contrastés et des différences de végétations associées. Nous présentons également une analyse des séquences et des procédures d'annotation en utilisant des logiciels publiquement disponibles et des scripts de python en utilisant l'environnent d'Obitools. Nous avons également développé un pipeline d'analyse bio-informatique adapté qui permet d'extraire correctement des renseignements fonctionnels et taxinomiques de ces bases de données. / The distribution of snow across the landscape in the Alps is one of the most important variables controlling the structure and function of mountain ecosystems. Changes in snow depth and duration can cause major changes in soil and climatic conditions, as well as the composition of plant communities and especially on the major biogeochemical cycles and consequently the structure and functioning of the ecosystem. We used the approach métatranscriptomique to try to understand the functional diversity and real activity expressed in Alpine soils by micro-organisms in response to different environmental constraints. Transcriptomics, and by extension, the métatranscriptomique, can be seen as full quantitative analysis of all genes expressed by one or more agencies or by the entire ecosystem. Using this approach involves first extracting RNA in good quality and good yield, then the conversion of RNA into cDNA by targeting mRNA fractions. The ability to assess metatranscriptome complex microbial communities under different environmental conditions is in itself a significant advance in our ability to link the structure and functions of communities with the genotypes of DNA (the sequence) and phenotype correspondence. In this study, we present the first use of the approach métatranscriptomique on the activities of eukaryotic microbial communities of alpine soil in two very contrasting locations called LSM (Lately snowmelt) and ESM (early snowmelt) which are characterized by contrasting climatic gradients and differences in vegetation associated. We present an analysis of sequences and annotation procedures using publicly available software and scripts using python programs and Obitools. We have also developed a pipeline of bioinformatics analysis adapted to correct extraction of information of the functional and taxonomic databases.

Stochastic fluctuations in mRNA and protein copy number between cells are inevitable during the process gene expression, even when cells carry identical chromosomes. Such fluctuations are able to impact the phenotypic fate of the cell, and are known to have greater impact when the copy number of the molecule involved is low. Additionally, up to 50% of proteins in Escherichia coli are present in the cell at a level of 10 molecules per cell or fewer (Taniguchi et al. 2010). As such, quantification of low copy number gene expression products and their distribution in cellular populations is key in understanding the process of gene expression. Currently, there are few techniques that allow investigation with the single cell and single molecule resolution required to study low copy number gene expression products. This work presents a novel method for protein quantification at the single molecule level, Quantitative HaloTag-TMR labelling, and uses the technique to quantify the absolute numbers of the low copy number RecB, RecC and RecD subunits of the bacterial DNA repair enzyme RecBCD, finding each subunit is present at between two and eight molecules per cell with mean numbers per cell of 4.9, 4.7 and 4.5 respectively. Additionally single molecule mRNA FISH was used to quantify the mRNA levels of recB and recD within cells, with means of 0.21 and 0.31 mRNA per cell being observed respectively. Finally this work presents a new method for use detecting both mRNA and protein simultaneously in individual cells by combining the HaloTag and FISH protocols to give HaloFISH. This work introduces two novel techniques that allow for single cell examination of gene expression, and investigates RecBCD expression at the single molecule level.

In Drosophila, early embryonic development is controlled by maternally loaded RNAs and proteins. For proper development to occur it is vital these maternal transcripts are post-transcriptionally regulated. SMAUG, a major post-transcriptional regulator, has been found to be responsible for the destabilization of two thirds of the unstable maternal transcripts upon egg activation (Tadros et al., 2007). smg mRNA is translationally repressed in stage 14 oocytes, but its translation is activated upon egg activation in a PAN GU kinase dependent manner. Here I show that redundant translational repression elements reside in the smg 3’UTR, and PUMILIO mediates repression through one of these elements. I also show that these elements are sufficient to cause translational repression in stage 14 oocytes. smg mRNA appears to be regulated post-initiation in stage 14 oocytes in a large repression complex which is similar to smg mRNA repression in a png mutant.

In Drosophila, early embryonic development is controlled by maternally loaded RNAs and proteins. For proper development to occur it is vital these maternal transcripts are post-transcriptionally regulated. SMAUG, a major post-transcriptional regulator, has been found to be responsible for the destabilization of two thirds of the unstable maternal transcripts upon egg activation (Tadros et al., 2007). smg mRNA is translationally repressed in stage 14 oocytes, but its translation is activated upon egg activation in a PAN GU kinase dependent manner. Here I show that redundant translational repression elements reside in the smg 3’UTR, and PUMILIO mediates repression through one of these elements. I also show that these elements are sufficient to cause translational repression in stage 14 oocytes. smg mRNA appears to be regulated post-initiation in stage 14 oocytes in a large repression complex which is similar to smg mRNA repression in a png mutant.

Recently, it has been suggested that neurodevelopmental abnormalities underlie schizophrenia. However, it has also been suggested that schizophrenia is a neurodegenerative disease as evidenced by a progressive worsening of symptoms over time. Neurodevelopmental abnormalities may, therefore, create a functionally compromised system that is more susceptible to neuronal atrophy and/or death caused by environmental factors such as stress (a known precipitant of acute psychotic episodes and exacerbant of schizophrenia). This hypothesis was tested using the putative neurodevelopmental model of schizophrenia described by Lipska 'et al'. (1993). The effects of neonatal hippocampal lesions on BDNF mRNA and NMDAR1 mRNA, factors involved in development, cell survival and cell communication, were investigated in adult rats following exposure to a physiological stressor. Apoptosis levels were also investigated in these rats to determine if neurodegeneration was present. Results demonstrate that BDNF mRNA was reduced in the prefrontal cortex and hippocampus of lesioned as compared to sham rats. Increased BNDF mRNA resulted from swim stress in both groups, but the increase in lesioned animals was more pronounced than controls. NMDAR1 mRNA was also reduced in the prefrontal cortex and CA3 and CA1 regions of the hippocampus in lesioned versus sham rats. There was an increase, however, in the dentate gyrus of lesioned versus sham rats. Swim stress increased NMDAR1 mRNA in the prefrontal cortex and decreased it in the hippocampus. There was also an increase in apoptosis in lesioned versus sham rats, with no significant increase in response to stress. Reductions in BDNF mRNA in lesioned versus control animals support the hypothesis that neurodevelopmental lesions may result in a system more susceptible to stressors. Reductions in NMDAR1 mRNA are in accordance with the NMDA glutamate receptor hypofunction theory of schizophrenia. It is possible that reductions in glutamate function can remove the inhibitory effect of GABA, thereby resulting in overexcitation of the system and a potential for neurodegeneration. Increased apoptosis supports the presence of neurodegeneration as an ongoing phenomenon. Even though the effect of acute stress on apoptosis was not significant, the very small increases demonstrated can have significant functional consequences over extended periods of time.

Establishment of cell polarity requires the involvement of several posttranscriptional regulatory mechanisms, including mRNA localization and translational control. A family of highly conserved RNA binding proteins in vertebrates, VICKZ (V̲g1RBP/V̲era, I̲MP-1, 2, 3, C̲RD-BP, K̲OC, Z̲BP-1) proteins, has been shown to act in these two processes. Previous studies of the posttranscriptional mechanisms mediated by VICKZ family members have been largely limited by the lack of genetic approaches in certain vertebrate systems. Identification of Imp, the Drosophila member of the VICKZ family, opened the possibility to use genetic approaches to investigate the roles of a VICKZ family member in mRNA localization and translational control. In this dissertation, we show that Imp is associated with Squid and Hrp48, two heterogeneous proteins (hnRNP) that complex with one another to regulate localized expression of gurken (grk). In addition, Imp binds grk mRNA with high affinity in vitro and is concentrated at the site of grk localization in midstage oocytes. Mutation of the Imp gene does not substantially alter grk expression, but does partially suppress the grk mis-expression phenotype of fs(1)k10 mutants. In contrast, overexpression of Imp in germ line cells results in mislocalization of grk mRNA and protein. The opposing effects of reduced and elevated Imp activities on grk expression suggest that Imp acts in regulation of grk expression, but in a redundant way. To further explore the mechanisms by which localized expression of grk is regulated by Imp, a deficiency screen was conducted to search for dominant modifiers of the dorsalized phenotype resulting from Imp overexpression. Twelve genomic regions were identified to contain dominant modifiers of the Imp overexpression phenotype. Further characterization of mutants of genes within these genomic regions led to identification of five modifiers, including cyclin E (cycE), E2f transcriptional factor 1 (E2f1), lingerer (lig), snail (sna) and mushroom body expressed (mub). E2f1 encodes a transcriptional factor that is involved in regulating the G1 to S phase transition during mitosis. Mutation of E2f1 results in altered grk mRNA and protein distribution within oocyte, revealing a role for this gene in regulation of grk expression. / text

Background: The naked mole-rat (NMR) is a long-lived and cancer resistant species. Identification of potential anti-cancer and age related mechanisms is of great interest and makes this species eminent to investigate anti-cancer strategies and understand aging mechanisms. Since it is known that the NMR expresses higher liver mRNA-levels of alpha 2-macroglobulin than mice, nothing is known about its structure, functionality or expression level in the NMR compared to the human A2M. Results: Here we show a comprehensive analysis of NMR- and human plasma-A2M, showing a different prediction in glycosylation of NMR-A2M, which results in a higher molecular weight compared to human A2M. Additionally, we found a higher concentration of A2M (8.3±0.44 mg/mL vs. and 4.4±0.20 mg/mL) and a lower total plasma protein content (38.7±1.79 mg/mL vs. 61.7±3.20 mg/mL) in NMR compared to human. NMR-A2M can be transformed by methylamine and trypsin resulting in a conformational change similar to human A2M. NMRA2M is detectable by a polyclonal antibody against human A2M. Determination of tryptic and anti-tryptic activity of NMR and human plasma revealed a higher anti-tryptic activity of the NMR plasma. On the other hand, less proteolytic activity was found in NMR plasma compared to human plasma.