Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Daniel Diviš Supervisors: Prof. Dr. Florence Apparailly, Directrice de Recherche Prof. PharmDr. Petr Pávek, Ph.D. (formal tutor) Title of diploma thesis: The role of miR-150 in the physiopathology of oligoarticular juvenile idiopathic arthritis Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatoid disease affecting children, and its pathological mechanisms are still poorly understood. Innate and adaptive immunity including myeloid cells play a major role in these processes. Epigenetic deregulations along with non-coding microRNAs have been reported in many inflammatory diseases. Moreover, preliminary results obtained by the research group of Prof. Florence Apparailly showed accumulation of intermediate monocytes along with the high expression of miR-150 in the synovial fluid of children affected by oligoarticular JIA. Based on these findings a hypothesis has been postulated suggesting that miR-150 could have a role in the pathogenesis of this disease and in the regulation of monocyte differentiation and function. To study the impact of miR-150 on monocytes from the peripheral blood of healthy donors, transfection experiments were performed to neutralize miR-150. The...

This thesis offers an insight in how information influence works in the digital modern age, through the use of strategic narratives in news reporting. The study uses a combination of a textual and narrative analysis in order to examine what kind of patterns are possible to distinguish in how the Russian state-funded news agencies RT and Sputnik are portraying Sweden in their reporting. Insights obtained by analyzing articles from the mentioned news outlets shows that four strategical ’master’-narratives can be found in the reporting: 1) Sweden is being ridiculed, 2) Sweden is portrayed as a collapsing state, 3) conspiracy theories about Sweden are made up and, 4) Russia is portrayed in friendly and peaceful terms. The analysis ultimately demonstrates how these four ‘master’-narratives can be understood as information produced in order to spread a negative image about Sweden and to promote a positive image of Russia. The study emphasizes the importance of continued research in the area and discusses the difficulties with addressing something that cannot be termed as fake news, but rather as fake narratives.

Oligodendroglial tumours originate from oligodendrocytes usually arising in the white matter and could be classified into grade-II oligodendrogliomas (OD) and anaplastic oligodendrogliomas (AOD, grade-III) according to the 2016 World Health Organization (WHO) grading scheme. ODs1 could be diagnosed by pathological and immunohistochemical analyses, however recent evidence suggests that they could be better diagnosed on the basis of defined genetic entities, such as the combined loss of chromosome 1p and 19q arms and IDH mutation. 1p/19q co-deletion is molecular hallmark of ODs and is clinically associated with better prognosis, response to chemo/radio-therapy and overall survival. Typical oligodendroglial histological features are strongly associated with 1p/19q loss and IDH mutation, which is critically important as diagnostic point of view. The examining of exclusive molecular signatures and transcriptome expression profiles added to histological class could compliment the classification of OD subtypes. In this regard, microRNAs (miRNAs, miRs) profiles could serve classifier signatures for tumour subsets. MiRNAs are 22nt short non-coding RNAs which are expressed endogenously and regulate diverse cellular process through negative control on gene expression at the posttranscriptional level by direct or imperfect interaction with their target mRNAs. MiRNAs are involved in regulating human tumorigenesis acting as either tumour suppressors or oncogenes. During the passage of tumorigenesis miRNA expression level is significantly increased or decreased compared to corresponding normal tissue. The same is observed with their mRNAs. Therefore, transcriptome profiling of human tumours could identify signatures associated with progression, diagnosis, prognosis and response to therapy. However, until recently the information regarding the expression of miRNAs and mRNA in oligodendroglial tumours is scarce. In this study we performed miRNA and mRNA differential expression profiling between grade II and grade III ODs using microarray based expression profiling platforms (723 transcripts and 41,000 genes, respectively). 7 cases for OD grade-II, and 7 for AOD grade-III, and 15 non neoplastic white matter (nnWM) samples were used after microdissection with no previous history of treatment. We performed a systematic evaluation of miRNAs and mRNAs expressions and determined miRNAs and putative target genes that are differentially expressed in grade III AOD, but not in grade II OD and in non-neoplastic white matter (nnWM). 1 ODs when used with ,,s\" will represent both OD and AOD. 50 miRNAs were overexpressed and 43 were down regulated in AOD-III, whereas 7 miRNAs showed significant reduction in expressions in OD-II group. 3 miRNAs were commonly down regulated in comparisons of both groups. The hsa-miR-23a was strongly upregulated and hsamiR-27a was strongly downregulated in AOD-III. The functions of hsa-miR-23a and hsa-miR- 27a were tested in human adult fibroblasts for cell proliferation assay and apoptosis detection. Cells treated with pre-miR-23a and pre-miR-27a showed 20% reduction in cell proliferation as compared with controls. Further, the functional relevance of miRNAs to their target mRNAs was validated for each group, using real time qPCR. 10 key-miRNAs from AOD were subjected to validation by qPCR. We were able to confirm 7 miRNAs (p? 0.05). Among these, 5 miRs (miR- 193a-3p, miR-24, miR-27a, miR-30a-5p and miR-30c) showed reduced expression whose target genes (CCND1, HDAC2, PDGFA and RAB-26) were upregulated. Whereas, 2 miRNAs likewise miR-301b and miR-378 were overexpressed whose target genes BCL2, FGF2, CD44 and PPP4R4 confirmed by qPCR (p? 0.05). Bioinformatics based gene ontology (GO), and networking analysis revealed that differential expression and targets are attributed to differentiation of embryonic stem cells, cell adhesion, angiogenesis and neurogenesis, resistance to apoptosis, protein-protein interactions and cell proliferation. It was possible to identify and validate miRNAs and their mRNA-targets potentially involved in the progression of oligodendrogliomas particularly in grade III-AOD. Collectively, this analysis provides new insights to malignant progression of oligodendroglial tumours and could compliment WHO-2016 diagnosis scheme and may provide predictive outcome in patients as well as decision to therapy. / Oligodendrogliomas originários de oligodendrócitos que geralmente surgem na substância branca podendo ser classificados em grau oligodendroglioma (II-OD), e anaplastic oligodendrogliomas (grau III-AOD). Os ODs2 podem ser diagnosticados por análises patológicas e imuno-histoquímicas, porém evidências recentes sugerem que poderiam ser melhor diagnosticados com base em assinaturas moleculares, como a deleção combinada dos cromossomas 1p e 19q - marcadores moleculares dos OD associados clinicamente a um melhor prognóstico, resposta à terapia e melhor sobrevida. As características histológicas típicas dos oligodendrogliomas também estão fortemente associadas à deleção de 1p/19q, que é criticamente importante como ponto de vista diagnóstico. Assim, os subtipos de gliomas podem ser fortemente diferenciados não somente em relação ao seu perfil histológico mas também com base em seu perfil de expressão genica e suas assinaturas moleculares exclusivas. Os microRNAs (miRNAs, miRs) emergiram como assinaturas moleculares para os diferentes graus. Os miRNAs são RNAs não codificantes, contendo em torno de 22 nucleótidos. São expressos endogenamente e regulam diversos processos celulares através do controle negativo da expressão gênica em nivel pós-transcricional e por interacção directa ou imperfeita com o RNAm-alvo. Os miRNAs estão envolvidos na regulação da tumorigenese humana atuando como supressores de tumour ou oncogenes. Durante o processo da tumorigenese o nível de expressão dos miRNAs é aumentado ou diminuído significativamente em comparação com tecido normal correspondente. O perfil de expressão de miRNA de tumores humanos poderia identificar assinaturas associadas com progressão, diagnóstico, prognóstico e resposta à terapia. Contudo, até recentemente a informação sobre a expressão de miRNAs em oligodendrogliomas é escassa. Neste estudo, avaliamos o perfil de expressão diferencial de miRNA e RNAm em ODs graus II e III usando plataformas de perfis de expressão baseadas em microarray (723 transcritos e 41.000 genes, respectivamente). Foram utilizados 14 casos de ODs microdissecados, sendo 7 OD grau II, e 7 AOD grau III (anaplasicos) sem histórico prévio de tratamento, além de 15 amostras de substancia branca não neoplásica (nnSB). Por meio de avaliações sistemáticas foram determinados miRNAs e mRNAs expressos em AOD grau III, mas não em OD grau II e em substancias brancas não neoplásicas (nnSB). 2 ODs when used with ,,s\" will represent both OD and AOD. Assim, foram encontrados 50 miRNAs com alta expressão e 43 miRNAs com baixa expressão em AOD-III, enquanto que 7 miRNAs apresentaram expressões reduzidas no grupo OD-II. Na comparação entre os dois grupos, 3 miRNAs apresentaram baixa expressão. A hsa-miR-23a mostrou alta expressão e a hsa-miR-27a apresentou uma diminuição de expressão importante em AOD III. A atividade dos hsa-miR-23a e hsa-miR-27a foram testadas em células de fibroblastos adultos humanos usando ensaios de proliferação celular e detecção de apoptose. As células tratadas com pre-miR-23a e pre-miR-27a mostraram 20% redução de proliferação celular em comparação com os controles. Para cada grupo, a relevância funcional dos miRNAs e seus mRNAs alvos foi validada utilizando qPCR. Dos 10 miRNAs submetidos a validação em grau III, foi possivel confirmar 7 miRNA(p<0,05). Entre esses, 5 miRs (miR-193a-3p, miR-24, miR- 27a, miR-30a-5p e miR-30c) mostraram expressão reduzida, cujos genes alvos (CCND1, HDAC2, PDGFA e RAB-26) apresentavam alta expressão. Enquanto que, 2 miRNAs como miR-301b e miR-378 apresentaram alta expressão cujos genes alvo BCL2, FGF2, CD44 e PPP4R4 foram confirmados por qPCR (p<0,05). Ferramentas de bioinformática (Gene Ontology) e a análises em rede revelaram que a expressão diferencial e os alvos são atribuídos à diferenciação de células-tronco embrionárias, adesão de celular, angiogênese e neurogênese, resistência à apoptose, interações proteína-proteína e proliferação celular. Foi possível identificar e validar miRNAs e RNAm-alvos potencialmente envolvidos na progressão de oligodendrogliomas. Coletivamente, esta análise fornece novos achados relacionados a progressão maligna de tumores oligodendrogliais e poderia facilitar o diagnóstico preciso e mais restritivo, o desfecho preditivo em pacientes, bem como auxiliar na decisão da terapia.

A reverse transcription reaction allows the production of complementary DNA (cDNA) using an RNA template and relies on polymerases displaying reverse transcriptase (RT) activity. This process, with major applications in both research and in medical diagnostics, is often limited by the nature of the RTs available. RNA secondary structure can prove problematic where mesophilic retroviral RTs are used while the alternative approach, using thermophilic DNA polymerases with RT activity, often results in error-prone cDNA production. <br /> This project recognised the need to study other possible sources of thermophilic RTs and outlines the study of four previously uncharacterised Group II Intronencoded proteins (IEP), with RT domains, from thermophilic bacteria. While cloning of the IEP genes and their expression on a small scale proved successful, difficulties were encountered when attempting purification. Despite a lack of overall purity, samples containing IEPs from Thermosinus carboxydivorans and Petrotoga mobilis were shown to have RT activity but characterisation of these IEPs was not carried out. However, an IEP from Bacillus caldovelox proved to be an excellent candidate for characterisation as successful purification was achieved. Enzyme engineering was also performed, fusing a Sac7d domain onto the C-terminus of this protein. These enzymes were shown to have optimum RT activity at 54ºC with activity still being displayed at 76ºC. Other studies on these enzymes showed that, unlike the retroviral RTs, the IEPs displayed no DNA-dependent DNA polymerase activity. The Sac7d fusion protein was also studied in terms of possible enhancements to the RT activity of an IEP. However, preliminary studies showed that, although this domain did not prove to be detrimental to the enzyme, it had little effect on improving the processivity of the RTs. <br /> Although this class of RT looks promising in terms of use as an alternative thermophilic RT, the IEPs studied in this report did incur major limitations during cDNA synthesis, which included lower than expected optimum reaction temperatures, very low fidelity and an inability to synthesise cDNA using complex RNA templates.

The National Health Service (NHS) continues to go through a period of considerable transition as health services change to meet the needs of a 21st century population. Staff are acknowledged as key to such processes. Staff well-being is a key concept in organisational change literature. For example, levels of staff well-being can be used to measure the success of organisational change. Existing literature has established that a number of different features of change are associated with staff well-being such as levels of control and demand, and social support. The study presented here extends these relationships to focus on how and why staff well-being is influenced during organisational transition. An ethnographic approach was used to observe two surgical units, both of which were undertaking transitions by relocating to new purpose-built facilities. Findings are arranged around three different themes and within each theme a number of aspects of the change were found to be driving effects on well-being: 1) information and communication during transition: the extent to which change-related communications were consultative/participatory, well-scheduled, transparent and incorporated job-related technical information; 2) the nature of the transition: working with ‘unsuitable’ patients, working in restrictive and disconnected work spaces and the fast-paced nature of work; 3) the impact of the transition on social relationships: the presence of support structures and changes to team dynamics. This investigation contributes to improving understanding of what affects staff well-being during change. A number recommendations for best practice are subsequently formulated.

Individuals or groups will form impressions of another based upon a series of traits, which may be relied upon when forming behaviour pattern towards others (Asch, 1946; Crocker and Major, 1989; Pinel, 1999; Albon, 2002; Corrigan and Wassel, 2008). These traits will depict the reception individuals receive within healthcare and may depend upon learnt and inherited ‘perceived’ ideals affecting the working and personal relationships experienced by positively diagnosed healthcare workers, predisposing stigma responses to others (Asch, 1946). A mixed method study investigating the potential existence of stigmatising values from student nurses towards positively diagnosed healthcare workers with Pulmonary Tuberculosis (PTB), Human Immunodeficiency Virus (HIV), Methicillin-resistant Staphylococcus Aureus (MRSA), Hepatitis C and Diabetes type 2, was undertaken. Analysis provided exploration of the stigmatising attitudes and values of 482 student nurses undertaking an education programme. Interpretation of the findings explored the participants views at course commencement, midpoint and completion considering variables of education (theoretical and clinical), personal and professional influences. Findings indicated that stigmatising values and attitudes return to those identified precourse and underpin the need for implementation of a change to nurse education within this area. The development of a longitudinal education model for healthcare workers considering disease processes and influencing factors psychologically, socially and physically, will provide opportunities to reduce the existence of stigmatisation for positively diagnosed healthcare workers.

Treatment and advice on amoebic dysentery from case studies onboard H.M.H.S. Lanfranc, May 1916.

Waterbirths have been available in the United Kingdom as a method of pain relief for childbirth for over two decades but the neonatal safety of birth in water remains unevaluated. Opponents of a waterbirth randomised controlled trial state randomisation would undermine women’s childbirth experience. In addition, little is known about midwives’ attitudes to waterbirths. This thesis addresses some of the lack of evidence by reporting the findings of two studies which had three aims: to investigate the feasibility of a waterbirth RCT to assess the effects of a waterbirth on the neonate, to explore women’s thoughts about participation and whether randomisation affects women’s satisfaction with their childbirth experience and to assess midwives’ attitudes to waterbirths. The first study involved a RCT with a ‘preference arm’. Eighty women were recruited: 60 in the RCT and 20 in the ‘preference arm’. Women were asked to complete questionnaires to assess their expectations for, and satisfaction with, their childbirth experience: at recruitment, after the birth and 6 weeks after the birth. Women in the randomised arm indicated willingness to partake but questioned midwives’ commitment to offering waterbirths. A Q Methodology study was undertaken to identify factors which influence midwives’ (n=31) attitudes towards waterbirths. Four factors were identified: Motivation, Risk Assessment, Confidence, Safety. Conclusion: It is feasible to organise a larger RCT to assess neonatal safety and women would be supportive. Strategies would be required to ensure midwives are confident and supportive of the waterbirth service.

The primary aim of the thesis was to report new cost-effectiveness evidence in the clinical area of heart disease. Following a review of published empirical work, this was achieved by undertaking three new cost-effectiveness studies: one in nurse-led secondary prevention clinics for coronary heart disease in primary care, one on cardiac resynchronisation therapy with or without an implantable cardioverter defibrillator in chronic heart failure, and the final one on a new drug therapy, nebivolol, compared with standard treatment in elderly patients with heart failure. The second aim of the thesis concerned the application of modelling methodology, with the intent being the provision of general recommendations in using Markov modelling approaches in economic evaluation conducted in the heart disease area. The focus was on extrapolation of cost-effectiveness of an intervention beyond a trial both in terms of the time horizon of the analysis and in relation to the population involved. Fundamental issues in parametric distribution functions and Markov modelling approaches have been revisited, with detailed consideration of which parametric distribution functions should be employed when extrapolating beyond a trial and how they could be adopted into model-based analyses. The need for further methodology investigations in this area is discussed in conclusion.

La progression naturelle des tumeurs consiste en une extension locale, puis à distance (métastase) par migration de cellules dans le sang et la lymphe vers des sites secondaires. Il est donc primordial de pouvoir détecter des cellules tumorales circulantes en plus de l’analyse morphologique et de l’immunocytochimie. De plus, deux technologies (cytométrie en flux et RT-PCR quantitative en temps réel) sont adaptées pour une analyse automatisée, rapide et sensible d’une très faible quantité de cellules. Le but de notre travail a été de mettre au point des systèmes de détection pour l’identification de cellules cancéreuses dans les épanchements péritonéaux et dans le sang. L’étude des biomarqueurs moléculaires apparaît comme une approche complémentaire intéressante pour améliorer l'efficacité du diagnostic dans ce type d'échantillons biologiques. Nous nous sommes intéressés à la mise en évidence de nouveaux marqueurs tumoraux qui pourront être utilisés pour le diagnostic précoce et le pronostic des cancers en utilisant les nouvelles techniques de biologie moléculaire. Il est probable que l'utilisation de multiples marqueurs moléculaires puisse permettre d’évoquer plus particulièrement certains types de cancers. Nous avons pu mettre en place une technique de PCR quantitative en temps réel, nettement plus sensible que la cytologie classique, et nous avons appliqué cette technique à l'étude de marqueurs tumoraux dans les liquides d’épanchement, mais aussi dans le sang pour rechercher et doser l’ARN messager. Nos résultats montrent que la cytométrie en flux adaptée à des lignées cellulaires ne l’est pas pour des prélèvements cliniques. Par la PCR quantitative, il a été possible de quantifier le niveau d’expression des marqueurs tumoraux étudiés en utilisant des plasmides de référence qui ont été préparés pour chaque gène. Plusieurs marqueurs permettent de différencier des épanchements malins et des épanchements bénins, mais surtout les antigènes CLDN4 et Ep-CAM étaient significativement plus élevés (68% et 57%, respectivement) chez les patients avec épanchements malins. L’ARN messager circulant de la CLDN4 était détectable et significativement plus élevée dans les sérums de patients atteints de cancer du sein (64% p<0,05). Les résultats indiquent que l'utilisation d'une combinaison de marqueurs comportant laclaudine 4 est plus susceptible de détecter des cellules malignes et d'être utiles pour le suivi de patients / The natural progression of tumors is a local extension, and remotely (metastasis) by migrating cells in the blood and the lymph to secondary sites. It is therefore essential to detect circulating tumor cells in addition to morphological analysis and immunocytochemistry. In addition, two technologies (flow cytometry and RT-PCR in real time) are suitable for a rapid and sensitive automated analysis of a very small quantity of cells. The aim of our work was to develop detection systems for identification of cancer cells in peritoneal effusions and blood. The study of molecular biomarkers appears as an attractive complementary approach to improve the efficiency of diagnosis in this type of biological samples. We are interested in the identification of new tumor markers that can be used for early diagnosis and prognosis of cancer using new techniques of molecular biology. It is likely that the use of multiple molecular markers can help to raise some specific types ofcancers. We were able to develop a quantitative PCR technique in real time, significantly more sensitive than conventional cytology, and we applied this technique to the study of tumor markers in effusions, but also in blood for detecting messenger RNA. Our results show that flow cytometry well-adapted to cell lines, is not unusable for clinical specimens. For quantitative PCR, it was possible to quantify the expression levels of tumor markers using reference plasmids prepared for each gene. Several markers can differentiate malignant and benign effusions, but especially CLDN4 and Ep-CAM antigens were significantly higher (68% and 57% respectively) in patients with malignant effusions.The circulating CLDN4 mRNA was detectable and significantly higher in the sera of patients with breast cancer (64% p <0.05). The results indicate that using a combination of markers including claudin 4 is more likely to detect malignant cells and be useful for monitoring patients