Nob1 (New Zealand obese 1) bezeichnet einen Adipositas-QTL auf Chr. 5 der Maus (LODBMI >3,3), der in einem Rückkreuzungsexperiment der Mausstämme NZO (adipös) und SJL (schlank) identifiziert wurde. Um Kandidatengene für Adipositas zu finden, wurden mehr als 300 Nob1-Transkripte mit Hilfe von Genexpressionsanalysen auf Unterschiede in stoffwechselrelevanten Geweben zwischen beiden Mausstämmen untersucht. Sieben Gene zeigten eine differentielle Expression: 2310045A20Rik, Tbc1d1, Ppp1cb, Mll5, Insig1, Abhd1 und Alox5ap. Die codierenden Bereiche dieser Gene wurden anschließend auf Sequenzunterschiede zwischen NZO und SJL untersucht. Nur im Gen Tbc1d1, das im Peak-Bereich des Nob1 lokalisiert ist, wurde eine SJL-spezifische Deletion von sieben Basen detektiert, die zu einer Leserasterverschiebung und einem vorzeitigen Abbruch des Proteins in der funktionellen Rab-GAP-Domäne führt (Loss-of-Function-Mutation). Interessanterweise wurde eine Variante von TBC1D1 (R125W) in Kopplungsanalysen mit Adipositas beim Menschen assoziiert (Stone et al., 2006). TBC1D1 zeigt eine hohe Homologie zu TBC1D4 (AS160), das im Insulinsignalweg eine wichtige Rolle spielt. In 17 weiteren Genen im Peak-Bereich des Nob1 wurde keine weitere SJL-spezifischen Mutation detektiert. Bei NZO-Tieren erfolgte die Tbc1d1-mRNA-Expression vorwiegend in glycolytischen Fasern des Skelettmuskels. Zudem wurden zwei gewebsspezifisch exprimierte Tbc1d1-Isoformen identifiziert, die sich durch alternatives Splicen der Exone 12 und 13 unterscheiden. Die im Rahmen dieser Arbeit gefundenen Ergebnisse machen Tbc1d1 zu einem plausiblen Kandidatengen für den Nob1-QTL. Welche Funktion Tbc1d1 im Glucose- und Fettstoffwechsel des Skelettmuskels hat, muss in weiteren Analysen untersucht werden. / Nob1 (New Zealand obese 1) has been identified as an obesity QTL on chromosome 5 (LODBMI >3,3) in a backcross experiment of obese NZO and lean SJL mice. To identify candidate genes for obesity expression profiling experiments with RNA from metabolic tissues were performed with more than 300 Nob1-genes. Seven genes showed differences in mRNA expression levels between both strains: 2310045A20Rik, Tbc1d1, Ppp1cb, Mll5, Insig1, Abhd1, and Alox5ap. Sequencing of the coding regions of these genes revealed a SJL-specific deletion of seven basepairs in the Tbc1d1 gene that is located in the peak region of Nob1. This mutation leads to a frameshift resulting in a truncated protein that lacks the important Rab-GAP-domain (Loss-of-Function-mutation). Interestingly, linkage analysis of the R125W-variant of TBC1D1 has been recently associated with human obesity. TBC1D1 shows high homology to TBC1D4 (AS160) that plays an important role in the insulin signaling pathway. No other SJL-specific mutations were detected in 17 further genes in the Nob1 peak region. In NZO mice Tbc1d1 mRNA is predominantly expressed in glycolytic fibres of skeletal muscle. Two isoformes were identified differing in alternative spliced exons 12 and 13 and showing a tissue specific mRNA expression. The results presented in this work make Tbc1d1 a very feasible candidate gene to be causal for Nob1. The function of Tbc1d1 in the metabolism of carbohydrates and fat has yet to be analyzed.

<p>Syftet med denna studie är att belysa situationen för asylsökande barn med uppgivenhetssymptom utifrån behandlarens perspektiv. Syftet är vidare att undersöka behandlarens upplevelser och erfarenheter av att vårda och stödja dessa barn.</p><p>Frågeställningarna som är i fokus berör den vård och stöd som behandlarna erbjuder till barnen och deras familjer, effekter av behandlingen samt specifika behandlingar som är särskilt verksamma i förhållande till de aktuella barnens tillstånd och behov.</p><p>Studien är av en kvalitativ design och den vetenskapliga positionen av konstruktivistisk karaktär. Det empiriska materialet består av intervjuer med nio behandlare som varit i direkt kontakt med barnen och deras föräldrar. Kommande tre teman har varit grundläggande i intervjuerna; symtombilden, vägen ut ur tillståndet och slutligen behandlarens reflektioner kring problemet/fenomenet.</p><p>Studiens teoretiska perspektiv som valdes i syfte att nå en ökad kunskap och förståelse kring fenomenet är; pervasive refusal syndrome, depressiv devitalisering, kvävande moderskap, hopplöshet hos barn, simulering samt munchenhausen by Proxy.</p><p>Studiens resultat och analys kom att visa att fenomenet apatiska flyktingbarn är mycket komplext i sig. Alla behandlare anser att en del barn är verkligt sjuka men att majoriteten av barnen avsiktligt framkallar detta tillstånd. Behandlare har kunnat bevittna flera tillfällen då barnet eller barnen kunde sitta utan hjälp samt gå och äta själva. Alla behandlare upplevde att det var svårt att samarbeta med en del av föräldrarna samt att det fanns ett starkt motstånd till behandlingsarbetet från föräldrarnas sida.</p>

Syftet med denna studie är att belysa situationen för asylsökande barn med uppgivenhetssymptom utifrån behandlarens perspektiv. Syftet är vidare att undersöka behandlarens upplevelser och erfarenheter av att vårda och stödja dessa barn. Frågeställningarna som är i fokus berör den vård och stöd som behandlarna erbjuder till barnen och deras familjer, effekter av behandlingen samt specifika behandlingar som är särskilt verksamma i förhållande till de aktuella barnens tillstånd och behov. Studien är av en kvalitativ design och den vetenskapliga positionen av konstruktivistisk karaktär. Det empiriska materialet består av intervjuer med nio behandlare som varit i direkt kontakt med barnen och deras föräldrar. Kommande tre teman har varit grundläggande i intervjuerna; symtombilden, vägen ut ur tillståndet och slutligen behandlarens reflektioner kring problemet/fenomenet. Studiens teoretiska perspektiv som valdes i syfte att nå en ökad kunskap och förståelse kring fenomenet är; pervasive refusal syndrome, depressiv devitalisering, kvävande moderskap, hopplöshet hos barn, simulering samt munchenhausen by Proxy. Studiens resultat och analys kom att visa att fenomenet apatiska flyktingbarn är mycket komplext i sig. Alla behandlare anser att en del barn är verkligt sjuka men att majoriteten av barnen avsiktligt framkallar detta tillstånd. Behandlare har kunnat bevittna flera tillfällen då barnet eller barnen kunde sitta utan hjälp samt gå och äta själva. Alla behandlare upplevde att det var svårt att samarbeta med en del av föräldrarna samt att det fanns ett starkt motstånd till behandlingsarbetet från föräldrarnas sida.

Att åldras kan innebära stora genomgripande förändringar i den äldre personens liv. Som sjuksköterska i hemsjukvård är erfarenheten att det behövs ökade kunskaper om hur den äldre klarar av åldrandet. Syftet med litteraturöversikten var att belysa äldre personers copingstrategier för att klara av åldrandet. Sökningar via databaserna resulterade i 11 artiklar med kvalitativ design som analyserats med fokus på syftet. Den kvalitativa analysen av artiklarna resulterade i flera copingstrategier vilka bildade ett huvudtema, Att skapa ny tillvaro och sex underteman, Att acceptera nya förhållanden, Att tänka positivt och kämpa, Att resignera inför omständigheterna, Att ta hjälp av andra, Att ha struktur i tillvaron samt Att hålla igång och använda tekniska lösningar. Åldrandet tillhör livet och innebär bland annat förluster eller hot om förlust vilka behövde klaras av genom att skapa ny tillvaro. Viktigast för de personer som vårdar äldre är att visa respekt för den äldres önskningar och att ha tid att lära känna den äldres behov samt vårda i en lugn miljö. Meningsfulla aktiviteter var betydelsefullt, dels för stimulans och distraktion men också för social samvaro. Kunskapen om äldres copingstrategier ger sjuksköterskan bättre förutsättningar att vårda äldre personer och stödja ett gott åldrande. / Aging can mean large changes for the older person’s life. The experience as a community care nurse is that there is a need for more knowledge of how the elderly cope with aging. The aim of this literature review was to investigate the strategies of coping with aging in the elderly. The review was based on 11 articles with qualitative design which had been focusing on the aim. The result of the quality analysis of the research articles resulted in several coping strategies as formed one mainthemes: To create a new existence and six subthemes: To accept new environments, To think in a positive way and fight, To resign ourselves circumstances, To accept help from others, To have a routine in day-to-day life and To keep active and use technical solutions. Ageing is past of life and losses or threat of loss is part of ageing and to be able to cope the need to create a new way is important. The literature review showed that the most important thing to consider for staff working with the elderly is to show respect for their wishes, to allow time to get to know their needs and to care for them in a calm environment. Meaningful activities were important to stimulate and distract but also for social interaction. The knowledge of the elderly persons way of coping gives the nurse a better way of caring for the individual and to support them in ageing.

Primary immunodeficiencies (PID) are genetic disorders characterized by increased susceptibility to infections and various degrees of immune dysregulation. With the expansion of massive parallel sequencing, an increasing number of defects in immune-related genes is being identified in PID. However, the biological impact of the found mutations is often unknown. It is necessary to devise methods to clarify their causality for disease development, which may also aid therapeutic decisions. One of the novel discoveries are gain-of-function mutations in STAT1 gene, resulting in chronic mucocutaneous candidiasis. Candidiasis may be ameliorated with antimycotics or with targeted JAK-STAT inhibitor, ruxolitinib. For our patient with a novel mutation in STAT1, we developed a simple test for the detection of phospho-STAT molecules in peripheral blood lymphocytes. The test confirmed the gain-of-function character of the identified mutation and was used to monitor ruxolitinib treatment efficacy. In the second patient, who presented with lymphadenopathy and immunodeficiency, the as yet undescribed mutation in CASP8 was found. We proved its loss-of-function property expressed as reduced caspase-8 and caspase-3 cleavage, impaired cellular apoptosis, and decreased NFB-related signaling. The third patient who...

Primary immunodeficiencies (PID) are genetic disorders characterized by increased susceptibility to infections and various degrees of immune dysregulation. With the expansion of massive parallel sequencing, an increasing number of defects in immune-related genes is being identified in PID. However, the biological impact of the found mutations is often unknown. It is necessary to devise methods to clarify their causality for disease development, which may also aid therapeutic decisions. One of the novel discoveries are gain-of-function mutations in STAT1 gene, resulting in chronic mucocutaneous candidiasis. Candidiasis may be ameliorated with antimycotics or with targeted JAK-STAT inhibitor, ruxolitinib. For our patient with a novel mutation in STAT1, we developed a simple test for the detection of phospho-STAT molecules in peripheral blood lymphocytes. The test confirmed the gain-of-function character of the identified mutation and was used to monitor ruxolitinib treatment efficacy. In the second patient, who presented with lymphadenopathy and immunodeficiency, the as yet undescribed mutation in CASP8 was found. We proved its loss-of-function property expressed as reduced caspase-8 and caspase-3 cleavage, impaired cellular apoptosis, and decreased NFB-related signaling. The third patient who...

Sudden cardiac death is largely attributable to sudden onset ventricular arrhythmias. Alterations in cardiac conduction, particularly the slowing of conduction velocity is one major factor in arrhythmogenesis. By understanding the mechanisms and factors that modulate cardiac conduction velocity, we can assess and perhaps mitigate the risk of arrhythmia in patients for whom slowed conduction is a arrhythmogenic substrate. Cardiac conduction has traditionally been described by cable theory, which predicts an inverse relationship between extracellular resistance and conduction velocity (CV). However, in studies that reduce extracellular resistance by inducing interstitial edema, there are conflicting results, with some labs showing increased CV when edema is induced with one agent, and others showing reduced CV when edema is induced with a different agent. In the first part of this dissertation, we present experimental data in support of ephaptic coupling (EpC), a theorized mechanism of conduction that resolves these apparent contradictions. In the later part of this dissertation, we address how changes in sodium concentration can alter conduction, despite conventional wisdom suggesting that it should not. We show that when sodium channels are impaired, such as by genetic mutation or pharmacologic blockade, that conduction is sensitized to changes in sodium concentrations that would not otherwise induce changes in CV. We go on to explore the mechanisms that modulate this sensitivity and present data that show it is a function of both EpC and outward potassium currents. Taken together, these data expand our understanding of the mechanics behind cardiac conduction and demonstrate that EpC has a clinically relevant impact on conduction and represents a new pathway to explore in regard to the treatment and management of arrhythmogenic and conduction disorders. / Doctor of Philosophy / In all large animals, life is sustained by the regular coordinated beating of the heart to pump blood throughout the body. Throughout this continuous activity, and even with minute-to-minute changes in heart rate, this electrically driven activity continues without major disruption. Until it doesn't. Major arrhythmias can occur suddenly, and without warning. Over the last century, we have begun to understand some of the reasons why heart, even an injured one, will work normally for hundreds of thousands of beats, and on the next fall into a life-threatening new pattern, and one of the most important of these reasons is the speed of conduction: the spread of electrical activation throughout the heart tissue. Understanding the mechanisms of conduction provides a way to assess and mitigate the risk of arrhythmias and may open up new avenues for treatment and prevention. This dissertation presents evidence for a previously theoretical mechanism of conduction called ephaptic coupling. We show that this electric field mediated form of conduction can be modulated with clinically used osmotic agents, and that it has a physiologically relevant impact on conduction. We also show that hyponatremia (i.e. low sodium), a condition that is traditionally thought to have minimal impact on cardiac conduction, because a significant modulator of conduction when sodium channel functions are impaired. As a great many drugs block sodium channels, this sensitization to hyponatremia and the factors that mediate it are underappreciated concerns that are relevant to a wide array of patients. The new findings presented in this dissertation advance our collective understanding of the mechanisms of cardiac conduction and provide evidence for new avenues of exploration in the prevention and management of arrhythmias and conduction disorders.

A range of biochemically diverse molecules located in the plasma membrane— such as proteins, glycans, and lipids—mediate cellular recognition events, initiation of signalling pathways, and the regulation of processes important for the normal development and function of multicellular organisms. Interactions mediated by cell surface receptors can be challenging to detect in biochemical assays, because they are often highly transient, and membrane-embedded receptors are difficult to solubilise in their native conformation. The biochemical features of low-affinity extracellular protein interactions have therefore necessitated the development of bespoke methods to detect them. Here, I develop a genome-scale cell-based genetic screening approach using CRISPR-Cas9 knockout technology that reveals cellular pathways required for specific cell surface recognition events. Using a panel of high-affinity monoclonal antibodies, I first establish a method from which I identify not only the direct receptor but also other required gene products, such as co-receptors, post-translational modi cations, and transcription factors contributing to antigen expression and subsequent antibody-antigen recognition on the surface of cells. I next adapt this method to identify cellular factors required for receptor interactions for a panel of recombinant proteins corresponding to the ectodomains of cell surface proteins to the endogenous surface receptors present on a range of cell lines. In addition to finding general cellular features recognised by many ectodomains, I also identify direct interaction partners of recombinant protein probes on cell surfaces together with intracellular genes required for such associations. Using this method, I identify IGF2R as a binding partner for the R2 subunit of GABAB receptors, providing a mechanism for the internalisation and regulation of GABAB receptor signalling. The results here demonstrate that this single approach can identify the molecular nature and cell biology of surface receptors without the need to make any prior assumptions regarding their biochemical properties.

During the past few decades, the landscape of available therapeutic interventions for cancer treatment has widely expanded, boosted mainly by immunotherapy progress and the precision oncology paradigm. The extensive use of pre-clinical models in cancer research has led to the discovery of new effective treatment options for patients. Despite the notable advancements, some cancer types have found minor benefits from the use of precision-oncology interventions. Characterized by a heterogeneous molecular landscape, bladder cancer is one of the most frequent cancer types in which standard-of- care treatments involve surgical operations accompanied by broad-spectrum chemotherapy. My research stems from the need for precision oncology interventions in bladder cancer and specifically focuses on the development of computational frameworks to guide the discovery of new therapeutic opportunities. This work first introduces the exploration of possible therapeutic interventions in 9p21.3 depleted bladder tumors through the analysis of an in-house large High-Content Drug Screening that tested 2,349 compounds. By combining cell count changes and morphological quantitative features extracted from fluorescence images, we nominate cytarabine as a putative candidate eliciting specific cytotoxic effects in an engineered 9p21.3 depleted bladder cancer model compared to an isogenic wild-type clone. Focusing on the development of computational methodologies to nominate robust context-specific vulnerabilities, I further describe PRODE (PROtein interactions informed Differential Essentiality), an analytical workflow that integrates protein-protein interaction data and Loss of Function screening data. I extensively tested PRODE against the most commonly used and alternative methodologies and demonstrated its superior performance when classifying reference essential and context-essential genes collected from experimental and literature sources. Furthermore, we applied PRODE to a real case scenario, seeking essential genes selectively in the context of HER2+ Breast Cancer tumors. Finally, I report the computational analyses performed on Patient-Derived Organoids (PDOs) established from a bladder cancer cohort. PDOs are demonstrated as informative models when assessing the therapeutic sensitivity of patients to drugs. Overall, this research highlights novel precision-oncology applications by ad-hoc computational analyses that address key open technical and biological challenges in the field of bladder cancer and beyond.

Chromatin remodeler proteins exert an important function in promoting dynamic modifications in the chromatin architecture, rendering the transcriptional machinery available to the condensed genomic DNA. Due to this central role in regulating gene transcription, deregulation of these molecular machines may lead to severe perturbations in the normal cell functions. Loss-of-function mutations in the CHD7 gene, a member of the chromodomain helicase DNA-binding (CHD) family, are the major cause of the CHARGE syndrome in humans. The disease is characterized by a variety of congenital anomalies, including malformations of the craniofacial structures, peripheral nervous system, ears, eyes and heart. In this context, several studies have already shown the importance of CHD7 for proper function of the neural stem cells (NSCs). Interestingly, we found that CHD7 mRNA levels are upregulated in gliomas, when compared to normal brain tissue, therefore, we hypothesized that CHD7 might have a role in the pathogenesis of these tumors. To investigate the possible oncogenic role of CHD7 in glioblastoma (GBM), we adopted gain- and loss-of-function approaches in adherent GBM cell lines. Using CRISPR_Cas9 genome editing, we found that CHD7 deletion suppresses anchorage-independent growth and reduces spheroid invasion in human LN-229 cells. Moreover, deletion of CHD7 delayed tumor growth and improved overall survival in an orthotopic xenograft glioma mouse model. Conversely, ectopic overexpression of CHD7 in LN-428 and A172 cells was found to increase cell motility and invasiveness in vitro and LN-428 tumor growth in vivo. RNAseq analysis showed that alterations of CHD7 expression levels promote changes in several molecular pathways and modulate critical genes associated with cell adhesion and locomotion. However, the mechanisms underlying the effects of CHD7 overexpression in glioma tissue are still not understood. Here, we also generated recombinant plasmid with functional CHD7 promoter activity reported by luciferase assay. This powerful tool should enable future studies to determine the direct targeting relationship between different signal transduction pathways and CHD7 geneexpression. In summary, our findings indicate that GBM cells expressing a high level of CHD7 may exist and contribute to tumor infiltration and recurrence. Further studies should warrant important clinical-translational implications of our findings for GBM treatment. / As proteínas remodeladoras de cromatina exercem importante papel, promovendo modificações dinâmicas na arquitetura da cromatina e dando acesso à maquinaria transcricional ao DNA genômico condensado. Devido à esta função central na regulação da transcrição gênica, a desregulação dessas máquinas moleculares pode levar a perturbações graves na função normal das células. Assim, por exemplo, mutações do tipo perda de função no gene CHD7, um membro da família \"chromodomain helicase DNA-binding\" (CHD), são a principal causa da síndrome de CHARGE em humanos. A doença é caracterizada por uma variedade de anomalias congênitas, incluindo malformações das estruturas craniofaciais, sistema nervoso periférico, orelhas, olhos e coração. Neste contexto, vários estudos já mostraram a importância da proteína CHD7 para o funcionamento normal de células-tronco neurais (NSCs). Curiosamente, descobrimos que os níveis de mRNA de CHD7 estão mais fortemente expressos em gliomas, quando comparados ao tecido cerebral normal, portanto, nós hipotetizamos que CHD7 poderia ter um papel na patogênese desses tumores. Para investigar o possível papel oncogênico de CHD7 em glioblastoma (GBM), utilizamos enfoques de ganho e perda de função em linhagens celulares aderentes de GBM. Utilizando a técnica de CRISPR_Cas9 para edição do genoma, demonstramos que a deleção do gene CHD7 suprime o crescimento independente de ancoragem e reduz a invasão de esferóides em células LN-229 humanas de GBM. Além disso, a deleção de CHD7 reduziu o crescimento do tumor e melhorou a sobrevida em modelo de injeção ortotópica xenográfica em camundongo. Por outro lado, verificou-se que a super-expressão ectópica de CHD7 nas células LN-428 e A172 aumenta não só a motilidade celular e a capacidade de invasão in vitro, mas, também, o crescimento do tumor de LN-428 in vivo. A análise de RNA-seq mostrou que o nocauteamento da sequência codificadora de CHD7 e sua super-expressão promovem alterações em diversas vias moleculares, modulando genes críticosassociados à adesão e locomoção celular. No entanto, os mecanismos subjacentes aos efeitos da super-expressão de CHD7 em tecidos de glioma ainda não são compreendidos. Neste trabalho, geramos um plasmídeo recombinante contendo um fragmento da região promotora de CHD7, o qual se mostrou funcional em ensaios de luciferase. Esta ferramenta permitirá que estudos futuros possam identificar a relação direta entre as diferentes vias de transdução de sinal e a expressão do gene CHD7. Em resumo, nossos achados indicam que células de GBM expressando um alto nível de CHD7 podem existir e contribuir para a infiltração e recorrência do tumor. Estudos posteriores deverão avaliar as possíveis implicações dos resultados apresentados neste trabalho para a translação clínica no tratamento de pacientes com GBM.