Bc. Klára Horáčková DIPLOMA THESIS Mechanisms of immune dysregulation leading to inflammatory bowel disease Abstract Inflammatory bowel disease (IBD) is a complex disorder characterized by chronic inflammation of the gastrointestinal tract. Classical IBD is a multifactorial disease with adulthood or later-childhood onset. However, children with very early onset IBD (VEO-IBD, before 6 years of age) are a specific cohort, whose pathology can be caused by severe genetic defects in genes connected to immune homeostasis in the gut. We aimed to identify the causal genetic variants in 20 pediatric patients diagnosed with IBD (age of onset from 3 to 154 months) using whole exome sequencing (WES). We evaluated several bioinformatical approaches for WES data analysis. This included a comparison of two methods of variant identification using VarScan2 or GATK4-based tools. Furthermore, we compared 4 gene lists ("virtual panels") for variant filtering, one of which was compiled purposefully for this thesis. We identified and validated via segregation analysis 5 causal variants in 4 genes (DUOX2 compound heterozygote, FOXP3, NLRP3 and NOD2) accounting for 20 % of the cohort. NOD2 (p.A755V) variant has already been reported in IBD cases, while DUOX2 (p.R1216W + p.A1131T), FOXP3 (p.H400L) and NLRP3 (p.V200M) were newly...

In the late 90’s, emerging evidence revealed that the brain is insulin-sensitive, highlighted by broad expression of brain-specific insulin receptors and reports of circulating brain insulin. Contemporary literature robustly supports the role of insulin signaling in normal brain function and suggests that insulin-related processes diminish with aging, evidenced by decreased signaling markers, reduced insulin receptor density, and lower levels of insulin transport across the blood-brain barrier. In the context of pathological cognitive decline, clinical trials using intranasal insulin delivery have reported positive outcomes on memory and learning in patients with mild cognitive decline or early-stage Alzheimer’s disease. However, while the importance of insulin and its related actions in the brain are robustly supported, the distinct mechanisms and pathways that mediate these effects remain unclear. To address this, I conducted a series of experiments exploring the impact of insulin on memory and learning in two models: primary hippocampal cell cultures and the Fisher 344 animal model of aging. These studies attempted to identify relationships between insulin receptor signaling, neuronal gene expression, glucose metabolism, and calcium homeostasis in the hippocampus using either expression of a constitutively active human insulin receptor or administration of intranasal insulin. The following dissertation summarizes this work and provides valuable insights into the potential pathways mediating these relationships. Of note, intranasal studies reported that insulin is able to significantly alter gene expression patterns in the hippocampus of both young and aged rats following chronic, repeated exposure to the ligand. In cell culture, constitutive insulin signaling correlated with significantly elevated neuronal glucose uptake and utilization, as well as with significant alterations in the overall expression and localization of the neuron-specific glucose transporter 3. Interestingly, continued activity of the insulin receptor did not appear to alter voltage-gated calcium channels in hippocampal neurons despite prior evidence of the ligand’s role in other calcium-related processes. The results reported in this manuscript suggest that in the brain, insulin may be involved in a myriad of complex and dynamic events dependent on numerous variables, such as age, length of the exposure, and/or the insulin formulation used. Nevertheless, this work highlights the validity of using insulin to ameliorate age-related cognitive decline and supports the need for further studies exploring alternative approaches to enhance insulin receptor signaling in the brain.

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Researchers have postulated that those with difficult temperament are at risk for difficulties with regulating emotions, are less tolerant of distressing stimuli, have characteristic difficulty coping with distress, and are (at some periods of development) more apt to experience clinically significant psychological symptoms. This study used exploratory factor analyses and structural equation modeling to compose and test a model that explained how emotion regulation, distress tolerance, and coping skills interact to explain how certain temperament features translate into psychological symptoms. Because those with difficult temperament were thought to be at a unique risk for psychological maladjustment, mean-based criterion were used to identify those with relatively difficult, typical, or easy temperament and then test whether the degree of between-group differences on study variables was statistically significant. Results of correlational and EFA analyses suggested that there were statistically significant differences between constructs that were correlated highly (i.e., distress tolerance, emotion regulation, and emotion dysregulation). Results of SEM analyses indicated that the relationship between difficult temperament and psychological maladjustment was explained partially by the way in which emotion regulation, emotion dysregulation, distress tolerance, and coping skills interact, with the strength of each mediating variable differing considerably. There were also differences in the power of the relationship between variables when correlational power was considered alone rather than in the context of the larger measurement and structural models. Future directions and implications are discussed.

For those with attention deficit hyperactivity disorder (ADHD), social interactions involving high levels of face-to-face interaction can raise stress levels and emotional dysregulation. Using animated avatar video models may mitigate potential emotional dysregulation while learning social skills in these populations. This study examined autonomic data of adolescents aged 7-13 diagnosed with attention deficit hyperactivity disorder (ADHD), n=5 during avatar animated video modeling (AAVM) of social and emotional skills. This was a replication study with the addition of biofeedback data collection and a change of population. Participants were given three Nearpod training modules with AAVM and multiple-choice quizzes on self-awareness, social awareness, and relationship skills. Using a multiple baseline design, we collected Social Emotional Learning (SEL) scores at baseline, and during each phase of intervention. During all phases, we collected heart rate and analyzed heart rate variability (HRV) metrics: standard deviation of N-N intervals (SDNN), high frequency (HF), low frequency (LF), and HF/LF ratio). We also collected real-time somatic data: muscle tension (EMG), skin conductance (SC), and skin temperature (temp). The somatic autonomic data were not analyzed as part of this thesis. Results suggest that persons with ADHD may benefit from avatar animated video modeling delivered instruction based on patterns in autonomic data, increases in scores on the targeted skills taught during instruction, and participant's expressions about this method of learning. In future research and practice the population for this content could be narrowed to age 8-12. Reliable but smaller and less obtrusive biofeedback devices are currently available, and having several accessible options is recommended.

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Misophonia is a condition characterized by a strong physiological, emotional, and behaviouralresponse to specific auditory stimuli, which have a significant negative impact on the wellbeingof affected individuals. The present investigation focuses on emotional dysregulation, which arises due to the triggering of specific auditory stimuli. Individuals with misophoniastruggle to regulate their emotions when exposed to sounds, leading to emotional reactions, including anger, anxiety, disgust, avoidance behaviour, fight or flight, and feeling overwhelmed. These reactions might even lead to violent impulses directed towards the source of the sound. Despite its growing recognition, little is yet known about misophonia, and experts have not established any clear boundaries or criteria for the condition to be considered adisorder. As such, it is not yet included in any classification systems for disorders. The aim of this study is to understand how individuals with misophonia experience emotional dysregulation, how do they describe their experience, what is it like to live with misophonia ona day-to-day basis, and how do individuals understand and cope with emotional dysregulation caused by misophonia? The study utilized a qualitative approach with semi-structured interviews as the data collection method. Thematic analysis was used to identify patterns and themes within the data. Participants stated that misophonia causes significant distress and disruption impacting emotional well-being and daily functioning. Validation of this new condition can be very helpful and make a positive impact in their social circle, and that lack of awareness and effective treatment may hold back seeking professional help.

Les cortico-surrénalomes (CCS) sont considérés comme des tumeurs malignes endocriniennes rares, associées à un pronostic sombre. Les trois mécanismes moléculaires les plus fréquemment altérés dans les CCS comprennent les mutations inactivatrices du gène suppresseur de tumeur TP53,la surexpression de IGF-II et l'activation constitutive de la voie de signalisation Wnt/β-caténine. En utilisant des modèles de souris transgéniques, nous avons montré que ces altérations, même combinées, ne sont pas suffisantes pour permettre la progression maligne.Nous avons précédemment identifié l'histone méthyltransférase EZH2 comme le modificateur d'histone le plus dérégulé dans les CCS. Nous avons également montré que sa surexpression est associée à une progression tumorale et à un mauvais pronostic. Cependant, les mécanismes sous-jacents de cette agressivité sont largement inconnus. Dans cette étude, nous avons cherché à identifier les gènes cibles de EZH2 dans les CCS, qui sont soient activés, soient réprimés. Ainsi, nous avons effectué une analyse bio-informatique des données du transcriptome de trois cohortes de patients porteurs de CCS. L’analyse montre une forte corrélation entre la surexpression de EZH2 et les gènes régulés positivement, suggérant un rôle majeur d’inducteur transcriptionnel de EZH2 dans les CCS. Nous avons montré que cette activité positive repose sur une interaction entre EZH2 et E2F1, qui entraîne la surexpression de gènes impliqués dans la régulation du cycle cellulaire et la mitose tels que RRM2,PTTG1 et PRC1/ASE1. Nous avons montré que l'inhibition de RRM2 par ARN interférent ou traitement pharmacologique avec le GW8510 inhibe la croissance cellulaire, la capacité à combler les blessures, la croissance clonogénique, la migration et induit l'apoptose des cellules H295R en culture. En revanche, l'expression du facteur pro-apoptotique NOV/CCN3 est diminuée dans les CCS, ce qui est corrélé au développement de tumeurs agressives. Nos analyses moléculaires montrent que l'inhibition de EZH2 augmente l'expression de NOV/CCN3, suggérant que la surexpression de EZH2 pourrait favoriser la progression maligne des CCS en inhibant les stimulateurs de l'apoptose. Le facteur NOV a déjà été identifié comme cible négative du récepteur nucléaire SF1 dans les cellules du CCS, bien que les mécanismes moléculaires à l'origine de cette inhibition n'aient pas été identifiés. De manière intéressante, dans le cancer de la prostate, l'expression de NOV est inhibée par le récepteur des androgènes AR, grâce au recrutement de EZH2 qui pose la marque répressive H3K27me3. Nous avons pu identifier une coopération similaire entre SF1 et EZH2 pour réprimer l'expression de NOV et bloquer ainsi l'apoptose dans les CCS.Au total, ces résultats identifient SF1 et E2F1 comme deux partenaires indépendants de EZH2, induisant la répression de facteurs pro-apoptotiques et l'activation des gènes du cycle cellulaire respectivement, conduisant ainsi à l'agressivité des CCS. / Adrenocortical carcinomas (ACC) are regarded as rare endocrinemalignancies associated with dismal prognosis. The three common molecularmechanisms predominantly altered in ACC include inactivating mutations of theTP53 tumor suppressor gene, overexpression of IGF-II and constitutive activationof the Wnt/β-catenin signaling pathway. Using transgenic mouse models, wehave shown that these alterations, even when combined together, were notsufficient to induce malignant progression.We previously identified the histone methyltransferase EZH2 as the mostderegulated histone modifier in ACC. We have also shown that its overexpressionis associated with tumor progression and poor prognosis. Yet, the mechanismsunderlying this aggressiveness are largely unknown. Here, we aimed to identifyEZH2 target genes in ACC, which are either activated or repressed.Thus, we conducted a bio-informatics analysis of transcriptome data fromthree cohorts of ACC patients. The analysis showed a strong correlation betweenhighly expressed EZH2 and positively regulated genes suggesting a major role of‘transcriptional inducer‘ for EZH2 in ACC. We have shown that this positiveactivity relies on an interaction between EZH2 and E2F1 that results in theupregulation of genes implicated in cell cycle regulation and mitosis such asRRM2, PTTG1 and PRC1/ASE1. We showed that Inhibition of RRM2 by RNAinterference or pharmacological treatment with GW8510 inhibits cellular growth,wound healing, clonogenic growth, migration and induces apoptosis of H295Rcells in culture.In contrast, expression of the pro-apoptotic factor NOV/CCN3 is decreasedin ACC, which is correlated with development of aggressive tumours. Ourmolecular analyses show that EZH2 inhibition increases expression ofNOV/CCN3, suggesting that EZH2 overexpression may also favour malignantprogression in ACC by inhibition of apoptosis stimulators. NOV has previouslybeen identified as a negative target of the nuclear receptor SF1 in ACC cells,although the molecular mechanisms underlying this inhibition were unidentified.Interestingly, in prostate cancer, NOV expression is inhibited by the androgenreceptor, through recruitment of EZH2 and deposition of the H3K27me3 mark.We have been able to identify a similar cooperation between SF1 and EZH2 tosuppress NOV expression and block apoptosis in ACC.Altogether, these findings identifiy SF1 and E2F1 as two independentpartners of EZH2, inducing repression of proapoptotic factors, and activation ofcell cycle genes respectively, thus leading to aggressiveness of ACC.