Equine infectious anemia virus (EIAV) is an important animal model to study the contribution of macrophages in viral persistence during lentiviral infections. EIAV is unique amongst the lentiviruses in that it causes a rapid, rather than the very slow disease progression, characteristic of other lentiviral infections. The accessory gene, S2, unique to EIAV, is an important determinant in viral pathogenesis. A functional S2 gene is required to achieve high-titer viremia and the development of disease in infected horses. Despite its essential role, the mechanisms by which S2 influences EIAV pathogenesis remain elusive. The goal of this research was to gain insight into the role of S2 in pathogenesis. To accomplish this goal we: (i) Examined the effects of EIAV and its S2 protein in the regulation of the cytokine and chemokine responses in macrophages, (ii) Assessed the influence of EIAV infection and the effect of S2 on global gene expression in macrophages and (iii) Identified host cellular proteins that interact with S2 as a starting point for the identification of host factors implicated in S2 function. The results from this study provide evidence for a role of S2 in enhancing a proinflammatory cytokine and chemokine response in infected macrophages. Specifically, S2 enhances the expression of IL-1 alpha, IL-1 beta IL-8, MCP-2, MIP-1 beta, IP-10 and a newly discovered cytokine, IL-34. Involvement of S2 in cytokine and chemokine dysregulation may contribute to disease development by optimizing the host cell environment to promote viral dissemination and replication. Microarray analyses revealed an interesting set of differentially expressed genes upon EIAV infection. Genes affected by EIAV were involved in the immune response, transcription, translation, cell cycle and cell survival. Finally, we used the yeast two-hybrid system to identify S2 host cellular interacting proteins. We identified osteosarcoma amplified 9 (OS-9) and proteasome 26S ATPase subunit 3 (PSMC3) proteins as interacting partners of S2. Additional evidence is needed to demonstrate the physiological relevance of these interactions in vivo. In summary, the results from this study contribute towards our understanding of the role S2 in disease expression and allow the formulation of new hypotheses as to the potential mechanisms of action of S2 during EIAV infection.

Children with attention-deficit/hyperactivity disorder (ADHD) experience frequent and persisting peer rejection, yet current social skills training is ineffective. The current study focused on emotion dysregulation as a possible mediator between ADHD symptoms and poor peer outcomes with oppositional defiant disorder (ODD) symptoms as a moderator. Participants included 145 elementary-age children ranging from 8-10 years old. Parents and teachers rated children’s ADHD and ODD symptoms as well as their social skills. Parents also rated children on their emotion regulation abilities. Children then participated in a three-hour playgroup with unfamiliar peers in six structured and unstructured tasks. Research assistants provided global ratings of emotion regulation and peer rejection during each of the six tasks. At the end of the playgroup, children and staff completed sociometric questions about each child. Using multiple raters and methods, observed emotion regulation was found to mediate between increased symptoms of ADHD and worse peer relations as rated by the playgroup staff members. There were limited findings of significant moderation by ODD. Emotion dysregulation may be a valuable target for intervention in order to improve peer relations for children with ADHD.

<p>Inflammatory breast cancer (IBC) is a rare and highly aggressive form of breast cancer that is characterized by survival signaling through overexpression and/or activation of the epidermal growth factor receptors EGFR/ErbB1 and Her2/ErbB2 and defects in the apoptotic program. The development of therapeutic resistance is a significant barrier to successful treatment in IBC, and thus, strategies targeting the mechanisms that drive drug resistance could prevent or reverse therapeutic resistance, significantly improving patient prognosis. Based on analysis of previously developed models of therapeutic resistant IBC, we hypothesized that apoptotic dysregulation and redox adaptive mechanisms were central to the drug resistant phenotype in IBC cells, and that targeting of these mechanisms could overcome therapeutic resistance. Our objectives to address this hypothesis were: 1. to develop and characterize an isotype-matched IBC cellular model to investigate the mechanisms of acquired therapeutic resistance; 2. to characterize IAP-specific small molecule inhibitors as a means of targeting the mechanism of apoptotic dysregulation in IBC; and 3. to characterize a novel redox modulatory combination as a means of targeting redox adaptive mechanisms in IBC.</p><p>Analysis of cell viability, proliferation, and growth parameters, evaluation of protein expression and signaling via western immunoblot, and measurement of reactive oxygen species (ROS), antioxidants, and apoptosis in patient-derived IBC cell lines and isogenic derivatives revealed that resistance to the ErbB1/2 inhibitor lapatinib was protective against other targeted agents and chemotherapeutics. Additionally, reversal of resistance was associated with enhanced ability to accumulate ROS and downregulation of anti-apoptotic and antioxidant proteins. Targeting of resistance mechanisms using small molecule IAP inhibitors and a redox modulatory strategy both effectively induced apoptosis in therapy resistant IBC cells. Together, these results confirm XIAP and the redox adaptive phenotype as promising therapeutic targets for IBC and demonstrate the feasibility of targeting those mechanisms in order to reverse therapeutic resistance.</p> / Dissertation

Huntington’s disease (HD) is caused by expression of the huntingtin gene containing an expanded CAG repeat. N-terminal mutant huntingtin protein (N-mHtt) accumulates in the nucleus and impairs transcription of a subset of genes through incorporation into transcriptional complexes or sequestration of proteins away from the promoter. CREB-binding protein (CBP) is a transcriptional co-activator and acetyltransferase (AT) that binds to N-mHtt. We hypothesized that overexpressing CBP fragments that lack a promoter association domain would block N-mHtt-mediated transcriptional dysregulation and toxicity. We found that overexpressing full-length CBP or CBP fragments did not reverse transcriptional dysregulation, but did decrease toxicity in a cell model of HD. Overexpressing fragments of CBP containing the AT domain increased toxicity in wild-type cells, while overexpressing a fragment lacking this domain had no effect. We conclude that excess AT activity was detrimental in wild-type cells, while overexpressing CBP or CBP fragments was protective in HD cells.

Diets high in saturated fatty acids are associated with the development of circadian dysregulation, obesity, and type 2 diabetes mellitus. Conversely, unsaturated fatty acids are now known to improve insulin sensitivity, reduce weight gain, and alleviate obesity-induced inflammation. The aforementioned effects of saturated and unsaturated fatty acids have also been identified in the hypothalamus; however, there is a paucity of studies regarding the role of unsaturated fatty acids in circadian rhythms. Therefore, a novel cell model was established to examine the effects of omega-3 fatty acids on circadian rhythms in hypothalamic neurons. The mHypoE-37 cell line expresses Bmal1, Per2, and Rev-erbα in a circadian manner. The saturated fatty acid, palmitate, was found to induce circadian dysregulation of the mHypoE-37 neurons, whereas the unsaturated fatty acid, docosahexaenoic acid, protected against palmitate-induced circadian changes. These studies are the first to identify the potential for unsaturated fatty acids to protect the circadian system.

Diets high in saturated fatty acids are associated with the development of circadian dysregulation, obesity, and type 2 diabetes mellitus. Conversely, unsaturated fatty acids are now known to improve insulin sensitivity, reduce weight gain, and alleviate obesity-induced inflammation. The aforementioned effects of saturated and unsaturated fatty acids have also been identified in the hypothalamus; however, there is a paucity of studies regarding the role of unsaturated fatty acids in circadian rhythms. Therefore, a novel cell model was established to examine the effects of omega-3 fatty acids on circadian rhythms in hypothalamic neurons. The mHypoE-37 cell line expresses Bmal1, Per2, and Rev-erbα in a circadian manner. The saturated fatty acid, palmitate, was found to induce circadian dysregulation of the mHypoE-37 neurons, whereas the unsaturated fatty acid, docosahexaenoic acid, protected against palmitate-induced circadian changes. These studies are the first to identify the potential for unsaturated fatty acids to protect the circadian system.

>Magister Scientiae - MSc / Kidney cancers, of which clear cell renal cell carcinoma comprises an estimated 70%, have been placed amongst the top ten most common cancers in both males and females. With a mortality rate that exceeds 40%, kidney cancer is considered the most lethal cancer of the genitourinary system. Despite advances in its treatment, the mortality- and incidence rates across all stages of the disease have continued to climb. Since the release of the Human Genome Project in the early 2000’s, most genetics studies have focused on the protein coding region of the human genome, which accounts for a mere 2% of the entire genome. It has been suggested that diverting our focus to the other 98% of the genome, which was previously dismissed as non-functional “junk DNA”, could possibly contribute significantly to our understanding of the underlying mechanisms of complex diseases.In this study a whole genome sequencing somatic mutation data set from the International Cancer Genome Consortium was used. The non-coding somatic mutations within the promoter, intronic, 5-prime untranslated and 3-prime untranslated regions of clear cell renal cell carcinoma-implicated genes were extracted and submitted to RegulomDB for their functional annotation.As expected, most of the variants were located within the intronic regions and only a small subset of identified variants was predicted to be deleterious. Although the variants all belonged to a selected subset of kidney cancer-associated genes, the genes frequently mutated in the non-coding regions were not the same genes that were frequently mutated in the whole exome studies (where the focus is on the coding sequences). This indicates that with whole genome sequencing studies a new set of genes/variants previously unassociated with the clear cell renal cell carcinoma could be identified. In addition, most of the non-coding somatic variants fell within multiple transcriptions factor binding sites. Since many of these variants were also deleterious (as predicted by RegulomDB), this suggests that mutations in the non-coding regions could contribute to disease due to their role in transcription factor binding site disruptions and their subsequent impact on transcriptional regulation. The substantial overlap between the genes with the most aberrantly methylated variants and the genes with the most transcription factor binding site disruptions signifies a potential link between differential methylation and transcription factor binding site affinities. In contrast to the upregulated DNA methylation generally seen in promoter methylation studies, all of the significant hits in this study were hypomethylated, with the subsequent up-regulation of the genes of interest, suggesting that in the clear cell renal cell carcinoma, aberrant methylation may play a role in activating proto-oncogenes, rather than the silencing of genes. When a cross-analysis was carried out between the gene expression patterns and the transcription factor binding site disruptions, the non-coding somatic variants and differential methylation profiles, the genes affected again showed a clear overlap. Interestingly, most of the variants were not present in the 1000genomes data and thus represent novel mutations, which possibly occurred as a result of genomic instability. However, identifying novel variants are always promising, since they epitomise the possibility of developing pioneering ways to target diseases. The numerous detrimental effects a single non-coding mutation can have on other genomic processes have been demonstrated in this study and therefore validate the inclusion of non-coding regions of the genome in genetic studies in order to study complex multifactorial diseases. / National Research Foundation (NRF) and DAAD

The path to an eating disorder (ED) always leads through a borderland, which, in this thesis, is referred to as disordered eating (DE) (Neumark-Sztainer, Wall, Eisenberg,Story, &amp; Hannan, 2006; Waaddegaard, Thoning, &amp; Petersson, 2003). In this borderland, people tend to make unhealthy eating choices, such as greatly reducing their food intake, self-inducing vomiting, or engaging in binge eating, but not to the extent that they would receive an ED diagnosis. Nevertheless, DE can have a strong negative effect on psychological health. Approximately 15%–52% of all adolescents, depending on the gender and the study’s focus, are found within the borderland between a healthy diet accompanied by psychological well-being and full-blown ED (e.g. Hautala et al., 2011; Herpertz-Dahlmann et al., 2008). While most of these individuals return to a more or less healthy diet after engaging in DE for some time, others continue to engage in DE and also tend to have trouble regulating their emotions, depression, and low self-esteem. For these reasons, DE itself, apart from being a springboard to EDs, is well worth exploring.At the outset of this thesis, an instrument assessing DE among 1265 adolescents (54.5% girls) was validated. This easily administered questionnaire, referred to by the acronym SCOFF (Morgan, Reid, &amp; Lacey, 1999), comprises five questions assessing possible eating disturbances that are all answered using a “yes”/“no” answer format. The results showed that more girls than boys suffered from DE, and that girls also suffered from more severe DE, which is in line with previous research (e.g. Hautala et al., 2008). Additionally, this assessment of the SCOFF gave rise to the question of whether a positive answer on only certain items (instead of the stipulated cut-off of two) is necessary for indicating the possible presence of DE among adolescents, such as the item assessing whether individuals had ever vomited because they felt uncomfortably full.To further explore DE among adolescents, a person-oriented approach to identify specific patterns of DE based on the subscales of the Eating Disorders Examination Questionnaire (EDE-Q) (restraint, eating, weight, and shape concerns) was used. There were six different DE patterns for both boys and girls. The associations of these patterns with emotion dysregulation, depressive symptoms, and self-esteem, which all are related to DE (e.g. Shea &amp; Pritchard, 2007; Svaldi, Griepenstroh, Tuschen- Caffier, &amp; Ehring, 2012), were also assessed. Four of the six girl clusters and five of the six boy clusters showed scores above the cut-off for a clinical ED on at least one of the four indicators. Furthermore, although the “non-problematic” pattern was substantial, including 50% and 76% of girls and boys, respectively, a large portion of adolescents were part of clusters reporting generally high levels of DE. This might partly have to do with my use of an overly permissive cut-off, but nevertheless indicates that a considerable amount of adolescents suffer from DE. Generally, individuals in the DE patterns showed worse emotion regulation, depressive thoughts, and self- esteem than did those in the “non-problematic” patterns. However, some exceptions were found, which emphasizes the utility of analyzing different patterns of DE, not merely severity. Specifically, both girls and boys belonging to the pattern characterized by scores well above the cut-off on shape and weight concerns reported the lowest levels of self-esteem. Moreover, girls and boys in the pattern with scores above the cut-off on restraint showed good emotion regulation skills, few depressive symptoms, and high self-esteem.In Study III, the possible links between adolescents’ and parents’ possible DE and emotion dysregulation were explored, alongside the possible impact of shared family meals on DE. This study further examined whether it is possible to predict DE among adolescents according to their parents’ behaviors. Both DE and emotion dysregulation were found to be more frequent among adolescents than among parents. Furthermore, both adolescents and parents showed weak but significant associations between DE and emotion dysregulation, and showed similarities regarding specific aspects of emotion regulation, although the associations were gender specific. For example, parental emotional strategies were associated with girls’ emotional strategies, impulse control, and emotional goals, but only with boys’ emotional strategies. The only factor that was (weakly) associated with DE and emotion regulation among adolescents was the number of dinners that they shared with the family. Additionally, parental ED was the only predictor of current adolescent DE.In summary, the results of this thesis showed that many adolescents, especially girls, suffer from DE as well as poor emotional regulation, depressive thoughts, and low self-esteem. This is a problem, especially given that existing instruments for evaluating DE do not seem optimal, especially for boys. For instance, answering “yes” to the question of ever having engaged in self-induced vomiting because you have felt too full is probably best followed by a visit to the school nurse. Furthermore, the results indicated the importance of viewing DE not as a singular problem, but as a collection of different problems, even among individuals of the same gender. These differences call for different strategies aimed at helping adolescents achieve a healthier diet. Finally, while the parental influence of DE was significant, more research is required,preferably in a Swedish or Nordic context, where parental responsibility is not as heavily reliant on the mother as in other countries.

Attention deficit/hyperactivity disorder (ADHD) is one of the most common childhood neurodevelopmental disorders and is characterized by excessive inattention and/or hyperactivity and impulsivity. There is evidence that many children with ADHD experience emotion dysregulation, but little is known about the mechanisms by which children with ADHD develop difficulties with emotion dysregulation. The goal of the present study is to identify early neural and environmental predictors of emotion dysregulation and determine whether these factors interact in contributing to later emotion dysregulation. In this study, children (aged 4-7) with ADHD symptoms and typically developing children participated. Measures of emotion socialization and neural measures of emotion reactivity and regulation were completed at the first visit. Follow-up was conducted 18 months later, and emotion dysregulation was assessed using parent report, child self-report, and observed affect during a frustration task. Supportive and unsupportive emotion socialization, distress reactions, and neural markers of reactivity and regulation (P1, N2, and P3) predicted later emotion dysregulation. Additionally, emotion socialization and neural markers during reactivity interacted in predicting later emotion dysregulation, such that neural markers predicted later emotion dysregulation in the context of low but not high quality emotion socialization. This study has implications for understanding mechanisms by which emotion dysregulation develops in children with ADHD symptoms and will aid in the development of targeted interventions for children with ADHD.

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