Genetic Cardiac Arrhythmias in the Young: From Population Trends to Cellular Mechanisms

Arrhythmic heart disease in the young often has a hereditary genetic basis and may carry a lifelong burden of increased morbidity and mortality. The presenting rhythm can be atrial and/or ventricular origin, and both the age of onset and associated risk of sudden cardiac death vary markedly. This is influenced in part by the genetic driver(s) of disease. Atrial fibrillation is one of the most malignant atrial arrhythmias that can negatively impact quality of life and lead to an increased risk of stroke, but generally not sudden death. Although ample research efforts have been dedicated to understanding the different facets of the more common acquired atrial fibrillation in older adults, relatively little is known about atrial fibrillation presenting in young patients with no known risk factors for acquired atrial fibrillation. The latter is thought to represent a potentially genetic arrhythmia condition. In contrast, ventricular-predominant arrhythmia syndromes classically presenting <40 years of age consist mainly of inherited conditions that can cause sudden death. These syndromes may be under-recognized due to their low prevalence, often non-specific and initially innocuous presentations, and concealed nature despite standard clinical testing. Once recognized and diagnosed, which may include cascade family screening, these patients can be treated by targeting the molecular mechanisms of the underlying ion channel dysfunction. There is increasing recognition that the mechanisms of genetic atrial and ventricular arrhythmias in the young are overlapping, making their combined study logical. This dissertation includes six retrospective studies and one systematic review and meta-analysis that collectively examine the incidence, disease mechanisms, and therapeutic outcomes of potentially genetic arrhythmia conditions in the young. First, we report a population study investigating the incidence of atrial fibrillation/flutter in young Canadians. We show a strong sex-predilection to lone AF in young males, but worse outcomes in affected females. By developing a rigorous definition of lone/idiopathic atrial fibrillation, we conclude that this population-level approach may be an effective methodology to identify young AF patients who may benefit from genetic discovery. Next, the focus turns to a population-level study of syncope in the young, as this is a very common symptom in genetic arrhythmia syndromes. Amongst 11,488 children with syncope presenting for emergency care, there was a low rate of hospitalization (2%) but a high burden of comorbidities and likelihood of re-presentation. Cardiac conditions were common, but mortality was extremely low (one potentially cardiac death amongst 11,488 syncopal patients at 1-year). These findings suggest that although syncope can be the sentinel symptom of a genetic arrhythmia syndrome, this population approach using administrative coding is likely too non-specific to be useful in identifying those rare children predisposed to sudden death. Finally, using a large international registry cohort, we examine a rare form of genetic ventricular-predominant arrhythmia caused by cardiac ryanodine receptor dysfunction, called catecholaminergic polymorphic ventricular tachycardia. We show that implantable cardioverter defibrillators are paradoxically associated with increased harm, chronotropic incompetence during exercise is a risk factor for arrhythmic events, and homology mapping is a useful tool to predict RyR2 variant pathogenicity in this disorder. Through deeper phenotypic analysis of the registry, we further identify one of the first cases of a novel genetic arrhythmia syndrome related to the ryanodine receptor, now being termed cardiac ryanodine receptor release deficiency syndrome.Collectively, the studies comprising this dissertation provide a comprehensive overview of the methodologies needed to address key question in this diverse and challenging population. Future efforts should focus on leveraging the centralized nature of the single-payer healthcare system in Canada to better link population level cardiac outcomes to deeply phenotyped and genotyped arrhythmia registry cohorts

Análise transcriptômica da bactéria Azospirillum brasilense FP2 e reanotação do genoma de Herbaspirillum seropedicae SmR1

Orientadora: Profa. Rose Adele MonteiroCoorientador: Prof. Emanuel Maltempi de SouzaTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciências (Bioquímica). Defesa : Curitiba, 25/06/2018Inclui referências: p. 75-90Resumo: Herbaspirillum seropedicae e Azospirillum brasilense são bactérias fixadoras de Nitrogênio, encontradas associadas à plantas de interesse agrícola e econômico para o país, como milho, arroz e trigo. São bactérias promotoras do crescimento vegetal, sendo utilizadas como biofertilizantes. Na primeira parte deste trabalho A. brasilense FP2 teve seu genoma montado novamente, reanotado e utilizado para o mapeamento dos resultados de RNA-Seq de Azospirillum brasilense cultivado em condições de fixação de nitrogênio. Os genes expressos diferencialmente foram identificados. Com esses dados obteve-se o panorama metabólico de FP2 crescida em condições de fixação de Nitrogênio. Foi possível comparar os resultados de A. brasilense com outras espécies fixadoras de Nitrogênio, em que se destaca a repressão da Montagem Flagelar em A. brasilense. Na segunda parte deste trabalho, o genoma de H. seropedicae SmR1 foi reanotado, utilizando dados de RNA-Seq. Identificamos 309 genes novos e atualizamos as classes funcionais dos genes. Também foi possível identificar 901 operons no genoma.Abstract: Azospirillum brasilense and Herbaspirillum seropedicae are diazotrophic bacteria, living associated with plants of agricultural and economic interest to the country, such as corn, rice and wheat. They are bacteria that promote plant growth and are used as biofertilizers. In the first part of this work A. brasilense FP2 had its genome re-assembled, reanoted and used for the mapping of the RNA-Seq results of Azospirillum brasilense cultivated under nitrogen fixation conditions. Differentially expressed genes were identified. With these data we obtained the metabolic panorama of FP2 grown under Nitrogen fixation conditions. It was possible to compare the results of A. brasilense with other Nitrogen fixing species, in which the repression of the Flagellar Assembly in A. brasilense is highlighted. In the second part of this work, the genome of H. seropedicae SmR1 was reanoted, using RNA-Seq data. We identified 309 new genes and upgraded the functional classes of the genes. It was also possible to identify 901 operons in the genome

Molecular Mechanisms Governing the Pharmacology of LDT409 for the Treatment of Metabolic Disease

The prevalence of metabolic diseases, including obesity, dyslipidemia, and type 2 diabetes, is dramatically increasing in the world; thus, it is important to develop effective, low cost, and safe medications to treat these diseases. The peroxisome proliferator-activated receptors (PPARa/g/b) are fatty acid sensors that play important roles in the regulation of lipid metabolism and glucose homeostasis. Herein, we report the characterization of novel compounds derived from phenolic lipids that are abundant in cashew nut shell liquid (CNSL), a by-product of the cashew nut industry. In fact, these compounds retained structural similarity to fatty acids that are known to endogenously activate PPARs. We identified several active phenolic lipid derivatives as single-, dual-, and/or pan-active PPAR agonists, with partial agonist activity and low micromolar potency. Detailed characterization of adipocyte and hepatocyte responses, and in vivo biodistribution studies in zebrafish embryos led to the identification of the lead compound, 23 (LDT409). LDT409 is a novel partial pan-PPAR agonist with potent and balanced affinity for PPARa and PPARg and weak binding affinity to PPARδ (Chapter 2). Moreover, we assessed that chronic treatment with LDT409 was effective at reversing diet-induced obesity and its complications in mice. LDT409 exhibited metabolically beneficial effects on lowering food intake and hyperlipidemia, while improving insulin sensitivity. Surprisingly, LDT409 normalized the HFD-induced weight gain back to chow-fed control mice via targeting these mechanisms: (i) decreasing food intake, (ii) increasing fatty acid utilization, and (iii) decreasing intestinal absorption of fat. We also demonstrated LDT409 ameliorated fatty liver disease and upregulated expression of fasting-associated target genes (e.g., Fgf21, Pdk4), suggesting that LDT409 may mimic fasting to ameliorate fatty liver disease (Chapter 3). Lastly, we explored whether LDT409 induced a “fasting phenotype” to prolong the catabolic state and found that LDT409 yielded a similar response to fasting phenotypically but by RNA-seq LDT409 had sexually dimorphic, wide-ranging effects on signaling pathways that extended beyond fasting- related genes (Chapter 4). In conclusion, LDT409 represents a fatty acid mimic that generates a uniquely favorable metabolic response for the treatment of multiple sequelae of metabolic disease including obesity, dyslipidemia, NAFLD, and type 2 diabetes.Ph.D.2025-06-26 00:00:0

Development of human brain tumor assembloids that mimic the interaction of normal and tumorous tissue to assess the impact of ionizing radiation

Although brain and other central nervous system (CNS) cancers are relatively rare, they have a high mortality rate [1] despite treatment with surgery, chemotherapy, and/or radiation therapy [2–5]. To further improve therapeutic approaches and to diminish the lethality of patients with brain and other CNS cancers, it is crucial to understand the treatment-induced cellular and molecular alterations in both normal brain and tumorous tissues, as well as their interactions. For this purpose, the main objective of this thesis was to generate a three-dimensional human cell culture model that simulates brain tumors, including early tumorigenesis, and that is suitable for investigating radiation-induced effects exerted on normal and tumorous brain tissue as well as their interaction. The brain tumor assembloid model generated in this thesis consisted of two connected parts with the same genetic background: the normal neural tissue was modeled using neural spheroids generated from human embryonic stem cells (hESCs), and the tumor-like tissue was modeled using genetically modified MYC overexpressing (MYCOE) cells, which were generated in an autologous setting from neural spheroids. Tumor initiation and promotion were simulated by overexpression of the oncogene MYC [6], which was stably integrated into the genome of single cells of the neural spheroid, and by the proliferation of these MYCOE cells within the neural spheroid thereafter. To balance the physiological cellular and morphological heterogeneity observed in vivo with the cellular and morphological similarity of replicate samples across experiments, a heterogeneous MYCOE cell population was isolated, aggregated into uniform spheres (MYCOE spheres), and fused with normal d100 neural spheroids, containing neuronal and glial cells, to simulate the tumor-like part of the generated brain tumor assembloid model. The proliferating MYCOE cells showed an immature neural phenotype, lower expression of tumor suppressor genes, as well as infiltrative and metastatic characteristics. In the assembloids, astrocytes from the neural spheroids extended their branches into the bordering MYCOE spheres and enveloped the infiltrated MYCOE cells within the neural spheroid. The assembloids are suitable for analyzing and interpreting radiation therapy effects using, for example, X-ray irradiation. Exposition of 1 or 3 Gy X-rays resulted in increased extend of MYCOE cell death, a lower number of infiltrating MYCOE cells, and shorter infiltration depth. The model bridges the gap between different model systems, is adaptable to specific tumor features, and offers a suitable possibility for human brain tumor modeling. A schematic overview of the brain tumor assembloid generation is shown in Figure 1