Dissertations / Theses on the topic 'Protein fibrillation'

Advances in recent nanoscience technologies have generated a new compilation of biocompatible, fluorescent nanoparticles derived from semiconductor quantum dots (QDs). QDs are extremely small in size and possess very large surface areas, which gives them unique physical properties and applications distinct from those of bulk systems. When exposed to biological fluid, these QDs may become coated with proteins and other biomolecules given their dynamic nature. These protein-QD systems may affect or enhance the changes in protein structure and stability, leading to the destruction of biological function. It is believed that these QDs can act as nucleation centers and subsequently promote protein fibril formation. Protein fibrillation is closely associated with many fatal human diseases, including neurodegenerative diseases and a variety of systemic amyloidoses. This topic of protein-QD interaction brings about many key issues and concerns, especially with respect to the potential risks to human health and the environment. Herein, the behavioral effects of dihydrolipoic acid (DHLA)-capped CdSe/ZnS (core/shell) QDs in hen egg-white lysozyme (HEWL) and human serum albumin (HSA) protein systems were systematically analyzed. This study gives rise to a better understanding of the potentially useful application of these protein-QD systems in nanobiotechnology and nanomedicine as a bioimaging tool and/or as a reference for controlled biological self-assembly processes.

Background: Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia. There is a need for new pharmacological treatment strategies since the current antiarrhythmic drugs have a modest efficacy and may have severe side effects. Cardioversion (CV) of AF offers an opportunity to study related conditions in sinus rhythm (SR) and during AF. Since catheter ablation of AF is a symptomatic treatment, it is important to have tools for measurement of arrhythmia-related symptoms. Aims: To evaluate the effect of atorvastatin on maintaining SR after CV of persistent AF. To assess if highsensitivity C-reactive protein (hsCRP) predicts the recurrence of AF after CV in a population randomized to treatment with either atorvastatin or placebo. To quantify the symptomatic effect of left atrial catheter ablation of AF. To assess if the restoration of SR by CV, in a population with persistent AF, affects sleep apnea. Methods: Paper I: A total of 234 patients were randomized to treatment with either high dose atorvastatin or placebo prior to CV. Paper II: In a pre-specified substudy which included 128 of the patients in study I, hsCRP was analyzed before and after CV. Paper III: Umea 22 Arrhythmia Questions (U22) is a questionnaire that quantifies paroxysmal tachycardia symptoms. A total of 105 patients underwent first-time pulmonary vein isolation and answered U22 forms at baseline and follow-up 304 (SD 121) days after ablation. Paper IV: Polysomnography was performed before and after CV in 23 patients with persistent AF scheduled for elective CV. Results: Paper I: An intention-to-treat analysis with the available data, by randomization group, showed that 57 (51%) in the atorvastatin group and 47 (42%) in the placebo group were in SR 30 days after CV (OR 1.44, 95%CI 0.85–2.44, P=0.18). Paper II: HsCRP did not significantly predict recurrence of AF at 30 days. However, after adjusting for treatment with atorvastatin, hsCRP predicted the recurrence of AF (OR 1.14, 95% CI 1.01–1.27). Six months after CV, hsCRP at randomization predicted recurrence of AF in both univariate analysis (OR 1.30, 95% CI 1.06–1.60) and in multivariate logistic regression analysis (OR 1.33, 95% CI 1.06– 1.67). Paper III: The U22 scores for well-being, arrhythmia as cause for impaired well-being, derived timeaspect score for arrhythmia, and discomfort during attack detected relevant improvements of symptoms after the ablation. U22 showed larger improvement in patients undergoing only one procedure than in patients who later underwent repeated interventions. Paper IV: Obstructive sleep apnea occurred in 17/23 patients (74%), and central sleep apnea in 6/23 patients (26%). Five patients had both obstructive and central sleep apnea. SR at follow-up was achieved in 16 patients. The obstructive apnea-hypopnea index, central apneahypopnea index, and the number of patients with obstructive or central sleep apnea did not differ before and after restoration of SR. Conclusions: Atorvastatin is not a treatment option with regards to maintaining SR after CV in patients with persistent AF. HsCRP was associated with AF recurrence 1 and 6 months after successful CV of persistent AF. U22 quantifies the symptomatic improvement after AF ablation with adequate internal consistency and construct validity. Both obstructive and central sleep apneas are highly prevalent in patients with persistent AF. Obstructive sleep apneas are unaffected by the CV of AF to SR.

The electrophysiological basis of atrial fibrillation (AF) is unknown. Regulators of G-protein signalling (RGS) act to deactivate G-protein signalling; they interact with the α subunit and accelerate intrinsic GTP-ase activity. RGS 4 negatively regulates inhibitory Gαi/o and Gαq/11 signalling. Absence of RGS 4 may increase G-protein gated inwardly rectifying potassium (GIRK) currents, shorten the atrial effective refractory period and promote atrial arrhythmia. The absence of RGS 4 results in continued Gαq/11 and thus calcium (Ca2+) signalling which may predispose to atrial arrhythmia. Does attenuation of RGS 4 promote AF? What is the mechanism? In vivo studies were performed in global RGS 4 knockout (-/-) and wild type (+/+) mice. Electrophysiological studies (EPS) were performed and ECG and EP parameters recorded. AF induction was attempted. Telemetry probes were inserted and conscious mouse ECGs were studied for AF and atrial ectopics (AE). Heart rate variability (HRV) was measured. Atrial cardiomyocytes were studied with patch-clamping and confocal microscopy. ECG and EP parameters were comparable. RGS 4 (-/-) developed AF. Conscious RGS 4 (-/-) were tachycardic, and had an enhanced bradycardic response to carbachol (CCh). ECG and HRV parameters of RGS 4 (+/+) and RGS 4 (-/-) without and with CCh were comparable. CCh-treated RGS 4 (-/-) had disrupted HRV, pauses and AE compared to RGS 4 (-/-) alone, but no AF. At patching, GIRK was not implicated but single cell restitution curves showed a steeper slope in RGS 4 (-/-). There was an increase in frequency of Ca2+ sparks in RGS 4 (-/-) compared to RGS 4 (+/+) in the absence and presence of endothelin-1 (ET-1). 2-aminoethoxydiphenyl borate (2-APB) abolished sparks. Sparks were reduced in amplitude in RGS 4 (-/-) in the absence of ET-1, but were longer in duration, time to peak and tau. A subset analysis showed that the left atrium had a greater frequency of Ca2+ sparks than the right atrium, and that these were comparatively longer in duration, time to peak and tau. RGS 4 (-/-) mice are predisposed to AF. There is good evidence that this is mediated via Gαq/11-IP3-Ca2+. RGS 4 is a potential therapeutic target in the treatment of AF. More recently RGS 6 has been found to be expressed in the heart and negatively regulates inhibitory Gαi/o signalling. EPS with AF induction were performed in RGS 6 (-/-) and RGS 6 (+/+) mice, with greater inducibility in RGS 6 (-/-) mice. It is possible that RGS 6 mediates its effects via Gαi-GIRK and could be a therapeutic target in the treatment of AF but further mechanistic studies are underway.

My PhD project investigated the regulation of human cardiac transient outward potassium current (Ito) by protein tyrosine kinases (PTKs) and the functional expression of transient receptor potential (TRP) channels in human atrial myocytes to make an advanced understanding of human cardiac electrophysiology and pathophysiology. The modulation of human cardiac Itoby PTKs was studied in human atrial myocytes and HEK 293 cells expressing hKv4.3 (coding human cardiac Ito). We found that the broad-spectrum PTK inhibitor genistein, the selective EGFR kinase inhibitor AG556, and the Src-family kinases inhibitor PP2 inhibited human atrial Itoand the inhibitory effect was countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Similar results were observed in hKv4.3-HEK cells. Interestingly, tyrosine phosphorylation of hKv4.3channels was reduced by genistein, AG556, and PP2,and the reduction was antagonized by orthovanadate. The mutant Y136F of hKv4.3 lost the inhibitory response to AG556, whileY108F lost the response to PP2.The double mutant Y108F-Y136F hKv4.3 failed to respond to both AG556 and PP2, and exhibited a dramatic reduction of tyrosine phosphorylation. These results indicate that native cardiac Itois regulated by both EGFR and Src family kinases. In the second part, we studied whether TRPC channels would mediate the nonselective cation current described previously in human atrial myocytes. It was found that TRPC1 channel activator thapsigargin activated the current, and the effect was suppressed by La3+or prevented by intracellular anti-TRPC1 antibody. Endothelin-1 and angiotensin II stimulated the current, andthe effect was inhibited by La3+and/or 2-APB. RT-PCR and Western blot analysis revealed that in addition to the TRPC1 channels mediating the nonselective cation current, the components of store-operated Ca2+channels (SOCs), STIM1 and Orai1 were abundantly expressed in human atria. The interaction of TRPC1, STIM1, and Orai1 was confirmed by co-immunoprecipitation. Interestingly, we found that protein expression of TRPC1 and STIM1, but not Orai1, was up-regulated in human atria with atrial fibrillation. The third part of the project determined whether TRPM7 channels were expressed in human atrial myocytes, since this channel was reported in human atrial fibroblasts, conferring atrial fibrosis in human atria with atrial fibrillation. We found a TRPM7 -like current which was potentiated by acidic pH, and inhibited by La3+and 2-APB, and a Ca2+-activated TRPM4 current. RT-PCR and Western blot analysis confirmed the expression of TRPM7 and TRPM4 channels in human atria. Moreover, we found TRPM7 protein, but not TRPM4 protein was significantly up-regulated in human atria with atrial fibrillation, suggesting the potential participation of TRPM7 channels in atrial remodeling of human atria with atrial fibrillation. Collectively, this PhD thesis project has demonstrated for the first time that human cardiac Itois modulated by EGFR kinase and Src kinases via phosphorylating Y136and Y108, respectively. TRPC1 channels mediate the nonselective cation current and SOCs.TRPM7 channels are expressed in human atrial myocytes. The up-regulation of TRPC1, STIM1, and TRPM7 channels in human atria with atrial fibrillation suggest that they are likely involved in atrial electrical and/or structure remodeling in patients with atrial fibrillation.
published_or_final_version
Medicine
Doctoral
Doctor of Philosophy

Les maladies dites ''conformationnelles'' sont caractérisées par un mauvais repliement des protéines qui, de ce fait, ne peuvent plus assurer leur fonction biologique. C'est le cas des amyloses, ces pathologies impliquent des protéines ayant la capacité de s'agréger pour former des structures spécifiques appelées « fibres amyloïdes ». Aujourd'hui, une trentaine de protéines humaines sont connues pour former ce type de fibres et notamment la protéine tau. Celle-ci est associée à plusieurs maladies neurodégénératives, regroupées sous le terme de « tauopathies », incluant la maladie d'Alzheimer. En conditions physiologiques, tau est associée aux microtubules et régule leur polymérisation. Dans les tauopathies, elle devient hyperphosphorylée et s'agrège dans les neurones sous forme de neurodégénérescences fibrillaires (NFTs) toxiques. Les protéines chaperons et particulièrement la protéine de choc thermique de 90 kDa, Hsp90, régule l'homéostasie de la protéine tau. L'interaction entre tau et Hsp90 implique différentes régions de la protéine tau dont celle contenant un hexapeptide de séquence VQIVYK. Ce court fragment est nécessaire et suffisant pour induire la fibrillation de la protéine tau entière in vivo. Cet hexapeptide est également capable, à lui seul, de former des fibres amyloïdes, in vitro, comparables à celles retrouvées in vivo. Nous avons donc choisi d'utiliser l'hexapeptide VQIVYK comme modèle d'étude de la fibrillation, in vitro, et testé l'effet de Hsp90 sur les processus agrégatifs du peptide. Nous avons démontré que Hsp90 interagit spécifiquement avec les structures amyloïdes formées par le peptide et qu'elle est capable d'inhiber à la fois la polymérisation et la dépolymérisation des fibres. Ce rôle antagoniste joué par Hsp90 permet la stabilisation d'espèces amyloïdes intermédiaires supposées moins neurotoxiques. Ces résultats confirment l'implication de Hsp90 dans les processus agrégatifs de la protéine tau et ouvrent de nouvelles perspectives thérapeutiques contre les pathologies neurodégénératives. De plus, cette étude apporte des éléments de réponse sur le fonctionnement des chaperons moléculaires vis-à-vis de leur protéine cliente
Conformational diseases are characterized by protein misfolding which causes a loss of biological activity. Amyloidosis is one of these diseases, and it involves the ability of proteins to self-aggregate into specific structures called “amyloid fibers”. At least thirty human proteins, including tau, are known to form amyloid fibers. The tau protein is linked to several neurodegenerative diseases called tauopathies, including Alzheimer’s disease. Tau is in physiological conditions associated with microtubules and regulates their polymerization. In tauopathies, tau becomes hyper-phosphorylated and aggregates into neurotoxic neurofibrillary tangles (NFTs). Molecular chaperones, and particularly the 90-kDa heat shock protein (Hsp90), regulate tau homeostasis. The interaction between tau and Hsp90 involves several tau regions including the sequence VQIVYK. This short fragment is necessary and sufficient on its own to induce aggregation of the full tau protein in vivo. In vitro this hexapeptide is also able to form amyloid fibers similar to those found in vivo. We therefore used this hexapeptide as an in vitro model to study the process of amyloid fibrillation and to test Hsp90’s effects on it. We demonstrated that Hsp90 interacts specifically with peptide fibrillar structures and that Hsp90 is able to inhibit both the polymerization and depolymerization processes. This antagonistic role for Hsp90 allows the stabilization of intermediate amyloid species that may display a lower neurotoxicity. These results confirm that Hsp90 is involved in tau’s aggregation process and paves the way for new therapeutic perspectives in neurodegenerative diseases. Our study also provides clues to the understanding of how molecular chaperones assist in the folding of their client proteins

Abstract The identification of risk factors for major adverse events after coronary artery bypass surgery is of main importance as it allows outcome prediction, facilitates preoperative patient selection and improves the quality of care. In the present clinical studies we have evaluated the impact of preoperative angiographic severity of a coronary artery disease and preoperative C-reactive protein (CRP) on the immediate outcome after coronary artery bypass surgery. We have reviewed the results of off-pump (OPCAB) versus conventional on-pump coronary artery bypass surgery (CCAB) in high risk patients. We have evaluated the impact of postoperative pulmonary artery blood temperature on the immediate outcome as well. In addition, we have investigated the incidence, timing and outcome of an atrial fibrillation (AF) related stroke after surgery. The multivariate analysis showed that among 2233 patients, the overall coronary angiographic score was predictive of postoperative death (p = 0.03; OR 1.027, 95% CI: 1.003–1.052) and of a low cardiac output syndrome (p = 0.04; OR 1.172, 95% CI: 1.010–1.218). The poor status of the proximal segment of the left circumflex coronary artery, the diagonal branches and the left obtuse marginal artery were most closely associated with adverse postoperative outcome. Patients (114/764) with a preoperative serum concentration of CRP ≥ 1.0 mg/dL had a higher risk of overall postoperative death (5.3% vs. 1.1%, p = 0.001), cardiac death (4.4% vs. 0.8%, p = 0.002), a low cardiac output syndrome (8.8% vs. 3.7%, p = 0.01). Among 179 high risk patients with an additive EuroSCORE6, the 30-day postoperative death and stroke rates were 7.5% and 6.0% in the OPCAB group, and 5.4% (p = 0.75) and 8.0% (p = 0.77) in the CCAB group, respectively. No significant differences were observed in other major outcome end-points between these non-randomised groups either. High pulmonary artery blood temperature on admission to the ICU among 1639 patients was significantly associated with an increased risk of overall postoperative death (p = 0.002), cardiac death (p = 0.03), and a low cardiac output syndrome (p < 0.0001), and was significantly correlated with prolonged length of the ICU stay (r = 0.095; p < 0.0001), and postoperative bleeding (ρ = –0.091; p = 0.001). Among 2,630 patients who underwent coronary artery bypass grafting (CABG), 52 (2.0%) experienced a postoperative stroke. Twelve out of these 52 patients (23.1%) died postoperatively. The ischemic cerebral event occurred after a mean of 3.7 days (0–33). In 19 patients (36.5%), atrial fibrillation preceded the occurrence of neurological complication. The angiographic severity of the coronary artery disease and the preoperative serum concentration of CRP predict postoperative outcome after a CABG operation. OPCAB can be performed safely in high-risk patients with results as satisfactory as those achieved with CCAB. CABG patients with a high pulmonary artery blood temperature on admission to the ICU seem to have a higher risk of postoperative adverse events. Atrial fibrillation occurring after coronary artery bypass grafting is a major determinant of a postoperative stroke.

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Esta tese de doutorado foi dedicada à investigação do modo como a técnica de espectroscopia de impedância elétrica (EIE) poderia ser usada para acompanhar os processos de mudanças conformacionais de macromoléculas biológicas, como proteínas e DNA. Para isso, usamos como sistemas modelos a proteína albumina do soro bovino (BSA), e a formação do complexo polianilina/DNA (PANI/DNA). Com a caracterização de soluções de DNA e BSA por EIE e sua modelagem elétrica convenientemente descrita pelo circuito de Randles (e sua variante), foram determinados os parâmetros relevantes para descrição dos fenômenos de desnaturação e de agregação da proteína e da precipitação do complexo PANI/DNA. As informações obtidas sobre a solubilidade desses últimos complexos são de grande utilidade para o entendimento dos mecanismos de interação entre cadeias de DNA e de polímeros condutores. Do mesmo ponto de vista da EIE, as sucessivas mudanças da conformação da proteína e os detalhes da cinética de sua agregação na interação com surfactantes foram adequadamente correlacionados com a característica elétrica do circuito de Randles das soluções correspondentes. Finalmente, estudos iniciais foram estendidos para a análise dos processos de fibrilação de proteínas. Para todos os problemas abordados, o uso da resistência de transferência de carga elétrica (RCT) (um parâmetro do circuito de Randles) nos permite sugerir ser a técnica de EIE apropriada para caracterizar as diferentes mudanças conformacionais envolvidas em fenômenos que resultam da interação de biomoléculas com moléculas de prova. Assim, ela se confirma como um método competitivo quando comparado ao uso da fluorescência e da absorção UV-Vis (técnicas rotineiramente adotadas para a análise desses problemas).
This doctoral thesis was devoted to the investigation of the technique of electrical impedance spectroscopy as an alternative method to assess conformational changes of biological macromolecules, such as proteins and DNA. For this, we used protein bovine serum albumin (BSA), and the formation of polyaniline (PANI)/DNA complexes as model systems. With the characterization of DNA and BSA solutions by Electrical Impedance Spectroscopy (EIS) and their electrical modeling conveniently described by the Randles circuit (and its variant), we determined the relevant characteristics of phenomena such as the denaturation and aggregation of proteins (BSA), and polymer/DNA complex formation (PANI/DNA). As a result of this approach we identified the existence of different interaction regimes between the chains of polyaniline and DNA molecules that are dependent on the concentration of PANI/DNA and the existence of equilibrium conditions which separate regions of precipitation/stability the PANI/DNA complex. Also from this point of view, the modes of interaction BSA / surfactants involved in the conformation changes well as typical stages associated with fibrillation kinetics were adequately correlated with the electric characteristic of the Randles circuit. In all studies carry out in this thesis, the analysis of the electric charge transfer resistance behavior (RCT) (a parameter of the Randles circuit) when confronted with the results obtained by standard techniques showed that the EIS presents reliable and some comparative advantages. These results allow us to provide an adequate and competitive alternative to conventional methods such as UV-Visible absorption, fluorescence and the use of probe molecules

Alzheimer's disease (AD) is a large and growing problem and while we today lack a full understanding of this disease, we know that the protein tau and the amyloid fibrils it forms play a central role in its development. We also know that these fibrils can have different morphologies in different diseases and that fibrils produced in vitro not necessarily adopt any of the morphologies found in patients. This means there is a need for more pathologically relevant fibrils in vitro to be able to understand this disease better. One approach to satisfy this need is to use fibrils found in patients as seeds and thus transfer their morphology to recombinantly purified protein. To facilitate this process this study has attempted to develop a way to differentiate between different fibril morphologies using a FRET based system. This involves fluorescent fusion proteins (tau-EXFPs) and fluorescent amyloid probes as well as seeding experiments with pseudo wild type tau (PWT) and tau with the P301L mutation. Greater differences in terms of fibrillation rates and ThT fluorescence between PWT and P301L was shown than previously reported between WT and P301L. They were also shown to differ in fibril morphology in TEM. The ThT fluorescence intensity was to a certain degree transferable from PWT to P301L by seeding. Furthermore, this study confirms that the tau-EXFP fusion protein can be incorporated into amyloid fibrils and strongly suggests that a FRET effect between EXFP and BTD14 (as well as X34 and ThT) can be achieved. It also demonstrates differences in FRET efficiency between PWT and P301L fibrils using FLIM. These results indicate that a FRET based approach could be a useful method to discern different fibril morphologies from each other, but further measurements and optimization are needed before this method could be reliably applied. The fusion proteins could also be used to investigate tau spreading in vivo, e.g. in D. melanogaster. To find suitable FRET partners to the fusion proteins, a ligand screen was conducted. This could be used as an alternative to the FRET method. With the right selection of fluorescent amyloid probes, a unique fingerprint for each fibril morphology could maybe be generated and fulfill the same intended function as the FRET method.

Despite major advances in the treatment and prevention of atherosclerosis the last several decades, cardiovascular disease still accounts for the majority of deaths in Sweden. With the population getting older, more obese and with rising numbers of diabetics, the cardiovascular disease burden may increase further in the future. The focus in cardiovascular disease has shifted with time from calcification and narrowing of arteries to the biological processes within the atherosclerotic plaque. C-reactive protein (CRP) has emerged as one of many proteins that reflect a low grade systemic inflammation and is suitable for analysis as it is more stable and easily measured than most other inflammatory markers. Several large prospective studies have shown that CRP is not only an inflammatory marker, but even a predictive marker for cardiovascular disease. C-reactive protein is associated with several other risk factors for cardiovascular disease including obesity and the metabolic syndrome. Our study of twenty healthy men during a two week endurance cross country skiing tour demonstrated a decline in already low baseline CRP levels immediately after the tour and six weeks later. In a study of 200 obese individuals with impaired glucose tolerance randomised to a counselling session at their health care centre or a one month stay at a wellness centre, we found decreased levels of CRP in subjects admitted to the wellness centre. The effect remained at one, but not after three years of follow-up. In a prospective, nested, case-referent study with 308 ischemic strokes, 61 intracerebral haemorrhages and 735 matched referents, CRP was associated with ischemic stroke in both uni- and multivariate analyses. No association was found with intracerebral haemorrhages. When classifying ischemic stroke according to TOAST criteria, CRP was associated with small vessel disease. The CRP 1444 (CC/CT vs. TT) polymorphism was associated with plasma levels of CRP, but neither with ischemic stroke nor with intracerebral haemorrhage. A study on 129 patients with atrial fibrillation was used to evaluate whether inflammation sensitive fibrinolytic variables adjusted for CRP could predict recurrence of atrial fibrillation after electrical cardioversion. In multivariate iv models, lower PAI-1 mass was associated with sinus rhythm even after adjusting for CRP and markers of the metabolic syndrome. In conclusion, lifestyle intervention can be used to reduce CRP levels, but it remains a challenge to maintain this effect. CRP is a marker of ischemic stroke, but there are no significant associations between the CRP1444 polymorphism and any stroke subtype, suggesting that the CRP relationship with ischemic stroke is not causal. The fibrinolytic variable, PAI-1, is associated with the risk of recurrence of atrial fibrillation after electrical cardioversion after adjustment for CRP. Our findings suggest a pathophysiological link between atrial fibrillation and PAI-1, but the relation to inflammation remains unclear.

Le proteine giocano un ruolo fondamentale nello svolgimento delle varie funzioni degli organismi viventi. Tuttavia, in condizioni patologiche, esse possono aggregarsi in fibrille tossiche che possono causare una grande varietà di disfunzioni neurodegenerative. Lo studio di proteine e fibrille potrebbe portare importanti innovazioni nella diagnosi e terapia delle varie malattie collegate ad esse. In questa tesi sono presentate alcune tecniche spettroscopiche, principalmente dicroismo circolare e dicroismo circolare plasmonico, utilizzate per studiare la struttura proteica ed identificare fibrille in vitro e in vivo. Il capitolo I è una introduzione, tratta dalla letteratura, dei due principali oggetti di questa tesi: fibrille e tecniche spettroscopiche. La prima parte di questo capitolo affronta l’evoluzione, le cause e la tossicità della fibrillazione, prendendo come esempio di malattia neurodegenerativa il Morbo di Parkinson. Nella seconda parte viene trattata la chiralità, la luce circolare polarizzata, il dicroismo circolare e le nanoparticelle, essendo queste le basi delle tecniche spettroscopiche usate in questo lavoro. Il Capitolo II è dedicato alle fibrille di alfa-siucleina. Questo capitolo illustra come è stato utilizzato il dicroismo circolare plasmonico nell’individuazione e differenziazione tra fibrille generate in vitro da alfa-sinucleina in forma integra ed in forma tronca. Lo stesso metolo d’analisi è stato inoltre applicato a fibrille estratte e purificate a partire da diversi sistemi biologici, quali cellule di neuroblastoma, omogenati cerebrali di topo e di pazienti umani. Il Capitolo III esplora due altre proteine, cioè l’insulina usata come modello semplice per fibrille e superossido dismutasi1. Fibrille di insulina con due sensi di avvolgimento, destro e sinistro, sono state generate in vitro in condizioni denaturanti e sono state analizzate mediante fluorescenza, dicroismo circolare, luminescenza circolarmente polarizzata e dicroismo circolare plasmonico. Sono stati usati anche coloranti fluorescenti come tioflavina T, congo red e bis-ANS. Come studio preliminare per una altra proteina che potrebbe subire fibrillazione, é stata considerata la superossido dismutasi 1. In particolare, la proteina nativa é stata confrontata con quella sintetizzata in presenza di cadmio. Le due forme sono state esaminate con dicroismo circolare nella regione dell’ultravioletto lontano al variare della temperatura, al fine di monitorare cambiamenti e differenze nella struttura secondaria.
Proteins play a vital role in all the processes taking place in a living organism. However, under pathological conditions, they may assemble as fibrils that are toxic and may cause a wide range of neurodegenerative disorders leading eventually to death. Studying proteins and fibrils can serve as a significant step toward new ways of diagnosis and therapy. This thesis proposes some spectroscopic techniques, mainly circular dichroism and plasmonic circular dichroism, to study proteins structures and to monitor fibril formation in vitro and in vivo. Chapter I is an introduction from literature of the two major subjects of this thesis: protein fibrils and spectroscopic techniques. The first part of this chapter delivers knowledge about the process, causes and toxicity of fibrillation taking Parkinson’s disease as an example of neurodegenerative disorder. The second part deals about chirality, circularly polarized light, circular dichroism and nanoparticles as they are the basics of the spectroscopic techniques used in this work. Chapter II is devoted to fibrils generated from alpha synuclein. This chapter illustrates how plasmonic circular dichroism was used to detect and differentiate between fibrils generated in vitro from full-length alpha synuclein and from a truncated form thereof. The technique was also applied to fibrils extracted and purified from different biological systems such as neuroblastoma cells and brain homogenates of mice and human patients. Chapter III explores two other protein systems: insulin, representing an easily available model for fibrils, and superoxide dismutase 1. Insulin fibrils with two opposite handedness were generated in vitro under denaturant conditions. These fibrils were then analyzed by fluorescence, circular dichroism, circularly polarized luminescence and plasmonic circular dichroism. Dyes such as thioflavin T, congo red and bis-ANS have been used. As a preliminary study for another system which may undergo fibrillation, superoxide dismutase 1 has been considered. In particular the native protein has been compared to the one produced in ambient with Cadmium excess. The two forms have been examined with far UV-CD spectroscopy at variable temperature to monitor changes and differences in the secondary structure.

Le proteine giocano un ruolo fondamentale nello svolgimento delle varie funzioni degli organismi viventi. Tuttavia, in condizioni patologiche, esse possono aggregarsi in fibrille tossiche che possono causare una grande varietà di disfunzioni neurodegenerative. Lo studio di proteine e fibrille potrebbe portare importanti innovazioni nella diagnosi e terapia delle varie malattie collegate ad esse. In questa tesi sono presentate alcune tecniche spettroscopiche, principalmente dicroismo circolare e dicroismo circolare plasmonico, utilizzate per studiare la struttura proteica ed identificare fibrille in vitro e in vivo. Il capitolo I è una introduzione, tratta dalla letteratura, dei due principali oggetti di questa tesi: fibrille e tecniche spettroscopiche. La prima parte di questo capitolo affronta l’evoluzione, le cause e la tossicità della fibrillazione, prendendo come esempio di malattia neurodegenerativa il Morbo di Parkinson. Nella seconda parte viene trattata la chiralità, la luce circolare polarizzata, il dicroismo circolare e le nanoparticelle, essendo queste le basi delle tecniche spettroscopiche usate in questo lavoro. Il Capitolo II è dedicato alle fibrille di alfa-siucleina. Questo capitolo illustra come è stato utilizzato il dicroismo circolare plasmonico nell’individuazione e differenziazione tra fibrille generate in vitro da alfa-sinucleina in forma integra ed in forma tronca. Lo stesso metolo d’analisi è stato inoltre applicato a fibrille estratte e purificate a partire da diversi sistemi biologici, quali cellule di neuroblastoma, omogenati cerebrali di topo e di pazienti umani. Il Capitolo III esplora due altre proteine, cioè l’insulina usata come modello semplice per fibrille e superossido dismutasi1. Fibrille di insulina con due sensi di avvolgimento, destro e sinistro, sono state generate in vitro in condizioni denaturanti e sono state analizzate mediante fluorescenza, dicroismo circolare, luminescenza circolarmente polarizzata e dicroismo circolare plasmonico. Sono stati usati anche coloranti fluorescenti come tioflavina T, congo red e bis-ANS. Come studio preliminare per una altra proteina che potrebbe subire fibrillazione, é stata considerata la superossido dismutasi 1. In particolare, la proteina nativa é stata confrontata con quella sintetizzata in presenza di cadmio. Le due forme sono state esaminate con dicroismo circolare nella regione dell’ultravioletto lontano al variare della temperatura, al fine di monitorare cambiamenti e differenze nella struttura secondaria.
Proteins play a vital role in all the processes taking place in a living organism. However, under pathological conditions, they may assemble as fibrils that are toxic and may cause a wide range of neurodegenerative disorders leading eventually to death. Studying proteins and fibrils can serve as a significant step toward new ways of diagnosis and therapy. This thesis proposes some spectroscopic techniques, mainly circular dichroism and plasmonic circular dichroism, to study proteins structures and to monitor fibril formation in vitro and in vivo. Chapter I is an introduction from literature of the two major subjects of this thesis: protein fibrils and spectroscopic techniques. The first part of this chapter delivers knowledge about the process, causes and toxicity of fibrillation taking Parkinson’s disease as an example of neurodegenerative disorder. The second part deals about chirality, circularly polarized light, circular dichroism and nanoparticles as they are the basics of the spectroscopic techniques used in this work. Chapter II is devoted to fibrils generated from alpha synuclein. This chapter illustrates how plasmonic circular dichroism was used to detect and differentiate between fibrils generated in vitro from full-length alpha synuclein and from a truncated form thereof. The technique was also applied to fibrils extracted and purified from different biological systems such as neuroblastoma cells and brain homogenates of mice and human patients. Chapter III explores two other protein systems: insulin, representing an easily available model for fibrils, and superoxide dismutase 1. Insulin fibrils with two opposite handedness were generated in vitro under denaturant conditions. These fibrils were then analyzed by fluorescence, circular dichroism, circularly polarized luminescence and plasmonic circular dichroism. Dyes such as thioflavin T, congo red and bis-ANS have been used. As a preliminary study for another system which may undergo fibrillation, superoxide dismutase 1 has been considered. In particular the native protein has been compared to the one produced in ambient with Cadmium excess. The two forms have been examined with far UV-CD spectroscopy at variable temperature to monitor changes and differences in the secondary structure.

Le proteine giocano un ruolo fondamentale nello svolgimento delle varie funzioni degli organismi viventi. Tuttavia, in condizioni patologiche, esse possono aggregarsi in fibrille tossiche che possono causare una grande varietà di disfunzioni neurodegenerative. Lo studio di proteine e fibrille potrebbe portare importanti innovazioni nella diagnosi e terapia delle varie malattie collegate ad esse. In questa tesi sono presentate alcune tecniche spettroscopiche, principalmente dicroismo circolare e dicroismo circolare plasmonico, utilizzate per studiare la struttura proteica ed identificare fibrille in vitro e in vivo. Il capitolo I è una introduzione, tratta dalla letteratura, dei due principali oggetti di questa tesi: fibrille e tecniche spettroscopiche. La prima parte di questo capitolo affronta l’evoluzione, le cause e la tossicità della fibrillazione, prendendo come esempio di malattia neurodegenerativa il Morbo di Parkinson. Nella seconda parte viene trattata la chiralità, la luce circolare polarizzata, il dicroismo circolare e le nanoparticelle, essendo queste le basi delle tecniche spettroscopiche usate in questo lavoro. Il Capitolo II è dedicato alle fibrille di alfa-siucleina. Questo capitolo illustra come è stato utilizzato il dicroismo circolare plasmonico nell’individuazione e differenziazione tra fibrille generate in vitro da alfa-sinucleina in forma integra ed in forma tronca. Lo stesso metolo d’analisi è stato inoltre applicato a fibrille estratte e purificate a partire da diversi sistemi biologici, quali cellule di neuroblastoma, omogenati cerebrali di topo e di pazienti umani. Il Capitolo III esplora due altre proteine, cioè l’insulina usata come modello semplice per fibrille e superossido dismutasi1. Fibrille di insulina con due sensi di avvolgimento, destro e sinistro, sono state generate in vitro in condizioni denaturanti e sono state analizzate mediante fluorescenza, dicroismo circolare, luminescenza circolarmente polarizzata e dicroismo circolare plasmonico. Sono stati usati anche coloranti fluorescenti come tioflavina T, congo red e bis-ANS. Come studio preliminare per una altra proteina che potrebbe subire fibrillazione, é stata considerata la superossido dismutasi 1. In particolare, la proteina nativa é stata confrontata con quella sintetizzata in presenza di cadmio. Le due forme sono state esaminate con dicroismo circolare nella regione dell’ultravioletto lontano al variare della temperatura, al fine di monitorare cambiamenti e differenze nella struttura secondaria.
Proteins play a vital role in all the processes taking place in a living organism. However, under pathological conditions, they may assemble as fibrils that are toxic and may cause a wide range of neurodegenerative disorders leading eventually to death. Studying proteins and fibrils can serve as a significant step toward new ways of diagnosis and therapy. This thesis proposes some spectroscopic techniques, mainly circular dichroism and plasmonic circular dichroism, to study proteins structures and to monitor fibril formation in vitro and in vivo. Chapter I is an introduction from literature of the two major subjects of this thesis: protein fibrils and spectroscopic techniques. The first part of this chapter delivers knowledge about the process, causes and toxicity of fibrillation taking Parkinson’s disease as an example of neurodegenerative disorder. The second part deals about chirality, circularly polarized light, circular dichroism and nanoparticles as they are the basics of the spectroscopic techniques used in this work. Chapter II is devoted to fibrils generated from alpha synuclein. This chapter illustrates how plasmonic circular dichroism was used to detect and differentiate between fibrils generated in vitro from full-length alpha synuclein and from a truncated form thereof. The technique was also applied to fibrils extracted and purified from different biological systems such as neuroblastoma cells and brain homogenates of mice and human patients. Chapter III explores two other protein systems: insulin, representing an easily available model for fibrils, and superoxide dismutase 1. Insulin fibrils with two opposite handedness were generated in vitro under denaturant conditions. These fibrils were then analyzed by fluorescence, circular dichroism, circularly polarized luminescence and plasmonic circular dichroism. Dyes such as thioflavin T, congo red and bis-ANS have been used. As a preliminary study for another system which may undergo fibrillation, superoxide dismutase 1 has been considered. In particular the native protein has been compared to the one produced in ambient with Cadmium excess. The two forms have been examined with far UV-CD spectroscopy at variable temperature to monitor changes and differences in the secondary structure.

Aggregation and fibrillation of proteins have a great importance in medicine and industry. Misfolding and aggregation are the basis of many neurodegenerative diseases like Alzheimer and Parkinson. Osmolytes are molecules that can accumulate within cells and act as protective agents and they can inclusively act as protein stabilizers when cells are exposed to stress conditions. Osmolytes can also act as protein stabilizers in vitro. In this work, two different proteins were studied, the ribosomal protein from Thermus thermophilus and the mouse prion protein. The existence of an unstructured N-terminal on the prion protein does not affect its stability. The effect of the osmolyte sucrose on the fibrillation and stabilization of these two proteins was studied through kinectic and equilibrium measurements. It was shown that sucrose is able to compact the native structure of S6 protein in fibrillization conditions. Sucrose affects also folding and unfolding kinetic of S6 protein, delaying unfolding and increasing folding rate constants. The mechanism of stabilization by sucrose is non-specific because it is distributed for all protein structure, as it was demonstrated by a protein engineering approach. Sucrose delays the process of formation and elongation of S6 and prion protein from mouse. This delay is the result of the compaction of the native structure refered above. However, cellular toxicity studies have shown that fibrils formed in the presence of sucrose are more toxic to neuronal cells.
A agregação e fibrilhação de proteínas têm um grande impacto no sector industrial e na medicina. O misfolding e a agregação de proteínas estão na origem ou são uma consequência de várias doenças neurodegenerativas como as doenças de Alzheimer e Parkinson. Para se protegeram de várias condições adversas as células utilizam vários mecanismos entre os quais a acumulação de osmólitos. Os osmólitos conseguem, entre outras funções, actuar no interior das células ao nível da estabilização de proteínas. Estudos in vitro demonstram que este efeito estabilizador dos osmólitos é também verificado fora do meio celular. Neste trabalho foram estudadas duas proteínas, a proteína ribossomal S6 de Thermus thermophilus e a proteína priónica de rato. A existência de um N-terminal não estruturado na proteína priónica não afecta a sua estabilidade. O efeito do osmólito sacarose na fibrilhação e estabilização destas duas proteínas foi analisado por medidas de equilíbrio e cinéticas. Foi demonstrado que a sacarose é capaz de compactar a estrutura nativa da proteína ribossomal S6 de T. thermophilus em condições que promovem a sua fibrilhação. A sacarose também afecta as cinéticas de folding e unfolding da proteína S6, diminuindo a velocidade de unfolding e aumentando a velocidade de folding. O mecanismo de estabilização pela sacarose não é específico, pois o seu efeito verifica-se globalmente em toda a estrutura proteica, tal como demonstrado usando uma estratégia de engenharia de proteínas. A sacarose atrasa o processo de formação e alongamento de fibras da S6 e da proteína priónica de rato. Este atraso no processo de fibrilhação da S6 resulta do efeito de compactação da estrutura nativa referido em cima. Porém, os estudos de toxicidade celular demonstraram que as fibras formadas na presença da sacarose são mais tóxicas para células neuronais.

碩士
國立臺灣大學
化學工程學研究所
99
L -arginine is a type of semi essential amino acid, and does not cause any harm on human being due to its biologically compatible property. While several lines of evidence has indicated that L arginine can effectively reduce protein aggregation, the precise interacting mechanism regarding the aggregation inhibition induced by L- arginine still remains elusive. The first part of this thesis is aimed at exploring the effects of L- arginine on the formation of amorphous aggregates using papain as the model protein system. Our data revealed that low concentration of guanidine hydrochloride (GdnHCl) could induce papain aggregation at acid condition (pH 2.0) with the highest level of aggregation observed at 0.7 M GdnHCl, which was taken as the aggregation condition used in our study. We found that, when a small amount of L- arginine (final concentration 0.02- 0.2M) were present in papain solution, the amount of papain aggregation was augmented. However L- arginine was observed to dose dependently attenuate the extent of papain aggregation upon futher increase of L- arginine (final concentration 0.3- 1.5M). Therefore, we postulate that, similar to GdnHCl, L- arginine not only perturbs the structure of papain but also has hydrophobic interactions with papain. In addition, in order to explore the ability of L- arginine to retain papain activity when its anti aggregation potency was observed, neutral pH and 65°C were used as the aggregation inductive condition. Our results demonstrated that, whereas 1M L-arginine was sufficient to decrease papain aggregation, only 10% of papain activity was retained. We hypothesize that papain active sites may exhibit higher conformational flexibility than the enzyme molecule as a whole, resulting in the finding that the loss of papain activity precedes noticeable conformational change of papain. As for the study of protein ordered aggregation, we used bovine serum albumin (BSA) as model protein. When incubating BSA at 65°C for hours, there was a considerable reduction in the α helical content accompanied by increases in β- sheet content in the BSA solution containing immature fibrils. We found that L- arginine dose dependently reduced the thioflavin T (ThT) fluorescence of BSA. However, as revealed by transmission electron microscopy (TEM), size exclusion chromatography (SEC), and dynamic light scattering (DLS) results, L-arginin did not prevent amyloid- like fibril formation by BSA. We concluded that L -arginine competed against ThT for binding sites on BSA amyloid -like fibrils, leading to biased results in ThT fluorescence measurements. Moreover, the use of ThT fluorescence assay to screen for potential inhibitors against amyloid fibrillation can give misleading results.

Heart rate is modulated by the opposing activities of sympathetic and parasympathetic inputs to pacemaker cardiomyocytes in the sinoatrial (SA) node. Parasympathetic activity on nodal myocytes is mediated by acetylcholine-dependent stimulation of M2 muscarinic receptors and activation of Gαi/o signalling. Although, regulators of G-protein signalling (RGS) proteins are potent inhibitors of Gαi/o signalling in many tissues, the RGS protein(s) that regulate parasympathetic tone in the SA node are unknown. Our results demonstrate that RGS4 mRNA levels are higher in the SA node compared to right atrium. Conscious freely moving RGS4-null mice showed a greater extent of bradycardia in response to parasympathetic agonists compared to wild-type animals. Moreover, anaesthetized rgs4-null mice had lower baseline heart rates and greater heart rate increases following atropine administration. Retrograde-perfused hearts from rgs4-null mice also showed enhanced negative chronotropic responses to carbachol, while isolated SA node myocytes showed greater sensitivity to carbachol-mediated reduction in the action potential firing rate. Finally, rgs4-null SA node cells showed decreased levels of G-protein-coupled inward rectifying potassium (GIRK) channel desensitization, and altered modulation of acetylcholine-sensitive potassium current (IKACh) kinetics following carbachol stimulation. Taken together, our studies establish that RGS4 plays an important role in regulating sinus rhythm by inhibiting parasympathetic signalling and IKACh activity. Following these results, we predicted that loss of RGS4 expression and function will result in increased levels of parasympathetic effector activity leading to increased susceptibility to atrial fibrillation. Susceptibility to atrial fibrillation (AF) depends strongly on parasympathetic activity. Since RGS4 inhibits parasympathetic / M2-dependent Gαi/o signalling in the SA node, we explored whether changes in RGS4 levels altered the susceptibility of atrial fibrillation. We found that, RGS4 levels were decreased in atria of tachypaced dogs prior to their development of chronic AF. Moreover, in vivo ECG recordings of anaesthetized mice showed greater susceptibility to AF while optical mapping of isolated atrial preparations using a voltage-sensitive dye revealed greatly increased susceptibility to rotor formation when RGS4 was ablated. Consistent with altered parasympathetic signalling in the myocardium of rgs4-null mice, IKACh evoked by carbachol application were greater in isolated atrial myocytes from rgs4-null mice. These IKACh changes were, as expected, associated with marked action potential duration shortening in response to parasympathetic activation, but not to slower conduction velocities. Together, our findings establish that RGS4 protects atrial tissues from excess parasympathetic signalling that predispose to atrial fibrillation.

Lo studio delle proprietà strutturali e funzionali della α1-glicoproteina acida (AGP) costituisce l’oggetto di questa tesi. L’AGP rappresenta, dopo l’albumina, la più abbondante proteina nel plasma. Essa appartiene alla famiglia delle lipocaline, un ampio gruppo di proteine caratterizzato da una struttura centrale a barile β (β-barrel) che funge da sito di legame per numerose molecole ad attività biologica. E’ stata dimostrata l’applicabilità biotecnologica dell’AGP nel campo dello sviluppo di nuovi biosensori e delle purificazioni industriali. Durante il mio dottorato, è stata condotta un’ampia analisi riguardante numerosi fattori ambientali (pH, stato ossidoriduttivo, temperatura), la quale ha mostrato che valori di pH debolmente o fortemente acidi e/o condizioni riducenti, inducono la concomitante denaturazione e aggregazione delle catene polipeptidiche in seguito all’aumento della temperatura. Da queste osservazioni si è passati alla caratterizzazione delle forme aggregate dell’AGP ottenute in specifiche condizioni di pH, temperatura e stato ossidoriduttivo. La capacità, di questi aggregati, di legare la Tioflavina T indica che tali forme aggregate sono di natura amiloide. Indagini più mirate sono state condotte sulla cinetica di formazione delle fibrille, da cui ricavare parametri cinetici utili nell’individuare un possibile modello di aggregazione. È importante notare che le analisi cinetiche non mostrano la presenza di una fase di nucleazione, il che esclude il modello di polimerizzazione dipendente da nucleazione. In questo modello, il passaggio limitante, è rappresentato dalla formazione di un nucleo che funge da innesco critico per l’intero processo di fibrillazione. Dati sperimentali hanno evidenziato la mancanza di questa fase, il ché suggerisce un meccanismo a cascata, in cui lo step critico è rappresentato dalla conversione del monomero stabile in monomero ‘attivato’, il quale è più propenso all’aggregazione e si assembla in strutture fibrillari.
α1-Acidglycoprotein (AGP) is an important member of the acute phase response involved in drug binding and modulation of the immune system. AGP belongs to the lipocalin superfamily, a wide group of proteins sharing a strikingly conserved β-barrel fold that serves as a binding site for a large number of hydrophobic and neutral molecules. Several biological functions have been associated to AGP, both in vivo and in vitro. AGP is able to bind and transport a wide number of hydrophobic molecules. Potential therapeutic uses of AGP in the treatment of immune diseases have been envisioned, including, for instance, reduction of histamine levels. In the light of these observations, a detailed knowledge of the structure and stability of AGP appears essential. Fourier-Transform infrared (FT-IR) spectroscopy has been extensively used in the investigation of AGP due to its sensitivity towards fluctuations within protein structures. A survey of several environmental conditions, including pH and disulfide redox state, has shown that strongly acidic and reducing environments induce concomitant denaturation and aggregation of the polypeptide chain following increases in temperature. My doctoral research has been undertaken to characterize AGP aggregates obtained in the presence of the disulfide-specific reducing agents. The binding of Thioflavin T dye indicate that the aggregates possess a cross-β motif, suggesting that they are amyloid in nature. A molecular interpretation on the fibrillation kinetics of AGP was proposed. The experimental data do not show a nucleation-dependent polymerization mechanism otherwise they suggest a downhill polymerization mechanism as described for transthyretin and serum albumin. In this model, an aggregation-prone species self-assembles to form the amyloid fibrils. Further support to the downhill polymerization mechanism was provided by the seeding experiments and a more accurate kinetic analysis of fibril formation.

Ion channels are membrane proteins that facilitate electrical signaling in important physiological processes, such as the rhythmic contraction of the heart. KCNQ1 is the pore-forming subunit of a voltage-gated potassium channel that assembles with the β-subunit KCNE1 in the heart to generate the IKs current, which is critical to cardiac action potential repolarization and electrical conduction in the heart. Mutations in IKs subunits can cause potentially lethal arrhythmia, including long QT syndrome, short QT syndrome, and atrial fibrillation. Each channel consists of four voltage-sensing domains and a central pore through which ions permeate. Voltage-dependent gating occurs when movement of voltage sensors cause pore opening/closing through coupling mechanisms. Although KCNQ1 by itself is able to form a voltage-dependent potassium channel, its assembly with KCNE1 is essential to generating the physiologically critical cardiac IKs current, characterized by a delay in the onset of activation, an increase in current amplitude, and a depolarizing shift in the current-voltage relationship. KCNE1 is thought to have multiple points of contact with KCNQ1 that reside within both the voltage-sensing domain and the pore domain, allowing for extensive modulation of channel function. Atrial fibrillation is the most common cardiac arrhythmia and affects more than 3 million adults in the United States. Much rarer, genetic forms of atrial fibrillation have been associated with gain-of-function mutations in KCNQ1, such as two adjacent mutations, S140G and V141M. Both mutations drastically slow channel deactivation, which underlies their pathophysiology. Deactivation slowing causes accumulation of open channels in the context of repeated stimulation, which abnormally increases the repolarizing K+ current, excessively shortens the action potential duration, and predisposes to re-entry arrhythmia such as atrial fibrillation. Although both mutations are located in the voltage-sensing domain, their mechanisms of action remain unknown. Understanding the gating mechanisms underlying deactivation slowing may provide key insights for the development of mechanism-based pharmacologic therapies for arrhythmias associated with KCNQ1 mutations. In addition to gain-of-function mutations, molecular activators of KCNQ1 can slow deactivation and increase channel activity. An existing problem in the pharmacologic treatment of arrhythmia is that many antiarrhythmic drugs do not have specific targets and cause undesired side effects such as additional arrhythmia. Thus, developing mechanism-based therapies may optimize clinical treatment for patients with specific forms of channel dysfunction. Two KCNQ1 activators, ML277 and R-L3, have been previously shown to slow current deactivation, but the underlying gating mechanisms remain known. Although these modulators are unlikely to serve directly as antiarrhythmic therapy, investigating their mechanisms will likely provide fundamental insights on channel modulation and guide future efforts to develop personalized therapies for arrhythmia, such as congenital long QT syndrome. Given the central importance of deactivation slowing in both pathophysiology and pharmacology, we focused on investigating gating mechanisms that underlie deactivation slowing. To this end, we utilized voltage clamp fluorometry, a technique that simultaneously assays for voltage sensor movement and ionic current through the channel pore. In Chapter 1, we begin our study by examining the gating mechanisms of KCNQ1 atrial fibrillation mutations in the absence of KCNE1. We show that S140G slows voltage sensor deactivation, which indirectly slows current deactivation. On the other hand, V141M neither slows voltage sensor nor current deactivation. This is followed by Chapter 2, where we examine the gating mechanisms underlying deactivation slowing by atrial fibrillation mutations in the presence of KCNE1. We show that both S140G and V141M slow IKs deactivation by slowing pore closing and altering voltage sensor-pore coupling. Based on these findings, we proposed a molecular mechanism in which both mutations disrupt the orientation of KCNE1 relative to KCNQ1 and thus impede pore closing, implying that future efforts to modulate KCNQ1 function can benefit from targeting the β-subunit. Finally, in Chapter 3, we explore the gating mechanisms underlying deactivation slowing for two small-molecule activators of KCNQ1. We show that ML277 predominantly slows pore transitions, whereas R-L3 slows voltage sensor deactivation, which indirectly slows current deactivation. Taken together, these studies guide future efforts to develop mechanism-based therapies for arrhythmia.

Heart rate changes mediate the embryotoxic effect of antiarrhythmic drugs in the chick embryo A significant increase in cardiovascular medication use during pregnancy has occurred in recent years but only limited evidence on its safety profile is available. We hypothesized that drug-induced bradycardia is the leading mechanism of developmental toxicity. We tested metoprolol, carvedilol, or ivabradine for embryotoxicity and their acute effect on chick embryonic model. We used video microscopy and ultrasound biomicroscopy. Significant dose-dependent mortality was achieved in embryos injected with carvedilol and ivabradine. In ED4 embryos, metoprolol, carvedilol and ivabradine reduced the heart rate by 33%, 27%, and 55%, respectively, compared to controls (6%). In ED8 embryos this effect was more pronounced with a heart rate reduction by 71%, 54%, 53%, respectively (controls 36%). Cardiac output decreased in all tested groups but only proved significant in the metoprolol group in ED8 embryos. The number of -adrenergic receptors showed a downward tendency during embryonic development but a negative chronotropic effect of tested drugs was increasingly pronounced with embryonic maturity. This effect was associated with reduced cardiac output in chick embryos, probably leading to premature death....