No description available.

No description available.

No description available.

Mitochondrial structure and function are inextricably linked ("structure-function"), with decrements in structure-function evident across diseases. Barriers to new therapies include a complete understanding of the underlying molecular culprits, as well as effective mitochondria-targeted therapies that mitigate injury. In these works, we investigate the role of cristae-shaping factors like cardiolipin in health and disease. In a series of studies, we tested the effects of the cell-permeable tetrapeptides, elamipretide and a postulated peptide, (arginine-tyrosine-lysine-phenylalanine; "RYKF"), on the recovery of mitochondrial structure-function after injury. Elamipretide is a clinical-stage compound currently under investigation for genetic and age-related mitochondrial diseases, yet the mechanism of action is not completely understood. We used a combination of physiological models, mitochondrial imaging, and biomimetic membrane studies to test the hypothesis that elamipretide and RYKF-cardiolipin interactions improved mitochondrial structure-function. Post-ischemic treatment with elamipretide sustained mitochondrial function across electron transport chain complexes. Endogenous RYKF expression similarly improved mitochondrial respiration after peroxide and hypoxia nutrient deprivation injuries. Using two parallel electron microscopy paradigms, we show elamipretide and RYKF treatment led to maintenance of mitochondrial ultrastructure and notably, improved cristae interconnectedness. Finally, we utilized a novel biomimetic membrane system to model the pathological mitochondrial membrane and found that elamipretide and RYKF both improved biophysical pressure-area relationships through a mechanism that appears to involve aggregating cardiolipin. Our data indicate that targeting pathophysiological mitochondrial membranes with cationic, lipophilic peptides can improve bioenergetics by sustaining cristae networks and support interdependent relationships between mitochondrial structure and function. / Doctor of Philosophy / Mitochondria, the powerhouses of the cell, form energy networks that produce over 90% of the body’s energy. Mitochondrial dysfunction is implicated across diseases, yet no FDA-approved treatments exist that improve mitochondrial energy production. In this study, we tested the effects of elamipretide, a peptide that localizes to mitochondria. Although elamipretide is currently in clinical trials for several diseases characterized by energetic deficiencies, its mechanism of action is not fully understood. Since mitochondrial structure and function are directly linked, we modeled heart attacks in cultured cells and rat hearts to test the hypothesis that elamipretide and a postulated analog, RYKF, glue damaged mitochondrial membranes back together to preserve structure and function during disease. In hearts subjected to a heart attack, elamipretide significantly protected mitochondrial energy production. Similarly, RYKF protected mitochondrial function in muscle cells exposed to peroxide stress. In damaged hearts imaged with electron microscopy, elamipretide and RYKF treatment significantly improved mitochondrial structure and notably, improved the interconnectedness of mitochondrial energy networks. Furthermore, elamipretide and RYKF improved the integrity of diseased mitochondrial membranes. Together, these data support our hypothesis that elamipretide and RYKF act as mitochondrial adhesion molecules to protect mitochondrial structure and sustain energy production during disease.

As part of the ongoing loss of global biodiversity, amphibian populations are experiencing declines and extinctions. A primary factor in these declines is the skin disease chytridiomycosis, which is caused by the fungus Batrachochytrium dendrobatidis (Bd). Recent research suggests that the amphibian skin microbiota has anti-Bd activity and may be an important factor in host disease resistance. However, little is known about the basic ecology of this host-microbe symbiosis, such as how much variation there is in microbial symbionts among host species and populations, and the nature of symbiont transmission, culturability, and function. My dissertation research addressed these basic questions in microbial ecology, as well as used a novel system to examine the long-standing ecological theory of community structure-function relationships. First, host-specificity, population-level variation and potential environmental transmission of the microbiota were examined by conducting a field survey of bacterial communities from bullfrogs, newts, pond water, and pond substrate at a single pond, and newts from multiple ponds. There was variation among amphibian host species and populations in their skin symbionts, and, in a host species-specific manner, amphibian skin may select for microbes that are generally in low abundance in the environment. Second, the culturability of amphibian skin bacteria was assessed by directly comparing culture-dependent and -independent bacterial sequences from the same individuals. Although less than 7% of the amphibian skin microbes were captured using R2A medium, most of the dominant bacteria were represented in our cultures, and similar patterns of diversity among four amphibian species were captured with both approaches. Third, the relationship between microbial community structure and function and selective forces shaping structure and function were examined in bullfrogs by tracking microbial community structure and function following experimental manipulation of the skin microbiota and pathogen exposure. Results of this study demonstrated that Bd is a selective force on cutaneous bacterial community structure and function, and suggest that beneficial states of bacterial structure and function may serve to limit infection and negative fitness consequences of Bd exposure. Using a combination of observational and experimental approaches, my dissertation contributes to understanding structure-function relationships of these complex symbiotic communities of vertebrates. / Ph. D.

NADPH-cytochrome P450 reductase is the electron transfer partner for the cytochromes P450, heme oxygenase, and squalene monooxygenase, and is a component of the nitric oxide synthases and methionine synthase reductase. P450 reductase shows very high selectivity for NADPH and uses NADH only poorly. Substitution of tryptophan 677 with alanine (W677A) has been shown by others to yield a 3-fold increase in turnover with NADH, but profound inhibition by NADP+ makes the enzyme unsuitable for in vivo applications. In the present study site-directed mutagenesis of amino acids in the 2'-phosphate-binding site of the NADPH domain, coupled with the W677A substitution, was used to generate a reductase that was able to use NADH efficiently in vivo without inhibition by NADP+. Of 11 single, double, and triple mutant proteins, two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold increase in catalytic efficiency (kcat/Km) with NADH. Inhibition by NADP+ was reduced by up to four orders of magnitude relative to the W677A protein and was equal to or less than that of the wild-type reductase. Both proteins were 2- to 3-fold more active than wild-type reductase with NADH in reconstitution assays with cytochrome P450 1A2 and with squalene monooxygenase. In a recombinant cytochrome P450 2E1 Ames bacterial mutagenicity assay the R597M/W677A protein increased the sensitivity to dimethylnitrosamine by approximately 2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay. In addition to providing a valuable tool for understanding the determinants of nucleotide cofactor specificity in this and related enzymes, these mutants might also lend themselves to creation of bioremediation schemes with increased enzymatic activity and robustness in situ, as well as cost-effective reconstitution of enzyme systems in vitro that do not require the use of expensive reducing equivalents from NADPH.

As optical designs become more sophisticated and incorporate aspheric and free form surfaces, the need to specify limits on mid-spatial frequency manufacturing errors becomes more critical, particularly as we better understand the effects of these errors on image quality. While there already exist methods based on Fourier analysis to specify these errors in most commercial interferometry software, the method of calculation and the power spectral density (PSD) results remain obscure to many in the optical design and manufacturing field. We suggest that the structure functions (SF) contains the same information as in the Fourier based PSD but in a way that is much more transparent to analysis, interpretation and application as a specification. The units of measure are more familiar and the concept behind the analysis is simpler to understand. Further, the information contained in the structure function (or PSD) allows a complete specification of an optical surface from the finest measurable detail of roughness to the overall figure. We discuss the origin of the structure function in the field of astronomy to describe the effects of air turbulence on image quality, the simple mathematical definition of the structure function and its easy means of calculation and how its results should be scaled depending on the location of the optical surface in a system from pupil to image plane. Finally, we give an example of how to write a specification of an optical surface using the structure function.

KirBac channels are prokaryotic homologs of eukaryotic inwardly-rectifying potassium channels, which have served as models for gaining insight into the structure of eukaryotic channels. This thesis focuses on the structure-function relationship in these channels. The first part of this study concerns a novel KirBac channel, KirBac9.2, which contains a unique amino acid sequence in the place of the canonical GYG selectivity filter. Although expressed and purified in a stable and functional form, the protein did not form well-diffracting crystals. Functional studies suggest that KirBac9.2 is non-selective for monovalent cations and a random mutagenesis screen identified a number of activatory mutants in the cytoplasmic domains of the channel. A full electrophysiological investigation of KirBac9.2 channel function is beyond the scope of this study. However, initial studies suggest that it is possible to record currents from KirBac9.2 channels reconstituted into lipid bilayers. The second part of this thesis investigates KirBac3.1, which is a classical KirBac channel containing the consensus GYG sequence for potassium selectivity. Five high resolution structures of a mutant channel are reported, which suggest a new feature in the gating mechanism of KirBac3.1 where a rotation of the cytoplasmic domains is linked to a change in the electrostatic environment of the cytoplasmic cavity. In addition, a functional study of the KirBac3.1 showed that the channel is highly pH sensitive.

Transient receptor potential vanilloid 3 receptor channel (TRPV3) is a thermosensitive ion channel expressed in skin keratinocytes. There, in a molecular complex with the epidermal growth factor receptor (EGFR) contributes to proliferation and terminal differentiation of keratinocytes, temperature detection, pain and pruritus. TRPV3 is activated by a number of exogenous compounds, such as carvacrol from oregano, thymol from thyme and eugenol from clove. Its unique feature is sensitization, TRPV3 channel activity successively increases upon repeated stimulation. The molecular basis of this process is not yet understood. One of the considered possibility is a direct phosphorylation of TRPV3 protein through signaling pathways involving EGFR and mitogen-activated protein kinase MAPK1 / MAPK3 (also called ERK2 / ERK1). In this thesis we investigated whether sensitization of TRPV3 which is expressed in a human cell line immortalized keratinocytes could be influenced by mutations on the predicted consensual phosphorylation sites for MAPK1 / MAPK3. We used electrophysiological patch-clamp technique and tested eight mutants, in which was threonine or serine replaced with aspartic acid mimicking phosphorylation. We identified six residues where the mutations influenced at least one of the functional...

L'électronique organique représente une alternative réaliste aux technologies conventionnelles à base de silicium par le design, la synthèse et la mise en oeuvre des matériaux organiques fonctionnels dans des dispositifs légers et flexibles. Les matériaux organiques, tels que les petites molécules ou les polymères organiques, sont avantageux pour leur faible coût, leur flexibilité et leur facilité de traitement. Grâce aux avantages liés à l'utilisation de matériaux organiques, en termes économiques et de gain de temps, l'électronique organique est devenue un domaine innovant qui s'applique aux technologies de l'énergie, de l'environnement, de la santé, de l'information et de la communication.L'électronique organique est issue de la découverte de polymères dotés de fonctionnalités semi-conductrices. Cependant, il ne faut pas négliger une autre classe de polymères exceptionnels, les polymères ferroélectriques. La nature électroactive des polymères ferroélectriques, qui sont également pyroélectriques et piézoélectriques, combinés aux avantages intrinsèques des polymères, les a désignés comme éléments constitutifs d’une gamme étendue de dispositifs électroniques organiques.La famille de polymères ferroélectriques la plus connue est celle du poly(fluorure de vinylidène) P(VDF) et de son copolymère avec le trifluoroéthylène, P(VDF-co-TrFE). La récupération d'énergie, le stockage et la détection de données, principales applications de l'électronique organique, peuvent potentiellement tous être réalisés avec ces matériaux fonctionnels exceptionnels. La ferroélectricité étant une propriété dépendant de la structure, il est indispensable de mieux comprendre les relations réciproques entre la structure et les propriétés ferroélectriques finales afin d'améliorer les applications existantes des polymères ferroélectriques en électronique organique et de promouvoir l'introduction du P(VDF-co-TrFE) dans de nouvelles applications.P(VDF-co-TrFE) en tant que polymère semi-cristallin possède des propriétés cristallines sensibles au traitement thermique. Puisque seules les régions cristallines contribuent a le commutation électronique de la polarisation et non les amorphes, le degré de cristallinité est un facteur clé pour moduler les propriétés ferroélectriques. En autre, l'orientation des cristallites ainsi que la présence de défauts dans les cristallites sont des paramètres cruciaux qui jouent un rôle important dans la définition des performances finales des dispositifs dans lesquels P(VDF-co-TrFE) est incorporé. Tel est l'objectif de cette thèse: atteindre une compréhension exhaustive des relations traitement-structure-fonction qui serviront d'outil pour moduler les performances des dispositifs ferroélectriques.De plus, les applications potentielles de P(VDF-co-TrFE) en électronique organique sont explorées en examinant sa mise en oeuvre dans: (1) des capteurs médicaux à cathéter piézoélectrique destinés à mesurer la fonction cardiaque et éventuellement à détecter maladies cardiaques et (2) dispositifs électroniques dans lesquels P(VDF-co-TrFE) est mélangé avec le polymère poly(3-hexylthiophène) semi-conducteur, P3HT. Ce dernier a déjà été appliqué dans les diodes à mémoire ferroélectrique non volatile et l’utilisation potentielle dans le champ de l’organique photovoltaïque est explorée. / Organic electronics represent a realistic alternative to conventional silicon-based technologies through the design, synthesis and implementation of functional organic materials into light and flexible devices. Organic materials, such as small molecules or organic polymers, are advantageous for their low-cost, flexibility and easy processing. Thanks to the economical and timesaving advantages, organic electronics have emerged as an innovative field with application in energy, environment, health, information and communication technologies.Organic electronics originates from the discovery of polymers with semiconducting functionalities. However, one should not neglect another class of outstanding polymers, the ferroelectric polymers. The electroactive nature of ferroelectric polymers, which are also pyroelectric and piezoelectric, combined with the intrinsic advantages of polymers have designated them as constituent elements of a widespread range of organic electronic devices. The most well-known family of ferroelectric polymers is that of poly(vinylidene fluoride), P(VDF), and its copolymers with trifluoroethylene, P(VDF-co-TrFE). Energy harvesting, data storage and sensing, main applications of organic electronics, can potentially all be realised using these exceptional functional materials.Since ferroelectricity is a structure-dependent property an insight into the interrelations between structure and final ferroelectric properties is indispensable in order to improve existing applications of ferroelectric polymers in organic electronics and to promote the introduction of P(VDF-co-TrFE) in new application fields. P(VDF-co-TrFE) as semi-crystalline polymer possess crystalline properties which are sensitive to thermal treatment. Since only the crystalline regions contribute to ferroelectric switching and not the amorphous ones, the degree of crystallinity is a key factor to modulate the ferroelectric properties. Moreover, crystallites orientation as well as the presence of defects within the crystallites are crucial parameters playing an important role in defining the final performance of the devices in which P(VDF-co-TrFE) is incorporated.Herein stands the aim of this thesis: reach an exhaustive understanding of processing-structure-function relationships that will serve as tool to modulate ferroelectric devices performances.Going one step further, the potential applications of P(VDF-co-TrFE) in organic electronics are explored by investigating it in: (1) medical piezoelectric catheter sensors for measuring cardiac function and eventually for detecting cardiac disease and (2) electronic devices in which P(VDF-co-TrFE) is blended with the semiconducting polymer poly(3-hexylthiophene), P3HT. The latter has already been applied in non-volatile ferroelectric memory diodes and the potential use in organic photovoltaics is explored.