Browsing by Subject "Mitochondria"

Now showing 1 - 14 of 14
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    Adipocyte protein carbonylation and oxidative stress in obesity-linked mitochondrial dysfunction and insulin resistance.
    (2011-09) Curtis, Jessica Marie
    Carbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. The antioxidant enzyme glutathione S-transferase A4 (GSTA4) catalyzes a major detoxification pathway for such reactive lipids but its expression was selectively down regulated in the obese, insulin resistant adipocyte resulting in increased protein carbonylation. The effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. Mitochondrial functions in adipocytes of lean or obese GSTA4 null mice were significantly compromised compared to wild type controls and were accompanied by an increase in superoxide anion. Silencing GSTA4 mRNA in cultured adipocytes resulted in increased protein carbonylation, increased mitochondrial ROS, dysfunctional state 3 respiration and altered glucose transport and lipolysis. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4- silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1 alpha subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production and a reduction in membrane potential without changes in mitochondrial number, area or density. These results suggest protein carbonylation plays a major instigating role in mitochondrial dysfunction and may be a linked to the development of insulin resistance in the adipocyte.
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    Biochemical and functional characterization of fatty acid transport proteins.
    (2009-07) Wiczer, Brian Michael
    The adipocyte fatty acid transport proteins (FATPs), FATP1 and FATP4, have been implicated in both lipid influx and storage and understanding their role in adipose tissue would gain insight into the persistence of metabolic disorders, such as type 2 diabetes. FATP1 was previously determined to be an acyl-CoA synthetase and work described in this thesis additionally explored the acyl-CoA synthetase activity of purified FATP4. FATP4 was found to be a more robust acyl-CoA synthetase than FATP1. Through the use of RNAi in cultured adipocytes, silencing the expression of either FATP1 or FATP4 results in cellular phenotype demonstrating improved insulin responsiveness. Interestingly, silencing FATP1 abolished insulin-stimulated long-chain fatty acid (LCFA) influx, whereas silencing FATP4 had no effect on LCFA influx despite its higher activity. Furthermore, the expression of FATP1 was demonstrated to be important for the activation of the AMP-activated protein kinase during insulinstimulated LCFA influx. In addition to the cytoplasmic localization of FATP1, it was also found to exhibit mitochondrial localization. Further analysis demonstrated a novel role in the regulation of TCA cycle function and mitochondrial energy metabolism, in part, through the interaction of FATP1 with the 2-oxoglutarate dehydrogenase complex, a rate-limiting step in the TCA cycle. This work shines light on how FATPs may play broader roles in metabolism that previously appreciated and the potential implications associated with such roles.
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    Biosynthetic and Energetic Lethality of Targeting Metabolic Plasticity for Cancer Treatment
    (2020-08) Ronayne, Conor
    Solid tumors are composed of numerous heterogeneous tissue types with a diverse molecular pathology. Uncontrolled replication and division, along with nutrient and oxygen gradients across the tumor, dictate dynamic intratumoral phenotypes that are reinforced by molecular hallmarks of cancer; largely shaping modern clinical treatment regimens. Importantly, deregulated energetics and reprogrammed tumor metabolism enable constitutive growth in challenging microenvironments. The ability of malignant cells to switch between numerous metabolic phenotypes (metabolic plasticity) allows for the generation of energy, appropriation of biosynthetic building blocks, and control of redox equilibrium. Hence, therapeutic targeting of metabolic plasticity with small molecules holds promise as a novel and enduring therapeutic strategy. In this regard, the current thesis work describes efforts toward developing novel small molecule mitochondrial pyruvate carrier inhibitors to induce bioenergetic and synthetic lethality in cancer cells.
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    Defining AMD disease mechanisms: a comparative analysis of proteins and mitochondrial DNA
    (2010-08) Karunadharma, Pabalu Pussellage Ranmini
    Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in the developed world. Current treatments are limited due to our inadequate understanding of the pathogenic events leading to AMD. Early clinical symptoms occur in the retinal pigment epithelium (RPE), suggesting RPE as the potential site of defect in AMD. This research evaluated the RPE proteome and mitochondrial DNA (mtDNA) to test the hypothesis that molecular changes in the RPE contributes to AMD. Human donor eyes categorized into four progressive stages of AMD were utilized in these investigations. Two proteomic analyses using 2D gel electrophoresis and mass spectrometry were performed to define changes in the RPE proteome. In the first proteomic study, analysis of the mitochondrial proteome revealed significant changes that suggested potential damage to mtDNA with AMD. These results prompted an analysis of mtDNA lesions associated with aging and AMD. These results suggest a potential link between mt dysfunction due to increased mtDNA damage and altered proteins and AMD pathology. In the second study, we tested the hypothesis that mt dysfunction is communicated to the nucleus via retrograde signaling and consequently alters the protein profile to reflect a major shift in metabolism and stress response. Our results suggest not only adjusted metabolism, response to stress and cellular redox regulation but also show major differences in the protein profile with AMD compared to aging. In summary, our investigations distinguished between normal and pathologic aging by identifying key macromolecules and pathways affected with each process. Furthermore, our data indicate a potential link between mitochondrial dysfunction and AMD pathology, thus providing a point of intervention for the treatment of AMD.
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    Distinct functions of autophagy kinases ULK1 and ULK2 in adipogenesis and adipocyte metabolism
    (2011-11) Ro, Seung-Hyun
    Autophagy, the catabolic process through which intracellular constituents are degraded in the lysosome under nutrient starvation or stress, has gained growing attention in the field of diabetes and obesity (Goldman S 2010; Ost A 2010; Beau I 2011; Kovsan J 2011). Despite the fundamental cellular function of autophagy in maintaining cellular energy homeostasis and survival under nutrient– or energy– deprived conditions and stress, the role of adipose autophagy in metabolism and metabolic diseases remains largely unknown. The goal of my study has been to better understand the function of autophagy in adipogenesis and in the regulation of adipocyte metabolism. My study has been focused on defining the role of ULK1 (Unc–51 like kinase 1, mammalian homolog of Atg1, hATG1) and its homologue ULK2 in the regulation of adipogenesis, metabolism and mitochondrial functions in adipocytes. ULK1 and ULK2 are key regulators of autophagy induction in mammalian cells (Kundu M 2009; Chang YY 2009; Ganley IG 2009; Hosokawa N 2009; Jung CH 2009). Knockdown of ULK1 or ULK2 inhibited autophagy in 3T3–L1 adipocytes, suggesting that they play important roles in autophagy in adipocytes. The knockdown experiment also revealed that ULK1 and ULK2 share key functions in lipolysis, mitochondrial respiration and protection of cells against oxidative stress. Despite these shared functions, their knockdown had different or even opposing effects on several metabolic parameters. Knockdown of ULK1 raised PPAR–γ level, facilitated differentiation of 3T3–L1 cells, increased the levels of GLUT4, insulin receptorβ(IRβ) and insulin receptor substrate–1 (IRS–1), and insulin–stimulated glucose uptake, and reduced fatty acid oxidation. By contrast, knockdown of ULK2 had opposite or no significant effects on these parameters. Through knocking down both ULK1 and ULK2, we found that ULK2 has a dominant effect over ULK1 in the regulation of adipogenesis. These results demonstrate that ULK1 and ULK2 have distinct functions in the regulation of adipogenesis and adipocyte metabolism, and that ULK2–dependent autophagy appears to be important for adipogenesis.
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    Distinct Physiological Mitochondrial Ca2+ Handling And Responses To Mitochondrial Ca2+ Overload Between The Left And Right Ventricles
    (2023-08) Sung, Jae Hwi
    Mitochondria are essential organelles in eukaryotic cells because they play critical roles in metabolism, intracellular ROS signaling, and cell fate decision-making. In energy-demanding organs including the heart, the role of mitochondria in energy metabolism is even more essential. To modulate mitochondrial function, Ca2+ acts as an important signaling molecule. Mitochondrial Ca2+ (mtCa2+) levels are regulated by various influx and efflux transporters such as the mitochondrial calcium uniporter complex (mtCU), the major mtCa2+ uptake pathway, and mitochondrial Na+/Ca2+/Li+ exchanger (NCLX), a major mtCa2+ efflux pathway. The mtCU is composed of the pore component MCU and regulatory components such as Mitochondrial Calcium Uptake 1-3 (MICU1-3) and Essential MCU Regulator (EMRE). MICU1 senses low cytosolic Ca2+ levels and inhibits mtCU activity as a gatekeeper. Many studies have shown that Micu1 deletion increases basal mtCa2+. EMRE is essential for MCU-mediated mtCa2+ uptake in metazoans via stabilization of the mtCU pore. Different stoichiometry of mtCU components and NCLX protein levels shape unique mtCa2+ kinetics among different types of tissues. In the heart, despite their differences in metabolism, mechanics, response to pathological stresses, and developmental origin, the right ventricle (RV) is often assumed to function similarly to the left ventricle (LV). Notably, though Ca2+ transport into mitochondria is well established to play a crucial role in matching cardiac energy production to demand and dysregulation of Ca2+ transport may contribute to fatal cardiac diseases including heart failure (HF), little is known regarding whether physiological and pathological mtCa2+ handling is distinct in the LV and RV. In addition, previous data from the lab reported that germline Micu1 deletion in mice led to mtCa2+ overload, a condition associated with multiple diseases including heart failure. At least in liver mitochondria, EMRE downregulation was reported to partially restore mtCa2+ homeostasis in MICU1 loss-mediated mtCa2+ overload, suggesting that EMRE downregulation can function as a time-dependent adaptation mechanism. However, in the heart, the adaptation mechanism to mtCa2+ overload has not been explored, and prior observation of more dramatic impairment in the RV than in the LV of mice with Micu1 deletion suggested ventricular differences that also have not been studied.To uncover whether the characteristics of mtCa2+ handling are different between the LV and RV in physiological and pathological conditions, firstly, I compared an array of parameters indicative of mtCa2+ dynamics and mitochondrial functions regulated by Ca2+ using isolated mitochondria from LV and RV free wall tissues in wild-type mice and healthy pigs. Here, I found that basal mtCa2+ levels were higher in RV mitochondria than in LV mitochondria. When successive boluses of Ca2+ were administered to isolated mitochondria, RV mitochondria took up fewer Ca2+ boluses, showing lower Ca2+ retention capacity. RV mitochondria displayed relatively more protein carbonylation, suggesting oxidative stress. Interestingly, ATP production rate was higher in RV mitochondria relative to LV mitochondria; however, only LV mitochondria exhibited an increase in ATP production rate in the presence of Ca2+. I also compared the protein expression of the subunits of the mtCU and the NCLX and found that levels of EMRE and NCLX were higher in the LV than the RV, potentially facilitating more dynamic Ca2+ transport in and out of LV mitochondria. Collectively, I found that mtCa2+ is calibrated to higher but more static levels in the RV, whereas in the LV basal mtCa2+ is lower to ensure dynamic range for physiological stimuli to increase mitochondrial bioenergetics, corresponding to the larger changes in workload experienced by the LV. Next, I compared pathological responses to mtCa2+ overload between the LV and RV as well as the possibility of time-dependent adaptation to mtCa2+ overload through downregulation of EMRE. To induce mtCa2+ overload in the heart, we generated tamoxifen (tmx)-inducible cardiac-specific Micu1 knockout mice (Micu1cKO). Here, I found by echocardiogram that LV function was reduced at 4 weeks post-tmx but was partially improved by 6 weeks post-tmx. However, RV function declined from 4 to 6 weeks post-tmx, without evidence of pulmonary arterial hypertension. To understand the differences in LV and RV response to mtCa2+ overload, we confirmed that loss of Micu1 at 1 week post-tmx resulted in mtCa2+ overload to a similar extent in both LV and RV mitochondria. Interestingly, at 7-9 weeks post-tmx, mtCa2+ and oxidative stress remained elevated in the RV mitochondria but returned to control levels in the LV mitochondria. Concurrently, EMRE protein level and mtCa2+ uptake rate were reduced in Micu1-deficient LV but not in the RV. Interestingly, in the LV, the activity of m-AAA proteases, which are known to degrade EMRE, was higher and levels of p-PKA, which can phosphorylate an m-AAA protease, were lower. Furthermore, using neonatal cardiomyocytes, I reproduced the LV-specific adaptation through EMRE downregulation in response to MICU1 loss-mediated mtCa2+ overload by using a H89, p-PKA inhibitor to augment m-AAA protease activity. Lastly, protein expression in human dilated cardiomyopathy (DCM) LV tissues suggest that a similar adaptation mechanism may occur in response to potential mtCa2+ overload induced by DCM. In summary, mtCa2+ overload results in a more pronounced impairment over time in the RV than the LV, due to compensatory EMRE reduction via increased m-AAA protease activity in the LV that is absent in the RV. To conclude, my dissertation research uncovered important differences between LV and RV cardiac mitochondria, showing that the LV maintains lower basal mtCa2+ levels to enable dynamic range in responsiveness to stimulation and that the RV is more susceptible to the mtCa2+ overload due to lack of a LV-specific adaptive response. Therefore, my studies provide new understanding of distinct ventricle-specific behaviors of LV and RV mitochondria in physiological and pathological conditions and establish a strong rationale to develop ventricular specific therapy targeting mitochondrial dysfunction.
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    Engineering mammalian mitochondrial genomes
    (2012-01) Yang, Yi-Wei
    Mutations in mitochondrial genomes can cause dysfunction of mitochondria and disease. Studying pathogenic mutations in mitochondrial genomes is difficult due to the lack of suitable tools for engineering mitochondrial genomes in mammalian cells. This project aims to solve some of the technical hurdles that will allow direct manipulation of mammalian mitochondrial genomes and characterization of the biological consequences of specific sequence changes in mammalian mitochondrial genomes in vivo. Mouse mitochondrial genomes were cloned and stably maintained in E. coli at low copy number. Using standard techniques of molecular biology, one full and three deleted mouse mitochondrial genome clones carrying a selection marker, γ-ori and yeast COX2 gene flanked by duplicated sequences were generated. These mouse mitochondrial genome clones were stably maintained as circular monomers being transformed into yeast mitochondria. The exogenous sequences used for cloning and screening were removed by homologous recombination in yeast mitochondria. Yeast Artificial Mitochondria (YAM) strains were generated that are yeast cells carrying only engineered mouse mitochondrial genomes in their mitochondria. In order to evaluate the biological consequences of engineered mouse mitochondrial genomes in mouse cells, an artificial cytoplast fusion method was developed for introducing isolated mitochondria into mouse tissue culture cells. This is the first method that can deliver large quantities of isolated mitochondria into mammalian tissue culture cells. The respiratory deficiency phenotype in the recipient cells was rescued by the introduced mouse mitochondria, indicating the procedure can preserve the biological activities of isolated mitochondria. Isolated YAM were introduced into mouse tissue culture cells by the artificial cytoplast cell fusion method. Interestingly, as engineered mouse mitochondrial genomes were actively replicated in yeast, these genomes did not replicate efficiently enough to be maintained in mouse cells in long-term culture. A competition between the rat mitochondrial genomes co-introduced from the artificial cytoplasts (generated from rat oocytes) and the mouse mitochondrial genomes carried in YAM could be the cause. In order to test the hypothesis that the mouse mitochondrial genomes could replicated more efficiently if the mitochondrial genomes from rat oocytes were eliminated from the same mouse cells, artificial heteroplasmic mouse cell lines were generated. By expressing mitochondrial-targeted XhoI endonuclease, the mitochondrial genomes from rat oocytes that contained a single XhoI site were selectively removed from the mouse cells whereas genomes without an XhoI site continued to replicate. The effect of expressing mitochondrial-targeted XhoI endonuclease on XhoI-negative engineered mouse mitochondrial genomes in mouse cells will be investigated in the future.
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    Gene Expression Within the Fluctuating Life Cycle Stages of Trypanosome Parasites
    (2019-05) Susa, Emily
    Trypanosoma cruzi and Trypanosoma brucei are vector-borne protozoan parasites that cause devastating disease to humans and livestock in South America, North America and Africa. Both parasites have complex life cycles which involve a variety of different environments and nutrient sources within mammalian hosts and arthropod vectors. Transitioning between life cycle stages requires a transformation of morphology, replicative ability, and metabolism, which requires remodeling of mitochondrial and nuclear gene expression. To better understand the complicated genetic factors involved in life stage differentiation in these organisms, we have investigated several aspects of the regulation of gene expression through life stage transitions. In T. brucei, we investigated the role of a putative endoribonuclease, EEP1, on the transition from the mammalian life stage to the insect life stage. Our results indicate that EEP1 does not play a role in the differentiation process, and may serve an entirely unique function. In T. cruzi, we examined the mitochondrial genome which plays a crucial role in metabolism and has been shown to exhibit life-stage specific remodeling in related species. Mitochondrial genome regulation must occur post-transcriptionally in the form of RNA editing, translational control, and stability. Significant changes were detected in mature mRNA abundance between several life stages, and these differences appeared to be correlated with nutrient availability and replication status. Overall, both of these studies provide further understanding of the regulatory processes that govern life cycle transitions in trypanosome parasites.
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    The importance of calcium cycling and mitochondria in the local onset of alternans in the heart
    (2014-05) Visweswaran, Ramjay
    Action potential duration (APD) alternans can be accompanied by alternans in intracellular calcium, leading to electromechanical alternans. Electromechanical alternans is considered a substrate for ventricular fibrillation, especially during pathophysiological conditions such as ischemia. The work in thesis seeks to elucidate the spatio-temporal evolution of alternans and to investigate the potential pathways through which they occur. High resolution mapping was used to simultaneously map membrane voltage and intracellular calcium in normal rabbit hearts. By mapping both parameters simultaneously in the same region of the heart, we were able to reveal that instability in calcium cycling plays a primary role in the development of EM alternans in the whole heart. Further, we were able to apply a special restitution portrait analysis to predict the onset of both calcium and APD alternans before it occurs. We also wanted to elucidate the mechanisms behind the increased incidences of arrhythmias during ischemia. By simulating ischemic and mitochondrial dysfunction in isolated rabbit hearts, we were able to show that mitochondrial stress caused by uncoupling of the mitochondria is responsible for early occurrence of both APD and calcium alternans in the heart, which in turn creates a substrate to ventricular arrhythmias. Thus, uncoupling of the mitochondrial network that occurs during ischemia might be the primary reason for increased incidences of arrhythmias in the heart during ischemia. Overall, this study improves our knowledge of alternans and their basic underlying mechanism which can be used in the development of better treatment and/or prevention strategies. Development of techniques to predict alternans before it occurs would be a valuable clinical tool, especially for use in implantable pacemakers paving the way for pre-emptive interventions. In addition, elucidating the mechanism or pathways of alternans formation would lead to targeted drug treatments to prevent alternans and thus, VF and sudden cardiac death.
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    Membrane Stabilizing Copolymer Poloxamer 188 in Preclinical Models of Acute Myocardial Infarction Reperfusion
    (2019-11) Chandra Shekar, Kadambari
    Coronary artery disease is the most common disease of the heart and number one cause of death worldwide, affecting millions annually. Acute myocardial infarction (AMI) is a classic manifestation of coronary artery disease and occurs when prolonged myocardial ischemia reaches a critical threshold, partially or completely occluding the coronary arteries, leading to necrosis of the adjacent tissue and subsequent scar formation. Despite recent advances in treatment strategies, risk of death by secondary cardiac events like hemorrhagic shock and cardiac arrest remain very high. Cellular injury after acute myocardial infarction occurs in two stages- ischemic injury, which occurs when there is a myocardial oxygen supply-demand mismatch, and reperfusion injury, which occurs with the sudden unrestrained return of blood to the oxygen deprived tissue. Some of the strategies to minimize reperfusion injury including ischemic pre-and post-conditioning, and therapeutic hypothermia have been successful in animal studies but have exhibited mixed results in clinical trials. Several cellular events that occur in ischemia including calcium overload and reactive oxygen species (ROS) generation, inflammation and myocardial contracture are further exacerbated during reperfusion injury. Mitochondria play a crucial role in augmenting the events of reperfusion injury by further increasing calcium overload, ROS induced ROS release and opening of the mitochondrial permeability transition pore, all of which triggers cell death pathways. Hence, there’s an urgent need for therapies that prevent mitochondrial dysfunction and mitigate reperfusion injury. Poloxamer 188 (P188) is the most studied member of the poloxamer family, comprised of non-ionic polymers made of a hydrophobic core, flanked by hydrophilic end chains. Due to its membrane stabilizing and anti-coagulant properties, P188 remains a favorable agent to prevent membrane damage in several disease models including sickle cell anemia, muscular dystrophy, cardiac arrest and acute myocardial infarction. The predominant theme of this dissertation revolves around understanding the events that occur at reperfusion after acute myocardial infarction and finding ways to combat this reperfusion injury with the use of P188. Here, we demonstrate for the first time the benefit of P188 administration in salvaging myocardial and mitochondrial function using a large animal model of AMI with current treatment procedures. We further investigate if this improvement in mitochondrial function transcends to the level of the two distinct mitochondrial subtypes in the heart. Finally, with the results from these studies, we examine if there’s a survival benefit with our model of AMI reperfusion. Results from this study will provide a better understanding of the events surrounding reperfusion injury within the distinctive subpopulations of mitochondria and underscore the immediate and long-lasting benefits of administering P188 promptly at reperfusion.
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    A Role for βcyto- and γcyto Actin at the Sarco/endoplasmic Reticulum-Mitochondrial Interface
    (2017-05) O'Rourke, Allison
    Skeletal muscle accounts for 40% of mass in adult humans. The primary function of skeletal muscle is to produce movement via contraction of the sarcomere. The sarcomere is the basic functional until of skeletal muscle and two of the key components in sarcomeric contraction are myosin and actin. Actin, though often referred to as a single entity, separates into two functional groups muscle (αsk-actin, αca-actin, αsm- and γsm-actin) and cytoplasmic (βcyto- or γcyto actin) actins. Though αsk-actin is the predominate isoform in adult skeletal muscle, specifically in the sarcomere, the ubiquitously expressed βcyto- and γcyto actin cytoplasmic actin isoforms are also present. As may be predicted by their ubiquitous expression patterns, βcyto- and γcyto actin, are enmeshed in a wide array of cellular functions including, migration, cell shape, cell division, vesicle trafficking, and organelle anchoring among others. Though collectively involved in many processes the unique and redundant role of both βcyto- and γcyto actin are not fully delineated. βcyto- and γcyto actin are highly conserved, with 99% homology of the coding sequence. The evolutionary pressure which has maintained both sequences suggests that they are both are necessary. The importance of having both βcyto- and γcyto actin was demonstrated through whole body knockouts. Whole body ablation of either βcyto- or γcyto actin proved lethal to the majority of animals during embryogenesis or within 24 hours after birth. The severity of the whole body phenotype while one cytoplasmic actin is still present illustrates the vital role each isoform has. The lethal nature of whole body knockouts of either cytoplasmic actin isoform made studies into tissue specific phenotypes difficult. One tissue of interest was skeletal muscle, because of the role cytoplasmic actins have in linking the sarcomere to the extracellular matrix. Previously, insight was gained into the functional significance of βcyto- and γcyto actin in skeletal muscle via muscle specific knockouts. Each knockout revealed the presence of a mild myopathy associated with muscle death and regeneration which worsened overtime. The focus of my thesis is to better understand how muscle specific ablation of either βcyto- or γcyto actin resulted in a progressive mild myopathy. Since, the link between either βcyto- and γcyto actin and the observed myopathy wasn’t readily apparent I investigated the known interactions of cytoplasmic actins in skeletal muscle. It was previously reported that cytoplasmic actin colocalizes with a host of structures in skeletal muscle including two which could affect cell viability: mitochondria and peri-z-disk region where the sarcoplasmic reticulum also localizes. My thesis advances understanding for how ablation of cytoplasmic actin isoforms leads to a mild myopathy. Firstly, I showed that both βcyto- and γcyto actin isoforms were present at the interface between mitochondria and sarcoplasmic reticulum. Secondly, I showed that βcyto- and γcyto actin knockout skeletal muscle had perturbations in sarcoplasmic reticulum and mitochondria morphology. Additionally, I showed that both cytoplasmic actin isoforms contribute to mitochondrial fission. Finally, I demonstrated that in skeletal muscle lacking βcyto- and γcyto actin, changes indicative of decreased sarcoplasmic reticulum function preceded the observed morphological changes. Though cytoplasmic actin isoforms have been localized to an array of structures in skeletal muscle, we chose to focus on two organelles which can affect cell viability. Further investigation into the source of the observed progressive myopathy revealed morphological aberrations in both the mitochondria and sarcoplasmic reticulum. The stable nature of skeletal muscle structure hindered further examination of the observed mitochondrial morphological phenotype so, we utilized a mouse embryonic fibroblast model. In mouse embryonic fibroblasts lacking either or both cytoplasmic actin isoforms, mitochondria are elongated and display a decrease in fission events. As mitochondria are a central regulator of cell health and because of the effect we observed βcyto- and γcyto actin have on mitochondrial dynamics, we initially hypothesized that mitochondria would be mildly functionally impaired. However, upon investigation of function it was not mitochondrial function which was impaired, but that an indicator of sarcoplasmic reticulum function was decreased. In sarcoplasmic reticulum a functional phenotype preceded the development of morphological changes supporting a potential role for sarcoplasmic reticulum dysfunction to act as a possible stressor linked to the observed mild progressive myopathy.
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    The Role of Lipocalin-2 in Exercise Induced Mitochondrial Metabolism in Thermogenic Adipose Tissue
    (2019-08) chohan, shaila
    Obesity is a major health issue faced by the present era. According to the CDC, obesity is not just a weight-gain problem; it can have serious deleterious effects on an individual’s physical, metabolic and psychological health. Dysfunctional adipose tissue is the major contributor to obesity and its associated metabolic syndrome. Brown adipose tissue (BAT) is a major thermogenic organ that regulates energy expenditure and is a potential target of drugs for combating obesity and type 2 diabetes. BAT is involved in non-shivering thermogenesis and is a site of glucose uptake and lipid oxidation. The mitochondrion plays a critical role in energy metabolism and it is a dynamic organelle that needs quality control while activated during thermogenesis. Exercise is a useful tool to activate brown adipose tissue. It helps stimulate the sympathetic nervous system to activate the mitochondrial oxidation of brown adipose tissue. When exercise activates adipose tissue, mitochondrial turnover is increased in BAT but decreased in beige adipose tissue. Lipocalin-2 is an adipokine that has a role in energy metabolism via activating BAT and white adipose tissue beiging. Our previous studies have demonstrated that Lcn-2 plays a key role in cold-induced thermogenesis. Herein, we sought to discover if Lcn-2 plays a role in exercise-induced thermogenesis and mitochondrial metabolism. We used a Lcn-2 KO mouse model to investigate the expression of thermogenic and mitochondrial genes. We showed that the expression levels of Ucp-1 were not significantly changed in BAT of Lcn-2 KO mice, i.e. Ucp-1 expression can be induced by exercise when Lcn-2 is not present. We also determined if Lcn-2 had any effects on mitophagy involving PINK-1/Parkin/P-62 system in BAT and iWAT in response to exercise. Interestingly, we found that Lcn-2 deficiency does seem to affect mitochondrial quality control as Lcn-2 KO does not show exercise-induced mitochondrial biogenesis but shows increased mitophagy in white adipose tissue, indicating that the beiging process is defective. We conclude that Lcn2 plays an important role in exercise-induced mitochondrial turnover and metabolism in brown and beige adipose tissue.
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    Timing, growth and homeostasis: an anthology of three novel players in Drosophila melanogaster
    (2013-08) Ghosh, Arpan
    Developmental timing, growth and homeostasis form the cornerstones that shape the genesis and subsequent maintenance of a healthy adult for all multicellular organisms. Understanding the mechanisms that regulate these processes has important implications both from the perspective of our understanding of basic biology and also for our understanding of complex biological disorders involving these processes. My work explores the mechanisms regulating developmental timing, homeostasis and growth using the model organism Drosophila melanogaster. In this thesis I report involvement of three novel players in the regulation of timing, homeostasis and growth in the Drosophila larvae.Firstly, my work unearths the mechanism by which the developmental gap gene, giant, regulates developmental timing in Drosophila larvae. While the effect of Giant (Gt) on larval developmental timing was long known, the mechanism by which Gt exerted this effect was not known. I find that Gt affects developmental timing by influencing the developmental fate of the prothoracicotropic hormone producing PG neurons that are essential for determining the timing of larval developmental transitions. Additionally, I show that Gt is required for PG neuron axon targeting. Secondly, I show that TGF-beta/Activin signaling mediated by the Activin-like ligand Dawdle (Daw) regulates sugar homeostasis, pH balance and mitochondrial metabolism in Drosophila larvae. Canonical signaling by Daw regulates sugar homeostasis primarily by affecting release of insulin in the larvae. The effect of Daw on pH is mediated independently by Daw's action on mitochondrial metabolism and production of metabolic acids. Interestingly, Daw affects both phenotypes in a dose-dependent manner, as demonstrated by both loss-of-function and over-expression/gain-of-function experiments, thereby providing evidence for a hormonal role of Daw in regulating systemic homeostasis. Lastly, I show that eukaryotic uracil salvaging enzyme uracil phospho-ribosyltransferase (UPRT), that was considered inactive in higher eukaryotes including Drosophila, is active in the Drosophila larvae. The Drosophila UPRT homologue, Krishah, can actively incorporate a uracil derivative (4TU) into RNA indicating that the enzyme is active in vivo. Interestingly, I find that Krishah is also essential for larval growth as knocking out the gene leads to impaired larval growth and increased larval and pupal lethality.