Mitochondrion is an endosymbiotic organelle synthesizing ~1% of its proteome, while remaining ~99% of the proteins are encoded by the nuclear genome and translated on the cytosolic ribosome. Therefore active mitochondrial biogenesis requires efficient protein transport destined for the different sub-compartments. Mitochondrion contains specialized translocation machineries in the outer and in the inner membrane known as TOM40 and TIM23-complex respectively. Import of a majority of mitochondrial proteome is mediated by inner membrane presequence translocase (TIM23 complex). However, the structural organization of Tim23-complex and mechanisms of mitochondrial inner membrane protein translocation is still elusive. Therefore, the present thesis addresses above elusive questions. Chapter 2 highlights the functional significance of different segments of Tim23 in regulating the conformational dynamics of the presequence-translocase- Tim23 is the central channel forming subunit of the presequence-translocase which recruits additional components for the assembly of the core complex. However the functional significance of different segments of Tim23 was not understood due to the lack of suitable conditional mutants. Our study has reported many conditional mutants from different segments of Tim23 which are precisely defective in the organization of the core complex and in the recruitment of the import motor component which enhances our understanding of protein translocation across mitochondrial inner membrane. Chapter 3 highlights the functional cooperativity among mtHsp70 paralogs and orthologs using Saccharomyces cerevisiae as a model organism- mtHsp70s are implicated in a broad spectrum of functions inside the mitochondria. In case of lower eukaryotes gene duplication event has given rise to multiple copies of Hsp70s thereby presenting an opportunity of division of function among these paralogs. The mitochondria of yeast Saccharomyces cerevisiae contains three Hsp70s, including Ssc1, Ssq1 and Ssc3 (Ecm10). The Ssc1 is essential for protein translocation and de novo protein folding functions while Ssq1 is needed for the Fe/S cluster biogenesis inside the mitochondria. Although it has been proposed earlier that, Ssc1 and Ssc3 possesses overlapping functions in protein translocation as a part of import motor in the Tim23-complex. However the physiological relevance and experimental evidences in favor above hypothesis was not established clearly. Our study has reported Ssc3 as an ‘atypical chaperone’ which cannot perform the generalized chaperone functions due to the conformational plasticity associated with both the domains of Ssc3 resulting into weaker client protein affinity, altered interaction with cochaperones and dysfunctional allosteric interface. Additionally, we have also highlighted the role of Nucleotide-binding domain in determining the functional specificity among Hsp70 paralogs and orthologs.

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Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Introduction: Doxorubicin (DOX), infamously known as the “red devil,” is considered the most effective antineoplastic drug utilized in oncologic practice today. However, its clinical use is hampered due to cumulative, dose-dependent cardiotoxicity, which can lead to reduced quality of life, irreversible heart failure and death. The mechanisms involved in the pathogenesis of cardiotoxicity have not been fully elucidated, but have previously been demonstrated to involve oxidative stress, calcium dysregulation and mitochondrial dysfunction. Since the mitochondria play a critical role in generation of reactive oxygen species, the maintenance of calcium homeostasis and are the most extensively damaged by DOX, they have become the main focus of novel therapeutic interventions. The morphology and function of these dynamic organelles are regulated in part by mitochondrial fission and fusion events, as well as mitochondrial quality control systems. Since mitochondrial morphology is often associated with crucial cellular functions, this study aimed to investigate the long-term effects of DOX on mitochondrial dynamics and the mitochondrial quality control systems, mitophagy and the ubiquitin-proteasome pathway (UPP). Additionally, since the mitochondria and the endoplasmic reticulum (ER) are two interconnected organelles, and both play a role in maintaining calcium homeostasis, this study further assessed the effects of chronic DOX treatment on ER function and calcium status. Materials and Methods: In order to fully establish the effect of chronic DOX treatment in vitro, two cardiac cell lines were utilized in this study. H9C2 cardiomyoblasts and humanderived Girardi heart cells were cultured under standard culture conditions until ± 70-80% confluency was reached, where after treatment commenced. Cells were treated daily with 0.2 and 1.0 μM of DOX for 96 and 120 hours in order to simulate chronic, cumulative cardiotoxicity. Cell viability and apoptotic cell death were assessed with the MTT assay and Caspase Glo 3/7 assays, respectively. The expression of proteins involved in mitochondrial dynamics, mitochondrial biogenesis, the ubiquitin-proteasome pathway, mitophagy and ER stress were determined with Western blotting. Organelle morphology was visualized with fluorescence microscopy, and flow cytometry was used to assess mitochondrial and ER load. In order to determine the oxidative capacity, stress and status within the cells following treatment, the Oxygen radical absorbance capacity (ORAC), Thiobarbituric acid reactive substances (TBARS) and Glutathione (GSH) assays were employed respectively. Finally, intracellular and mitochondrial calcium was assessed and quantified with superresolution structured illumination microscopy (SR-SIM) and flow cytometry respectively. Results: DOX significantly reduced cell viability and increased apoptosis in both in vitro cardiac cell models. This study further demonstrated that the expression of mitochondrial fusion proteins, Mfn 1 and Mfn 2 were significantly downregulated, whilst the regulators of fission, Drp1 and hFis1, were significantly elevated, therefore shifting the balance of mitochondrial dynamics towards fission. Unopposed and elevated mitochondrial fission was clearly evident from the morphology of these organelles, which displayed short, highly fragmented mitochondria with a dispersed network following treatment. Chronic DOX also downregulated the regulator of mitochondrial biogenesis, PGC-1α, thus inhibiting the formation of new, functional mitochondria. The E3 ligases, MARCH5 and Parkin were highly upregulated following treatment, indicating activation of the UPP and mitophagy. Although chronic DOX stimulated K48 ubiquitination following treatment, it inhibited the catalytic activity of the 26S proteasome, therefore blocking proteasomal degradation. Although the antioxidant capacity (measured as ORAC) was significantly enhanced by both concentrations of DOX, an increase in oxidative stress status was shown following DOX treatment. In this regard lipid peroxidation significantly increased, while redox status of the endogenous antioxidant, glutathione, significantly decreased. Additionally chronic DOX treatment induced ER stress, which lead to an increase in cytosolic and mitochondrial calcium. In response to ER stress, the unfolded protein response (UPR) was then stimulated. Discussion: Results from this study indicate that chronic DOX treatment disrupts the balance of mitochondrial dynamics, favouring mitochondrial fission. Mitochondrial fragmentation is mediated by the downregulation of fusion proteins regulated by the E3 ubiquitin ligase, MARCH5 as well as by the increase in mitochondrial calcium. Mitochondrial fission results in mitophagy, an adaptive response to protect the cardiac cell against damaged mitochondria. This study also indicates that during chronic DOX-induced cardiotoxicity ER stress and the UPR are induced, which is possibly responsible for the disruption in calcium homeostasis. The inhibition of mitochondrial biogenesis coupled with elevated mitophagy as observed in this chronic study, elucidates a plausible mechanism whereby DOX induces mitochondrial dysfunction. Unregulated mitochondrial fragmentation and inhibited mitochondrial biogenesis are known to regulate various cardiomyopathies, therefore since both these effects are induced by chronic DOX treatment suggests a mechanism whereby cardiotoxicity, and ultimately heart failure are produced. This study provides new insight into the role of chronic DOX plays in altering mitochondrial dynamics and mitochondrial quality control systems. Further investigations targeted at limiting mitochondrial fission may reduce the cardiovascular side effects associated with DOX. / AFRIKAANSE OPSOMMING: Inleiding: Doksorubisien (DOX), ook bekend as die “rooiduiwel,” word beskou as die mees effektiewe anti-neoplastiese middel wat tans in onkologie praktyke gebruik word. Die kliniese gebruik hiervan word gerem deur die kumulatiewe dosis-afhanklike kardiotoksisiteit wat tot verlaagde lewenskwaliteit, onomkeerbare hartversaking, en tot die dood kan lei. Die meganismes wat by die kardiotoksiese patogenese betrokke is, is nog onbekend, maar die meganisme het moontlik te doen met oksidatiewe stres, kalsiumwanregulering en mitochondriale wanfunksionering. Omrede die mitochondria ‘n kritieke rol in die vorming van reaktiewe suurstofspesies speel, asook die handhawing van kalsiumhomeostase en die mees beskadigde organelle deur DOX, het die hooffokus na nuwe terapeutiese intervensies verskuif. Die morfologie en funskie van hierdie dinamiese organelle word gereguleer deels deur mitochondriale fragmentering en fussie, asook mitochondriale kwaliteitsbeheersisteme. Omrede mitochondriale morfologie geassosieer is met noodsaaklike sellulêre funksies, het hierdie studie gepoog om die langtermyneffkte van DOX op mitochondriale dinamika en die mitochondriale kwaliteitsbeheersisteme, mitofagie en die ubikwitien-proteosoomweg (UPW) te ondersoek. Siende dat die mitochondria en die endoplasmiese retikulum (ER) twee interverweefde organelle is, en beide ‘n rol speel in die handhawing van kalsiumhomeostase, het hierdie studie verder die effekte van chroniese DOX behandeling op ER funksie en kalsiumstatus ondersoek. Materiaal en Metodes: Om die effek van chroniese DOX behandeling in vitro te verstaan in hierdie studie, is twee hartsellyne gebruik. H9C2 kardiomioblaste en menslike Girardi hartselle is onder standaardtoestande tot ± 70-80% konfluensie bereik is gekweek, waarna behandeling begin is. Selle is daagliks met 0.2 en 1.0 μM DOX vir 96 en 120 uur behandel om chroniese en kumulatiewe kardiotoksisiteit n ate boots. Selvatbaarheid en apoptotiese seldood is onderskeidelik ondersoek deur middel van die MTT en Caspase Glo 3/7 toetse. Die proteïenuitdrukking betrokke by mitochondriale dinamika, mitochondriale biogenese, die ubikwitien-proteosoom weg, mitofagie en ER stres is deur middel van westerse afblatting bepaal. Organelmorfologie is deur middel van fluoresensie mikroskopie gevisualiseer, en vloeisitometrie was gebruik om die aantal mitochondria en ER lading te bepaal. Om die oksidatiewe kapasiteit, stres en status binne die selle na behandeling te bepaal, is die ORAC, TBARS en GSH toetse onderskeidelik gebruik. Laastens was die intrasellulêre en mitochondriale kalsium ondersoek en gekwantifiseer met superresolussie gestruktureerde illuminasie mikroskopie (SR-SIM) en vloeisitomerie. Resultate: DOX het selvatbaarheid betekenisvol verlaag en apoptose in beide in vitro kardiale selmodelle verhoog. Hierdie studie het verder aangetoon dat die uitdrukking van mitochondriale fussie proteïene, Mfn 1 en Mfn 2 betekenisvol afgereguleer is, terwyl die reguleerders van fragmentering, Drp1 en hFis1, betekenisvol verhoog is en daardeur die balans van mitochondriale dinamika na fussie verskuif. Onverhinderde en verhoogde mitochondriale fragmentering is duidelik sigbaar deur die morfologie van die organelle, wat as kort, hoogsgefragmenteerde mitochondria met ‘n verspreide netwerk na behandeling vertoon. Chroniese DOX het ook die mitochondriale biogenese reguleerder, PGC-1α, afgereguleer en daardeur die vorming van nuwe, funksionele mitochondria geinhibeer. Die E3 ligase, MARCH5 en Parkin is hoogs opgereguleer na behandeling, wat aktivering van UPW en mitofagie aantoon. Alhoewel chroniese DOX K48 ubikwitinering na behandeling gestimuleer het, het dit die katalitiese aktiwiteit van die 26S proteasoom geinhibeer en dus die proteosomale degradasie geblokkeer. Antioksidantkapasiteit en oksidatiewe status was betekenisvol na behandeling wat gevolglik tot hoë vlakke oksidatiewe skade binne die selle gelei het. Addisioneel het chroniese DOX behandeling ER stres geïnduseer wat tot ‘n toename in sitosoliese en mitochondriale kalsium gelei het. In reaksie op die ER stres is die UPW gestimuleer. Bespreking: Resultate van hierdie studie het aangetoon dat chroniese DOX behandeling die balans van mitochondriale dinamika onderbreek en sodoende mitochondriale fragmentering bevoordeel. Mitochondriale fragmentering word gemediëer deur die afregulering van fussie proteïene wat deur die E3 ubikwitienligase, MARCH5, gereguleer word, en ook deur die toename in mitochondriale kalsium. Mitochondriale fragmentering induseer mitofagie, ‘n aanpassingsreaksie om die hartselle teen beskadigde mitochondria te beskerm. Hierdie studie toon verder ook dat gedurende chroniese DOX-geïnduseerde ER stres, word die UPW ook geïnduseer, wat moontlik dan verantwoordelik is vir die ontwrigting van kalsiumhomeostase. Die inhibering van mitochondriale biogenese gekoppel met verhoogde mitofagie soos waargeneem in hierdie studie, verklaar ‘n moontlike meganisme waardeur DOX mitochondriale wanfunksionering veroorsaak. Ongereguleerde mitochondriale fragmentering en geinhibeerde mitochondriale biogenese is bekend om verskeie kardiomiopatieë te reguleer. Omrede beide hierdie effekte geinduseer word deur chroniese DOX behandeling kan dit moontlik ‘n meganisme voorstel waarby kardiotokiese en uiteindelik hartversaking ontwikkel. Hierdie studie bied nuwe insig in die rol wat chroniese DOX speel in die wysiging van mitochondriale- dinamika en kwaliteitskontrole sisteme. Verdere ondersoeke wat die mitochondriale fragmentering kan verminder mag moontlik die kardiovaskulêre newe-effekte wat met DOX behandeling geassosieer is, verlaag. / National Research Foundation (NRF)

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Les insectes forestiers évoluent dans des écosystèmes particuliers caractérisés par leur longévité et leurs dimensions spatiales. Certains insectes forestiers, en particulier les défoliateurs, montrent des patterns de pullulations cycliques, parfois associés à un développement spatial précis. Zeiraphera diniana présente deux caractéristiques intéressantes (1) une temporelle et (2) une spatiale : (1) une très forte régularité dans la périodicité de ses pullulations observée depuis plus d’un millénaire. Les densités de population fluctuant drastiquement tous les 8 à 10 ans dans les Alpes, provoquant des défoliations impressionnantes sur de grandes surfaces forestières (2) le développement spatial du pic de pullulation suit un pattern de “traveling waves” avec une initiation toujours située dans une région Française appelée Briançonnais. En tant que première étude sur la génétique des populations de la tordeuse du mélèze à l’échelle de son aire de pullulation, cette thèse donne des éléments descriptifs sur les caractéristiques génétiques spatiales, avec une vision phylogéographique et historique. De plus, cette étude propose un regard sur la dynamique des populations complexe de cet insecte, en testant les prédictions génétiques correspondent aux modèles et hypothèses de dynamique existants. / Forest insects evolve in particular ecosystems characterized by their longevity and their spatial dimensions. Some populations of forest insects, in particular defoliators, exhibit a pattern of cyclic outbreaks that can be associated with particular spatial development. Zeiraphera diniana exhibits two interesting characteristics, a (1) temporal one and a (2) spatial one : (1) really high regularity in outbreak periodicity observed for more than a thousand years. Population densities fluctuate dramatically with outbreaks every 8 to 10 years in the Alps, causing spectacular defoliation of large stands of larch forests (2) the outbreak spatial development follows a travelling wave pattern always initiated from an area located in a French area called Briançonnais. As the first study on population genetics of the larch budmoth all over its outbreak range, this PhD gives descriptive elements on the spatial genetic characteristics of the insect, with an insight in its phylogeography and past history. This study furthermore gives a spatio-temporal insight in the complex population dynamics observed, by testing genetic predictions corresponding to existing population dynamics models and hypotheses.