Organelles sequester biological phenomena within the cell, and allow an additional layer of complexity to life. The presence and maintenance of these organelles is crucial for cellular function. Two of the most expansive and complex organelles are the mitochondria and endoplasmic reticulum. These organelles contribute energy, protein folding and secretion, lipids, calcium regulation, and various other metabolites to the biology of the cell. Importantly, these organelles accumulate damage and cannot be derived de novo, therefore must be inherited and maintained in a functioning state. The study of these organelle quality control processes serves as the basis for my thesis. We use the budding yeast as a model organism to uncover conserved pathways affecting organelle, and ultimately cellular homeostasis. In yeast we find mitochondrial inheritance is critical for cell survival. Furthermore, not only is inheritance critical, but inheritance of a certain threshold of functional mitochondria appears critical in maintaining normal lifespan in yeast, identifying mitochondria as an aging determinant. By examining mutants that negatively affect mitochondrial inheritance in yeast, we established a role for phosphatidylcholine biosynthesis in organelle maintenance and inheritance. Glycerophospholipid biosynthesis plays a clear role not only in mitochondrial inheritance but also in that of the endoplasmic reticulum. We use insights gained from yeast to guide research into a human disease caused by similar glycerophospholipid biosynthetic deficiency.

The following dissertation discusses the role of Aβ42 dependent hyperactivation of AMPK mediating synaptic loss through coordinated Mff-dependent mitochondrial fission and Ulk2-dpendent mitophagy in dendrites of PNs. In Chapter 1, I provide a brief background on Alzheimer’s disease and the cellular and molecular mechanisms that have been relevant to the pathogenesis of the disease including disruption on mitochondrial homeostasis and autophagy. In Chapter 2, I discuss the findings of my main project describing the role of Aβ42 induced mitochondrial remodeling leading to synapse loss in vitro and in vivo in part by hyperactivation of CAMKKII-AMPK. Chapter 3 covers a review article that I participated in in examining the role of mitochondria in various ND. In Chapter 4, I discuss about a project I was involved in in examining the mechanism behind maintaining mitochondrial morphology in axon versus dendrite and its functional consequence. In Chapter 5, I end the dissertation by highlighting key findings, potential future studies, and concluding remarks.

Peroxiredoxins are small ubiquitous cysteine-containing proteins that exhibit high reactivity to hydrogen peroxide. Apart from their role as antioxidants, detoxifying hydrogen peroxide to water, peroxiredoxins have been implicated in other cellular processes, such as protein folding and signalling. Using S. cerevisiae as a model organism, we utilised a variety of techniques to examine previously unexplored links between peroxiredoxins and mitochondrial function. Firstly, we characterised the role of Gpx3 in yeast mitochondria. Proteomic work revealed the presence of Gpx3 in the mitochondrial intermembrane space (IMS) and we characterised when, how and why Gpx3 can be found within the mitochondria. We showed that cells lacking Gpx3 have aberrant mitochondrial morphology and defective protein import capacity and inner membrane potential upon H2O2 stress. Gpx3 translocates to the IMS via a targeting sequence encoded from a non-AUG codon. This provides a novel and unique molecular mechanism that protects mitochondria from the exceptional oxidative stress which their activity imposes. Secondly, we focused on the role of Tsa1 upon protein aggregation-induced stress. Previous studies using the proline analogue AZC to cause protein misfolding revealed that protein aggregates are localised adjacent to mitochondria and mitochondrial ROS are generated in response. We questioned what effect this might have on mitochondrial function and we showed that upon AZC treatment there is a drop in respiratory rate, dependent on Tsa1. We questioned whether Tsa1, like other peroxiredoxins, is involved in regulating signalling cascades and we showed that cells that are lacking Tsa1 have alterations in the activity of the cAMP/PKA pathway. In parallel, we looked for differences both in the proteome and the transcriptome to understand what is the cause of the lethality of a tsa1 strain upon protein aggregation stress. We propose a mechanism where Tsa1 mediates a transcriptional response to protein misfolding stress via the activity of the heat shock transcription factor, Hsf1. Finally, we focused on the role of the mitochondrial peroxiredoxin Prx1. Under conditions where the mitochondrial matrix is oxidised, either genetically or by chemical addition, we showed than an apoptotic pathway is activated, dependent on the redox state of thioredoxin, Trx3. We showed that Trx3 can interact with Prx1 and loss of Prx1 also stops the induction of cell death. Analysis of the interactome of Trx3 unraveled the involvement of Bxl1/Ybh3, the yeast BH3 domain-containing protein and Aim9, a previously uncharacterised protein with kinase-like motifs, in the progression of cell death. The data presented in this thesis widens our understanding of the function of peroxiredoxins and their involvement in the regulation of cellular cascades that ensure correct mitochondrial function and responses to stress.

After naïve lymphocytes are activated by foreign antigens, they yield cellular progeny with diverse functions, including memory cells, effector cells, and precursors of germinal center B cells. However, it remains unclear whether a naïve lymphocyte is capable of generating daughter cells with multiple fates or multiple naive cells are activated and each give rise to daughter cells with different cell fates. This dissertation analyzes the role of asymmetric cell division in the generation of effector lymphocytes and maintenance of progenitor cells. Our data provide evidence that daughter cells exhibit differential mitochondrial stasis and inherit different amounts of glucose transporters, which is coupled to distinct metabolic and transcriptional program in the sibling cells. To uncover the links between mitochondrial stasis, transcription network reprogramming and cell fate, we perturbed mitochondrial clearance with pharmacological and genetic approaches. I found that the treatments, which impaired mitochondrial function, increased the differentiation of B cells and T cells into effector subsets. Thus, we hypothesize that mitochondrial stasis could be a trigger for effector cell differentiation. To further explore the mechanism for aged mitochondria-induced shifts in transcriptional and metabolic programs, we used reactive oxygen species (ROS) scavengers and glycolysis inhibitors to demonstrate that mitochondria function and the expressions of lineage-specific transcription factors crosstalk through ROS-mediated signaling and activating AMPK. ROS scavenger treatments helped to maintain the progenitor population and suppressed the differentiation of effector subsets, whereas effector cell differentiation was boosted in the AMPK-α1 knockout. These results suggest mitochondrial stress-induced ROS is required for repressing Pax5 and increasing IRF4. In addition to showing mitochondrial stasis’ connection to cell fate, this dissertation also demonstrates the linkage between phosphatidylinositol-3-kinases and glucose transporter 1 (Glut1) in establishing polarity in dividing cells and in transcriptional reprogramming. In sum, this dissertation suggests that asymmetric mitochondrial stasis and nutrient up-take could be part of the driving force of cell fate owing to self-reinforcement and reciprocal inhibition between anabolism and catabolism. These results shed light on the deterministic mechanism of effector cell differentiation and provide clues to the basis of maintenance of self-renewal by activated lymphocytes. These findings could be beneficial for producing memory cells and preventing effector cell exhaustion phenotype in a chronic infection or in cancer microenvironment.

Parkinson's disease is a progressive neurodegenerative disorder of the central nervous system, manifesting with both motor and non-motor symptoms. Autosomal recessive mutations in the FBXO7 gene have been identified to cause a rapidly progressing early-onset form of PD. Canonically, FBXO7 functions as a substrate-recruiting subunit of the SCF-type E3 ubiquitin ligase. However, it also has a variety of other atypical functions, such as cell cycle regulation, proteasome regulation, and mitophagy. The overall aim of this research was to characterise the functional role of FBXO7 in various in vitro and in vivo PD models. The models examined included FBXO7 shRNA knockdown SH-SY5Y cell lines, FBXO7 CRISPR knockout SH-SY5Y cell lines, primary patient fibroblasts with a FBXO7 mutation, and MEFs and tissues from a Fbxo7 KO mouse. My analysis of fibroblasts from a patient without FBXO7 expression revealed several interesting phenotypes. Briefly, the patient fibroblasts proliferated slower due to increased apoptosis and lower CDK6 and cyclin D1 expression, which led to fewer cells progressing through the G1 phase of the cell cycle. My experiments showed that these cells also had mitochondrial respiration defects, exhibiting lower basal respiration, ATP production, maximal respiration and spare capacity, in addition to complex I, III and IV deficiencies. Patient fibroblasts also had significantly lower levels of 12S and 16S ribosomal mRNA transcripts, which are necessary for the translation of mitochondrially encoded subunits of complexes I, III, and IV. Similar phenotypes were also observed in MEFs from a Fbxo7 KO mouse model, indicating conservation between human and mouse FBXO7 in regulating mitochondria, cell death and proliferation. In a tissue-specific KO mouse model of PD, where FBXO7 expression was ablated in the dopaminergic neurons, I analysed proteins regulated by FBXO7 which might be responsible for cell loss in the substantia nigra. I discovered that RPL23, a regulator of MDM2, was ubiquitinated by SCFFbxo7 using K48 chain linkages, promoting its degradation by the proteasome. This suggests that misregulation of the MDM2:p53 axis may underlie the cell loss observed in this conditional Fbxo7 KO mouse model. In conclusion, these results elaborate on the role of FBXO7 in mitochondrial biology, and identify a new ubiquitination substrate of FBXO7 in a mouse model of PD. It is hoped that by elucidating the potential pathogenic mechanisms of FBXO7 in rare familial forms of the disease, it will be possible to translate findings to the more prevalent sporadic forms of Parkinson's disease as well.

Complex I (NADH:ubiquinone oxidoreductase), located in the mitochondrial inner membrane, is a major electron entry point to the respiratory chain. It couples the energy released from electron transfer (from NADH to ubiquinone) to the concomitant pumping of protons across the membrane, to generate an electrochemical proton motive force. Mammalian complex I is composed of 45 subunits, 14 of which comprise its simpler bacterial homologues. It is encoded by both the mitochondrial and nuclear genomes, and pathological mutations in both sets of subunits result in severe neuromuscular disorders such as Leigh syndrome. Several structures of mammalian complex I from various organisms have been determined, but the limited resolutions of the structures, which typically refer to poorly characterised enzyme states, has hampered detailed analyses of mechanistic features. The first part of this thesis describes development of a method for purifying complex I from the genetically amenable and medically relevant model organism Mus musculus (mouse), in a pure, stable and active state. The enzyme from mouse heart mitochondria was then comprehensively characterised, to ensure the presence of all the expected subunits and co-factors, and to define its kinetic properties. The second part of this thesis describes structural studies by single particle electron cryomicroscopy (cryo-EM) on the purified mouse enzyme in two distinct states, the 'active' and 'de-active' states. The active state was determined to 3.3 Å resolution, the highest resolution structure of a eukaryotic complex I so far. Subsequently, comparison of the two mouse structures, together with previously determined mammalian and bacterial structures, revealed variations in key structural elements in the membrane domain, which may be crucial for the catalytic mechanism. Moreover, in the high-resolution active mouse complex I structure a nucleotide co-factor was observed bound to the nucleoside kinase subunit NDUFA10. Finally, complex I from the Ndufs4 knockout mouse model, which recapitulates the effects of a human mutation that causes Leigh syndrome, was purified and subjected to kinetic and proteomic analyses. Following cross-linking and preliminary structural studies, it was concluded that the detrimental effects of deleting NDUFS4 are due to lack of stability of the mature complex.

The O-linked N-acetylglucosamine post-translational modification (O-GlcNAcylation) is the dynamic and reversible attachment of N-acetylglucosamine to serine and threonine residues of target proteins. It is abundant in metazoa, involving hundreds of proteins linked to a plethora of biological functions with implications in human diseases. The process is catalysed by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), that add and remove the sugar moiety, respectively. Ogt gene knock-out is embryonic lethal in a range of animal models, hampering the study of the biological role of O-GlcNAc. O-GlcNAcylation of nuclear and cytoplasmic proteins has been extensively studied, however little is known about the role of O-GlcNAc in mitochondria. A previous report suggested the presence of a mitochondrial OGT isoform (mOGT) in human cell lines in addition to the well-characterised nucleocytoplasmic one (ncOGT). Since this report more than one decade ago, this putative mOGT has not been studied further. Similarly, hundreds of O-GlcNAcylated nucleocytoplasmic proteins have been identified by high-throughput proteomic screens, whereas only a few mitochondrial proteins have been detected. Nevertheless, several studies suggest that altered O-GlcNAc signalling affects mitochondrial function and morphology, with potential clinical implications. The aim of this thesis work was to study and characterise the biological role of mOGT and determine the mitochondrial O-GlcNAc proteome. Firstly, the presence of mOGT in human cell lines and mouse tissues was investigated. Surprisingly, analysis of genomic sequences indicates that this isoform cannot be expressed at protein level in most of the species analysed, except human and some primates. In fact, the putative mOGT cDNA in most of the genomes analysed contains a stop codon that excludes the presence of such isoform. In addition, mOGT was not detected at protein level in a wide range of human cell lines. Knock-down experiments and Western blot analysis of all the predicted OGT isoforms suggested the expression of only a single OGT isoform. In agreement with this, overexpression of ncOGT in HEK 293 suspension cells led to increased O-GlcNAcylation of mitochondrial proteins, suggesting that ncOGT is necessary and sufficient for the generation of the mitochondrial O-GlcNAc proteome. These data point to a model where O-GlcNAc cycling of mitochondrial proteins occurs in the cytosol, followed by their import into mitochondria. Alternatively, ncOGT itself might be transported into mitochondria where it can take part to O-GlcNAc cycling inside the organelle. In parallel, some advance in determining the O-GlcNAc mitochondrial proteome has been undertaken. Different mitochondrial fractionation protocols, combined with O-GlcNAc enrichment methods have been explored in order to map novel glycosylation sites on mitochondrial proteins. A novel technique established in our research group, employing a bacterial OGA orthologue as a bait to trap O-GlcNAcylated proteins, has been applied to crude mitochondrial fractions allowing the identification of several hits, although site mapping has not been yet achieved. The second chapter describes the work that has been done to improve and optimise novel O-GlcNAc inhibitors previously designed in the laboratory, called goblins. The original objective was to make these molecules cell-permeable and possibly target them to mitochondria in order to inhibit mOGT. Several strategies were explored to deliver the compounds into living cells, including the use of transfection reagents and covalent linkage to linear cell-penetrant peptides. The above methods did not achieve cellular uptake, although recently designed cyclic cell-penetrant peptides, linked to fluorescein, were internalised by HeLa cells with immediate diffuse nucleocytoplasmic staining. These molecules will be linked to goblins aiming to use the inhibitors for cell biology studies. A different approach, based on the permeabilisation of Drosophila embryos, enabled the penetration of goblins into the organisms with consequent reduction of global O-GlcNAc levels. This method allowed the use of these novel bisubstrate inhibitors in vivo for the first time, with potential applications in studying the role of O-GlcNAc in Drosophila development and possibly for future therapeutic purposes after further development of the scaffold.

As mitocôndrias são organelas semiautonômicas, portadoras do próprio DNA, o mtDNA e responsáveis pela produção de energia celular na forma de ATP, através do processo de fosforilação oxidativa. Atualmente, diferentes tipos de doenças, como distrofias musculares e diversos tipos de câncer, estão associadas à alteração nas moléculas de mtDNA. Na década de 70 um modelo a partir do cultivo celular com brometo de etídio (EtBr) foi desenvolvido com o objetivo de se criar uma linhagem celular depletada de cópias de mtDNA. Desde então os mais variados estudos foram realizados e diversos tipos celulares foram submetidos à depleção do mtDNA. Este projeto teve como objetivos criar um modelo de cultivo celular somático na espécie bovina com depleção de cópias de mtDNA para investigar a resposta da célula a esta condição; avaliar como as células depletadas se comportam na ausência de EtBr, além da utilização destas células no processo de reprogramação celular por indução gênica na tentativa de avaliar o efeito do numero de cópias de mtDNA na indução na espécie bovina. Para tanto foram desenvolvidos três experimentos; Experimento 1- Depleção de mtDNA a partir da utilização do brometo de etídeo; Experimento 2 Repleção do mtDNA; e Experimento 3 Utilização de células bovinas depletadas no sistema de reprogramação nuclear. Todos os experimentos foram avaliados quanto a quantidade de cópias de mtDNA e expressão gênica para os genes Bax, Bcl2 e Tfam. Ademais, os experimentos 1 e 2 foram avaliados quanto a viabilidade celular e apenas o experimento 1 foi avaliado quanto ao crescimento e morfologia celular. O experimento 1 foi avaliado durante o cultivo celular nos períodos D0, D4, D7, D10 e D13, com os grupos experimentais controle (EtBr-C) e tratado com 100 ng/mL de brometo de etídio (EtBr-T), quanto a núero de cópias do mtDNA, o grupo EtBr-T diferiu do grupo EtBr-C (P=0,0459), apresentando menor número de cópias de mtDNA; menor taxa crescimento celular (P<0,05), porém sem alteração na morfologia celular, e na expressão dos genes descritos acima. No experimento da repleção, não houve diferença no número de cópias de mtDNA, entre os grupos EtBr-T e EtBr-R, indicativo de que as células atingiram o estado rho 0 ou que necessitam de mais tempo para ativar a replicação do mtDNA; quanto a viabilidade celular, houve diferença entres os grupos, quanto a expressão gênica, com aumento do Bax e do Bcl-2 para o grupo EtBr-T; O grupo EtBr-R apresentou queda do Bcl-2; para o Tfam houve aumento para o grupo EtBr-T e uma queda para o grupo EtBr-R. Quanto ao experimento 3, não foi possível observar sinais de pluripotência, porém foi detectada uma queda na quantidade de mtDNA dos dois grupos tratados por EtBr (EtBr com e sem Stemcca) e o grupo controle com Stemcca. Para analise de expressão gênica, não houve diferenças entre os grupos em relação ao Tfam. Quanto ao Bax, os grupos controle com Stemcca, controle sem Stemcca e EtBr sem Stemcca não diferiram, e o ultimo também não apresentou diferença quando comparado ao grupo EtBr com Stemcca. Para o Bcl-2, os grupos controle sem Stemcca e EtBr com Stemcca não apresentaram diferenças entre si; o grupo controle sem Stemcca não apresentou diferença quando comparado aos grupos controle com Stemcca e EtBr sem Stemcca. Concluindo, este trabalho no nosso conhecimento, descreve pela primeira vez a produção de células bovinas Rho 0 e discute sobre a relação da função mitocondrial e o processo de reprogramação celular. / Mitochondria are semi autonomic organelles which present their own DNA (mtDNA); are in charge of cell energy production as ATP through oxidative phosphorylation. Currently, different types of diseases like muscular distrofy; different types of cancer are associated to alterations of mtDNA molecules. In the 70\'s a model based on cell culture with ethidium bromide (EtBr) was developed in order to create a cell line depleted of mtDNA. Since then, a variety of studies were realized; diverse cell types were submited to mtDNA depletion. This project had as objective creating a model of somatic cell culture in bovine species with depletion of mtDNA copies, in order to investigate cell response to this condition; to analyze depleted cell behavior in the absence of EtBr, besides using this depleteded cell in a reprogramming cell process by genic induction in order evaluate the effect of the number of mtDNA copies during induction in bovine species. Therefore three experiments were developed: Experiment-1 Depletion of mtDNA using ethidium bromide. Experiment-2 repletion of mtDNA; Experiment-3 usage of depleted bovine cells in reprogramming nuclear system. Cell experiments were analyzed according to the quantity of mtDNA copies; genic expression for Bax, BCl2; Tfam genes. Also, experiments 1; 2 were analyzed on cell viability; only experiment 1 was analyzed regarding cell morphology; growth. Experiment-1 was analyzed during cell culture on periods D0, D4, D7, D10, D13, with control experimental groups (EtBr-C),; treated with 100 ng/mL ethidium bromide (EtBr-T); relating to mtDNA quantification the EtBr-T group differed from EtBr-C (P=0,0459) presenting a smaller number of mtDNA copies; smaller growth rate (P<0,05); although there was no differences on cell morphology as there was also no difference related to genic expression of the previous stated genes. Repletion experiment showed no differences about the number of mtDNA copies between EtBr-T; EtBr-R groups, indicating this cells reached Rho0 state or that they need more time to activate mtDNA replication; about cell viability, there were no differences among the groups; relating to genic expression there was an increase of Bax; BCl-2 for EtBt-T group; EtBr-R group showed decrease of BCl-2; for Tfam there was an increase for EtBr-T group; a decrease for EtBr-R. Relating to Experiment-3 it was impossible to notice signs of pluripotency, but we could see a decrease in the amount of mtDNA in both groups treated with EtBr (EtBr with; without STEMCCA) as in control group with STEMCCA. Genic expression analysis didn\'t show differences related to Tfam. Regarding to BAX, both control groups (with; without STEMCCA); EtBr without STEMCCA didn\'t differ from each other,; the last one also didn\'t show any difference when compared to EtBt with STEMCCA group. For BCl-2, control group without STEMCCA; EtBr with STEMCCA didn\'t show differences among each other; control group without SEMCCA didn\'t show differences when compared to control group with STEMCCA; EtBr without STEMCCA. Concluding, this work, regarding our knowledge, describes for the first time, production of bovine Rho0 cells; debates about the relationship among mitochondrial function; the process of cell reprogramation.

Cu/Zn Superoxide Dismutase (SOD1), is a ubiquitous antioxidant enzyme with several emerging roles outside of its canonical function. SOD1 is also emerging in central roles in cancer and neurodegenerative pathologies. Little is known about SOD1 regulation, particularly at a post-translational level. Post-translational modifications (PTMs) play an important role in enabling proteins to rapidly respond to their environment. Therefore, identifying specific PTMs involved in protein regulation represents a powerful opportunity to interfere with any associated pathologies. This work employs proteomics to identify mechanisms of post-translation regulation on cell survival signaling proteins. We focused on SOD1, which protects cells from oxidative stress. We found that acylation of K122 on SOD1, while not impacting SOD1 catalytic activity, suppressed the ability of SOD1 to inhibit mitochondrial metabolism at respiratory complex I. We found that deacylase depletion increased K122 acylation on SOD1, which blocked suppression of respiration in a K122-dependent manner. In addition, we found that acyl-mimicking mutations at K122 decreased SOD1 accumulation in mitochondria, initially hinting that SOD1 may inhibit respiration directly within the intermembrane space (IMS). However, surprisingly, we found that forcing the K122 acyl mutants into the mitochondria with an IMS-targeting tag did not recover their ability to suppress respiration. Moreover, we found that suppressing or boosting respiration levels toggled SOD1 in or out of the mitochondria, respectively. These findings place SOD1-mediated inhibition of respiration upstream of its mitochondrial localization. Interestingly, we also found that K122 acyl mutants were sufficient to prevent mitochondrial accumulation of the G93A SOD1 clinical mutant. We observed increased autophagic activity in G93A expressing cells compared to WT or G93A/K122-acyl mimic double mutants, and found that this double mutant was just as prone to aggregate as G93A SOD1—suggesting that SOD1 aggregation is more toxic when in the mitochondria. We observed increased protein turnover rates in cells expressing SOD1 G93A, in support of increased autophagy. Lastly, deletion-rescue experiments show that a respiration-defective mutant of SOD1 is also impaired in its ability to rescue cells from toxicity caused by SOD1 deletion. Together, these data suggest a new interplay between SOD1 acylation, metabolic regulation, SOD1 aggregate toxicity, and SOD1-mediated cell survival.

Prevalence of diabetes, especially type 2 diabetes mellitus (T2DM) is increasing worldwide. Millions of people are already affected by T2DM and estimates predict over half a billion people will likely be suffering from the disease by 2030. T2DM is associated with an increased risk of developing cardiovascular disease. Cardiovascular dysfunction is the leading cause of mortality among type 2 diabetics. Treatment for T2DM has changed over time. Though it was once known as insulin independent, a large portion of type 2 diabetics are now treated with insulin injections. However, type 2 diabetics treated with insulin are more likely to suffer from heart complications. Due to this, we sought to determine the specific effect of insulin and insulin-induced ceramide accrual on heart mitochondrial bioenergetics. To do so we used both in vitro and in vivo models. H9c2 cardiomyocytes and adult male mice were treated with insulin with or without the ceramide biosynthesis inhibitor myriocin. Mitochondrial bioenergetics were determined in permeabilized cardiomyocytes and myocardium. In this study we demonstrate that insulin induced ceramide accrual in both isolated cardiomyocytes and whole murine myocardium. We further found that insulin treatment is sufficient to disrupt mitochondrial respiration in both models. Inhibition of the ceramide accrual rescued mitochondrial respiration, indicating that ceramide is necessary for the insulin-induced alterations in heart mitochondrial respiration. These results suggest that insulin has a role in the development of heart complications associated with T2DM due to cardiomyocyte mitochondrial disruption. They also implicate ceramide as a possible mediator in the development of insulin-related heart disorders.