Mitochondrial dysfunction causes moderate to profound hearing loss both in isolation and as a feature of multi-systemic mitochondrial disease. The m.1555A > G mitochondrial DNA (mtDNA) variant is associated with a predisposition to aminoglycoside ototoxicity and maternally inherited non-syndromic deafness. However, the reasons for the highly variable penetrance of the associated hearing loss have not yet been fully resolved. Aminoglycosides are a recognised modifier factor of the hearing loss, but cannot account for all hearing impaired carriers in multi-generational pedigrees, implicating additional co-segregating genetic factors. By identifying and characterising the c.3G > A SSBP1 variant as a nuclear modifier of m.1555A > G the work detailed in this thesis extends our understanding of mitochondrial-nuclear interactions in human disease. To ascertain the frequency of the m.1555A > G variant in patients with suspected mitochondrial hearing loss we surveyed the laboratories within the United Kingdom that undertake genetic testing for this variant. We determined that the variant was not found more frequently in patients with known hearing impairment providing further evidence that m.1555A > G does not cause hearing loss in isolation. These results strengthened the case for nuclear genetic modifiers as important contributors to m.1555A > G pathogenesis. We next identified a multi-generational family that transmitted the m.1555A > G variant with variable clinical penetrance of hearing loss. In addition, a cohort of sporadic individuals carrying m.1555A > G was used to test the hypothesis that a conserved genetic mechanism accounted for the phenotype in all carriers. To this effect, we undertook whole exome sequencing in selected familial and sporadic carriers of m.1555A > G, identifying a heterozygous start loss mutation in the core mtDNA replisome protein gene, SSBP1, that co-segregated with the m.1555A > G variant and the phenotype in the family. The SSBP1 variant lead to a perturbation of mtDNA metabolism, and was associated with multiple mtDNA deletions and mtDNA depletion in skeletal muscle. Fibroblasts from these patients also showed mitochondrial network fragmentation and reduced intra-mitochondrial protein synthesis in keeping with the co-existing m.1555A > G variant, leading to reduced proliferation rates under conditions of forced mitochondrial respiration. Our findings provide an explanation for the variable clinical penetrance of the disorder within these m.1555A > G carriers and highlight the importance of trans-acting modifiers in mitochondrial disease.

Les maladies mitochondriales regroupent un ensemble de pathologies liées à un déficit de la chaînerespiratoire mitochondriale. Au laboratoire, nous focalisons notre intérêt sur les mitochondriopathies liées à undéfaut de stabilité de l’ADN mitochondrial (ADNmt), qui se traduit par des délétions multiples et/ou unedéplétion (diminution du nombre de copies). Ces pathologies sont caractérisées par une importantehétérogénéité clinique et génétique et sont secondaires à des mutations dans des gènes nucléaires codantpour des protéines impliquées dans le maintien de l’ADNmt. De nos jours, la recherche des gènesresponsables d’instabilité de l’ADNmt s’avère négative chez plus de 70% des malades, d’où un grand intérêtpour améliorer les techniques d’identification des mutations et la recherche de nouveaux gènes impliquésdans ces pathologies / Non communiqué

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Bipolar disorder is a psychiatric disorder with alarming rates of morbidity and mortality. Since the pathophysiology of the disease is not well understood, it is difficult to develop treatments or even explain why the current treatments are successful. An increasingly popular hypothesis is that mitochondrial dysfunction plays a role. This paper examines the relationship between mitochondrial dysfunction and bipolar disorder by examining the following: (i) mitochondrial complex I dysfunction and oxidative damage, (ii) mitochondrial complex I dysfunction, epigenetic modifications, and treatment with lithium, (iii) post-mortem brain studies, (iv) the mtDNA common deletion, (v) calcium, (vi) comorbidity with mitochondrial disorders, (vii) lactate and intracellular pH levels, (viii) phosphocreatine, (ix) apoptosis, and (x) inositol. These studies point to a definitive correlation between the bipolar disorder and mitochondrial dysfunction, but it is too soon to determine causation. Further research is needed.

abstract: The ability to manipulate the interaction between small molecules and biological macromolecules towards the study of disease pathogenesis has become a very important part of research towards treatment options for various diseases. The work described here shows both the use of DNA oligonucleotides as carriers for a nicotine hapten small molecule, and the use of microsomes to study the stability of compounds derived to treat mitochondrial diseases. Nicotine addiction is a worldwide epidemic because nicotine is one of the most widely used addictive substances. It is linked to early death, typically in the form of heart or lung disease. A new vaccine conjugate against nicotine held within a DNA tetrahedron delivery system has been studied. For this purpose, several strands of DNA, conjugated with a modified dTpT having three or six carbon atom alkynyl linkers, have been synthesized. These strands have later been conjugated to three separate hapten small molecules to analyze which conjugates formed would be optimal for further testing in vivo. Mitochondrial diseases are hard to treat, given that there are so many different variations to treat. There is no one compound that can treat all mitochondrial and neurodegenerative diseases; however, improvements can be made to compounds currently under study to improve the conditions of those afflicted. A significant issue leading to compounds failing in clinical trials is insufficient metabolic stability. Many compounds have good biological activity, but once introduced to an animal, are not stable enough to have any effect. Here, several synthesized compounds have been evaluated for metabolic stability, and several showed improved stability, while maintaining biological activity. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2016

The lactate level assesment in various body fluids plays an important role in the diagnostics of mitochondrial disorders in children. However, the interpretation of lactate level is often difficult due to its unspecificity and variability even in particular mitochondrial disorders. Three specific aims have been stated in this PhD Thesis: 1. To analyse the role of lactate examination in the differential diagnosis between children with mitochondrial disorders and children with other diseases. 2. To study the lactate level differences in various mitochondrial syndromes. 3. To characterise the clinical and laboratory data of neonates with mitochondrial disorders and to suggest new diagnostic algorhytms. Clinical and laboratory data from patients hospitalized in the Department of Pediatrics were collected. Laboratory methods were provided in the cooperation with the Mitochondrial laboratory of the Department of Pediatrics and Institute of Inherited Metabolic Disorders. The study with lactate levels in 107 patients documented that brief seizures lasting less than 2 minutes did not increase lactate concentration in the CSF. CSF-lactate was a relialable marker in differential diagnosis in the children with mitochondrial disorders against children with epilepsy. 2. The severity of particular phenotype is more...

The lactate level assesment in various body fluids plays an important role in the diagnostics of mitochondrial disorders in children. However, the interpretation of lactate level is often difficult due to its unspecificity and variability even in particular mitochondrial disorders. Three specific aims have been stated in this PhD Thesis: 1. To analyse the role of lactate examination in the differential diagnosis between children with mitochondrial disorders and children with other diseases. 2. To study the lactate level differences in various mitochondrial syndromes. 3. To characterise the clinical and laboratory data of neonates with mitochondrial disorders and to suggest new diagnostic algorhytms. Clinical and laboratory data from patients hospitalized in the Department of Pediatrics were collected. Laboratory methods were provided in the cooperation with the Mitochondrial laboratory of the Department of Pediatrics and Institute of Inherited Metabolic Disorders. The study with lactate levels in 107 patients documented that brief seizures lasting less than 2 minutes did not increase lactate concentration in the CSF. CSF-lactate was a relialable marker in differential diagnosis in the children with mitochondrial disorders against children with epilepsy. 2. The severity of particular phenotype is more...

Dysfunction within complex I (CI) of the mitochondrial electron transport system has been implicated in a number of disease states ranging from cardiovascular diseases to neuro-ophthalmic indications. Herein, we provide three novel approaches to model and study the impacts of injury on the function of CI. Cardiovascular ischemia/reperfusion (I/R) injury has long been recognized as a leading contributor to CI dysfunction. Aside from the physical injury that occurs in the tissue during the ischemic period, the production of high levels of reactive oxygen species (ROS) upon reperfusion, led by reverse electron transport (RET) from CI, causes significant damage to the cell. With over 700,000 people in the US set to experience an ischemic cardiac event annually, the need for a pharmacological intervention is paramount. Unfortunately, current pharmacological approaches to treat I/R related injury are limited and the ones that have shown efficacy have often done so with mixed results. Among the current approaches to treat I/R injury antioxidants have shown some promise to help preserve mitochondrial function and assuage tissue death. The studies described herein have provided new, more physiologically matched, methods for assessing the impact of potential therapeutic interventions in I/R injury. With these methods we evaluated the efficacy of the coenzyme-Q derivative idebenone, a proposed antioxidant. Surprisingly, in both chemically induced models of I/R and I/R in the intact heart, we see no antioxidant-based mechanism for rescue. The mechanistic insight we gained from these models of I/R injury directed us to further examine CI dysfunction in greater detail. Through the use of two cutting edge genetic engineering approaches, CRISPR/Cas9 and Artificial Site-specific RNA Endonucleases (ASRE), we have been able to directly edit the mitochondria to accurately model CI dysfunction in disease. The use of these genetic engineering technologies have provided first in class methods for modeling three unique mitochondrial diseases. The culmination of these projects has provided tremendous insight into the role of CI in disease and have taken a significant step towards elucidating potential therapeutic avenues for targeting decrements in mitochondrial function. / Doctor of Philosophy / Within the mitochondria, “the powerhouse of the cell,” exists a series of five enzyme complexes that produce 90% of the energy for our cells need to function. The largest of these enzymes, complex I (CI), plays an important role in ensuring proper mitochondrial function. Injury to CI contributes to a number of diseases, but surprisingly few options exist to treat complex I. One of the most prevalent forms of CI dysfunction can be seen in ischemia/ reperfusion injury, a form of which is most commonly recognized as a heart attack. Surprisingly, the American Heart Association reports that in the next year over 700,000 people in the US will suffer from an ischemic event. With such a profound impact on the population, the need for new therapeutic developments is extremely high. Some current therapeutic approaches have been shown to be effective at treating cardiac dysfunction, but few address the dysfunction that occurs in the mitochondria. Here we test both a method for modeling these ischemia/reperfusion-based injuries and a potential therapeutic for treating these injuries within the context of CI dysfunction. We further evaluate CI dysfunction by using both established genetic engineering approaches as well as a completely new method to model CI disease. Through the use of two cutting edge genetic engineering approaches, we have been able to directly edit components of the mitochondria to accurately model CI dysfunction in disease. The use of these genetic engineering technologies have provided a first-in-class method for modeling three unique mitochondrial diseases. The culmination of these projects has provided tremendous insight into the role of CI in disease and have taken a significant step towards elucidating potential therapeutic avenues for targeting decrements in mitochondrial function.

Mitochondria are a key organelle within human cells, with functions ranging from ATP synthesis to apoptosis. Changes in mitochondrial function are associated with many diseases, as well as 'natural' processes like ageing. Mitochondria have a unique evolutionary origin, as the result of an endosymbiotic relationship between a bacterium and an archaeal cell. Therefore, the phylogenetic history of the mitochondrial proteome is also unique within the total human proteome. A new description of the genes encoding the human mitochondrial proteome - IMPI (Integrated Mitochondrial Protein Index) 2017 - provided an opportunity for exploration of mitochondrial proteome history and the application of this knowledge to the understanding of gene function, disease and ageing. To facilitate the exploration of the mitochondrial proteome, I created a manually curated dataset of 190,097 predicted orthologues of the 1,550 IMPI 2017 human genes across 359 species, using reciprocal best hit analysis as the basis for orthologue prediction. I used this to explore gene history and the potential for phylogenetic profiling to predict the function of uncharacterised genes. This inspired the use of phylogenetic profiling within two phyla of animals, to link presence and absence of metabolic genes to the function of mitochondrial transporters. Potential transport substrates were predicted for two groups of uncharacterised mitochondrial carriers. I also used the dataset to identify features of genes associated with monogenetic disease, as well as differences between recessive and dominant disease genes. A similar orthologue identification method was used to explore the total sequenced viral proteome for potential orthologues of mitochondrial proteins. This showed that a range of mitochondrial proteins are shared with viruses, potentially facilitating the co-opting of mitochondrial function during viral infection of eukaryotic cells. I then used orthology to explore the conservation of residues linked to protein acetylation and identify a link with lifespan in warm-blooded vertebrates. In conclusion, I have used orthology to further the understanding of human mitochondrial proteome history and developed applications of this information. For example, phylogenetic features of disease genes are being used as part of a wider pipeline to predict mitochondrial disease genes. Furthermore, predicted substrates of the SLC25A14/30 mitochondrial carriers are being tested. My dataset provides further opportunities to explore the evolution and function of the mitochondrion.