This thesis focuses on toxicity and occurrence of hydroxylated polybrominated diphenyl ethers (OH-PBDEs) in Baltic Sea biota. The aims were to assess OH-PBDEs potency for disruption of oxidative phosphorylation (OXPHOS) and determine their and related compounds exposure in Baltic blue mussel, herring and long-tailed duck. A method for analysis of OH-PBDEs in herring and long-tailed duck plasma was also evaluated. Relevant OH-PBDEs were tested in vitro for OXPHOS disruption, using a classic rat mitochondrial respiration assay and a cell mitochondrial membrane potential assay. All compounds were found to disrupt OXPHOS either by protonophoric uncoupling and/or via inhibition of the electron transport chain. 6-OH-BDE47 and 6-OH-BDE85, were identified as particularly potent OXPHOS disruptors. Strong synergism was observed when OH-PBDEs were tested as a mixture corresponding to what is present in Baltic blue mussels. Baltic blue mussel is main feed for several species of mussel feeding sea ducks which have decreased dramatically in numbers. To assess long-tailed ducks exposure to brominated substances, liver tissue from long-tailed ducks wintering in the Baltic Sea and blue mussels were analysed. The result confirms that long-tailed duck are exposed to OH-PBDEs via their diet. However, low concentrations were found in the duck livers, which suggest low retention of these compounds despite daily intake. How the nutritional value of blue mussels as feed for sea ducks are affected by OH-PBDE exposure still needs further studies. Other species of sea ducks foraging on Baltic blue mussels during summer months can also be more exposed due to seasonal variation in primary production. Herring sampled in the Baltic Proper and Bothnian Sea, were found to contain OH-PBDEs and high levels of their methylated counterpart, MeO-PBDEs. As demethylation of MeO-PBDEs is known to occur in fish, MeO-PBDEs may pose as additional source for more toxic OH-PBDEs in herring and their roe. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.</p>

Metallothioneins (MTs) are small, non-enzymatic proteins that are involved in cellular detoxification and metal homeostasis because of their high cysteine content. MTs have also been identified as one of the vast number of adaptive responses to mitochondrial respiratory chain (RC) deficiencies. Aside from this, numerous other studies have linked MTs to several mitochondrion-linked components, including reactive oxygen species (ROS) and oxidative stress, apoptosis, glutathione, energy metabolism and nuclear- and mitochondrial DNA transcription regulation. However, most of the reports concerning the putative link between MTs and mitochondria are from in vitro studies and relatively little supportive in vivo evidence has been reported. Information on the involvement of MTs with respiratory chain function is especially limited. Is was therefore the aim of this study to investigate the involvement of MTs in mitochondrial respiration and respiratory chain enzyme function by using an MT knockout (MTKO) mouse model, which was treated with the irreversible complex I inhibiting reagent, rotenone. The aim was achieved by implementing three objectives: firstly, the RC function was investigated as a complete working unit; secondly, the functional and structural properties of single units (enzymes) of the RC were investigated utilising enzyme activity assays and BN- PAGE/western blot analysis; and thirdly, the possible effect of MTs on mtDNA copy number was investigated. While some tendencies of variation in RC enzyme activity and expression were identified, no significant effect on the overall mitochondrial respiratory function, or any significant differences in the relative mtDNA copy number of MTKO mice were observed. Thus it is concluded, while MTs have in this study revealed relatively small changes in respiratory chain function, which may still prove to have biological ignificance in vivo, the exact nature of the putative role of MTs in mitochondrial respiration or oxidative phosphorylation remains undefined. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2012.

Metallothioneins (MTs) are small, non-enzymatic proteins that are involved in cellular detoxification and metal homeostasis because of their high cysteine content. MTs have also been identified as one of the vast number of adaptive responses to mitochondrial respiratory chain (RC) deficiencies. Aside from this, numerous other studies have linked MTs to several mitochondrion-linked components, including reactive oxygen species (ROS) and oxidative stress, apoptosis, glutathione, energy metabolism and nuclear- and mitochondrial DNA transcription regulation. However, most of the reports concerning the putative link between MTs and mitochondria are from in vitro studies and relatively little supportive in vivo evidence has been reported. Information on the involvement of MTs with respiratory chain function is especially limited. Is was therefore the aim of this study to investigate the involvement of MTs in mitochondrial respiration and respiratory chain enzyme function by using an MT knockout (MTKO) mouse model, which was treated with the irreversible complex I inhibiting reagent, rotenone. The aim was achieved by implementing three objectives: firstly, the RC function was investigated as a complete working unit; secondly, the functional and structural properties of single units (enzymes) of the RC were investigated utilising enzyme activity assays and BN- PAGE/western blot analysis; and thirdly, the possible effect of MTs on mtDNA copy number was investigated. While some tendencies of variation in RC enzyme activity and expression were identified, no significant effect on the overall mitochondrial respiratory function, or any significant differences in the relative mtDNA copy number of MTKO mice were observed. Thus it is concluded, while MTs have in this study revealed relatively small changes in respiratory chain function, which may still prove to have biological ignificance in vivo, the exact nature of the putative role of MTs in mitochondrial respiration or oxidative phosphorylation remains undefined. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2012.

Mood disorders are serious diseases. Nevertheless, their pathophysiology is not sufficiently clarified. Biological markers that would facilitate the diagnosis or successful prediction of pharmacotherapy are still being sought. The aim of the study was to find out whether mitochondrial functions are affected by antidepressants, mood stabilizers and depression. Our research is based on recent hypotheses of mood disorders, the advanced monoamine hypothesis, the neurotrophic hypothesis, and the mitochondrial dysfunction hypothesis. We assume that impaired function of mitochondria leads to neuronal damage and can be related to the origin of mood disorders. Effects of antidepressants and mood stabilizers on mitochondrial functions can be related to their therapeutic or side effects. In vitro effects of pharmacologically different antidepressants and mood stabilizers on the activities of mitochondrial enzymes were measured in mitochondria isolated from pig brains (in vitro model). Activity of monoamine oxidase (MAO) isoforms was determined radiochemically, activities of other mitochondrial enzymes were measured spectrophotometrically. Overall activity of the system of oxidative phosphorylation was measured electrochemically using high- resolution respirometry. Methods were modified to measure the same...

Apesar da melhoria no sistema de produção e cultivo dos embriões in vitro, cerca de 60% dos oócitos que entram no sistema, não atingem o estágio de blastocisto e a qualidade dos embriões obtidos é bastante variável quando comparadas com embriões produzidos in vivo. Este bloqueio pode ser afetado por íons inorgânicos, tampões, aminoácidos e composição da atmosfera gasosa. Partindo-se da premissa que há influência da mitocôndria sobre a ativação da morte celular programada levou-nos a formular a hipótese que ausência de fragmentação nuclear nos embriões antes das 72 hpi está relacionada com a ausência do potencial de membrana mitocondrial e a inibição da OXPHOS pela utilização de bloqueadores leva a manutenção de baixos níveis de potencial de membrana mitocondrial e baixas taxas de fragmentação nuclear nos embriões. Embriões foram produzidos in vitro mediante maturação durante 22 horas, fecundação e cultivo 18 horas após a inseminação (hpi). Decorridas 24hpi realizou-se o cultivo com 0% de oxigênio, a fim de bloquear o processo de OXPHOS. Após 48 hpi realizou-se o feeding do meio de cultivo (SOF) com inibidores da OXPHOS (antimicina A e/ou oligomicina, cianeto de potássio) em diferentes doses. O número de embriões 8 células foi determinado às 80 hpi, mesmo momento em que foram realizadas as técnicas de JC-1 e TUNEL. Verificou-se as 168 hpi o efeito dos tratamentos no desenvolvimento embrionário. Os resultados obtidos com a inibição da OXPHOS após 48 hpi com oligomicina e/ou antimicina A nas doses utilizadas não alterou a capacidade do embrião atingir o estádio de 8 células. Entretanto, esta inibição inviabilizou o desenvolvimento até o estádio de blastocisto. O tratamento com KCN permitiu o desenvolvimento até o estádio de 8 células e a blastocisto em taxas semelhantes ao controle. A inibição do cultivo na ausência do O2 inviabilizou o processo de cultivo. Já os resultados obtidos quanto ao &#936;mm e TUNEL evidenciam que os tratamentos dos embriões antimicina e/ou oligomicina levaram a um aumento do &#936;mm e fragmentação nuclear na maioria dos embriões testados. Portanto, não foi possível testar a hipótese de que o &#936;mm é necessário para o estabelecimento da MCP, todavia, foi observada uma correlação positiva entre &#936;mm e fragmentação nuclear. / Although in vitro embryo production has been improved in the last 2 decades, about 60% of the oocytes do not reach the blastocyst stage and embryo quality is very variable when compared with in vivo produced embryo. This developmental block can be affected by inorganic ions, buffers, aminoacids and gaseous atmosphere composition. The knowledge that there influence of mitochondria on the activation of the programmed cellular death led to formulate the hypothesis that nuclear fragmentation absence in embryos before 72 post insemination is related with absence of mitochondrial membrane potential and OXPHOS blockage by inhibiting agents, would cause the maintenance of low levels of mitochondrial membrane potential and low rates of embryo nuclear fragmentation. Embryos were cultured in vitro for 18 hours post insemination (hpi) and after 24 hpi, they were submitted to 0% oxygen culture, in order to block the OXPHOS process. At 48 hpi, feeding was performed with SOF medium containing OXPHOS inhibitors (antimycin A and/or oligomycin, potassium cyanide) in different concetrations. The numbers of 8 cell embryos were estimated at 80 hpi, the same moment that they were submitted to JC-1 probes and TUNEL for mitochondrial membrane potential and DNA damages evaluation, respectively. At 168 hpi the effect of the treatments was verified on embryonic development. The results obtained with the OXPHOS inhibition after 48 after hpi using oligomycin and/or antimycin A did not modify embryo capacity to reach 8 cell stage. However, this inhibition prevented development to the blastocyst stage. KCN treatment allowed development up to the 8 cell stage and blastocyst similar to controls. The absence of O2 prevented embryo development. The &#936;mm and TUNEL results showed that antimycin and/or oligomycin treatment increased &#936;mm and nuclear fragmentation in the majority of the embryos tested. In conclusion, it was not possible to test the hypothesis that &#936;mm is necessary to the establishment of MCP, but a positive correlation between &#936;mm and nuclear fragmentation was observed.

Human essential hypertension affects over 75 million people in the United States, and can lead to death due to its several serious health complications such as hypertension-related cardiovascular disease. The purpose of this research was to understand how hypertension could cause physiological changes to the microcirculation, specifically the PO2 dependence of oxygen consumption (VO2) in skeletal muscle of normotensive and hypertensive rats. The Spontaneously Hypertensive Rat (SHR) strain was used as the diseased model, and Wistar-Kyoto (WKY) rats were used as controls to conduct this study. The SHR strain develops hypertension between 5-6 weeks after birth with an average systolic blood pressure of 150 mmHg. By arresting blood flow using an objective-mounted inflatable airbag, PO2 measurements were obtained along with an oxygen disappearance curve (ODC), which was used to calculate VO2 over various ranges of physiological PO2 values. PO2 and VO2 curves were analyzed based on Hill’s equation to fit the data and describe the PO2 dependence of VO2. When compared to the healthy Wistar-Kyoto rats, the SHRs exhibited a higher Vmax, or maximum rate of oxygen consumption. The average maximal rate of consumption by the hypertensive animal models could be a consequence of a “mitochondrial uncoupling” or some disconnect in the mitochondrial oxygen consumption and the normal corresponding ATP production. In conclusion, this project demonstrated that in situ muscle tissue from hypertensive and normotensive rats had a PO2 dependence of oxygen consumption over a wide range of physiological PO2 values and the hypertensive rats consumed oxygen at a higher maximal rate.

Philosophiae Doctor - PhD / Numerous studies on mammalian spermatozoa have reported large variations in the dimensions of the main sperm structural components, namely the head, midpiece and flagellum. These variations in sperm architecture are believed to be adaptations for functioning of spermatozoa in complex environments outside the male reproductive system. The midpiece of the mammalian permatozoon contains a varied number of mitochondria, but the reason for the marked difference in the size and structure of this sperm component is not clear. This study confirmed the variations in the sperm morphometry of seven selected mammalian species and revealed unique features of the sperm midpiece and sperm mitochondria of these seven species. Evaluation of several sperm kinematic parameters revealed the unique swimming characteristics of the different spermatozoa. The importance of using standardized motility parameters was highlighted as well as the assessment of different subpopulations of spermatozoa in order to produce more reliable and comparable data. Investigating the role of sperm mitochondria in human sperm metabolism indicated that these organelles are related to sperm function in terms of sperm motility. Furthermore, it was suggested that glycolysis and mitochondrial respiration are linked processes and that both are important for the maintenance of human sperm motility. By optimizing and employing standardized experimental procedures and analysis techniques, this study was able to confirm the species specificity of almost all the sperm parameters evaluated, while also elucidating the phylogenetic relatedness of the non-human primate species. In conclusion, the present study has confirmed that the various midpiece morphometry parameters are related to the remaining sperm morphometry parameters as well as to the sperm kinematic parameters. These proposed associations between the various sperm parameters were used to explain the sperm velocity of two hypothetical and morphologically different sperm structures. Therefore, the results of the current study support the idea of co-evolution between sperm components in mammalian spermatozoa and propose that the midpiece morphometry parameters that are selected for in these spermatozoa are midpiece volume, total number of mitochondrial gyres, thickness of the mitochondrial sheath and mitochondrial height. / South Africa

Huntington's disease (HD) is an autosomal dominant hereditary neurodegenerative disease characterized by motor, cognitive and behavioral disorders. HD is caused by expansion of CAG triplet (cytosine-adenosine-guanine) located in a gene on the short arm of the fourth chromosome. This expansion encodes an aberrant polyglutamine chain in the protein huntingtin. Physiological and mutated huntingtin (in case of HD) are expressed in almost all tissues and influences many cellular functions. The prevalence of HD in population is about 1 per 10.000. The disease is currently incurable and its mechanisms are not sufficiently understood. Besides affecting the central nervous system HD also affects peripheral tissues, including skeletal muscles. HD disrupts mitochondrial function and damages oxidative phosphorylation system, which has the task of producing energy in the form of ATP in cells. Research of transgenic minipig model for HD could help elucidate the mechanisms of disease's pathogenesis and potential therapeutic strategy. In this diploma thesis, immunodetection with help of specific antibodies to detect changes in amount of 14 selected mitochondrial proteins in skeletal muscle tissue of three age groups of transgenic HD minipigs - 24, 36 and 48 months old was used. Gradual progression in reduced...

The evolution and adaptation of microorganisms is so rapid it can be seen in the time frame of days. The root cause for their evolution comes from selective environmental pressures that see organisms with beneficial mutations survive otherwise deadly encounters or outperform members of its population who fail to adapt. This does not always result in strict improvement of the individual as in the case of antibiotic resistant bacteria who often display fitness tradeoffs to avoid death (see Reviews [1-3]). For example, when an ampicillin resistance gene (ampC) containing plasmid that is occasionally found in the wild was transformed into S. typhimurium the bacteria had slower growth and impaired invasiveness [4]. In another example, capreomycin use with mycobacteria resulted in lower binding of the drug to the ribosome through mutations in rRNA methylase TlyA 16S rRNA, which decreases the overall fitness of the mycobacteria [5]. The evolutionary interactomes between bacteria and antibiotics do not end there, as antibiotic resistant bacteria often accumulate compensatory mechanisms to regain fitness. These range in effect with some altering individual cellular pathways and others having systemic affects [1]. My work has focused on the intersection of diabetes and related antibiotic resistant bacterial infections. Diabetes is one of the leading health issues in the United States, with over 10% of the adult population and over 26% of the elderly diagnosed (American Diabetes Association) [6]. Herein I further characterize the molecular pathways involved in diabetes, through the study of PAS kinase (PASK) function. PAS kinase is a serine-threonine protein kinase which regulates the pathways disrupted in diabetes, namely triglyceride accumulation, metabolic rate (respiration), adiposity and insulin production and sensitivity [7-9]. In this study I specifically focus on the effects of PAS kinase and its substrate, USF1/Cbf1p, and how their altered metabolic deficiencies can be suppressed using yeast cells. Through this study I further characterized the molecular function of USF1/Cbf1p through the identification of putative co-transcriptional regulators, identify novel genes involved in the regulation of respiration, and uncover a function or a previous uncharacterized protein, Pal1p. Part of the diabetes healthcare challenge results from the wide range of diseases that are associated with diabetes, including obesity [10, 11], renal failure [12, 13], neuropathies and neurodegeneration [14, 15], endocrine dysfunctions [16, 17], and cancers [18]. In addition, diabetes is a leading cause of lower limb amputations, due to poor circulation and the prevalence of ulcers [19-21], many of which are antibiotic resistant [22-25]. Phage therapy, based on the administration of bacterial viruses, is a viable option for the treatment of these diseases, with our lab recently isolating bacteriophages for several clinical cases. In the second half of my thesis, I present the study of the adaptation of bacteriophages to their hosts as well as report contributions of local ecology to their evolution.

The complexes of the oxidative phosphorylation (OXPHOS) system in the inner mitochondrial membrane are organised into structural and functional super-assemblies, so-called supercomplexes. This type of organisation enables substrate channelling and hence improves the overall OXPHOS efficiency. ATP synthase associates into dimers and higher oligomers. Within the supercomplex of ATP synthasome, it interacts with ADP/ATP translocase (ANT), which exchanges synthesised ATP for cytosolic ADP, and inorganic phosphate carrier (PiC), which imports phosphate into the mitochondrial matrix. The existence of this supercomplex is generally accepted. Experimental evidence is however still lacking. In this thesis, structural interactions between ATP synthase, ANT and PiC were studied in detail. In addition, the interdependence of their expression was examined either under physiological conditions in rat tissues or using model cell lines with ATP synthase deficiencies of different origin. Specifically, they included mutations in the nuclear genes ATP5E and TMEM70 that code for subunit ε and the ancillary factor of ATP synthase biogenesis TMEM70, respectively, and a microdeletion at the interface of genes MT-ATP6 and MT-COX3 that impairs the mitochondrial translation of both subunit a of ATP synthase and subunit Cox3...