Cancer remains a global pandemic and is rapidly overtaking other diseases as the no.1 killer in developing nations. Photodynamic therapy (PDT) has been advanced as a minimally invasive cancer therapy. In addition, the emergence of harmful microbes with increasing resistance to drugs has prompted the employment of photodynamic antimicrobial chemotherapy (PACT) as a promising alternative to combat antibiotic resistance. In PDT and PACT, a photosensitizer (dye/drug) upon activation by light transfers energy to molecular oxygen producing singlet oxygen which kills cells. There is increased attention and research into more selective and non-aggregated photosensitizers that will better PDT in treating cancer. This research work is focused on design and synthesis of non-aggregated asymmetric phthalocyanines (dyes) tagged with mitochondrial targeting vehicles to maximize selectivity and photo-killing of tumor cells. Chapter 1 presents a brief review of the current status of PDT and treatment of cancer. The three components of PDT namely, light, oxygen and the photosensitizer, are briefly discussed giving a concise overview of the development of each of them in bettering PDT as an alternative to cancer therapy. Chapter 2 outlines the design, synthesis and characterization of two non-aggregated symmetric ZnPc isomers that have improved water solubility due to incorporation of triethylene glycol groups. The extension of the max absorption to near-IR via non-peripheral substitution on the Pc macrocycle is reported, while comparing the photophysical characteristics of both isomers. Chapter 3 details the improved selectivity of photosensitizers by conjugating ZnPcs to rhodamine B, a delocalized lipophilic cation, which targets the mitochondria of the cell. This conjugation achieved 70% more cell death suggesting that incorporation of rhodamine improved cellular uptake and localization of the photosensitizers which is crucial. Chapters 4 and 5 cover the design, synthesis, characterization, and photodynamic therapy evaluation of ZnPc and phosphorous phthalocyanines. Introduction of phosphorous as an electron deficient central atom promoted a 42 nm bathochromic shift relative to the corresponding ZnPc isomer. Additionally, the effect of peripheral and non-peripheral substitution on phototoxicity of these new compounds is studied and reported. Chapter 5 also gives concluding remarks, and future directions of this work.

Biogeographers have long searched for common processes responsible for driving diversification in the Holarctic region. Although terrestrial flora and fauna have been well studied, much of the marine biogeographic work addresses patterns and processes occurring over a relatively recent timescale. A prerequisite to comparative biogeographic analysis requires well-resolved phylogenies of similarly distributed taxa that diverged over a similar timeframe. The overall aim of my Ph.D. thesis was to address fundamental questions in the systematics and biogeography of a family of Holarctic fish (Osmeridae) and place these results in a broad comparative biogeographic framework. With eight conflicting morphological hypotheses, the northern hemisphere smelts have long been the subjects of systematic disagreement. In addition to the uncertainty in the interrelationships within this family, the relationship of the Osmeridae to several other families remains unclear. Using DNA sequence data from three mitochondrial and three nuclear genes from multiple individuals per species, I reconstructed the phylogenetic relationships among the 6 genera and 15 osmerid species. Phylogenetic reconstruction and divergence dating yielded a well-resolved phylogeny of the osmerid genera and revealed several interesting evolutionary patterns within the family: (1) Hypomesus chishimaensis and H. nipponensis individuals are not reciprocally monophyletic, suggesting that they are conspecific and H. chishimaensis is a recently evolved freshwater ecotype that invaded the Kuril Islands following the last glaciation, (2) The trans-Pacific sister relationships in Hypomesus based on lateral line scale counts are not supported, implying that this phenotype evolved in parallel on each side of the North Pacific Ocean, (3) The Plecoglossidae are the Osmeridae sister group, (4) Over half of the characters from previous studies show evidence of parallel evolution; however, 27 traits reflect ancestral relationships, (5) Multiple divergences within the Osmeridae date to both the mid-Miocene cooling period and the Pliocene Bering Seaway opening, suggesting these events were important in the evolution of these fishes, and (6) Divergences in many marine taxa for which dated phylogenies are available are also correlated with these time periods. Future research should target additional Holarctic marine taxa for further comparative analysis.

Les mitochondries sont les organites qui produisent la quasi-totalité de l'énergie consommée par les cellules animales. Une de leurs particularités est de posséder leur propre génome (ADN mitochondrial ou ADNmt) qui, avec la collaboration indispensable du génome du noyau (ADNnu), code pour les enzymes responsables de la production d'énergie. Pour que ces organites fonctionnent adéquatement, la co-adaptation des génomes nucléaire et mitochondrial a donc été fortement sélectionnée au cours de l'évolution. Chez les animaux, l'ADNmt est, contrairement à l'ADNnu, transmis uniquement par la mère. L'hypothèse principale pour expliquer cette transmission maternelle est qu'en ayant un seul type d'ADNmt, la cellule évite un conflit génomique dans l'association intime entre les protéines codées par l'ADNnu et l'ADNmt. Autrement dit, la sélection naturelle doit nécessairement prévenir toute modification du dialogue entre les mitochondries et le noyau, ce qui risquerait de provoquer l'apparition de phénotypes indésirables. Exceptionnellement, un seul système mitochondrial «défie» les lois naturelles de transmission des mitochondries chez les animaux, soit celui observé chez les moules marines et d'eau douce. Les espèces possédant ce système atypique, qui est désigné sous le nom de double transmission uniparentale (doubly uniparental inheritance ou DUI), sont caractérisées par la présence de 2 ADNmt distincts qui sont hérités de façon maternelle (ADNmt F) ou paternelle (ADNmt M). Généralement, les femelles ne contiennent que le génome F, tandis que les mâles contiennent le génome F dans leurs tissus somatiques et le génome M dans leur gonade. Les divergences observées entre les ADNmt F et M chez les moules peuvent atteindre plus de 20%. Les produits des deux différents ADN mitochondriaux peuvent-ils interagir normalement avec les protéines d'un seul système génétique nucléaire (en raison de la co-adaptation intergénomique), ou certaines fonctions mitochondriales ont-elles été compromises dans une ou l'autre des lignées (c'est-à-dire, lignée paternelle M ou maternelle F)? Le grand attrait du système de double transmission uniparentale est qu'il est le seul modèle animal où deux variantes mitochondriales fortement divergentes coexistent et s'adaptent simultanément à un génome nucléaire. Cette situation se traduit probablement par la conservation d'une intégrité relative de certaines portions du génome mitochondrial afin de maintenir une cohésion fonctionnelle dans les interactions structurales entre les peptides mitochondriaux et nucléaires. L'objectif principal de mon doctorat était d'étudier l'évolution moléculaire des génomes mitochondriaux F et M chez les espèces du genre Mytilus et de vérifier si les gènes nucléaires et les gènes F et M mitochondriaux qui interagissent pour former le système de la phosphorylation oxydative co-évoluent. Spécifiquement, l'objectif premier du chapitre 1 a été d'analyser les ADNmt M et F complets chez trois espèces de moules marines (Mytilus edulis, M trossulus, et M galloprovincialis) afin de vérifier si leur évolution dans un environnement nucléaire commun (ou différent) se traduit par la conservation (ou non) de certaines de leurs portions (c'est-à-dire, évolution congruente des génomes M et F). Pour ce faire, les ADNmt ont été amplifiés par PCR et séquencés à l'aide d'un séquenceur d'ADN automatique. Les principaux résultats ont effectivement démontré que le fait de co-exister dans un même environnement nucléaire contraint les ADNmt F et M à expérimenter des pressions sélectives similaires, ce qui se traduit par une évolution congruente des différentes portions des ADNmt M et F (c'est-à-dire la corrélation positive des patrons de taux de substitution protéiniques). L'objectif du chapitre 2 a été de déterminer, chez les spermatozoïdes, si le remplacement d'un génome M par un génome F avait un effet sur les capacités métaboliques des mitochondries. L'étude visait à déterminer si la co-évolution du génome nucléaire avec deux types d'ADNmt différents avait été favorisée dans une où l'autre des lignées. Pour ce faire, des dosages enzymatiques (activité des complexes mitochondriaux I+Ill, II et IV et de la citrate synthase) ont été réalisés par spectrophotométrie. Les résultats ont démontré que la combinaison «ADNmt récemment-masculinisé -ADNnu» (qui est essentiellement une combinaison ADNmtF -ADNnu) performe mieux au niveau enzymatique que la combinaison «ADNmt M -ADNnu». Le troisième volet a été consacré à l'étude de l'évolution moléculaire des protéines interactives du système énergétique (c'est-à-dire, produits nucléaires et mitochondriaux en contact physique) afin de vérifier si les portions génétiques qui interagissent co-évoluent. Pour ce faire, le gène nucléaire codant pour le transporteur mobile d'électron cytochrome c (CYC) et des gènes mitochondriaux et nucléaires codant pour des sous-unités protéiques des complexes III (ADNmt = CYTB; ADNnu = C3S6) et IV (ADNmt = COX1 et COX2; ADNnu = COX4) du système de la phosphorylation oxydative ont été amplifiés par PCR et séquencés à l'aide d'un séquenceur d'ADN automatique. Les données moléculaires semblent démontrer que la co-évolution entre le génome F et le génome nucléaire est mieux couplée, ce qui vient appuyer les résultats enzymatiques du chapitre 2. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : ADN mitochondrial, Génomique comparative, ADN nucléaire, Mytilus, Co-évolution intergénomique.

The lower oxygen limit (LOL) in plants marks the oxygen (O2) level where the metabolism shifts from being predominantly aerobic to anaerobic; recent work has shown that respiratory-based indicators of this metabolic shift are well-correlated with changes in chlorophyll a fluorescence signals. The physiological and biochemical changes at the root of this relationship have not been well-described in the literature. The processes involved are spatially separated: chlorophyll fluorescence is associated with the lightdependent reactions and emanates from the chloroplasts whereas aerobic respiration and fermentation occurs in the mitochondria and cytosol, respectively. Evidences outlined in this thesis are used to suggest the mechanistic link between these three regions of the cell is a fluid exchange of cellular reductant. When mitochondrial respiration is inhibited as a result of inadequate O2, used as a terminal electron acceptor, glycolytic reductant in the form of NADH accumulates in the cytosol. Reductant imbalances between the cytosol and organelles can be adjusted indirectly using translocators. Excess chloroplastic reductant is used to reduce the plastoquinone (PQ) pool via NADPH-dehydrogenase, a component of the chlororespiratory pathway, effectively decreasing the photochemical quenching (qP) capacity thereby inducing a switch from minimum fluorescence (Fo) to a higher relative fluorescence (F) value where qP < 1. Subjecting dark-adapted photosystems to low-intensity light increased Fo to a slightly higher F value due to a lightinduced reduction of the oxidized PQ pool when the O2 was above the LOL, but decreased F as a result of a PSI-driven oxidation of the already over-reduced PQ pool when the O2 was below the LOL. Low O2 was also shown to increase violaxanthin deepoxidation and non-photochemical quenching (qN), likely a reflection of the overreduced state of the photosystems and associated pH decrease. Dynamic controlled atmosphere (DCA) is a fluorescence-based controlled atmosphere (CA) system that sets the optimum atmosphere for fruits and vegetables based on a product’s fluorescence response. Experiments in this thesis on the relationship between O2, temperature, light, metabolism, pigmentation and chlorophyll fluorescence were used to interpret the physiology behind fluorescence changes, suggest improved DCA techniques and outline potentially profitable avenues for future research.

This thesis is a series of investigations into the regulation of skeletal muscle mitochondrial metabolism. Novel regulatory mechanisms regarding mitochondrial fatty acid oxidation are continually being identified and alterations in skeletal muscle mitochondrial metabolism have been implicated in the pathogenesis of type II diabetes (T2DM). Therefore, advancing our basic understanding of mitochondrial regulatory processes is required to provide insight into the progression of T2DM. In study one, the utilization of knockout mice for the putative mitochondrial fatty acid transport protein FAT/CD36, showed that mitochondrial FAT/CD36 plays a functional role in mitochondrial long chain fatty acid (LCFA) oxidation. Specifically, FAT/CD36 was found to be located on the outer mitochondrial membrane (OMM) upstream of acyl-CoA synthetase. In study two, it was observed that in rat muscle, malonyl-CoA (M-CoA) inhibition kinetics of carnitine palmitoyltransferase I (CPT-I) display a more physiological IC50 in permeabilzed muscle fibre bundles (PmFB) compared to isolated mitochondria. These data suggest that the cytoskeleton may have a role in regulating M-CoA inhibition. Additionally, a significant effect of LCFA-CoA on M-CoA inhibition kinetics was observed. These data indicate that M-CoA content does not need to decrease to promote an increase in CPT-I flux. Finally, in a model of T2DM (ZDF rat), submaximal ADP-stimulated respiration rates and the content of adenine nucleotide translocase 2 (ANT2) content were depressed compared to lean control animals. Resveratrol treatment in ZDF rats recovered these declines concomitantly with improving insulin-stimulated skeletal muscle glucose uptake and the cellular redox state. A number of novel findings are presented, specifically, 1) a functional role for mitochondrial FAT/CD36 in mitochondrial LCFA oxidation was confirmed and the topology of this protein along the OMM is expanded upon, 2) M-CoA inhibition kinetics of CPT-I were re-evaluated in PmFB and a regulatory role of LCFA-CoA on M-CoA inhibition kinetics is established, and 3) submaximal ADP-stimulated respiration rates and ANT2 content are depressed in the ZDF rat and resveratrol supplementation prevents these decrements.

No description available.

The number of non-parasitic (brook) lamprey species in the genus Lampetra is underestimated since isolated populations are generally considered one species due to their relatively conserved body form. The phylogeographic and phylogenetic structure was estimated among and within Lampetra species along the Pacific coast of North America (presumed to represent Lampetra richardsoni; L. pacifica – which is currently regarded as a junior synonym of L. richardsoni; L. ayresii; and L. hubbsi) using up to three mitochondrial and three nuclear genetic markers. These data show that L. richardsoni as currently recognized is polyphyletic when lampreys (some of which show up to 8 K2P% sequence divergence) from Siuslaw River and Fourmile Creek (Oregon) and Mark West, Paynes, and Kelsey creeks (California) are included; Lampetra pacifica is a valid species; the population from Kelsey Creek almost certainly represents a new species; and those from Siuslaw, Fourmile, and Mark West may also be distinct species.

The mitochondrial small-subunit ribosomal RNA (mt. SSU rRNA = rns) gene appears to be a reservoir for a number of group I and II introns along with the intron- encoded proteins (IEPs) such as homing endonucleases (HEases) and reverse transcriptases. The key objective for this thesis was to examine the rns gene among different groups of ophiostomatoid fungi for the presence of introns and IEPs. Overall the distribution of the introns does not appear to follow evolutionary lineages suggesting the possibility of rare horizontal gains and frequent loses. Some of the novel findings of this work were the discovery of a twintron complex inserted at position S1247 within the rns gene, here a group IIA1 intron invaded the ORF embedded within a group IC2 intron. Another new element was discovered within strains of Ophiostoma minus where a group II introns has inserted at the rns position S379; the mS379 intron represents the first mitochondrial group II intron that has an RT-ORF encoded outside Domain IV and it is the first intron reported to at position S379. The rns gene of O. minus WIN(M)371 was found to be interrupted with a group IC2 intron at position mS569 and a group IIB1 intron at position mS952 and they both encode double motif LAGLIDADG HEases referred as I-OmiI and I-OmiII respectively. These IEPs were examined in more detail to evaluate if these proteins represent functional HEases. To express I-OmiI and I-OmiII in Escherichia. coli, a codon-optimized versions of I-OmiI and I-OmiII sequences were synthesized based on differences between the fungal mitochondrial and bacterial genetic code. The optimized I-OmiI and I-OmiII sequences were cloned in the pET200/D TOPO expression vector system and transformed into E. coli BL21 (DE3). These two proteins were biochemically characterized and the results showed that: both I-OmiI and I-OmiII are functional HEases. Detailed data for I-OmiII showed that this endonuclease cleaves the target site two nucleotides upstream of the intron insertion site generating 4 nucleotide 3’overhangs.

The use of mouthwash is a common complement to oral care. However, the physiological implication of this use, besides of effects on oral hygiene, is poorly known. The research of the gut micro flora and its implications on the host is a very active area of research today. Many important connections between the gut micro flora and obesity and diabetes have been found. These billions of bacteria are part of the immune system, they produce essential vitamins and they make inaccessible polysaccharides more digestible to the host, just to mention a few of their symbiotic roles for the host. A less explored area is the micro flora in the oral cavity. On the back of the tongue, anaerobic bacteria can reduce dietary nitrate to nitrite which then further can be reduced to nitric oxide, NO. NO is important in several important biological functions, e.g. as a signal substance, vasoregulation, mucus production and antibacterial effects. Vegetables as beetroot and spinach are dietary sources with a high nitrate content. Also drinking water and processed meats can be of relevance. Nitrite is added to processed meat for the prevention of botulism but also adds taste and color.   Experiments on humans indicate that mitochondrial efficiency increases after nitrate load, manifested as a decreased oxygen demand during physical exercise. This can also be relevant under conditions where the mitochondrial function is impaired, such as in diabetes and cardiovascular diseases. First a pilot study was made to evaluate the nitrate reducing effect from the antibacterial mouthwash. The mouthwash proved very effective. The concentrations of nitrate and nitrite in saliva was analyzed by HPLC and saliva from the antibacterial treatment showed greatly reduced concentrations of nitrite and high concentrations of nitrate. Saliva from placebo mouthwash showed high concentrations of nitrite and low concentrations of nitrate as expected. To study the importance of oral bacteria on metabolism, we performed a randomized, cross-over double-blinded study with 19 healthy males between 22-43 years. During two separate three-day periods they used an antibacterial and placebo mouthwash, respectively. On the fourth day their basal metabolic rate (BMR) was measured with an indirect calorimetric system. Moreover, samples from saliva, urine and blood were collected but these results are not included in this thesis. An earlier, unpublished study has demonstrated that nitrate administration reduces the basal metabolic rate. Accordingly, our aim was to study potential effects on the basal metabolic rate following reduction of the number of oral bacteria by aid of antibacterial mouthwash. Our hypothesis was that the reduced availability of nitrite would decrease the availability of NO in the body and manifest as an increased basal metabolic rate. The results from indirect calorimetry measurements showed no significant difference between placebo and antibacterial mouthwash, but there may be confounding factors. Further study is needed to assess the potential effects on host metabolism by these bacteria.

The objective - To identify Zemaitukai mtDNA D-loop region and genetic relationships between Zemaitukai and some other breeds based on mtDNA sequence variation. Methods - To perform a phylogenetic analysis of 10 Zemaitukai, mtDNA D-loop 49 horses in the 20 different horse breeds sequence were included from GenBank (http://www.ncbi.nlm.nih.gov/GenBank). DNA was extracted from hair roots using the DNEasy® Tissue Kit (Qiagen). 1,280 bp fragment of mtDNA D-loop were amplify out in PTC-100™ termocycler. Amplified products were sequenced on a LI-COR® 4200S-2 automated sequencer. Sequencher v 4.1.4. software package was used to generate the actual DNA sequence for each of the animals. Multiple alignments of sequences were performed with CLUSTAL X 1.8 (Thompson et al., 1997). The Neighbor-joining tree (Saitou and Nei, 1987) of mtDNA sequences was constructed from Jukes-Cantor distances, performed on the pairwise deletion using the MEGA software (Kumar et al., 1993). Results –Within Zemaitukai breed 19 polymorphic sites were detected and 6 haplotypes. At the individual level 4 zemaitukai haplotypes were private to one individuals, 1 haplotype-between 2 individuals and 1 haplotype was shared between 4 individuals. On the Neighbor-joining tree showed one Zemaitukai haplotype closer genetic relationships to the Icelandic and to the Norwegian Fiord horse haplotypes. Zemaitukai breed formed a separate branch with the ancient DNA haplotype. The phylogenetic analysis reflects the presence... [to full text]