付記する学位プログラム名: 霊長類学・ワイルドライフサイエンス・リーディング大学院 / 京都大学 / 新制・課程博士 / 博士(理学) / 甲第24471号 / 理博第4970号 / 新制||理||1709(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 伊谷 原一, 教授 平田 聡 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Pantosteus, a subgenus of Catostomus, includes the mountain sucker (Catostomus playthyrnchus), whose speculated older origins in the Miocene/Pliocene can provide insight into the ancient geographical events of western North America. We believe that major geologic events influencing the diversification of mountain suckers include the rise of the Colorado Plateau, the connections between the ancient Snake River system and the Lahontan system and subsequently the connection of the Snake River system to the Columbia Basin, dispersal of mountain suckers across the continental divide, as well as the Pleistocene Bonneville flood. If this is true, we should see evidence of geologic separation and timing through studying the phylogenetics of the mountain sucker. In order to clarify relationships of the mountain sucker with respect to other Pantosteus species, we examined cytochrome b (cyt b) sequences for 144 mountain suckers, 24 other Pantosteus species, and ten outgroup species. Phylogenetic relationships among haplotypes were constructed based on maximum likelihood and Bayesian criterion. In an effort to provide better resolution at some nodes, we also sequenced additional mitochondrial genes (ND1, ND2, ATPase, ND4L, ND4, ND5, ND6, and cyt b) for a subset of 44 individuals taken from the major clades obtained from the cyt b phylogentic analyses. Trees from this data set were also constructed under maximum likelihood and Bayesian criterion. All phylogenetic analysis revealed that mountain sucker are paraphyletic, with two major clades of mountain suckers separated by other members of the subgenus Pantosteus. One clade included two sub-clades, one from the upper Snake River drainage/northern Bonneville/Green River drainage Basins and the other from the southern Bonneville Basin. The other major clade included sub-clades from the Lahontan Basin, Columbia River Basin, and Upper Missouri River Basin. Molecular clock analysis revealed that Pantosteus likely split from Catostomus during the Miocene and that major speciation events within Pantosteus occurred during the Pliocene and Pleistocene. Genetic structuring and gene flow estimates for mountain sucker populations, with groupings based on major drainage basins, were calculated with AMOVA and Fst estimates in Arlequin and revealed that most of the genetic structuring was explained by variation among drainage basins with limited gene flow occurring between drainage basins. Based on this study, the role of the Colorado Plateau's geologic history in the evolution of the mountain sucker remains unclear. However, all other geologic events as discussed in this study seem to have played a significant role in the evolution of the mountain sucker.

The genetic structure of juvenile green turtles (Chelonia mydas) foraging on the east coast of central Florida is not well understood, nor has it been examined over time. In the last three decades, the dramatic increase in the number of green sea turtle nests in Florida, in association with other population parameters, has led to this species being down-listed under the Endangered Species Act from “endangered” to “threatened” in the northwest Atlantic. However, it was unclear if the exponential growth in Florida nest numbers had any influence on the genetic structure of juveniles in nearby foraging aggregations. To understand this potential impact mixed-stock analysis was conducted using mitochondrial DNA fragments that were over 800 base pairs long on samples taken from juveniles captured from 2002-2005 and 2016-2018 in the central Indian River Lagoon and Trident Submarine Basin in Port Canaveral. Results indicate the sampled foraging sites are genetically distinct habitats. In both sites, recruitment from Florida nesting beaches remained low despite increases in nesting while contributions from rookeries in Costa Rica and Mexico dominated both foraging aggregations across time. Haplotype diversity and nucleotide diversity decreased at both foraging sites over time. The foraging sites shared the two most frequently occurring haplotypes, but also had haplotypes that were unique to the site or sample period. Our results highlight the need for broader sampling of rookeries and foraging aggregations to understand the impacts of nesting increases in one rookery on juvenile diversity. Future studies should include all life stages of green turtles to enhance understanding of both the census population and effective population to better inform conservation policies necessary for a continued recovery.

No description available.

No description available.

Fibromyalgi (FM) är en kronisk sjukdom där hjärnans förmåga att hantera smärtsignaler betraktas vara rubbad. Symtomen omfattar bl.a. smärta i minst 11 av 18 smärtpunkter och annan värk, ofunktionell sömn, stelhet, trötthet, svaghetskänsla i musklerna, hjärtarytmi, ömhet och stickningskänsla i huden. Symptomen kan vara relaterade till musklerna, endokrina och immunologiska system. FM saknar diagnostiska markörer som kan analyseras och användas för diagnostik av den kroniska sjukdomen. Syftet med denna pilotstudie är att studera några biomarkörer i saliv som samlats i Salivette® från kvinnliga FM patienter, och relatera resultatet till den av patienten upplevda smärtan. Patienter och kontrollgrupp (KG) i studien (FM; n=7, 43±12 år och KG; n=6, 42±14 år) fick besvara frågor gällande bl.a. medicinering, smärta och infektion. Biomarkörer som kvantifierades var fritt cirkulerande mtDNA, urinsyra och IgA. Totalprotein analyserades för att normalisera IgA värdet. Elektrolytanalys utfördes för att granska salivprovets rimlighet. Resultaten visade att medelvärdena av nämnda biomarkörer är högre hos FM patienter än hos KG. Regressionen mellan IgA och urinsyra hos FM är svagt positiv. Det finns ingen korrelation mellan kvoten IgA/totalprotein och urinsyra hos FM. pH i saliv bland FM var lägre än hos KG. Medelvärdet av prov från dag ett och två hos FM i förhållande till den upplevda smärtan under den senaste veckan visar inga signifikanta samband relaterat till kvantifierade biomarkörer. För utveckling av denna studie och inom området krävs det en bättre standardisering av forskningsmetoderna, ett större antal deltagare och en utökad förståelse om sjukdomens variation hos patienter beroende på livsstil och anamnes. / Fibromyalgia (FM) is a chronic disease in which the brain's ability to manage pain signals are considered to be disturbed. Symptoms include pain in at least 11 of 18 tender points and other pain, non-functional sleep, stiffness, fatigue, weakness in the muscles, cardiac arrhythmia, tenderness and knitting feelings in the skin. The symptoms may be related to the muscles, endocrine and immune system. FM lack of diagnostic markers that can be analyzed and used for the diagnosis of the chronic disease. The purpose of this pilot study is to study some biomarkers in saliva collected with Salivette® by female FM patients, and relate the results to the perceived pain by the patient. Patients and control group (KG) in the study (FM; n = 7, 43±12 years KG; n = 6, 42±14 years) were asked to answer questions regarding e.g. medication, pain and infections. The quantified biomarkers are free circulating mtDNA, uric acid and IgA. Total protein was analyzed for the normalization of the IgA value. Electrolyte analysis was performed to examine the plausibility of the sample. The results showed that the average values of the biomarkers is higher for FM patients than for KG. The regression between IgA and uric acid of the FM is slightly positive. There is no correlation between the ratio of IgA /total protein and uric acid of the FM. The saliva pH among FM was lower than in KG. The average of samples from day one and two of the FM in relation to the perceived pain during the last week show no significant associations related to the quantified biomarkers. For the development of this study and in this area it is required a better standardization of research methods, a larger number of participants and an increased understanding of the disease variability in patients depending on lifestyle and medical history.

MtDNA mutations in mammalian cells are implicated in cellular ageing and encephalomyopathies, although mechanisms involved are not completely understood. The mitochondrial genetic bottleneck has puzzled biologists for a long time. Approximate models of genetic bottleneck proposed in the literature do not accurately model underlying biology. Recent studies indicate mitochondrial morphology changes during cellular aging in culture. In particular, the rates of mitochondrial fission and fusion are shown to be in tight balance, though this rate decreases with age. Some proteins involved in mitochondrial morphology maintenance are implicated in apoptosis. Hence, mitochondrial genetic and morphologic dynamics are critical to the life and death of cells. By working closely with experimental collaborators and by utilizing data derived from literature, we have developed stochastic simulation models of mitochondrial genetic and morphologic dynamics. Hypotheses from the mitochondrial genetic dynamics model include: (1) the decay of mtDNA heteroplasmy in blood is exponential and not linear as reported in literature. (2) Blood heteroplasmy measurements are a good proxy for the blood stem cell heteroplasmy. (3) By analyzing our simulation results in tandem with published longitudinal clinical data, we propose for the first time, a way to correct for the patient's age in the analysis of heteroplasmy data. (4) We develop a direct model of the genetic bottleneck process during mouse embryogenesis. (5) Partitioning of mtDNA into daughter cells during blastocyst formation and relaxed replication of mtDNA during the exponential growth phase of primordial germ cells leads to the variation in heteroplasmy inherited by offspring from the same mother. (6) We develop a “simulation control” for experimental studies on mtDNA heteroplasmy variation in cell cultures. Hypothesis from the mitochondrial morphologic dynamics model: (7) A cell adjusts the mitochondrial fusion rate to compensate for the fluctuations in the fission rate, but not vice versa. A deterministic model for this control is proposed. Contributions: extensible simulation models of mitochondrial genetic and morphologic dynamics to aide in the powerful analysis of published and new experimental data. Our results have direct relevance to cell biology and clinical diagnosis. The work also illustrates scientific success by tight integration of theory with practice. / Ph. D.

The study of the process of speciation is instrumental to understanding the species diversity observed today. Diverging populations are intriguing, because speciation has not reached an endpoint, yet the process that may eventually lead to distinct species can be studied. Systems that contain many putative species and/or parallel divergences, such as many species flocks and species pairs, are extraordinary examples of divergence and therefore are critical to the understanding of the speciation process. A "miniature" species flock of pupfish (Cyprinodon variegatus) discovered in lakes on San Salvador Island, Bahamas has evolved in less than 6 000 years, and is, therefore, important to the study of the pace of evolutionary processes. The San Salvador Island pupfish flock is composed of a normal form, which resembles coastal C. variegatus, and bulldog and bozo morphs, which diverge ecologically and morphologically from the normal morph. In Chapter 1, I sequenced the mtDNA control region and used haplotype frequency analyses to assess the level of differentiation between sympatric normals and bulldogs sampled from Osprey Lake and Little Lake on San Salvador Island. The bozo morph was too rare to include in the study. I also included samples of normals that occur in lakes without bulldog and bozo morphs to assess any differences between lakes on the island. All haplotype frequency comparisons for sympatric normals and bulldogs were highly significant, which suggests these morphs are distinct populations in sympatry and, therefore, have characteristics of biological species. Further, an estimation of Time for Speciation supports geological data that suggest this fauna is very young (6 000 years). The San Salvador Island pupfish species flock is, therefore, the youngest known species flock and presents an important model system for the study of how morphological and ecological divergence can promote speciation in Cyprinodon. In Chapter 2, I first compared the San Salvador Island pupfishes to other Bahamian C. variegatus populations to assess the level of inter- and intra-island pupfish population differentiation in the Bahamas. The mtDNA control region was sequenced for bulldogs and normals from San Salvador Island and normals sampled from New Providence and Exuma Islands. San Salvador Island bulldogs were found to be distinct from all normal populations sampled, and comparisons of shared haplotypes suggest they originated on San Salvador Island rather than any of the other islands sampled. This was intriguing, because a "bulldog-like" morph has recently been observed in a lake on New Providence Island, which suggests parallel divergences may be occurring throughout the Bahamas. I also sequenced the mtDNA cytochrome b gene to assess the phylogeography of C. variegatus. Populations were sampled from the Bahamas and the east coast of North America, and the results suggest the Bahamas were only recently colonized by the Southern coastal lineage of C. variegatus. A distinct Northern lineage of C. variegatus, which may warrant species designation, was also supported by the cytochrome b data. Overall, the results supported a San Salvador Island origin for the Little Lake and Osprey Lake bulldog morphs, and also suggest the Bahamian C. variegatus populations are very young. / Master of Science

Abstract Cellular energy is produced by the mitochondria via oxidative phosphorylation. In addition to nuclear DNA; the mitochondrion contains circular mitochondrial DNA (mtDNA) molecules. MtDNA is maternally inherited and encodes 37 genes that are crucial for the energy production of the cell. Mutations in the mtDNA cause mitochondrial diseases that manifest as maternally inherited energy metabolism disorders. Common symptoms include diabetes mellitus, myopathy, sensorineural hearing impairment, eye and vision problems, epilepsy and brain manifestations (encephalopathy). Mitochondrial mutations are often heteroplasmic; cells and tissues contain a mix of healthy and mutated mtDNA. The percentage of mutated mtDNA contributes to the severity of symptoms. Mitochondrial DNA also contains numerous polymorphisms; some of which have been reported to be non-neutral, thus contributing to the occurrence of common diseases. Whole mtDNA sequences were obtained from patients with diabetes mellitus (64), epilepsy (79) and unknown mitochondrial disease (66) using conformation-sensitive gel electrophoresis and direct sequencing. Whole mtDNA sequences of a Finnish family with ataxia were also obtained. Restriction fragment length analysis and cloning were used for heteroplasmy quantification. Whole mitochondrial genomes were organized into phylogenetic trees. All nonsynonymous mutations were analyzed with pathogenicity predicting algorithms (SNAP, PolyPhen-2, PMut, SIFT Blink). Non-neutral risk mutations were identified in diabetes mellitus and epilepsy patients. These patients had maternal relatives with diabetes, epilepsy and/or sensorineural hearing impairment. M. 3010A>G and m.16189T>C were found in increased frequency in diabetics and the haplogroup U5b variant m.15218A>G was detected more often among patients with epilepsy. These mutations were predicted to be deleterious in effect. Mitochondrial haplogroup V was found in increased frequency in matrilineal diabetes mellitus patients. We identified an m.8993T>C mutation in a Finnish family with ataxia. This mutation caused an adult-onset ataxic phenotype; previous studies have reported only juvenile onset phenotypes. Novel and rare mtDNA mutations were discovered in patients with an unspecified mitochondrial disease phenotype; these included an insertion m.7585insT and a novel MTTT mutation m. 15933G>A. This thesis emphasizes the importance of full mtDNA sequencing in patients with a suspected mitochondrial disease; novel mutations remain undetected if only the most common mutations are screened. In addition, the increasing importance of non-neutral mtDNA risk variants is supported by the findings of this thesis. In the future, individualized genetics and information on personal risk alleles will become even more important for maintaining health on a personal level. / Tiivistelmä Mitokondriot ovat energiaa tuottavia soluelimiä. Mitokondrioissa on oma rengasmainen mitokondriaalinen DNA (mtDNA), joka esiintyy solussa useana kopiona. MtDNA periytyy vain äidin kautta, joten kaikille lapsille periytyy sama mitokondriaalinen DNA. MtDNA koodaa 37:ää geeniä, jotka ovat tärkeitä solun energiantuotannolle. Geenimuutos mtDNA:ssa voi aiheuttaa äidiltä periytyvän mitokondriotaudin. Mitokondriotaudit ovat energia-aineenvaihdunnan sairauksia, joissa tavallisia oireita ovat diabetes mellitus, lihasoireet (esimerkiksi lihasten ennenaikainen väsymys, myopatia), sydänlihasoireet, maksaoireet, silmä- ja näköoireet, aistimistyyppinen kuulovika sekä aivo-oireet, kuten epilepsia. Oireet vaihtelevat huomattavasti, ja sama mutaatio voi aiheuttaa hyvin erilaisia taudinkuvia. Vakavimmillaan mitokondriotauti voi johtaa kuolemaan jo varhaislapsuudessa. Mutaation prosenttiosuus eli heteroplasmia-aste on usein oireiden vakavuutta määrittelevä tekijä. Mitokondriaalinen DNA muuntuu nopeasti evoluution aikana, joten siinä esiintyy paljon normaalia vaihtelua (polymorfioita). Osa näistä polymorfioista on kuitenkin todettu lievästi haitallisiksi, ja ne lisäävät riskiä sairastua kansanterveydellisesti yleisiin sairauksiin, kuten diabetekseen. Kartoitimme koko mitokondriogenomin muutokset eri potilasryhmiltä, joihin kuului diabetesta, epilepsiaa ja ataksiaa sairastavia potilaita. Lisäksi tutkittiin potilaita, joilla epäiltiin mitokondriotautia. Keskeiset käytetyt menetelmät olivat DNA:n rakenteellisia muutoksia havaitseva geelielektroforeesi ja sekvensointi. Määritimme heteroplasmian käyttäen restriktioentsyymianalyysia sekä kloonausta bakteerisoluihin. Järjestimme potilaiden mtDNA-sekvenssit fylogeneettisiksi puiksi ja kaikki proteiinin koodausta muuttavat geenimuutokset analysoimme haitallisuutta ennustavilla tietokoneohjelmilla (SNAP, PolyPhen-2, PMut, SIFT BLink). Diabetesta sairastavilla potilailla, joilla myös äidinpuoleisessa suvussa esiintyy diabetesta, havaitsimme useammin m.3010A>G- ja m.16189T>C-geenimuutoksia kuin väestöllä keskimäärin. Tutkimustulos tukee aikaisemmin julkaistuja tutkimustuloksia m.16189T>C-geenimuutoksen haitallisuudesta. Epilepsiapotilailta löytyi m.15218A>G-geenimuutos kahdessa U5a1-haploryhmän alatyypissä. Patogeenisyysanalyysien mukaan nämä geenimuutokset olivat haitallisia. Mitokondriaalinen haploryhmä V havaittiin useammin diabetesta sairastavilla kuin terveillä henkilöillä. Tutkimukseen valittiin potilaita, joiden äidinpuoleisilla lähisukulaisilla esiintyi yhtä tai useampaa seuraavista: diabetes, epilepsia tai aistimistyyppinen kuulovika. Väitöstutkimuksessa todetaan lisäksi, että m.8993T>C-mutaatio aiheuttaa aikuisiällä alkavaa ataksia-oireistoa. Kyseinen mutaatio on aiemmin yhdistetty vain lapsuusiän taudinkuviin. Kuvasimme uuden insertiomutaation (m.7585insT) kardiomyopatiasuvussa sekä uuden MTTT-geenin mutaation (m.15933G>A) tuntematonta mitokondriotautia sairastavalla potilaalla. Väitöskirjatutkimuksen tulokset osoittavat, että on tärkeää tutkia koko mitokondriogenomi, kun kyseessä on tuntemattomaksi jäänyt mitokondriaalinen taudinkuva. Uudet, tautia aiheuttavat, mtDNA:n geenimuutokset voivat jäädä tunnistamatta, jos tutkitaan ainoastaan raportoidut, tunnetut, mutaatiot. Lisäksi voi todeta, että mitokondriogenomissa esiintyy lievästi haitallisia geenimuutoksia tai niiden yhdistelmiä, jotka saattavat lisätä riskiä sairastua kansanterveydellisesti merkittäviin sairauksiin, kuten diabetekseen.

Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting mostly as early-onset mitochondrial encephalo-cardio-myopathies. Mutations in four nuclear genes can result in isolated deficiency of ATP synthase, all sharing a similar biochemical phenotype - pronounced decrease in the content of fully assembled and functional ATP synthase complex. The thesis summarises studies on two distinct causes of ATP synthase deficiency. First is TMEM70 protein, a novel ancillary factor of ATP synthase, which represents most frequent determinant of severe inborn deficiency of ATP synthase. TMEM70 is a 21 kDa protein of the inner mitochondrial membrane, facilitating the biogenesis of mitochondrial ATP synthase, possibly through TMEM70 protein region exposed to the mitochondrial matrix, but the proper regulatory mechanism remains to be elucidated. In TMEM70-lacking patient fibroblasts the low content of ATP synthase induces compensatory adaptive upregulation of mitochondrial respiratory chain complexes III and IV, interestingly by a posttranscriptional mechanisms. The second type of ATP synthase deficiency studied was mtDNA m.9205delTA mutation affecting maturation of MT-ATP8/MT-ATP6/MT-CO3 mRNA and thus biosynthesis of Atp6 (subunit a) and Cox3...