京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20926号 / 理博第4378号 / 新制||理||1629(附属図書館) / 京都大学大学院理学研究科地球惑星科学専攻 / (主査)教授 生形 貴男, 教授 酒井 治孝, 教授 田上 高広 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

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Type 2 diabetes is a metabolic disorder characterized by increased glucose concentrations in the blood due to decreased insulin sensitivity. The worldwide incidence of diabetes has increased remarkably over the last two decades. Obesity, due to increased consumption of calorie dense diets, and sedentary life styles, is commonly cited as a primary cause. However, many epidemiological studies have established a relationship between insulin resistance and exposure to environmental chemicals such as persistent organic pollutants (POPs). The mechanisms by which POPs alter metabolism remain poorly understood, although their lipophilic nature suggests a role in adipose tissue function. The Tischkau lab has established a relationship between Aryl hydrocarbon Receptor (AhR) activation by different types of POPs and increased risk of insulin resistance. This dissertation, therefore, explored the effects of AhR activation by POPs on adipose tissue function. Adipose tissue regulates systemic glucose and lipid metabolism through production of hormones and cytokines that regulate appetite and energy homeostasis. It is well-known that impaired adipose function promotes systemic insulin resistance. The first specific aim examined the hypothesis that activation of AhR suppresses adipogenesis by lowering the rate of pre-adipocyte differentiation. Adipogenesis is a process by which mesenchymal stem cells (MSCs) and pre-adipocytes differentiate into mature adipocytes. Limitations in adipogenesis and accumulation of ectopic lipid have significant roles in decreasing insulin sensitivity. Thus, I hypothesized that POPs contribute to systemic insulin resistance by lowering the rate of MSCs and preadipocyte differentiation; the resulting large, poorly-functioning adipocytes increase serum lipids and promote lipid deposition in other tissues. MSCs derived from mouse bone marrow and pre-adipocytes were treated with different concentrations of AhR agonist, β-Naphthoflavone (BNF), and levels of transcripts associated with adipocyte differentiation were determined by using quantitative PCR. Oil red O staining and lipid content were observed to examine differentiation into mature adipocytes. Genes that promote adipogenesis, including peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein alpha (CEBPα), fatty acid binding protein 4 (FABP4), and adiponectin were downregulated in MSCs treated with BNF. Moreover, accumulation of triglycerides was decreased after BNF treatment. Recombinant lentivirus vector-mediated AhR knockdown blocked the effects of BNF on adipogenesis. Therefore, activation of AhR by exogenous ligands inhibits adipogenesis leading to impaired fat storage. Limitations in adipogenesis promotes accumulation of the excess lipid in non-fat tissue such as liver, muscle, and heart leading to decrease the insulin sensitivity and disrupt energy homeostasis. The second specific aim examined effects of AhR activation on circadian clock regulation in adipose tissue. A circadian clock essentially regulates systematic energy homeostasis; the central clock in the suprachiasmatic nucleus (SCN) works with the local clocks in peripheral tissues such as liver, muscle, and adipose tissue to regulate whole-body metabolism. The Tischkau lab has previously shown that AhR interacts with the core machinery of the circadian clock. Activation of AhR by environmental toxicants leads to a dampening of the rhythm expression of core clock genes or an alteration in the timing of their peak expression, which subsequently promotes metabolic disorders such as glucose insensitivity and hyperlipidemia. Given the importance of appropriately timed adipose tissue function to regulation of energy homeostasis, this study focused on mechanisms by which AhR may influence clock-controlled mature adipose tissue activity. Lipolysis is a clock-regulated process in adipose tissue that provides the necessary energy during periods of fasting and exercise. Thus, I hypothesized that AhR activation in adipose tissue would impair lipolysis by altering molecular circadian clock function. AhR activation was proposed to dampen adipose rhythms, leading to a decreased lipolysis rate during the absence of food, and subsequently, increased glucose concentrations in the blood. C57BL/6 mice were injected with vehicle or 50 mg/kg body weight of the AhR agonist, BNF, 48 hours after release into constant darkness. Mice were sacrificed, and epididymal adipose tissue was collected every 6 hours over a 24 hour period. Real-Time RT-qPCR was used to measure mRNA expression of genes responsible for lipolysis. To examine effects of AhR activation in vitro; mouse pre-adipocytes, 3T3-L1 cells, were differentiated into mature adipocytes for 12 days. Cells were then starved for 24 hours with DMEM media containing 1% FBS to induce lipolysis in the presence of 100, 200, 300 µM of BNF. RNA was then extracted and mRNA expression for genes responsible for circadian clock and lipolysis were determined by RT-qPCR. Alterations were observed in rhythms of core clock genes in wild type mice injected with BNF compared to wild type mice injected with vehicle. Rhythms of key enzymes controlling lipolysis including hormone sensitive lipase (HSL) and adipose triglycerides lipolysis (ATGL) was changed in wild type mice injected with BNF compared to wild type mice injected with vehicle. These effects were blocked in AhR deficient mice, suggesting that these effects were AhR dependent. Liver glycogen was decreased in mice injected with BNF compared to wild type mice injected with vehicle after 12 hour of food restriction but not in AhR null mice. Activation of AhR led to decreased expression of lipolysis genes in adipose tissue at CT6 (middle of the rest phase) as well as in 3T3-L1 cells. Recombinant lentivirus vector-mediated AhR knockdown blocked the effects of BNF on lipolysis in 3T3-L1 cell line. These data establish a link between environmental toxicants and impaired lipolysis, specifically by altering rhythms of clock genes in adipose tissue. In response to the decreased available energy from impaired lipolysis, the body increases glycogenolysis, thereby degrading more glycogen to provide the necessary energy. This process may lead to increased glucose level in the blood and development of type 2 diabetes. The data from this study suggest that activation of AhR by BNF increases the risk of insulin resistance and type 2 diabetes by impairing adipogenesis. Reduced adipogenesis likely decreases adipocyte capacity to capture triglycerides from the blood. These effects may disturb energy homeostasis and contribute to the development of metabolic syndrome. This study also establishes a link between environmental toxicants and impaired lipolysis, specifically by altering rhythms of clock genes in adipose tissue. In response to the decreased available energy from impaired lipolysis, the body increases glycogenolysis, thereby degrading more glycogen to provide the necessary energy. This process may lead to increased glucose level in the blood and development of type 2 diabetes. All together, these data suggest that environmental pollutants result in adipose tissue dysfunction by reducing adipogenesis and lipolysis. Therefore, activation of AHR by its exogenous ligands may increase the risk of insulin resistance and type 2 diabetes by impairing adipose tissue function. In particular, activation of AHR by exogenous ligands leads to impairment of free fatty acids storage during feeding and release during fasting to disturb energy homeostasis.

No description available.

No description available.

Understanding thickness variation and facies transitions in the mudstone-rich part of the Upper Middle Jurassic (Callovian) Entrada Sandstone depositional system is critical for constraining the paleogeography and evaluating the economic potential of Utah's Entrada Sandstone. Facies of the Entrada Sandstone in south-central Utah are dominated by mudstone-rich intertidal facies that were widespread within the Jurassic seaway. Intertidal deposits interfinger basinward with subtidal ooid-bearing shoals and bars, and landward supratidal sabkha, and erg-margin eolian deposits. Three sections were measured to improve understanding of the lateral and vertical facies transitions. Variations in thickness indicate the rate of developing accommodation space was high along the southwestern shoreline and relatively low along the northeastern shoreline during Callovian time. Although accommodation space was highest in the west, sediment supply from the west kept pace with, and eventually outpaced subsidence. In the east, sediment supply was significant but at one time was outpaced by subsidence, creating a complete range of facies, from subtidal to supratidal deposits. Along this eastern shoreline, erg-margin coastal dunes associated with the larger erg to the east eventually prograded westward. The variation in subsidence, sediment supply, and sediment source makes sequence stratigraphic correlation difficult. Reservoir-quality sandstones are associated with muddy sections of the Entrada Sandstone within the San Rafael Swell. Porosity and permeability of the facies in this area indicate excellent reservoir potential in three of eight facies that were studied. Porosities of these potential reservoirs ranged from 11-22%, with permeabilities ranging from 44-430md. These high quality reservoir facies are surrounded by muddy, low reservoir-quality rocks, creating conditions amenable to the development of stratigraphic hydrocarbon traps. Based on further study and a modern analog at the north of the Gulf of California, Hicks and others' (2010) depositional model for the Entrada Sandstone of south-central Utah has been modified to include newly interpreted facies. This improved depositional model may have predictive power in exploring for stratigraphic and combination traps within the Entrada system of Utah and analogous depositional systems throughout the world.

[pt] Estudos paleoambientais sobre reconstruções climáticas no pretérito ajudam a compreender os processos biogeoquímicos atuais e, portanto, fornecem subsídios ao atual cenário de mudanças globais. As principais ferramentas utilizadas nesses estudos envolvem indicadores que requerem a integração de informações geoquímicas, geomorfológicas, oceanográficas e biológicas. No presente trabalho, dois testemunhos (RJ13-01 e RJ13-02) foram coletados na plataforma continental do Rio de Janeiro na isóbata de 100-m e datados através dos métodos de 210Pb e 14C, englobando, respectivamente, 14,5 cal kyr BP e 5,3 cal kyr BP. O objetivo principal do trabalho foi investigar o efeito de variáveis climáticas e ambientais sobre o aporte de material autóctono e alóctono na plataforma continental do Rio de Janeiro. Foram considerados dados geofísicos, granulometria, elementos traços (V, Cr, Mn, Ni, Cu, Zn, As, Sr, Ag, Cd, Sn, Ba, Tl, Pb e U), elementos majoritários (Ca, Si, Al e Fe), nitrogênio total (TN), carbono orgânico total (TOC), carbonato de cálcio (CaCO3), hidrocarbonetos alifáticos (n-alcanos) e hidrocarbonetos aromáticos (HPAs), assim como consideradas razões entre elementos e compostos que indicam a introdução de material continental e biogênico, razões redox e de produtividade, além de marcadores de tipo de vegetação e de combustão. Os resultados ressaltaram três fases deposicionais na área estudada: a primeira fase vai de 14,5 cal kyr BP à 7,5 cal kyr BP, e se caracteriza pela ocorrência de granulometria média a grosseira, fragmentos de conchas, alto teor de Si (27 porcento), alto aporte de material biogênico Ca/Al (3,00), altos valores (30,5) de ACL (tamanho médio da cadeia n-alcanos) e alta contribuição de HPAs pirogênicos (33 porcento), por volta de 8,0 cal kyr BP, indicando maior incidência de clima seco na maioria dos períodos, além de ser marcada por níveis do mar mais baixos. A segunda fase, entre 7,5 cal kyr BP e 4,5 cal kyr BP, mostrou influência das Águas Centrais do Atlântico Sul (SACW), marcada por um máximo de elevação do nível do mar, eventos de seca com maior intensidade de incêndios naturais com alta contribuição de HPAs pirogênicos de 5-6 anéis (68 porcento), entre 6,1 cal kyr BP e 4,1 cal kyr BP, e alto aporte de material continental Al/Ca (0,69); Fe/Ca (0,27) e alta produtividade. A terceira fase cobre o Holoceno superior, a partir de 4,5 cal kyr BP até o presente. Nessa fase, foram encontrados fluxos mais elevados de matéria orgânica, entre 3,2 cal kyr BP a 3,3 cal kyr BP (RJ13-01) e 3,4 cal kyr BP (RJ13-02), como indicado por TN, TOC, Ni, Cu, Zn, HA e HPAs, e um maior aporte continental, devido aos altos valores de razões redox-sensíveis a partir de 2,5 cal kyr BP no RJ13-01 e ao longo do testemunho RJ13-02. Esses fatores provavelmente são decorrentes do deslocamento da Zona de Convergência Intertropical (ITCZ) mais ao sul, deixando o clima mais úmido na Amazônia, a intensificação do SAMS trazendo umidade para SE do país através da SACZ. Porém, também podem refletir um evento de menor nível do mar, que influenciou nos níveis de oxigênio na coluna d água. Por outro lado, indicadores de clima seco (ACL, HPAs pirogênicos de 5-6 anéis) sugerem que houve oscilações entre clima úmido e seco ao longo do Holoceno superior. Desta forma, os indicadores orgânicos e inorgânicos considerados nos testemunhos da plataforma continental do RJ possibilitaram avaliar e corroborar dados de outros estudos sobre os eventos climáticos ocorridos nos últimos 14 mil anos no SE do Brasil, e suas relações com fatores como: variações no nível do mar, mudanças na ITCZ, intensificação da SAMS influenciando a SACZ, mudanças nos padrões de ventos e na Corrente do Brasil; influência da SACW mais no sentido S-W do sistema de ressurgência de Cabo Frio; variações climáticas com registros de incêndios naturais e variações da vegetação; além de registros que podem ser relacionados à ocupação humana. / [en] Paleoenvironmental studies on climate reconstructions in the past help to understand the current biogeochemical processes and therefore become increasingly important in the current scenario of global changes. The main parameters used in these studies involve proxies that require the integration of geochemical, geomorphological, oceanographic and biological information. In this study two cores (RJ13-01 and RJ13-02) were collected on the continental shelf of Rio de Janeiro in the isobath 100-m and dated by 210Pb and 14C methods covering14.5 cal kyr BP and 5.3 cal kyr BP, respectively. The main goal of this work was to investigate the effect of climatic and environmental variables on the autochthonous and allochthonous material inputs in the continental shelf of Rio de Janeiro. For this, geophysical properties, grain size, trace elements (V, Cr, Mn, Ni, Cu, Zn, As, Sr, Ag, Cd, Sn, Ba, Tl, Pb and U), major elements (Ca, Si, Al and Fe), total nitrogen (TN), total organic carbon (TOC), calcium carbonate (CaCO3), aliphatic hydrocarbons (n-alkanes) and polycyclic aromatic hydrocarbons (PAHs) were considered jointly with continental and biogenic input proxies, redoxsensitive and productivity proxies besides vegetation type and combustion proxies. Results highlighted three periods: the first period (from 14.5 to 7.5 cal kyr BP) with the occurrence of mean grain size of the sediment, shell fragments, high content of Si (27 percent), high input of biogenic material Ca/Al (3.00) and high ACL (average chain length) values (30.5). In addition to high contribution of pyrogenic PAHs (33 percent) around 8.0 cal kyr BP indicating higher incidence of dry weather in most periods; marked by a low sea level. The second period between 7.5 cal kyr BP and 4.5 cal kyr BP showed the influence of the South Atlantic Central Waters (SACW) marked by a maximum transgression of the sea, drought events with greater intensity of wildfires with high contribution of pyrogenic PAHs of 5-6 rings (68 percent) between 6.1 and 4.1 cal BP kyr BP and high input continental materials Al/Ca (0.69); Fe/Ca (0.27) and high productivity. The third period covers the late Holocene from 4.5 cal kyr BP to present. In this period, higher fluxes of organic matter were found between 3.2 cal kyr BP to 3.3 cal kyr BP (RJ13-01) and 3.4 cal kyr BP (RJ13-02), as indicated by TN, TOC, Ni, Cu,Zn, n-alkanes and PAHs, and higher continental input due to high redox-sensitive proxies from 2.5 cal kyr BP in RJ13-01 and along RJ13-02. These factors are probably related to the displacement further south of the Intertropical Convergence Zone (ITCZ), which brought humidity to Amazon, the intensification of SAMS bringing humidity to SE of Brazil trought SACZ. Also has been marked by the decrease in sea level which influenced the oxygen levels in the water column. On the other hand, dry climate indicators (ACL, pyrogenic PAHs with high molecular weight) suggest that there were fluctuations between wet and dry climate along the late Holocene. Thus, the organic and inorganic proxies considered on the cores of continental shelf of Rio de Janeiro allowed to evaluate and corroborate the data of other studies of climate events over the past 14,000 years in SE Brazil and their relation to the facts such is the variations in the sea-level, chances in ITCZ, the intensification of SAMS and SACZ, changes in wind pattern, and in the Brazilian current; influence of the SACW further S-W of the Cabo Frio upwelling system; and climate variations with records of wildfires and variation on the vegetation; in addition to records that could be related to human occupation.

This study was done to determine the effectiveness of a commercially available bioaugmentation product, BiOWiSHTM-Aqua FOG, for remediating petroleum-contaminated sandy soil. Biodegradation enhancement by BiOWiSHTM-Aqua FOG was evaluated in laboratory microcosms by directly measuring total petroleum hydrocarbon (TPH) and indirectly using respirometry. Attempts were made to enrich hydrocarbon-degrading bacteria in BiOWiSHTM-Aqua FOG, and the resulting enrichment cultures were screened using respirometry as well. Potential hydrocarbon-degrading bacteria in BiOWiSHTM-Aqua FOG were isolated. Experiments were performed at bench-scale using microcosm bottles containing sand contaminated with either motor oil or No. 2 diesel fuel. The microcosms were incubated at 25oC under aerobic conditions. TPH measurements of soil in the microcosms at 0, 25 and 56 days indicated that the addition of 500-ppm BiOWiSHTM-Aqua FOG improved biodegradation of the motor oil-contaminated soil by 45%. However, BiOWiSHTM-Aqua FOG did not have a measurable effect on biodegradation in the diesel-contaminated soil. In the respirometry experiments, BiOWiSHTM-Aqua FOG and two hydrocarbon-enriched BiOWiSHTM-Aqua FOG cultures were evaluated indirectly by the measurement of microbial carbon dioxide production and oxygen uptake using a MicroOxymaxTM respirometer. The respirometry experiments showed that in the six-day period following motor oil-contamination of soil, the addition of BiOWiSHTM-Aqua FOG substantially improves biodegradation rates. The added organisms in the product out-performed the indigenous organisms in the 5-6 days following contamination of the soil. The CO2 production observed in the BiOWiSHTM microcosms contaminated with motor oil was much greater than CO2 production without motor oil, which confirms that the observed metabolism can be attributed to motor oil biodegradation rather than metabolism of other organic material in the soil. Enriched consortia consistently generated far less CO2 than microcosms with the 500 ppm BiOWiSHTM-Aqua FOG. Stoichiometric calculations suggested that BiOWiSHTM-Aqua FOG removed approximately 1400 ppm TPH (14%) from the soil in 6.5 days, while an enrichment culture of BiOWiSHTM-Aqua FOG only reduced TPH levels by 459 ppm (5%). This result suggests that increased biodegradation rate in bioaugmented soil is aided by biodiversity in the augmenting inoculum. A potential hydrocarbon-degrading candidate organism was isolated from the product and cultured on Bushnell-Haas agar and plate-count agar (PCA). While at least two distinct colony types were successfully grown on media with motor oil, these same colonies appeared on Bushnell-Haas agar with no apparent carbon source, and survived repeated transfers onto this same medium. Therefore, their status as hydrocarbon-degraders is inconclusive. More thorough enrichment work could be pursued, especially using soil samples collected from petroleum-contaminated sites.

Cyclophilin-40 (CyP40) promotes the formation of the gel shift complex containing baculovirus aryl hydrocarbon receptor (AhR), AhR nuclear translocator (Arnt) and dioxin response element (DRE). CyP40 was found to play a role in the AhR signaling since when the CyP40 content in MCF-7 cells is reduced, up-regulation of cyp1a1 and cyp1b1 by 3-methylcholanthrene (3MC) is also reduced, suggesting that CyP40 is essential for maximal AhR function. The CyP40 region containing amino acids 186-215, but not the peptidylprolyl cis-trans isomerase and tetratricopeptide repeat domains, is essential for forming the AhR/Arnt/DRE complex. CyP40 is found in the cell nucleus after 3MC treatment and appears to promote the DRE binding form of the AhR/Arnt heterodimer. Coprecipitation data suggests CyP40 binds weakly to AhR, but not Arnt. We report on the progress of applying bioluminescence resonance energy transfer and chromatin immunoprecipitation techniques to further elucidate the role of CyP40 in the aryl hydrocarbon receptor signaling pathway.

Upon ligand binding, the aryl hydrocarbon receptor (AhR) translocates into the nucleus and dimerizes with its partner Ah receptor nuclear translocator (Arnt). The AhR/Arnt heterodimer binds to the enhancer element DRE to regulate target gene expression. It is known that the formation of the ligand-dependent AhR/Arnt/DRE complex requires protein factors in vitro. The first aim is to determine whether two other Hsp90-associated proteins present in rabbit reticulocyte lysate (RRL), namely CyP40 and Hsp70, play any role in forming the AhR/Arnt/DRE complex. Fractionation and immunodepletion experiments revealed that Hsp70 is not necessary for the formation of this complex. In contrast, CYP40 is involved in forming the complex since (1) immunodepletion of CyP40 from a RRL fraction reduces the intensity of the AhR-Arnt-DRE complex by 48% and (2) recombinant human CyP40 alone causes the formation of this complex. In addition, CyP40-interacting proteins appear to be essential for the full CyP40 effect on the AhR gel shift complex. The second aim is to determine the role of β-tubulin in Amt-dependent signaling pathways. From the insect Sf9 cytosol, β-tubulin enriched fraction (F5) was isolated which suppresses the AhR/Arnt/DRE complex formation in a gel shift assay. Tubulin enriched from pig brain had a similar inhibition of the AhR gel shift complex, suggesting that β-tubulin in F5 is likely responsible for the action. Using the TALON resin, β-tubulin was co-precipitated with the baculovirus 6His-Arnt, showing that β-tubulin interacts with Arnt. β-tubulin was examined to decide its role in the hypoxia inducible factor-1α (HIF-1α) signaling which is also Arnt-dependent. Gel shift data using HIF-1α and Arnt showed that F5 suppressed the formation of the HIF-1α/Arnt/HRE complex. Subsequently the Sf9 β-tubulin was cloned and about 95% of its full-length sequence was identified. The amino acid sequence of Sf9 β-tubulin shares high sequence identity with human β-tubulin. Upon transient transfection of a plasmid containing a human β-tubulin cDNA into MGF7 or Hep3B cells, the HRE-driven luciferase activity was clearly suppressed. In conclusion, we have evidence supporting that β-tubulin inhibits the Arnt-dependent signaling and the mechanism may involve the interaction between Arnt and β-tubulin.