京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13329号 / 論医博第2197号 / 新制||医||1044(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊達 洋至, 教授 森本 尚樹, 教授 川口 義弥 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23066号 / 医博第4693号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 溝脇 尚志, 教授 藤田 恭之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Obesity is a growing pandemic that claims close to three hundred thousand lives per year in the United States alone. Despite strong interest and investment in potential treatments, obesity remains a complex and challenging disorder. In the study of obesity, mouse models have been excellent tools that help in understanding the function of different genes that contribute to this disease of energy homeostasis. However, it was surprising when disfunction in primary cilia was found to be linked to syndromic obesity. To understand the role of primary cilia in obesity, a growing subset of GPCRs have been identified to selectively localize to the organelle. Several of which have known roles in energy homeostasis. In some examples, ciliary GPCRs appear to dynamically localize to the organelle; such is the case of GPR161 and smoothened in the hedgehog signaling pathway. Thus, we were interested to see if other GPCRs dynamically localize to the primary cilia as part of their regulation of energy homeostasis. For example, the GPCR MCHR1 selectively localizes to the cilia and is involved in energy homeostasis. Although much is known about the expression of the receptor in the brain, how its ciliary subcellular localization impacts its roles in energy homeostasis is unknown. Observing neuronal cilia in vivo remains a difficult task as some of the available tools such as tagged alleles rely on overexpression of ciliary protein which may impact function. Additionally, most of the work is done in vitro, leaving much to be discovered about neuronal cilia in vivo. In this thesis, we show that using a newly constructed reporter allele mCherryMCHR1, we can see ciliary expression of MCHR1 in the brain of developing and adult mice; more specifically in the ARC and PVN. Subsequently, using a novel Artificial intelligence analysis approach, we measured the length and composition of MCHR1 positive cilia under physiological conditions associated with MCHR1 function. Although in this work we are reporting no changes in dynamic localization of MCHR1 in the hypothalamus specifically, we are not excluding the potential for changes in other regions of the brain or under other conditions; and we are suggesting that pharmacological approaches may help highlight potential ciliary GPCR dynamic localization.

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>Magister Scientiae - MSc / Nanotechnology is a rapidly growing field of research. Due to major innovations brought about by developments in nanotech, several consumer products are currently available containing nanomaterials. The increase of nanomaterial production and use is accompanied by the increased potential of human, plant and animal exposure to these nanomaterials. As a relatively new nanomaterial, carbon quantum dots (CQDs) are being extensively used and researched due to its unique properties. Although many studies have assessed the toxic potential of CQDs, and found them to exhibit low toxicity, there is lack of work assessing the effects on the immune system. In the present study, RAW 264.7 murine macrophages were used as model to assess the immunomodulatory potential of CQDs. RAW cells exposed to varying concentrations of CQDs (0-500μg/ml), showed that CQDs caused a reduction at cell viability. In the absence of a mitogen CQDs, induced an inflammatory response by stimulating the release of various cytokines and chemokines such as, TNFα, MIP-1α, MIP-1β, MIP-2, IP-10, G-CSF, GM-CSF, and JE.

Background: A single infusion of ketamine has rapid antidepressant properties, although the drawback is a lack of sustained effect. A previous study showed a rapid enhancement (within 2 hours) in ventral tegmental area (VTA) dopamine (DA) neuron population and locus coeruleus (LC) norepinephrine (NE) firing and bursting activity following a single ketamine administration. The current study investigated whether these changes are present 24 hours after a single administration and if they are maintained with repeated administration. Additionally, we examined dorsal raphe nucleus (DRN) serotonin (5-HT) neurons to assess the effects of single and repeated ketamine administration on these neurons. Methods: Ketamine (10 mg/kg, i.p.) was administered to male Sprague Dawley rats once or repeatedly (3 times/week) for 2 weeks. After single and repeated administration of ketamine, electrophysiological recordings were done in the VTA, LC and DRN in anesthetized rats, 24 hrs, 3 or 7 days post-administration. Spike frequency, bursting, and for VTA neurons, spontaneously active neurons/trajectory were assessed. Results: In the VTA, LC and DRN, 24 hrs after ketamine was injected acutely there was no significant difference between controls and treated animals in all parameters assessed. However, after repeated administration, there was an increase in bursting and number of spontaneously discharging neurons per tract of VTA DA neurons as well as an increase in frequency of discharge of LC NE neurons. While the increased number of spontaneously discharging neurons per tract had dissipated after 3 days, the enhanced bursting was still present but dissipated after 7 days. As for LC NE neurons, the increased frequency of discharge was no longer present after 3 days. No significant differences in the firing of DRN 5-HT neurons were observed between controls and treated animals even after ketamine was administered repeatedly. Conclusion: These results indicate that repeated but not acute administration of ketamine maintained the increase in population activity of DA neurons and firing activity of NE neurons.

<p>Excess sodium (Na) intake is directly related to hypertension and an increased risk of developing many chronic diseases, but there is currently no method to directly quantify Na retained in the body. Because of this, the locations of Na storage and its exchange mechanisms are not well known. This information is critical for understanding the impact of increased Na intake in modern diets. In order to non-invasively quantify Na in bone and soft tissue, a compact deuterium-deuterium (DD) neutron generator-based <i>in vivo</i> neutron activation analysis (IVNAA) system was developed. MCNP was used to design a custom irradiation assembly to maximize Na activation in hand bone while minimizing dose. In order to test the system, live pigs were used. Two 100% efficient high purity germanium (HPGe) detectors collected Na-24 counts over 24 hours post irradiation. From the pig studies, a two-compartment model of exchange was developed to quantify Na in bone and in soft tissue. The right legs of four live pigs, two on a low Na diet and two on a high Na diet, both for 14 days, were irradiated inside the customized irradiation cave for 10 minutes (45 mSv dose to the leg) and then measured with the HPGe detectors. The spectra were analyzed to obtain the net Na counts at different time points. Analysis shows exponential decrease of Na in the leg during the first one hour of measurement, while the change was minimal at the second hour, and the counts were stabilized at the second and third 2 hour measurements, taken 7 and 21 hours post irradiation. Bone Na and soft tissue Na concentrations were calculated using calibration lines created with bone and soft tissue equivalent Na phantoms as well as the parameters obtained from the two-compartment model. The results show that the difference in bone and soft tissue Na between the pigs on high vs low Na diets was significant. With these results, we conclude that DD neutron generator-based IVNAA can be used to accurately quantify Na in bone and soft tissue <i>in vivo </i>and the system is a potential valuable tool for nutrition studies.</p>

Current epidemiological data showing that the use of the injectable contraceptive progestin Depotmedroxyprogesterone acetate (DMPA) is associated with increased HIV-1 acquisition is controversial. However, animal and ex vivo data reveal plausible biological mechanisms whereby MPA may increase HIV-1 acquisition. Relatively high levels of endogenous progesterone (P4) found in the luteal phase of the menstrual cycle have also been linked to increased HIV-1 acquisition in animal, clinical and ex vivo models. One of the central hypotheses of the present study was that the mechanism of MPA-induced increase in HIV-1 infection occurs via a different mechanism to that of the luteal phase. Furthermore, MPA has been shown to activate both the glucocorticoid receptor (GR) and its target, the progesterone receptor (PR) isoform B (PR-B), which are both transcription factors and regulate genes involved in immune function. Both the GR and PR are expressed in the cervix, the primary site of heterosexual HIV-1 infection. PR is regulated by endogenous estrogen (E2), of which the concentrations fluctuate throughout the menstrual cycle, and GR expression also varies in response to stress hormones, leading to conditions of varied relative levels of GR/PR. The immune-related consequences of changing the relative levels of GR and PR-B are not well understood. Therefore another hypothesis of this study was that changing the relative levels of GR/PR-B modulates HIV-1 infection and immunomodulatory gene expression in response to the GR/PR agonist, MPA. Since GR and PR-B recognize similar DNA target sequences and may regulate the same genes at the same time, the final hypothesis of the present study was that GR and PR-B reciprocally modulate each other’s activity, through possible association. To investigate the effects of exogenous hormones on HIV-1 infection and mechanisms thereof, peripheral blood mononuclear cells (PBMCs) and TZM-bl cervical cells were used as model systems for HIV-1 infection. These cells were stimulated with P4 and E2 at concentrations mimicking the menstrual cycle phases or with levels of MPA at the upper range of peak serum levels detected in DMPA users. Cells were infected with the R-tropic HIV-1 infectious molecular clone, HIV-1Bal_Renilla and luciferase assays were used to measure HIV-1 infection. Levels of HIV-1 CD4 receptor and CCR5 co-receptor protein or mRNA were measured by flow cytometry or qPCR, respectively, while activation of CD4+ T cells using the activation marker CD69 was measured by flow cytometry in PBMCs. To investigate the effects of changing GR/PR-B levels on HIV-1 infection and immune gene regulation, GR/PR levels were altered in End1/E6E7 immortalized endocervical and HeLa/TZM-bl cervical carcinoma cells by GR siRNA knockdown with or without the simultaneous over-expression of PR-B, and cells were stimulated with MPA or the GR agonist Dexamethasone. mRNA expression iii of key immunomodulatory genes in End1/E6E7 and HeLa cells was measured by qPCR. The modulation of GR activity by PR-B was assessed by promoter-reporter assay in COS1 and U2OS cells over-expressing GR and PR and stimulated with GR- and/or PR-specific ligands. Association of GR and PR-B was measured by co-immunoprecipitation in COS1 and MCF-7 cells, while co-localization of GR and PR-B was measured by confocal microscopy and super-resolution structured illumination microscopy in COS1 cells. MPA significantly increased HIV-1 infection in both PBMCs and TZM-bl cells, while luteal phase hormones did so to a lesser extent. However, MPA but not luteal phase hormones increased the ratio of CD4+/CD8+ T cells in PBMCs. MPA but not luteal phase hormones also increased CCR5 protein expression on CD4+ T cells in PBMCs and total CCR5 mRNA expression in TZM-bl cells. In addition, MPA but not luteal phase hormones increased activation of CD4+ T cells in PBMCs. Using a GR antagonist or GR siRNA, it was shown that the GR but not PR-B is required for MPA-, but not luteal phase hormone-induced increased HIV-1 infection in PBMCs and TZM-bls. The presence of PR-B altered the anti-inflammatory, GR-mediated regulation of some key immunomodulatory genes, including GILZ and IL-6, in End1/E6E7 and HeLa cells in response to MPA. In general, basal (unliganded) expression of immunomodulatory genes exhibited a pro-inflammatory profile in the presence of PR-B. Co-immunoprecipitation assays showed that GR and PR-B appeared to associate. Confocal microscopy suggested GR and PR co-localized in the nucleus in response to GR- and/or PRspecific ligands, while super-resolution microscopy showed that co-localization occurred in select regions within the nucleus. Taken together, MPA increases HIV-1 infection in a manner different from that of luteal phase hormones, most likely involving increased CD4+ T cell frequency (CD4+/CD8+ ratio), activation and increased expression of CCR5 on CD4+ T cells, and requiring the GR. Furthermore, PR-B modulates GR-mediated immune function gene regulation, via potential association and region-specific nuclear co-localization. This suggests that the relative levels of GR/PR may play an important role in determining the inflammatory and immune responses and HIV-1 infection in HIV-1 target cells, both in DMPA users and women not using hormonal contraception.

<p><b><i>Background and Objective:</i></b> Pb is a well-known toxic metal that can accumulate in bones over time and still threatening large populations nowadays, even those who are environmentally exposed to it. Strontium (Sr) is a metal directly related to bone health and has been used in the treatment of osteoporosis disease as a supplement. Manganese (Mn) is an essential nutrient in the body, yet excessive Mn is toxic and affecting many organ systems. Another toxic metal, mercury (Hg), has been poising different populations primarily through seafood consumptions, especially inducing neurological disorders in infants and fetuses. Even though significant associations between the above metal exposures and health outcomes have been recognized over the decades, the current technologies are limited in assessing cumulative long-term exposures <i>in vivo</i> to evaluate such associations further. Bone and toenail are appropriate biomarkers to reflect long-term exposure due to the longer half-life of these metals in them than in the traditional biomarkers. Therefore, this work evaluated the usefulness of portable x-ray fluorescence (XRF) technology on <i>in vivo</i> quantification of Pb and Sr in bone, and Mn and Hg in toenail.</p> <p><b><i>Materials and Methods:</i></b> The portable XRF device was calibrated by using the Pb- and Sr-doped bone-equivalent phantoms, and Mn- and Hg-doped nail-equivalent phantoms, correspondingly in different projects. Seventy-six adults (38-95 years of age, 63 ± 11 years) from Indiana, USA, were recruited to participate in this study. For the <i>in vivo</i> bone measurements, each participant was measured at the mid-tibia bone using the portable XRF and K-shell XRF system (KXRF). We estimated the correlation between the bone Pb concentration measured by both devices to evaluate the use of the portable XRF in the bones. Using the portable XRF, the bone Sr exposure of the study population were simultaneously assessed with the bone Pb exposures. Besides, we analyzed the mid-tibia bone Sr data of a Chinese population, which were measured with the same portable XRF device by our research group. We also examined the extent to which the detection limit (DL) of the portable XRF was influenced by scan time and overlying soft tissue thickness for both Pb and Sr. </p> <p>For the exposure assessment of Mn and Hg in toenails, we first established system calibrations and determined the DL with phantoms. In order to validate the portable XRF in a population study, the recruited participants were measured at the big toenail by the device, and their toenail clippings were analyzed by the inductively coupled plasma spectrometry (ICP-MS). Besides, we analyzed the toenail data of an occupationally-exposed population, collected by our collaborators in Boston. A portable XRF device with the same model as ours was used in that study. </p> <p><b><i>Results:</i></b> The uncertainty of <i>in vivo</i> individual bone measurement increased with higher soft tissue thickness overlying bone, and reduced with extending measurement time. With thickness ranging from 2 to 6 mm, the uncertainty of a 3-minute <i>in vivo</i> measurement ranged from 1.8 to 6.3 ug/g (ppm) for bone Pb and from 1.3 to 2.3 ppm for bone Sr. Bone Pb measurements via portable XRF and KXRF were highly correlated: R=0.48 for all participants, and R=0.73 among participants with soft tissue thickness < 6 mm (72% of the sample). A trend of different bone Sr concentrations was observed across the races and sexes. </p> <p>The DL of the portable XRF with 3-minute toenail measurements was 3.59 ppm for Mn and 0.58 ppm for Hg. The portable XRF and ICP-MS measurements were highly correlated in the occupational populations for both Mn (R = 0.59) and Hg (R = 0.75). A positive correlation (R = 0.34) was found for toenail Mn measurements in the environmentally-exposed population, while a non-significant correlation was observed for toenail Hg due to the extremely low-level of Hg (Mean = 0.1 ppm) in the study population. </p> <p><b><i>Discussion and Conclusion:</i></b> The portable XRF could be a valuable tool for non-invasive <i>in vivo</i> quantification of bone Pb and Sr, especially for people with thinner soft tissue; and of toenail Mn and Hg, especially for people with moderate- to high-level exposures. </p>

Background: Construct validity refers to the degree to which tests that claim to measure a “construct” (i.e., an inferred concept that is intangible regarding an individual’s health or internal state such as a disease, or postulated attribute) are truly reflective of that specific construct. It is suggested that construct validity is an important concept in preclinical research, as it may help reduce misinterpretations of study results allowing for better ability to predict the success of clinical translation of preclinical studies. However, there is a lack of empirical evidence to confirm its impact on preclinical research efficacy. Objectives: (I) Conduct a scoping review of the construct validity literature as it relates to the design of in vivo animal studies. (II) Conduct an overview of systematics reviews evaluating the application and reporting of construct validity within systematic reviews of in vivo animal studies. Methods: For the scoping review, we searched Embase, MEDLINE, and Google Scholar. Eligibility criteria was intentionally broad as we included any article that mentioned construct validity in preclinical in vivo research. Further review of citations was performed on eligible studies that provided substantial discussion on construct validity. For the overview, we searched MEDLINE, Embase, and TOXLINE for systematic reviews of preclinical in vivo interventions. The outcomes of interest were the prevalence of systematic reviews that mentioned construct validity and the prevalence of reviews that assessed construct validity. Results: The literature searches for the scoping and overview yielded 3657 and 2356 articles, respectively. After screening 372 and 444 met inclusion criteria for the scoping and overview. Six codes were generated (theory; mechanism; matches the human condition; measures what it reports to; experimental conditions; and outcomes) from the content analysis for the definition of construct validity. Of the 444 systematic reviews, seven mentioned construct validity, but only three used the term construct validity directly. None of the systematic reviews assessed construct validity. Discussion/Conclusion: Construct validity was not defined uniformly among studies suggesting it is not clearly understood. There was limited reporting on construct validity in systematic reviews and entirely no assessment of it; this may reflect a lack of awareness of this concept. Future research should aim to find a consensus on the definition of construct validity in order to develop tools and frameworks to help researchers assess construct validity.