Introduction- Exercise-induced plasma volume (PV) expansion is typically associated with an increase in plasma albumin content. Increased hepatic albumin synthesis, a transcriptionally regulated process, is thought to contribute to the increase in albumin content. Objective- We tested the hypothesis that exercise training induces an increase in albumin gene expression in relationship to the increase in PV. Methods and Results- 40 adult male Sprague-Dawley rats weighing between 245-350 grams were randomly assigned to one of four groups: cage control (CC); sham exercise 10 min/day at 48% VO2max (NE); continuous exercise training, 60 min /day at 72% VO2max (LI); and high intensity, intermittent exercise training, 8 bouts of 4 min at 98% VO2max followed by 5 min at 48% VO2max (HI). The training period lasted for two weeks with 12 training sessions with equalized training volumes in the exercise groups. 24 hours after the last training session the rats were anesthetized and a jugular catheter was placed for collecting blood samples during PV determination by a dilution of a labeled-albumin molecule (Texas Red albumin). The liver and red quadriceps (RQ) muscle tissue was then removed, flash frozen, and stored for later analysis. The training protocol produced a significant increase in RQ citrate synthase activity (p < 0.05). PV increased in proportion to the exercise intensity (p < 0.05) averaging 23.6 ± 2.7 ml•kg-1 body weight in the CC group and 26.6 ± 1.3 ml•kg-1 body weight in the HI group. Albumin mRNA expression determined by real time polymerase chain reaction (PCR) increased 2.2 ± 0.1 and 2.9 ± 0.2 fold following LI and HI exercise training, respectively. Conclusion- These data support the hypothesis that, during exercise-induced PV expansion, albumin gene expression is increased and contributes to an increase in plasma albumin content and PV.

Subcutaneous (sc) and visceral (vis) adipose tissue (AT) contribute to the variability in pathophysiological consequences of obesity and adverse fat distribution. To gain insights into the molecular mechanisms distinguishing vis and sc fat, we compared the transcriptome during differentiation of immortalized adipocytes from murine epididymal (epi) and inguinal (ing) AT. RNA was extracted on different days of adipogenesis (−2, 0, 2, 4, 6, 8) and analyzed using ClariomTM D mouse assays (Affymetrix) covering >214,900 transcripts in >66,100 genes. Transcript Time Course Analysis revealed 137 differentially expressed genes. The top genes with most divergent expression dynamics included developmental genes like Alx1, Lhx8, Irx1/2, Hoxc10, Hoxa5/10, and Tbx5/15. According to pathway analysis the majority of the genes were enriched in pathways related to AT development. Finally, in paired samples of human vis and sc AT (N = 63), several of these genes exhibited depot-specific variability in expression which correlated closely with body mass index and/or waist-to-hip ratio. In conclusion, intrinsically programmed differences in gene expression patterns during adipogenesis suggest that fat depot specific regulation of adipogenesis contributes to individual risk of obesity.

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Conventional methods of body fluid identification use labor-intensive, technologically diverse techniques that are performed in a series, not parallel, manner and are costly in terms of time and sample. Furthermore, for some frequently encountered body fluids, such as saliva or vaginal secretions, no confirmatory technique exists. Terminally differentiated cells, such as blood lymphocytes or epithelial cells lining the oral cavity, have a unique pattern of gene expression, which is evinced by the presence and relative abundance of specific mRNA species. If the type and abundance of mRNAs can be determined in a stain or tissue sample recovered at the crime scene, it would be possible to definitively identify the tissue or body fluid in question. Advantages of an mRNA-based approach, compared to conventional biochemical analysis, include greater specificity, simultaneous and semi-automated analysis though a common assay format, improved timeliness, decreased sample consumption and compatibility with DNA extraction methodologies. In this report, we demonstrate that RNA is stable in biological stains and can be recovered in sufficient quantity and quality for analysis using reverse transcriptasepolymerase chain reaction assay (RT-PCR). We have identified sets of candidate tissuespecific genes for body fluids and tissues of forensic interest, namely blood, saliva, semen, vaginal secretions, menstrual blood, urine, skin, muscle, adipose, and brain. We also report the identification of a new housekeeping gene for use in mRNA based assays. Select body fluid-specific genes have been incorporated into multiplex PCR and real-time PCR assays. These assays allow for the positive identification of blood, saliva, semen,vaginal secretions, and/or menstrual blood in a stain. The final task of this work was the molecular characterization of mRNA degradation patterns in biological stains, which not only has fundamental importance in possibly revealing mRNA degradation pathways in dried biological stains, but may ultimately lead to better assay design strategies for mRNA markers for forensic use. An mRNA-based approach described in this report could allow the facile identification of the tissue components present in a body fluid stain and could conceivably supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory.

Receptor-interacting protein kinase 1 (RIPK1) is a major upstream mediator of inflammation and cell death. These processes are key to common inflammatory diseases such as atherosclerosis, where macrophages play an important role in their progression. Closely linked to the expression of downstream genes, long non-coding RNAs (lncRNAs) are critical to controlling cellular processes in health and disease. As post-transcriptional regulatory mechanisms for RIPK1 are largely unknown, this project seeks to study the stability of Ripk1 mRNA and RIPK1 protein, along with Ripk1 mRNA interactions with relevant lncRNAs under various conditions. Using transcription and translation inhibitors, we determined that both Ripk1 mRNA and RIPK1 protein are relatively unstable with half-lives of approximately 3 h. Their turnover in macrophages is further influenced by the timing and duration of inflammation. We also implemented a novel RNA pull-down procedure to capture Ripk1 mRNA and attached lncRNAs for next-generation sequencing. Through differential expression analysis, we discovered significant upregulation of known lncRNA AC125611 and novel lncRNA MSTRG.5894.1 in Ripk1-targeted samples subject to inflammation. MSTRG.7477.1 was upregulated during necroptosis, while MSTRG.5684.5 was upregulated during both inflammation and necroptosis. GapmeR-mediated knockdowns of AC125611 and MSTRG.5684.5 under inflammatory conditions resulted in decreased Ripk1 mRNA expression and RIPK1 protein expression, respectively. Meanwhile, MSTRG.7477.1 knockdowns were connected to decreased RIPK1 at both the mRNA and protein levels. Our research ultimately advances the current understanding of RIPK1 regulation by focusing on Ripk1 mRNA-lncRNA associations and turnover of its mRNA and protein in macrophages, paving the way for future investigations into their capacity to act as therapeutic targets.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24192号 / 医博第4886号 / 新制||医||1060(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 萩原 正敏, 教授 森信 暁雄, 教授 遊佐 宏介 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24971号 / 医科博第153号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 村川 泰裕, 教授 竹内 理, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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