abstract: It has been repeatedly shown that females have lower stability and increased risk of ankle injury when compared to males participating in similar sports activities (e.g., basketball and soccer), yet sex differences in neuromuscular control of the ankle, including the modulation of ankle stiffness, and their contribution to stability remain unknown. To identify sex differences in human ankle stiffness, this study quantified 2- dimensional (2D) ankle stiffness in 20 young, healthy men and 20 young, healthy women during upright standing over a range of tasks, specifically, ankle muscle co-contraction tasks (4 levels up to 20% maximum voluntary co-contraction of ankle muscles), weight-bearing tasks (4 levels up to 90% of body weight), and ankle torque generation tasks accomplished by maintaining offset center-of-pressure (5 levels up to +6 cm to the center-of-pressure during quiet standing). A dual-axial robotic platform, capable of perturbing the ankle in both the sagittal and frontal planes and measuring the corresponding ankle torques, was used to reliably quantify the 2D ankle stiffness during upright standing. In all task conditions and in both planes of ankle motion, ankle stiffness in males was consistently greater than that in females. Among all 26 experimental conditions, all but 2 conditions in the frontal plane showed statistically significant sex differences. Further analysis on the normalized ankle stiffness scaled by weight times height suggests that while sex differences in ankle stiffness in the sagittal plane could be explained by sex differences in anthropometric factors as well as neuromuscular factors, the differences in the frontal plane could be mostly explained by anthropometric factors. This study also demonstrates that the sex differences in the sagittal plane were significantly higher as compared to those in the frontal plane. The results indicate that females have lower ankle stiffness during upright standing thereby providing the neuromuscular basis for further investigations on the correlation of ankle stiffness and the higher risk of ankle injury in females. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2020

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23102号 / 医博第4729号 / 新制||医||1050(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 花川 隆, 教授 髙橋 良輔, 教授 伊佐 正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Two groups of male and female rats were given the same dose of alcohol using an artificial rearing procedure on postnatal days 4-10. One group received the alcohol in a condensed manner each day which caused cyclic blood alcohol concentrations (BACs) with high peaks. A second group received the alcohol in a uniform manner over each day which resulted in moderate, stable BACs. Two control groups consisted of male and female rats artificially reared but not exposed to alcohol and rats reared normally by dams. All rats were raised to 90 days of age and then tested for spatial navigation ability in the Morris water maze, which involved locating a hidden underwater platform using distal extramaze cues. Neither the alcohol treatments nor the artificial rearing had any effects on performance of adult male rats relative to suckle controls in this task. In contrast, the condensed alcohol exposure but not the uniform alcohol exposure resulted in detrimental performance in the Morris water maze by adult female rats. When the ability to locate and escape onto a visible platform was examined, there were no differences between the female groups given condensed alcohol exposure or artificially reared on milk solution alone. Thus, exposure to high BACs during the brain growth spurt has a lasting and selective detrimental effect on spatial navigation learning in adult female but not adult male rats.

Reactive strength index-modified (RSImod) is a measure of lower body explosiveness calculated by dividing jump height by time to takeoff. RSImod is different between stronger and weaker athletes and between males and females. The purpose of this study was to evaluate differences in RSImod between males and females while controlling for maximal strength and lower body explosiveness. Forty-three female and fifty-eight male Division-I athletes performed countermovement jumps on a force plate during unloaded (0kg) and loaded (20kg) conditions. We used an ANCOVA to test whether RSImod is different between sexes conditioning on relative maximum strength (PFa) and average RFD 0-200ms (RFD200) measured during the isometric midthigh pull (IMTP). Differences of 0.087 (95% CI: 0.040 - 0.134; p = 0.0005) and 0.075 (95% CI: 0.040 - 0.109, p < 0.0001) were observed for RSImod between sexes in unloaded and loaded conditions, respectively. A male with PFa of 186 (grand mean of the sample) and RFD200 of 6602 N/s (grand mean of the sample) is predicted to have 28% greater RSImod than a female of similar PFa and RFD200. Maximum strength development should be a primary aim of training in female athletes, in addition to other trainable factors, such as stiffness and RFD.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第17955号 / 人健博第9号 / 新制||人健||1(附属図書館) / 30785 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 黒木 裕士, 教授 坪山 直生, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Binding parameters of soluble Type I and Type II receptors were assessed in hippocampus of adult, adrenalectomized, male and female rats. No sex differences in the number of either Type I or Type II receptors could be demonstrated between gonadally intact animals. When females treated with 17β-estradiol benzoate (10 μg/day) were compared with males, a statistically significant reduction in Type II receptors was observed in the females; progesterone produced no further decrease in receptor numbers. The amount of tissue-associated corticosteroid-binding globulin in gonadally intact animals (perfused with dextran-saline) was twice as great in females as males. Sex-dependent differences in these gonadally intact rats were found in the affinity, measured as the dissociation constant (Kd), of both the Type I and Type II receptors. For both receptors, affinity in cytosols from females was reduced. The difference for the Type II receptor was slight, but the Kd value of the type I receptor was several-fold higher in females. The difference in affinity was evident with both natural and synthetic steroid ligands. There appears to be little, if any, difference in affinity between the hippocampal Type I and the Type II receptors in females. This suggests that the occupancy of Type I receptors in females is substantially less than that of males at low circulating concentrations of corticosteroids.

Migraine is a common, episodic neurological disorder that includes headache, nausea and hypersensitivity to sensory stimuli. During the headache phase of migraine, migraine patients can be especially hypersensitive to thermal stimuli. The unpredictable and episodic nature of migraine makes it difficult to treat and much of the mechanism of migraine has yet to be elucidated. A T44A substitution in casein kinase 1δ is inherited with migraine with aura. A transgenic mouse model suggests that animals with this mutation exhibit increased sensitivity to thermal stimuli after injection with nitroglycerin (NTG). We performed behavior assays that measure animal responses to thermal stimuli, after injection with NTG, a known migraine-inducer in human migraine patients. Female animals with the CK1δ-T44A mutation are more sensitive than wildtype littermates, suggesting a sex difference emerges in pain sensitivity in animals that express the CK1δ-T44A but not in wildtype siblings. Female CK1δ-T44A animals are more sensitive to the effects of NTG on pain than male CK1δ-T44A mice. This indicates a potential sex hormone related pain response. Since estrogen is implicated in both migraine and pain response, we test the thermal sensitivity of heterozygous ERβKO/+ and CK1δ-T44A; ERβKO/+ mice compared to wildtype and CK1δ-T44A mice. Overall thermal sensitivity is decreased before stress of injection in both male and female ERβKO/+ and CK1δ-T44A: ERβKO/+ mice. This demonstrates that ERβ is necessary for thermal nociception in untreated mice. However, after injection with saline or NTG, animals of all genotypes responded to thermal stimuli similarly. This suggests that estrogen signaling through ERβ is likely not part of the pathway of NTG-induced thermal sensitivity or that one copy of ERβ is sufficient for NTG-induced thermal sensitivity. Since ERβ is fully functional in CK1δ-T44A mice and CK1δ-T44A mice have wildtype thermal sensitivity at baseline, we can conclude that CK1δ-T44A does not modulate ERβ to affect thermal sensitivity in untreated animals.

Post-traumatic stress disorder (PTSD) is a debilitating disorder with a prevalence rate of approximately 5%. Unfortunately, this disorder is commonly associated with another debilitating disorder, alcohol use disorder (AUD). Of the 5% of people diagnosed with PTSD, 30%-59% also suffer from AUD. Currently, there are limited effective treatment options for those suffering from comorbid PTSD/AUD. Previous research has suggested that biological sex differentially impacts PTSD comorbid with AUD, however, the underlying mechanisms are enigmatic. The goal of this study was to better understand the underlying mechanisms that mediate sex differences in a rodent model of comorbid PTSD/AUD by analyzing specific behavioral tasks and changes in neuronal function of specific brain regions. Chronic inflammation has been implicated in PTSD and AUD respectively, with differences between sexes being observed. Females tend to express elevated levels of inflammation in both disorders compared to males in brain regions such as, the hippocampus, amygdala, and prefrontal cortex. Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine that is released during neuronal inflammation. To further examine these sex differences, a comorbid PTSD/AUD rodent model was implemented using restraint stress (RS) and chronic intermittent ethanol use (CIE). Following the exposure to RS and CIE a fear conditioning procedure was implemented to assess changes in future stress sensitivity. The fear conditioning paradigm was accomplished by conditioning the animal to pair a tone with a foot shock, followed by extinction of that behavior in a different context where the animal received the tone but no foot shock. Thereafter, the animal was placed back in the context they received the foot shock, known as context renewal, but acquired no tone or foot shock. The behavior in these different contexts was analyzed to test memory and stress sensitivity. Brain tissue was collected to analyze TNF-α protein expression in regions associated with learning, memory, and addiction such as, the prelimbic cortex (PrL), infralimbic cortex (IfL), and the hippocampus. The results of the fear conditioning revealed that the females froze more altogether compared to the males, and there was more freezing of the females with RS and CIE during context renewal. It is expected that TNF-α protein expression will be significantly elevated in females when compared to males, regardless of treatment group. Females exposed to RS and CIE will have significantly higher TNF-α levels when compared to all other treatment groups. Finally, increases in TNF-α protein expression will be region specific with the PrL and IfL regions exhibiting significantly greater expression than the hippocampus. This study will aid in better understanding the sex differences and lead to better treatment options that are sex-dependent for those diagnosed with comorbid PTSD/AUD.

<p>Cytochrome P450s (CYPs) are enzymes that are found throughout the three domains of life. They function in the metabolism of endogenous and exogenous compounds. CYPs are extensively studied in mammalian systems due to their importance in drug metabolism and are highly expressed in detoxification organs like the liver and intestine. Fish CYP3s are not well understood. CYP3s have diversified in fish and subfamilies A, B, C and D constitute the CYP3 clade in fish. In this study, CYP3C1, CYP3C2, CYP3C3 and CYP3C4 in zebrafish (<em>Danio rerio</em>) and CYP3B4, CYP3B5 and CYP3B6 in medaka (<em>Orzyias latipes</em>) were quantified in hepatic and extrahepatic organs. CYP3C genes were quantified throughout development. All CYP3B and 3C isoforms were detected in all organs except CYP3B4 in male organs and in female brain. CYP3C1-C3 were maternally acquired and expressed in all embryonic stages. Higher expression of some of the isoforms occurred in the liver and intestine of zebrafish and medaka. This is indicative of a possible role in xenobiotic metabolism. Differences in expression between males and females gonad was observed, suggesting a possible role for estrogen in gene regulation. Further research will contribute to characterizing the upstream response elements in order to understand whether estrogens or other compounds are responsible for CYP3 regulation in fish. This knowledge will contribute to understanding the potential function these unique families of CYPs serve for fish.</p> / Master of Science (MSc)

Early growth response 1 (Egr1) is a transcription factor critical for learning and memory in the hippocampus and pituitary cell differentiation. Egr1 has been shown to extend continuation of the long-term potentiation in the hippocampus and is credited for forming long-term memories. The somatotrophs in the pituitary produce growth hormone and are found to be decreased in Egr1KO mice. These animals are also found to be sterile due to a decrease in LHB, which blocks ovulation. All previous studies have evaluated these physiological processes with complete Egr1KO research strains or antisense oligonucleotides, up until now, no data specific to individual type of cells has been generated. In an attempt to focus on the understanding of the functions of Egr1 gene in neural cell lineage, we are using an Egr1cKO Nestin-Cre model. Nestin allows for targeting neuronal lineage specific cells. In Chapter 1, we provide a systemic view of Egr1 gene and Nestin-Cre as a system for generating conditional knockout mouse strains. The Chapter begins with the identification of Egr1 gene and its protein structure, then proceeds to grasp its link to memory with behavior testing. The critical role of Egr1 in the pituitary and what cell populations are affected is also described. The same goes for Nestin-Cre, along with its limitations and understanding how to account for them in a study. The Egr1cKO Nestin-Cre system is the best form to understand neurological cell populations with Egr1 removal. In Chapter 2 and Chapter 3, we employ the Egr1cKO Nestin-Cre mouse model to understand cell-specific knockout of Egr1 in the nervous system by evaluating the hippocampus and pituitary. We explore learning and memory through behavioral tests and ribonucleic acid sequencing (RNA-seq) analysis to understand gene expression changes with Egr1 removal. Females showed higher activity during behavior tests, with more movement in the elevated plus maze and lower freezing times during the contextual fear conditioning. RNA-seq had higher changes in females than males but was not affected by the Nestin-Cre system overall. The same RNA-seq changes in the pituitary gland were present, with females having higher genomic differentiation. Females had growth-specific pathways altered by Nestin-Cre. / Doctor of Philosophy / Genetics has become a very important forerunner in scientific research. One gene that has become important in many different research arenas is Early growth response 1 (Egr1). This particular gene is critical for learning, memory, and cell changes in the pituitary. In Chapter 1, we have analyzed the current research landscape of information on Egr1 in its functions with learning and memory, as well as the pituitary. Most previous studies that have been completed only evaluate this gene by its removal from the entire body. This leaves a large gap in information about how this gene functions with specific cell types. To limit the type of cells from which Egr1 has been removed, we have selected Nestin-Cre, a tool to remove genes from neuronal stem cells. The capabilities and limitations of this tool have also been explained in this chapter, along with how the two together can accomplish a cell-specific knockout of Egr1. In Chapter 2, we have constructed an experiment with behavioral tests for mice, along with RNAseq data from the hippocampus to evaluate what changes have occurred in the Egr1cKO Nestin-Cre model. Female mice are more active in the behavioral test, including the elevated plus maze (EPM) and Contextual fear conditioning (CFC), than male mice. The same holds for differences in the RNAseq data as well. In Chapter 3, the pituitary of Egr1cKO Nestin-Cre mice is the main focus. We evaluated RNAseq data and determined growth rates of transgenic mice. The mice had different growth rates over twelve weeks between the controls and the knockout. The RNAseq data also revealed many differences between males and females. Female mice had specific growth genes effects by the knockout of Egr1