AIM & HYPOTHESIS: We hypothesized that the piezoelectric knife increases bone remodeling. The effect of a piezotome 2 prototype with different puissance and modulation settings on bone biology was compared under different bone resorption and/or bone formation conditions using an ex-vivo live bone organ culture MATERIALS & METHODS: We utilized an ex-vivo mouse calvarial bone culture system a n d piezocision surgical techniques with variable puissance and modulation settings under conditions of static versus dynamic, and formation versus resorption in order to decipher cellular, molecular, biological and genetic perturbations induced by piezocision surgery. In addition, we tested its impact on calvarial bone organ cultures in the presence of drugs such as bisphosphonate (zolendronic acid, ZOL) and insulin or its impact on bone healing with bone graft material such as Enamel matrix derivative (Emdogain). RESULTS: The impact of the piezoelectric knife extends beyond the immediate site of mineralized bone defect/damage. The impact is ~6 times greater than the apparent initial mineralized bone injury, 1.6 mm2, and extends to immediate surrounding local periosteal and endosteal cell layers, 11 mm2, leaving the mineralized bone layer intact. There were also major differences between the static and dynamic model systems in bone defect viii healing, resorption and new bone formation. There are also differences between the piezoelectric knife and bur in bone resorption and formation models and in both treated before or at the same time of the injury with bisphosphonate. In the calvarial defect repair model the defect closed much faster with the piezoelectric knife than with the bur. In a comparative study with controls versus piezotome or bur, and piezotome versus bur complete genomic analysis of changes revealed a large number of major differences in genomic expression levels. CONCLUSION: The extended effect of piezocision on the surrounding bone lining cells of the bone defect including de-vitalization of the proximal osteocytes within the intact mineralized bone matrix thet primes the bone to undergo rapid turnover. Piezocision incorporates high-frequency vibrational energy that can be transmitted a significant distance within the mineral phase and exploits the inherent bone cellular response mechanism(s) as defined by the global genearray analysis. / 2020-06-13T00:00:00Z

This thesis is composed of two topics both concerning diverse and obligatory trypanosomatid parasites. First part deals with identification of new Trypanosoma species identified in blood meal of tsetse flies caught in Dzanga-Sangha Protected Areas, Central African Republic, and identification of feeding preferences of tsetse flies. The second part concerns extraordinary monoxenous trypanosomatid Paratrypanosoma confusum which constitutes the most basal branch between free-living Bodo saltans and parasitic trypanosomatids. This thesis helped to elucidate morphology and biology of this deep branching trypanosomatid. Using genome and transcriptome sequencing and comparative bioinformatics approaches enabled search for ancestral genes shared with free-living bodonids and confirmed genome streamlining in trypanosomatids.

How chromatin-remodeling complexes modulate gene networks to control organ-specific properties is not well understood. For example, Baf60c (Smarcd3) encodes a cardiac-enriched subunit of the SWI/SNF-like BAF chromatin complex, but its role in heart development is not fully understood. We found that constitutive loss of Baf60c leads to embryonic cardiac hypoplasia and pronounced cardiac dysfunction. Conditional deletion of Baf60c in cardiomyocytes resulted in postnatal dilated cardiomyopathy with impaired contractile function. Baf60c regulates a gene expression program that includes genes encoding contractile proteins, modulators of sarcomere function, and cardiac metabolic genes. Many of the genes deregulated in Baf60c null embryos are targets of the MEF2/SRF co-factor Myocardin (MYOCD). In a yeast two-hybrid screen, we identified MYOCD as a BAF60c interacting factor; we showed that BAF60c and MYOCD directly and functionally interact. We conclude that Baf60c is essential for coordinating a program of gene expression that regulates the fundamental functional properties of cardiomyocytes.

In vertebrates, PTH receptor 1 (PTH1R) plays a pivotal role in control of bone development and homeostasis; however, how it is regulated is poorly defined. Here we report that Kindlin-2 binds to and modulates PTH1R to regulate bone mass and PTH actions. Deleting Kindlin-2 expression using the 10-kb mouse Dmp1-Cre severely impairs the anabolic effects of intermittent PTH on bone in adult mice with or without ovariectomy. Of particular interest, Kindlin-2 and Pth1r double heterozygous mice (Dmp1- Cre; Kindlin-2 f/+ ; Pth1r f/+ ), but not either singly heterozygous mice (Dmp1- Cre; Kindlin-2 f/+ or Dmp1-Cre; Pth1r f/+ ), display severe osteopenia and fail to increase bone mass in response to administration of intermittent PTH. Mechanistically, Kindlin-2 interacts with the C-terminal cytoplasmic region of PTH1R. When overexpressed, this region efficiently inhibits the endogenous PTH/PTH1R signaling in osteoblasts, which is reversed by introduction of a point mutation that abolishes the Kindlin-2 interaction. Furthermore, Kindlin-2 loss inhibits PTH-induced CREB phosphorylation and cAMP production in vitro and in bone. PTH upregulates, while estrogen deficiency downregulates, expression of Kindlin-2 in vitro and in bone. Collectively, we demonstrate that interplay between Kindlin-2 and PTH1R regulates bone mass by modulating PTH1R and provide a potential therapeutic target for metabolic bone diseases

Obesity is characterized by the accumulation of excess adipose tissue and has become a global health burden as the rates of obesity in both adults and children continue an upward trend year after year. Obesity is an important public health concern as it has been linked to increased prevalence of comorbidities such as type 2 diabetes, fatty liver, cardiovascular complications and cancer. Adipocytes are exquisitely sensitive to energy demands, quickly responding by releasing fatty acids, storing excess calories as triglycerides and/or secreting adipokines. The ability of the adipocyte to carry out its important functions requires perpetual remodeling of the extracellular matrix (ECM) surrounding the adipocyte. Metabolic dysfunction and fibrosis arise when the healthy balance of remodeling becomes dysregulated. The long-term goal of this project was to gain a deeper understanding of the events occurring within an adipocyte in the context of weight gain. I hypothesized that the adipocyte would play an active role in remodeling of the ECM. To test this hypothesis, I fed C57/Bl6 mice either high fat diet (HFD) or chow diet for 8, 20 and 34 weeks, at which time, the perigonadal adipose tissue was digested to isolate the adipocyte fraction and RNA-seq analysis was performed. My data demonstrate that adipocytes responded to their obesogenic, pro-inflammatory environment through upregulation of ECM-related genes after only 8 weeks of HFD coupled with declining expression of mitochondrial genes and increasing genes associated with endoplasmic reticulum (ER) stress after 20 and 34 weeks of HFD. Overall, these data give a novel view into the dysfunctional state of isolated adipocytes over a time course of HFD and response to the changing microenvironment. MICAL2 is an atypical actin-modulating protein that has been shown to be involved in the regulation of MRTFA/SRF signaling and in cancer progression. I demonstrated for the first time that MICAL2 expression is enriched in the stromal vascular fraction of adipose tissue and is upregulated with HFD and pro-fibrotic stimulus, TGF-β1. I also demonstrated that MICAL2 is an anti-adipogenic and pro-fibrogenic protein. Altogether, the novel biology uncovered suggests a role for MICAL2 in adipose tissue remodeling which warrants further investigation. / 2021-06-14T00:00:00Z

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20276号 / 医博第4235号 / 新制||医||1021(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 齊藤 博英, 教授 楠見 明弘, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Previous research has provided evidence in support of apoptotic osteocytes playing a role in the bone remodeling process. In this study, we examined the regional and quadrantal variations of apoptotic and viable osteocytes in cyclically loaded and unloaded samples. Left tibias of C57 Black 6 Taconic mice (C57Bl/6) were cyclically loaded for either 2 weeks or 5 weeks, with the right tibias being used as controls. After loading, tibias were resected, processed, and then stained using either a TUNEL stain, to show apoptotic osteocytes, or a 2.0% methyl green solution, to reveal viable cells. Cross-sectional images from each tibia were then captured and analyzed in each region (distal, midshaft and proximal) and quadrant (cranial, lateral, caudal, and medial) by counting the number of osteocytes, both apoptotic and viable, and subsequently calculating the percentages and densities of those osteocytes. Individual analysis of each sample group showed that the 5 week loaded bones, with the most statistically significant p-values, had the most regional variations within the samples, specifically showing decreased apoptotic and viable osteocytes in the lateral quadrants. Comparative analysis revealed a statistically significant higher percentage and density of apoptotic osteocytes in 5 week loaded samples compared to all other samples. This provides further quantitative evidence in support of apoptotic osteocytes playing a role in bone remodeling.

The study presented attempts to prove the concept that mechanical changes in the structure of a bone can be predicted for a specific exercise by a subject specific model created from CT data, MRI data, EMG data, and a physiologic FE model. Previous work generated a subject specific FE model of a femur via CT and MRI data as well as created a set of subject specific biomechanical muscle forces that are required to perform a single leg extension exercise. The FE model and muscle forces were implemented into a single leg extension FE code (ABAQUS) along with a specialized bone remodeling UMAT. The UMAT updated the mechanical properties of the femur via a damage-repair bone remodeling algorithm. The single leg extension FE code was verified by applying walking loads to the femur and allowing the system to equilibrate. The results were used to apply the appropriate walking loads to the final FE simulation for the single leg extension exercise. The final FE simulation included applying the single leg extension loads over a one year period and plotting the change in porosity at various regions of the femoral neck. Although only two regions were found to generate valid results, the data seemed counterintuitive to Wolff’s Law which states that bone adaptation is promoted when the material is stressed. The model was successful in creating a subject specific model that is capable of predicting changes in the mechanical properties of bone. However, in order to generate valid FE model results, further understanding of the bone remodeling process and application via a FE model is required.

Studies are currently being performed to determine the effects of bisphosphonate treatments on the structure and density of bone tissue. One of the pathways for gaining a better understanding of the effects of this and other treatments involves creating a computer simulation. Theory suggests that bone tissue structure and density are directly related to the manner in which the tissue is loaded. Remodeling is the process in which bone tissue is resorbed in areas of low stress distributions, and generated in areas of high stress distributions. Previous studies have utilized numerical methods and finite element methods to predict bone structure as a result of stress distributions within the tissues. The Finite Element method was chosen for this study. This study was done on a canine (beagle) rib. The goal of this study was to develop an FEA model of the rib that would be used in conjunction with a bone remodeling algorithm, to model the behavior of the bone tissue. Appropriate boundary conditions, loads, and loading cycles were determined from literature, and applied. Respiration was assumed as the dominating activity; therefore the muscles involved in respiration were the primary source of the rib loading. The model also included an integrated UMAT sub-routine, which utilized data from the FEA model to iterate bone tissue densities and structures. The model closely predicted the porosities of the bone tissue, when compared to actual tissue samples, as well as what literature describes.

Indiana University-Purdue University Indianapolis (IUPUI) / Previous research indicates that overall, bone turnover decreases with age. The effects of aging on the remodeling of the mandibular condyle have not been studied. As part of the temporomandibular joint, the mandibular condyle (MC) is exposed to a unique pattern of loading. As such, data obtained from studies of other bones may not be applicable to accurately explain the behavior of trabecular bone of the mandibular condyle and its relationship to aging. Recent research has led to the finding that cortical bone turnover in the mandible is much higher than that seen in tibia, and that both sites decline with age. The purpose of this study was to histomorphometrically quantify the effects of aging on the bone remodeling of the canine MC, and to determine whether site-specific differences due to age occur in the dynamic and static parameters between MC and tibial condyle (TC). Fluorochrome labels were used to mark sites of bone formation in ten old and five young dogs. Specimens were obtained from one MC and one TC for each dog and were prepared for analysis of static and dynamic histomorphometric indices. Mineral apposition and bone formation were totally absent in the old group, as shown by the lack of fluorochrome labels. In the young group, they were significantly higher in the MC than in the TC (p < 0.01). Aging resulted in a significant increase of volume density of the subchondral bone (p < 0.05). Trabecular bone volume was not significantly affected by age in the samples studied. The results indicate that bone remodeling is markedly higher in MC than in the TC in young dogs. With aging, it declines to zero in both sites. Whether the loss of remodeling activity in these elderly animals indicates that they are fully adapted to their mechanical environment or that they have lost the intrinsic ability to remodel, remains to be determined.