Mathematics in Software Reliability and Quality Assurance

This monograph concerns the mathematical aspects of software reliability and quality assurance and consists of 11 technical papers in this emerging area. Included are the latest research results related to formal methods and design, automatic software testing, software verification and validation, coalgebra theory, automata theory, hybrid system and software reliability modeling and assessment

Anelastic-like nature of the rejuvenation of metallic glasses by cryogenic thermal cycling

Cryogenic thermal cycling (CTC) is an effective treatment for improving the room-temperature plasticity and toughness in metallic glasses. Despite considerable attention to characterizing the effects of CTC, they remain poorly understood. A prominent example is that, contrary to expectation, the stored energy in a metallic glass first rises, and then decreases, as CTC progresses. In this work, CTC is applied to bulk metallic glasses based on Pd, Pt, Ti, or Zr. The effects on calorimetric and mechanical properties are evaluated. Critically, CTC-induced effects, at whatever stage, are found to decay over about one week at room temperature after CTC, returning the properties to those of the as-cast glass. A model is proposed for CTC-induced effects, treating them as analogous to the accumulation of anelastic strain. The implications for analysis of existing data, and for future research on CTC effects, are highlighted

Impact of Fibronectin Knockout on Proliferation and Differentiation of Human Infrapatellar Fat Pad-Derived Stem Cells

Fibronectin plays an essential role in tissue development and regeneration. However, the effects of fibronectin knockout (FN1-KO) on stem cells’ proliferation and differentiation remain unknown. In this study, CRISPR/Cas9 generated FN1-KO in human infrapatellar fat pad-derived stem cells (IPFSCs) was evaluated for proliferation ability including cell cycle and surface markers as well as stemness gene expression and for differentiation capacity including chondrogenic and adipogenic differentiation. High passage IPFSCs were also evaluated for proliferation and differentiation capacity after expansion on decellularized ECM (dECM) deposited by FN1-KO cells. Successful FN1-KO in IPFSCs was confirmed by Sanger sequencing and Inference of CRISPR Edits analysis (ICE) as well as immunostaining for fibronectin expression. Compared to the GFP control, FN1-KO cells showed an increase in cell growth, percentage of cells in the S and G2 phases, and CD105 and CD146 expression but a decrease in expression of stemness markers CD73, CD90, SSEA4, and mesenchymal condensation marker CDH2 gene. FN1-KO decreased both chondrogenic and adipogenic differentiation capacity. Interestingly, IPFSCs grown on dECMs deposited by FN1-KO cells exhibited a decrease in cell proliferation along with a decline in CDH2 expression. After induction, IPFSCs plated on dECMs deposited by FN1-KO cells also displayed decreased expression of both chondrogenic and adipogenic capacity. We concluded that FN1-KO increased human IPFSCs’ proliferation capacity; however, this capacity was reversed after expansion on dECM deposited by FN1-KO cells. Significance of fibronectin in chondrogenic and adipogenic differentiation was demonstrated in both FN1-KO IPFSCs and FN(–) matrix microenvironment

Physiological and genetic manipulation of adventitious rooting in Prunus spp

Many species from economically important genera remain rooting recalcitrant, prohibiting the commercialisation of many species in forestry and horticulture, and hindering genetic improvement by conventional breeding or recombinant DNA technology, where vegetative propagation is often used to preserve the genetic fidelity of elite progeny. Two cherry species (Prunus avium and P. padus) were used as models in this study to investigate the physiological and genetic manipulation of adventitious rooting. Mature trees are typically more difficult to propagate vegetatively than their juvenile counterparts. For some trees, micropropagation can circumvent certain effects of ageing and maturation, restoring shoot vigour and rooting, but the mechanism(s) involved have not been elucidated. During micropropagation, subculture interval was found not to be the predominant factor promoting the 'apparent rejuvenation' of mature P. avium tissue. 'Apparently rejuvenated' ex vitro and hedged (putatively) mature P. avium trees were treated with gibberellins predicted to have a range of structural related activities. GA7 improved the rooting of cuttings from hedged (putatively) mature cherry, but not from ex vitro trees. Methodology to regenerate adventitious shoots from P. avium leaf explants was developed, (putative) transgenic P. padus plants were produced by an Agrobacterium tumefaciens mediated strategy. Auxin redistribution in planta is postulated to require a component of active transport; inhibition of the predominantly basipetal transport has profound effects on rooting. The putative function of the Arabidopsis thaliana AtAUX1 gene is that of a cellular auxin influx carrier, possibly, as described by the chemiosmotic hypothesis. This thesis examined the hypothesis that transformation with the AtAUX1 gene would enhance the delivery of the root-inducing signal to improve rooting of P. padus, a species which is rooting recalcitrant and more or less obligate on exogenous auxin for this process. However, all six, constitutively expressed, Cauliflower Mosaic Virus 35S promoter driven, 35S::AtAUX1, transgenic shoot lines had reduced rooting

In vivo Partial Reprogramming by Bacteria Promotes Adult Liver Organ Growth without Fibrosis and Tumorigenesis

Ideal therapies for regenerative medicine or healthy aging require healthy organ growth and rejuvenation, but no organ-level approach is currently available. Using Mycobacterium leprae (ML) with natural partial cellular reprogramming capacity and its animal host nine-banded armadillos, we present an evolutionarily refined model of adult liver growth and regeneration. In infected armadillos, ML reprogram the entire liver and significantly increase total liver/body weight ratio by increasing healthy liver lobules, including hepatocyte proliferation and proportionate expansion of vasculature, and biliary systems. ML-infected livers are microarchitecturally and functionally normal without damage, fibrosis, or tumorigenesis. Bacteria-induced reprogramming reactivates liver progenitor/developmental/fetal genes and upregulates growth-, metabolism-, and anti-aging-associated markers with minimal change in senescence and tumorigenic genes, suggesting bacterial hijacking of homeostatic, regeneration pathways to promote de novo organogenesis. This may facilitate the unraveling of endogenous pathways that effectively and safely re-engage liver organ growth, with broad therapeutic implications including organ regeneration and rejuvenation

ディペンダブルコンピュータシステムのモデルベース性能評価に関する研究

広島大学(Hiroshima University)博士(工学)Doctor of Engineering in Information Engineeringdoctora

Stem cell matrix and cartilage regeneration

Cartilage defects caused by injuries of the knee affect about 900,000 Americans annually, resulting in more than 200,000 surgical procedures. Cartilage repair remains a major challenge due to its limited healing capacity. Current cell-based therapy using autologous chondrocyte implantation has been developed for decades and promising results have been observed in clinic. However, the shortage of autologous chondrocytes and their uncertain long-term effectiveness have led researchers to find alternative solutions. Stem cells from various tissues have been shown to be potential sources of chondrocytes. Among them, synovium-derived stem cells (SDSCs) have been suggested as tissue-specific stem cells for chondrogenesis. However, a major obstacle challenging the cartilage tissue engineering is cell senescence, which is due mainly to extensive ex vivo passaging and elderly donors. A reconstructed ex vivo microenvironment that can maintain or enhance stemness is urgently needed for facilitating large-scale tissue engineering. To facilitate ex vivo expansion, the conventional methods for stem cell expansion were extensively investigated. We first compared the influence of low- and high-seeding density on human SDSC stemness during ex vivo expansion. Low-density seeding expansion yielded SDSCs with enhanced proliferative and multi-differentiation capacities compared to high-density seeding though it is not highly efficient. Downregulation of ERK1/2 and JNK and upregulation of p38 might be attributed to the retained stemness under low-density expansion. We next compared the impact of hypoxia, fibroblast growth factor-2 (FGF-2) supplementation and a novel approach of SDSC-deposited decellularized extracellular matrix (DECM) on SDSC stemness. DECM expansion enhanced greater SDSC proliferation while retaining stem cell characteristics, compared to FGF-2 supplementation alone. The combination of hypoxia, FGF-2 and DECM contributed to the highest cell number in SDSC expansion, indicating their synergistic effects. Although the chondrogenic index was comparable between DECM expansion and FGF-2 supplementation, which were much higher than expansion on plastic flask alone, the observations that FGF-2 induced hypertrophic marker genes suggested the superiority of DECM in enhancing SDSC self-renewal while retaining stemness. Other potential cell sources for depositing DECM were also evaluated. We found that, besides SDSCs, adipose- or urine-derived stem cells and dermal fibroblasts could also deposit the DECM, which could enhance SDSC self-renewal and chondrogenic potential without concomitantly enhancing adipogenic and osteogenic potentials. These findings suggest that, given an optimal DECM substrate, the chondrogenic potential within the tissue-specific SDSC could be substantially enhanced. We further characterized human fetal synovial fibroblasts as fetal SDSCs as they possessed the multi-lineage differentiation capacities and mesenchymal stem cell surface marker expression. Fetal SDSC-derived DECM expansion not only increased cell number and enhanced chondrogenic potential, it also lowered SDSC senescence marker expression while enhancing MSC marker expression compared to expansion on plastic flasks alone. As cell senescence is a limiting factor for tissue regeneration, we then investigated whether the DECM derived from fetal SDSCs referred to as a young stem cell microenvironment could be used for rejuvenating adult SDSC. We found that fetal SDSC-derived DECM (FE) was superior to adult SDSC-deposited DECM (AE) in promoting SDSC proliferation and chondrogenic potential. Further investigation revealed that unique proteins in FE might be responsible for the rejuvenation effect and advantageous proteins in AE might contribute to differentiation more than proliferation. Compared to AE, the lower elasticity of FE yielded expanded SDSCs with lower elasticity, which could be responsible for the enhancement of chondrogenic differentiation. MAPK and noncanonical Wnt signals were also actively involved in DECM-mediated SDSC rejuvenation. The young and healthy microenvironment provided by fetal SDSCs could serve as a fountain of youth for adult SDSC rejuvenation. Finally, we tested whether the DECM expansion system would also be beneficial to the chondrocyte-like nucleus pulposus cell (NPC) rejuvenation from human herniated discs and whether fetal DECM is superior to adult DECM. Although both SDSC and NPC deposited DECMs (SECM and NECM) significantly enhanced NPC proliferation, only NECM expanded NPCs manifested the increased redifferentiation capacities after chondrogenic induction. NECM is better than SECM in functioning as an expansion system in vitro by promoting NPC proliferation and redifferentiation. In conclusion, we have demonstrated that the DECM deposited by human primary cells or stem cells serves as an ex vivo expansion system for maintaining self-renewal and differentiation potential, which could greatly benefit the future generation of cell-based therapy for cartilage and intervertebral disc regeneration

Aspects of Physical Aging and Solid State Processing of Polymeric Glasses

This thesis focuses on unconventional methods to improve the mechanical properties of glassy polymers such as PMMA, Tritan™ Copolyester, and epoxy-based thermosets by influencing the intrinsic mechanical behavior through solid-state processing. The solid-state processing includes pre-stress, mechanical rejuvenation, and mechanical work hardening that cause changes in microscopic conformation structure and dynamics of glassy polymers and enhance properties such as non-linear deformation behavior and low and high velocity fracture. To utilize such unconventional techniques, one needs to understand fundamental origins of dynamics in polymeric glasses where the chain segments are not completely frozen and the segmental diffusion displays high degree of intermolecular cooperativity. We demonstrate how fundamental principles of polymer physics can be applied to improve fracture toughness of polymeric glasses. Emphasis is placed on structure-process-property relationships of these systems. In Chapter 2 long-term effects of physical aging and solid state processing are monitored through dynamic mechanical properties of an amorphous glassy polymer. These phenomena are investigated through dynamic mechanical testing that evaluates in-situ the evolution of the storage modulus with time during annealing and physical aging. Comparisons are made on samples with different thermal histories and mechanical treatment. The results are discussed in context to an aging rate obtained from the various thermal and mechanical treatments. We demonstrate that there is apparent work hardening of glassy polymers. The effect of strain rate, dwell time and material are compared and the permanence of the processing is investigated. In Chapter 3, we investigate the effect mechanical rejuvenation on the fracture toughness of epoxy-based thermosets and correlate the kinetics of the recovery of fracture toughness to compression based ductility parameters and dynamic mechanical analysis (DMA)-based aging rates. We also investigate the effect of molecular additives, antiplasticizers, on the structural recovery rate of the epoxy after mechanical rejuvenation. Chapter 4 studies the optimization of prestressed poly (methyl methacrylate) (PMMA), through equibiaxial compression with increasing amounts of shear and simple shear. To suppress inherent large radial crack growth associated with simple shear prestress, orientation is superimposed to minimize crack growth. These prestressed states are compared at both low velocity and ballistic rates. To investigate the low velocity impact dependence on rate, a strain energy density term is used to remove the dependence of geometry. Lastly, to reduce scatter in ballistic date, a master curve is developed to collapse all data regardless of boundary conditions, rate of impact and materials. In Chapter 5 we examine the correlation between ductility parameters based on dynamic mechanical data and fracture toughness and other non-linear mechanical properties. This chapter focuses on the model system poly (methyl methacrylate) and the relationship of these ductility parameters to other engineering properties for a range of temperatures and strain rates

Stem Cells in Treatment of Coronary Heart Disease and Its Monitoring: Tissue Engineering and Clinical Evaluation

Cardiovascular and coronary heart diseases involve molecular and tissue level damage of blood vessels and heart. Coronary Heart Disease and heart failure are the leading cause of mortality worldwide. Stem cell transplantation is emerging as a new treatment option. Stem cells are capable to reach and settle down at damaged cardiac tissue. This stem cell option also repairs the myocardial infarction area in heart or vascular territories and ultimately reduces the infarct-related mortality. Non-invasive cardiovascular imaging monitors the real-time status of cardiovascular remodeling or differentiated stem cell autografting. Cardiac magnetic resonance imaging (MRI) and bioluminescence are robust non-invasive monitoring techniques to visualize cardiovascular structure changes due to myocardial dysfunction or restorative myocardial recovery. The present chapter highlights the sources, types, delivery methods of stem cells in cardiovascular treatment, advantages and current limitations of stem cell monitoring, scopes of ultra-high field cardiac 900 MHz MRI and bioluminescence methods applied in stem cell transplantation, to translate stem cell molecular events into clinical success and evaluation of rejuvenation rate with future perspectives. In conclusion, right choice of stem cells, pluripotent stem cell delivery, transplantation and real-time monitoring of stem cell trafficking enhances the stem cell therapeutic efficacy in cardiac engraftment and differentiation

Spatial Distribution of Macrophages During Callus Formation and Maturation Reveals Close Crosstalk Between Macrophages and Newly Forming Vessels

Macrophages are essential players in the process of fracture healing, acting by remodeling of the extracellular matrix and enabling vascularization. Whilst activated macrophages of M1-like phenotype are present in the initial pro-inflammatory phase of hours to days of fracture healing, an anti-inflammatory M2-like macrophage phenotype is supposed to be crucial for the induction of downstream cascades of healing, especially the initiation of vascularization. In a mouse-osteotomy model, we provide a comprehensive characterization of vessel (CD31+, Emcn+) and macrophage phenotypes (F4/80, CD206, CD80, Mac-2) during the process of fracture healing. To this end, we phenotype the phases of vascular regeneration-the expansion phase (d1-d7 after injury) and the remodeling phase of the endothelial network, until tissue integrity is restored (d14-d21 after injury). Vessels which appear during the bone formation process resemble type H endothelium (CD31hiEmcnhi), and are closely connected to osteoprogenitors (Runx2+, Osx+) and F4/80+ macrophages. M1-like macrophages are present in the initial phase of vascularization until day 3 post osteotomy, but they are rare during later regeneration phases. M2-like macrophages localize mainly extramedullary, and CD206+ macrophages are found to express Mac-2+ during the expansion phase. VEGFA expression is initiated by CD80+ cells, including F4/80+ macrophages, until day 3, while subsequently osteoblasts and chondrocytes are main contributors to VEGFA production at the fracture site. Using Longitudinal Intravital Microendoscopy of the Bone (LIMB) we observe changes in the motility and organization of CX3CR1+ cells, which infiltrate the injury site after an osteotomy. A transient accumulation, resulting in spatial polarization of both, endothelial cells and macrophages, in regions distal to the fracture site, is evident. Immunofluorescence histology followed by histocytometric analysis reveals that F4/80+CX3CR1+ myeloid cells precede vascularization