Mesenchymal stem cells are more and more widely used in tissue engineering due to their pluripotency and no relative ethical problems. Traditional characterization techniques to detect mesenchymal stem cell states include flow cytometry, gene expressing profiling and immunohistochemistry. However, these methods can only provide transient and low level information from current RNA or protein levels about mesenchymal stem cells, which may cause problems when predicting the possible downstream lineages they will commit into. We have developed chromatin immunoprecipitation (ChIP)-based epigenetic technique to detect mesenchymal stem cell states. For the systems we tested, this epigenetic assessor successfully characterized cell state changes and gave similar results obtained from gene expression profiling or protein expression assay. This epigenetic technique can provide information about mesenchymal stem cells states from a more fundamental chromatin level, which is promising for predicting future lineages from current states.

New synthetic advances in the control of nanoparticle size and shape along with the development of new surface modifications facilitates the growing use of nanomaterials in biomedical applications. Of particular interest are functional and biocompatible nanomaterials for sensing, imaging, and drug delivery. The goal of this research is to tailor the function of nanomaterials for biomedical applications by improving the biocompatibility of the systems. Our work demonstrates both a bottom up and a post synthetic approach for incorporating stability, stealth, and biocompatibility to metal based nanoparticle systems. Two main nanomaterial projects are the focus of this dissertation. We first investigated the development of a green synthetic procedure to produce gold nanoparticles for biological imaging and sensing. The size and morphology of gold nanoparticles directly impact their optical properties, which are important for their function as imaging agents or their use in sensor systems. In this project, a synthetic route based on the natural process of biomineralization was developed, where a designed protein scaffold initiates the nucleation and subsequent growth of gold ions. To gain insight into controlling the size and morphology of the synthesized nanoparticles, interactions between the gold ions and the protein surface were studied along with the effect of ionic strength on interactions and then subsequent crystal growth. We are able to control the size and morphology of the gold nanoparticles by altering the concentration or identity of protein scaffold, salt, or reducing agent. The second project involves the design and optimization of metal organic framework nanoparticles for an external stimulus triggered drug delivery system. This work demonstrates the advantages of using surface coatings for improved stability and functionalization. We show that the addition of a polyethylene glycol surface coating improved the colloidal stability and biocompatibility of the system. The nanoparticle was shown to successfully encapsulate a variety of small molecule cargo. This is the first report of photo-triggered degradation and subsequent release of the loaded cargo as a mechanism of stimuli-controlled drug delivery. Each of the aforementioned projects demonstrates the design, synthesis, and optimization of metal-based systems for use in biomedical applications. / Ph. D.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20400号 / 工博第4337号 / 新制||工||1672(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 中條 善樹, 教授 秋吉 一成, 教授 古賀 毅 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Globalization in the business environment today is something that affects almost every firm around the world. It has given opportunities not only to multinational enterprises (MNE) but also to small and medium enterprises (SME). In this study SME food service firms has been one of the main subjects and we find it interesting to investigate their internationalization process in the globalized market. This has been investigated with the help of a network approach and the main purpose of the thesis is to gain a deeper understanding in how SMEs in the food industry use a network approach when going into the international marketplace. The study used a qualitative case study as research method and the data has been gathered and analyzed from an interview with the CEO and founder of a food service firm in Sweden. The findings from the study suggest that it is crucial for SME food service firms to use their networks when expanding internationally. Furthermore, it is important to have a well thought out expansion strategy, including agents in the target market extending to strong market research. Our findings also suggest that SME characteristics are not fully applicable for SMEs in the food service industry. In addition to above, the findings also suggest that internal factors, for example a global mindset, is the strongest force in stimulating firms in the food service industry to start an internationalization process.

Metabolic engineering has enabled studying microorganisms by the modification of their genetic material and analysis of their metabolism for the isolation of microbial strains capable of producing high yields of high value chemicals and biofuels. In this research, novel tools were developed to improve genetic engineering of microbial cells. In this matter, λ-PCR (lambda-PCR) was developed enabling the construction of plasmid DNA. This technique allows DNA assembly and manipulation (insertion, substitution and/or deletion) at any location of a vector. λ-PCR addresses the need for an easy, highly-efficient, rapid and inexpensive tool for genetic engineering and overcoming limitations encountered with traditional techniques. Then, novel synthetic small RNA (sRNA) regulators were designed in a cell-free-system (in vitro) in order to modulate protein expression in biosynthetic pathways. The ability of the sRNAs to regulate mRNA expression with statistical significance was demonstrated. Up to 70% decrease in protein expression level was achieved by targeting specific secondary structures of the mRNA with antisense binding regions of the sRNA. Most importantly, a sRNA was identified capable of protein overexpression by up to 65%. An understanding of its mechanism showed that its mRNA target region(s) likely lead to occlusion of RNase E binding. This mechanism was translated for expression of a diaphorase enzyme, which has relevance to synthetic biology and metabolic engineering in in vitro systems. Results were successful, showing a greater than 75% increase in diaphorase expression in a cell-free protein synthesis reaction. Next, Raman spectroscopy was employed as a near real-time method for microbial phenotyping. Here, Raman spectroscopy was used in combination with chemometric analysis methods through RametrixTM Toolboxes to study the effects of environmental conditions (i.e. illumination, glucose, nitrate deprivation, acetate, sodium chloride and magnesium sulfate) on the phenotypic response of the cyanobacterium Synechocystis sp. PCC6803. The RametrixTM LITE Toolbox for MATLAB® enabled processing of Raman spectra and application of principal component analysis (PCA) and discriminant analysis of principal components (DAPC). Two studies were performed. PCA and DAPC produces distinct clustering of Raman spectra, representing multiple Synechocystis phenotypes, based on the (i) presence of glucose in the growth medium, (ii) illumination, (iii) nitrate limitation, and (iv) throughout a circadian rhythm growth cycle, in the first study. The second study focused on the phenotypic response based on (i) growth in presence of acetate, (ii) presence of high concentrations of sodium chloride and (iii) magnesium sulfate starvation. RametrixTM PRO was applied for the validation of the DAPC models through leave-one-out method that allowed calculation of prediction accuracy, sensitivity and selectivity for an unkown Raman spectrum. Statistical tests (ANOVA and pairwise comparison) were performed on Raman spectra to identify statistically relevant changes in Synechocystis phenotypes. Next, comparison between Raman data and standardized analytical methods (GF-FID, UPLC, spectrometric assays) was established. Overall, good correlation were obtained (R > 0.7). Finally, genomic DNA libraries were enriched to isolate a deoxynivalenol detoxifying enzyme. To do this, library fragments from microorganisms was generated through oligonucleotide primed polymerase chain reaction (DOP-PCR) and transformed in a DON-sensitive yeast strain. Rounds of subculture were performed in the presence of DON and ferulic acid in order to isolate a strain capable of enzymatic degradation of DON. / Doctor of Philosophy / Metabolic engineering is the use of genetic engineering to modify microorganisms in order to produce high yields of valuable commodity chemicals. The goal of this research is to develop new methods to improve genetic modification and selection of microbial cells. The specific objectives were to: (i) develop new tools for DNA assembly and manipulation, (ii) utilize small synthetic RNA to control protein expression level, (iii) use Raman spectroscopy to study phenotypic responses to environmental changes and (iv) enrich for microorganisms that detoxify dangerous toxins. First, a new technique for DNA assembly, named λ-PCR (lambda-PCR), was developed. This method allows the easy manipulation of plasmid DNA with high-efficiency and low-cost compared to traditional techniques. Second, novel synthetic small RNA (sRNA) regulators were designed in a cell-free-system in order to modulate (downregulate or overexpress) fluorescent protein expression. Next, Raman spectroscopy was used to assess phenotypic response of cyanobacterial cells to different environmental modifications (light settings, salts, sugar, etc…). Finally, genomic library was used to discover and characterize enzymes capable of degrading a mycotoxin.

The presented work is concerned with the development and application of an ultrasonic detection scheme suitable for the monitoring of muscle dynamics with high temporal - down to 5 µs - and spatial resolution - down to 0.78 µm. A differential detection scheme has been developed to monitor the variations of the velocity of longitudinal polarized ultrasound waves travelling in contracting and relaxing muscle, compensating for variations of the path length by referencing to a frame. The observed time dependent variations of the time-of-flight of the ultrasonic waves caused by variations in the muscle and in addition by minor deformations of the enclosure are detected each separately and synchronously and are evaluated differentially. Beside of the detected increase of the speed of sound observed for contracted muscle with respect to the relaxed state of about 0.6%, the recovery time from maximum isometric contraction is quantified and relaxation processes are observed for the recovery phase following the isometric contraction. The developed ultrasonic calliper was employed to monitor both, the brain controlled and externally excited muscle dynamics with sampling intervals down to 10 ms synchronously with signals relating to the excitation. Monitored are the activation, hold, and relaxation phase for maximum voluntary isometric contraction of the gastrocnemius muscle. A so far not reported post tetanus overshoot and subsequent exponential recovery are observed. Both are attributed to the muscle as suggested by combined monitoring with EMG and are modelled with a lumped mechanical circuit containing an idealized bidirectional linear motor unit, ratchet, damper, and springs. Both, the rapid contraction and relaxation phases require a high order filter or alternatively a kernel filter, attributed to the nerve system as suggested by external electric stimulation. The respective response function is modelled by an electrical lumped circuit. Together with a reaction time and occasionally observed droops in the hold phase, both adjusted empirically, the monitored response is represented in close approximation by the combined electrical and mechanical lumped circuits. The respectively determined model parameters provide a refined evaluation scheme for the performance of monitored athletes. Valuable parameters relate to the latent period, the muscle response time, the activation and deactivation dynamics, a possible droop and other instabilities of the hold phase, and parameters characterizing the relaxation phase including the observed post tetanus overshoot and subsequent contraction. Monitored and modelled are also the different processes involved in active muscle dynamics including isotonic, isometric, and eccentric contraction or stretching. The developed technology provides time sequential observation of these processes and registration of their path in the extension and force parameter space. Under suitable conditions the closed-loop cycles of mind controlled human muscle movements proceed along characteristic lines coinciding with well identifiable elementary processes. The presentation of the monitored processes in the extension and force parameter space allows the determination of the mechanical energy expenditure for the observed different muscle actions. An elementary macroscopic mechanical model has been developed, suitable to express the basic features of the monitored muscle dynamics.

Designers often look for inspiration in their environment when exploring possible solutions to a given problem. However, many studies have reported that external stimuli may constrain designers’ imagination and limit their exploration to similar solutions, a phenomenon described as design fixation. Inspiration and fixation effects are traditionally studied with a similar experimental paradigm, which has produced a complex web of findings and explanations. Yet, when analysing the experiments and their findings closely, it becomes clear that there is considerable variation in how studies are conducted and the results they produce. Such variation makes it difficult to formulate a general view of how external stimuli affect the design process, and to translate the research findings into education and practice. Moreover, it raises questions about the reliability and effectiveness of the traditional experimental method. This thesis reports on a collection of studies that examine how design inspiration and fixation research is done and how it can be improved. It explores the research area by reviewing the literature and analysing data from a workshop; describes the research method by scrutinising experiments and their procedures; and explains the variation in research findings by testing experimental procedures empirically and suggesting new interpretations. My main findings are that: abstract stimuli can inspire or fixate designers to different degrees depending on how explicitly the stimuli are represented; external stimuli can inhibit the exploration of ideas that would otherwise be explored; the effect of experimental instructions varies depending on how encouraging the instructions are; and the way participants represent and elaborate ideas can moderate fixation results. Whilst this thesis offers insights into design practice and education, its main contribution is to design research, where it represents a fundamental material for those who are new to inspiration and fixation research, and for those who are already expert.

The presented work is concerned with the development and application of an ultrasonic detection scheme suitable for the monitoring of muscle dynamics with high temporal - down to 5 µs - and spatial resolution - down to 0.78 µm. A differential detection scheme has been developed to monitor the variations of the velocity of longitudinal polarized ultrasound waves travelling in contracting and relaxing muscle, compensating for variations of the path length by referencing to a frame. The observed time dependent variations of the time-of-flight of the ultrasonic waves caused by variations in the muscle and in addition by minor deformations of the enclosure are detected each separately and synchronously and are evaluated differentially. Beside of the detected increase of the speed of sound observed for contracted muscle with respect to the relaxed state of about 0.6%, the recovery time from maximum isometric contraction is quantified and relaxation processes are observed for the recovery phase following the isometric contraction. The developed ultrasonic calliper was employed to monitor both, the brain controlled and externally excited muscle dynamics with sampling intervals down to 10 ms synchronously with signals relating to the excitation. Monitored are the activation, hold, and relaxation phase for maximum voluntary isometric contraction of the gastrocnemius muscle. A so far not reported post tetanus overshoot and subsequent exponential recovery are observed. Both are attributed to the muscle as suggested by combined monitoring with EMG and are modelled with a lumped mechanical circuit containing an idealized bidirectional linear motor unit, ratchet, damper, and springs. Both, the rapid contraction and relaxation phases require a high order filter or alternatively a kernel filter, attributed to the nerve system as suggested by external electric stimulation. The respective response function is modelled by an electrical lumped circuit. Together with a reaction time and occasionally observed droops in the hold phase, both adjusted empirically, the monitored response is represented in close approximation by the combined electrical and mechanical lumped circuits. The respectively determined model parameters provide a refined evaluation scheme for the performance of monitored athletes. Valuable parameters relate to the latent period, the muscle response time, the activation and deactivation dynamics, a possible droop and other instabilities of the hold phase, and parameters characterizing the relaxation phase including the observed post tetanus overshoot and subsequent contraction. Monitored and modelled are also the different processes involved in active muscle dynamics including isotonic, isometric, and eccentric contraction or stretching. The developed technology provides time sequential observation of these processes and registration of their path in the extension and force parameter space. Under suitable conditions the closed-loop cycles of mind controlled human muscle movements proceed along characteristic lines coinciding with well identifiable elementary processes. The presentation of the monitored processes in the extension and force parameter space allows the determination of the mechanical energy expenditure for the observed different muscle actions. An elementary macroscopic mechanical model has been developed, suitable to express the basic features of the monitored muscle dynamics.:Table of Contents Chapter 1 1. Introduction 1 1.1 Monitoring of muscle biomechanics 1 1.2 Detection methods in biomechanics 2 1.3 Ultrasound in biomechanical application 5 1.4 Skeletal muscle 6 1.5 Activation of skeletal muscle 8 1.6 Catatonus effect 10 Chapter 2 2. Concepts and methods in ultrasonic motion monitoring 12 2.1 Ultrasound 12 2.2 Specific concepts of the developed ultrasonic detection scheme 16 2.2.1 Time-of-flight 17 2.2.2 Cross correlation 18 2.2.3 Concepts of cross correlation 19 2.2.4 Chirp technique 19 Chapter 3 3. Ultrasonic monitoring of the muscle extension 21 3.1 Data analysis 21 3.2 Application of the developed monitoring scheme 23 3.2.1 Fast signal and data acquisition mode 23 3.2.2 Monitoring with off-line evaluation 24 3.2.3 Method 26 3.2.4 Data evaluation 27 3.3 Quasi-continuous monitoring scheme 28 3.3.1 Slow with on-line data processing and display 29 3.3.2 Fast with data storage only 30 3.4 Monitoring with on-line evaluation 34 3.4.1 Application involving monitoring of athletic performance 36 3.4.2 Data evaluation 37 3.4.3 Summary 42 3.5 Comparative study of pre and post physical loading session 43 3.5.1 Method 43 3.5.2 Results 44 3.5.3 Summary 45 Chapter 4 4. High resolution monitoring of the velocity of ultrasound in contracting and relaxing muscle 47 4.1 Methods 49 4.2 Results and evaluation 51 4.2.1 Poission’s ratio for isometrically contracted muscle 52 4.3 Summary 53 Chapter 5 5. Monitoring of muscle dynamics, muscle force, and EMG 56 5.1 Synchronous monitoring of muscle dynamics with muscle force 56 5.1.1 Force-length dynamics under all-out isometric contraction 56 5.1.1.1 Method 56 5.1.1.2 Result and evaluation 58 5.1.2 Force-length dynamics of equal holding monitoring 62 5.1.2.1 Method 62 5.1.2.2 Results and evaluation 63 5.1.3 Summary 67 5.2 Synchronous monitoring of muscle movement with EMG 69 5.2.1 Method 69 5.2.2 Results and evaluation 70 5.3 Synchronous monitoring of muscle movement, EMG and muscle force 73 5.3.1 Method 73 5.3.2 Results and evaluation 74 5.3.3 Summary 77 Chapter 6 6. Monitoring of skeletal muscle dynamics under isometric contraction and modelling of the non-linear response including post tetanus effects 80 6.1 Method 82 6.2 Data analysis 82 6.3 Results and evaluation 82 6.3.1 Mechanical model 83 6.3.2 Equations relating to modelling 85 6.3.3 Comparison of experimental results and modelling 91 6.3.4 Electrical lumped circuit 93 6.4 Summary 100 Chapter 7 7. Lumped Circuit Model and Energy Transfer for quasi-static approximation 101 7.1 Basic muscle model and biomechanical processes 102 7.1.1 Muscle model 102 7.1.2 Force in the muscular motoric processes 104 7.2 Method 104 7.3 Results of experimental observations of muscle action 106 7.3.1 Muscle force and closed-loop contraction dynamics 106 7.3.2 Muscle work considerations 109 7.4 Summary 110 Chapter 8 8.1 Ultrasonic calliper 112 8.2 Interpretation of sound velocity variation in muscle 114 8.3 Monitored muscle dynamics 118 8.4 Isometric muscle action and tetanus effect 121 8.5 Quasi-static muscle action 125 8.6 Summarizing statement with a moderate outlook 126 References 128 Acknowledgements 140 Selbständigkeitserklärung 141