京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18513号 / 医博第3933号 / 新制||医||1006(附属図書館) / 31399 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 羽賀 博典, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13214号 / 論医博第2164号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 安達 泰治, 教授 江藤 浩之 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Recent technologies in micro-devices for investigation of functional biology in a controlled microenvironment are continually growing and evolving. In particular, electric-field mediated microfluidic platforms are evolving technologies that have significant applications in drug delivery and cell migration investigations. Although drug delivery has had several successes, in some areas, it continues to be a challenge; in recent years, the positive impact of electric fields is being explored. The primary objectives of the dissertation are to design, fabricate, and employ two novel microfluidic platforms for drug delivery and cell migration in the presence of electric fields. Description of iontophoretic carboplatin delivery into the MDA-MB-231 triple-negative breast cancer cells and investigation of neutrophil electro taxis are two main aims of the dissertation. Transdermal drug delivery systems such as iontophoresis are useful tools for delivering chemotherapeutics for tumor treatment not only because of their non-invasiveness but also due to their lower systematic toxicity compared to other drug delivery systems. While iontophoresis animal models are commonly being used for the development of new cancer therapies, there are some obstacles for precise control of the tumor microenvironment's chemoresistance and scaffold in the animal models. We employed experimental and computational approaches, the iontophoresis-on-chip and the fraction of tumor killed mathematical model, for predicting the outcome of iontophoresis treatment in a controlled microenvironment. Also, precise control over the cell electromigration is a challenging investigation which we will address in the second aim of the dissertation. Here, we developed a microfluidic platform to study the consequences of DC electric fields on neutrophil electromigration (electrotaxis), which has an application of directing neutrophils away from healthy tissue by suppressing the migration of neutrophils toward pro-inflammatory chemoattractant. / Doctor of Philosophy / Recent technologies in the micro-scale medical devices for diagnosis and treatment purposes are continually growing and evolving. Microfluidic platforms are reproducible devices with the dimensions from tens to hundreds of micrometers for manipulating and controlling fluids. In particular, electric-field mediated microfluidic platforms, are developing technologies that have significant applications in drug delivery and biological cell directional movement investigations. Although drug delivery has had several successes, in some areas, it continues to be a challenge. In recent years, the positive impact of electric fields is a significant advancement in drug delivery techniques. Transdermal drug delivery systems such as iontophoresis are useful tools for delivering chemo drugs for tumor treatment not only because of their sensitivity but also to their lower systematic toxicity compared to injection or oral drug delivery. While iontophoresis animal models are conventional for the development of new cancer therapies, there are some obstacles to precise control of the tumor scaffold in the animal models. We also developed a novel microfluidic platform to study the consequences of DC electric fields on white blood cells' (WBC) directional movement, which has an application of directing WBC away from healthy tissue by suppressing the damage of WBC accumulation in healthy organs.

No description available.

The basis for many biological processes such as cell division and differentiation, immune responses, and tumor metastasis depends upon the cell's ability to migrate effectively. A mathematical model for simulating cell migration can be useful in identifying the underlying contributing factors to the crawling motions observed in different types of cells. We present a cell migration model that simulates the 2D motion of amoeba, fibroblasts, keratocytes, and neurons according to a set of input parameters. In the absence of external stimuli the pattern of cell migration follows a persistent random walk which necessitates for several stochastic components in the mathematical model. Consequently, the cell metrics which provide a quantitative description of the cell motion varies between simulations. First we examine different methods for computing the error observed between the output metrics generated by our model and a set of target cell metrics. We also investigate ways of minimizing the variability of the output by varying the number of iterations within a simulation. Finally we apply finite differences, Hooke and Jeeves, and Nelder-Mead minimization methods to our nonlinear stochastic function to search for optimal input values.

Thesis (M.Sc.) -- University of the North, 1982 / Refer to the document

The adult kidney consists of hundreds of thousands of fine epithelial tubules as functional units called nephrons. Nephrons have U-shaped tubules: loops of Henle that descend from the cortex to the medulla. This radial arrangement is critical to maintain water homeostasis in the kidney. Although Henle’s loops are crucial to renal physiology, the cue(s) they uses to navigate to the medulla are not understood. In this thesis, I investigate how the loop of Henle elongates during mouse renal development and show that it is probably guided to the medulla by diffusible, heparin-binding molecules. I used immumohistochemistry (IHC) on cryosections of embryonic kidneys to study the natural anatomy of the Henle’s loop. I used a low-volume culture system to allow embryonic kidneys (both natural and tissue-engineered) to form loops of Henle ex vivo and manipulated their direction of growth. Time-lapse imaging of Lgr-5 EGFP embryonic kidneys demonstrated the movement of the apex of the loop which suggested the idea of guidance cue(s) acting on the loop of Henle. Cut-and-paste experiments showed that loops appeared to be attracted to maturing collecting duct. Co-culture with an exogenous tubule inducer suggested the embryonic spinal cord as another source to attract the loops. Using raTAL (rat thick ascending loop of Henle) and 6TA2 (embryonic collecting duct cells) cell lines, I designed and performed a cell migration assay to test whether raTAL was attracted to 6TA2 cells. raTAL cells were notably attracted to 6TA2 cells compared to other cell lines. raTAL cells were also attracted to 6TA2-conditioned medium, which indicated that raTAL cells were attracted by secreted molecule(s). To begin to characterise those secreted molecule(s), heparin-binding protein-coated beads were used in the cell migration system and showed that at least one critical guidance factor is heparin-binding. From this study, I found that the apex of the Henle’s loop does move and loops are attracted by secreted molecule(s) possibly from the collecting duct. Although target molecule(s) were unidentified, this study provides the first mechanistic information about the guidance of the loop of Henle. Moreover, this was the first study of guidance of epithelial tubule shafts (rather than tips) adding to our understanding of general tubule morphogenesis.

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

Injury, infection and tissue malfunction are triggers of inflammation which if not regulated may acquire new characteristics that result in pathological outcomes. Since innate immunity plays a key role in the resolution of acute inflammation knowledge of the regulation of this component of the host response is relevant to understanding processes in disease progression and therefore has potential clinical benefits. In this thesis I have applied zebrafish as a model organism to investigate the response of innate immune cells to qualitatively distinct inflammatory signals in the absence of adaptive immunity. Using a zebrafish embryo wound injury model I have investigated leukocyte migration profiles by in vivo imaging. In response to wound alone leukocytes migrated to the site of injury with predominantly random walk behaviour. However, the addition of lipopolysaccharide (LPS) enhanced recruitment and influenced the directionality of leukocyte migration to the wound. I demonstrate that leukocyte dynamic behaviour is also dependent on the location of the cells. The LPS enhanced directionality and reduced the random walk behaviour of the leukocytes, and these effects were ablated in the presence of the p38 mitogenactivated protein kinase (MAPK) specific inhibitor SB203580. Cytokine gene profiling in adult zebrafish leukocytes reveals that LPS can stimulate a pro-inflammatory response via the activation of p38 MAPK characteristic of mammalian innate immune responses. It is documented in mammalian innate immune cells that LPS can modulate Notch mediated signalling and thereby cell function. Using zebrafish with null mutations in Notch, which provide an unbiased in vivo model, I have investigated the influence of Notch signalling on leukocyte recruitment and demonstrate that migration to a wound injury is reduced. However, this effect is due to decreased cell numbers and not altered function as the Notch signalling inhibitor DAPT had no effect of recruitment to wound injury. The defect in myelomonocyte numbers was also present in adult zebrafish and this was partially compensated for by an increase in lymphocytes. The experimental results that I report here highlight zebrafish as a model 2 organism for studying the function and regulation of innate immunity. The unique optical translucency, which permits in vivo imaging of host responses in real-time, facilitates the analysis of the innate immune response to different inflammatory signals and immune modulators.

Satellite cells are adult stem cells that contribute to hypertrophy and repair in muscles. It is hypothesized that in muscular dystrophy, the satellite cells population is depleted at a very early age, due to repeated muscle damage and repair. Satellite cell transplantation is a potentially useful therapy for muscle diseases, but the lack of an efficient delivery system has hindered its application. The presented work focuses on two specific aims that address the need for more effective cell delivery methods for cell-based therapy. In Specific Aim 1 enhanced tissue culture techniques, such as heat stress, are used to increase cell survival in satellite cell transplantation studies. Also addressed within this specific aim are methods to label and evaluate performance using real-time PCR techniques.Although much work remains to enhancing the viability of in vitro expanded myoblasts derived from satellite cells, a second important hurdle is the systemic delivery of satellite cells to multiple sites (all muscles, in the case of muscular dystrophies). In vitro and in vivo experiments are being undertaken to explore the physiological role of cell signaling systems involved in directed migration and to determine if these chemokine and growth factors can be manipulated to enhance efficacy of cell-based therapies involving skeletal muscle satellite cells. Specific Aim 2 addresses migration of satellite cells to sites of injury and methods to track transplanted cells within the host. Presented here is the use of FAST SPECT II imaging of 111-Indium oxine radiolabeled satellite cells. The long lifetime of 111-indium oxine and the ability to quantify label using FAST SPECT imaging techniques make this technique ideal for in-vivo tracking of transplanted satellite cells for week long studies. Without in-vivo imaging techniques cell fate studies require sequential animal sacrifice with histological sectioning. This not only increases the number of animals used but also adds a significant inter-animal variability to their assessment. The determination of cell fate after transplantation will have a major impact on cell therapy for treatment of muscle disease as well as other stem cell therapies.