Generation and Characterization of Knockout Mice and Analysis of Patient Material Demonstrates a Role for Tissue Inhibitor of Metalloproteinases 4 (Timp4) in the Cardiovascular System and Tumor Metastasis

This thesis focuses on tissue inhibitor of metalloproteinases 4 (TIMP4) which is the newest member of a small gene and protein family of four closely related endogenous inhibitors of extracellular matrix (ECM) degrading enzymes. Existing data on TIMP4 suggested that it exhibits a more restricted expression pattern than the other TIMPs with high expression levels in heart, brain, ovary and skeletal muscle. These observations and the fact that the ECM is of special importance to provide the cardiovascular system with structural strength combined with elasticity and distensibility, prompted the present molecular biologic investigation on TIMP4. In the first part of the study the murine Timp4 gene was cloned and characterized in detail. The structure of murine Timp4 genomic locus resembles that in other species and of the other Timps. The highest Timp4 expression was detected in heart, ovary and brain. As the expression pattern of Timp4 gives only limited information about its role in physiology and pathology, Timp4 knockout mice were generated next. The analysis of Timp4 knockout mice revealed that Timp4 deficiency has no obvious effect on the development, growth or fertility of mice. Therefore, Timp4 deficient mice were challenged using available cardiovascular models, i.e. experimental cardiac pressure overload and myocardial infarction. In the former model, Timp4 deficiency was found to be compensated by Timp2 overexpression, whereas in the myocardial infarct model, Timp4 deficiency resulted in increased mortality due to increased susceptibility for cardiac rupture. In the wound healing model, Timp4 deficiency was shown to result in transient retardation of re-epithelialization of cutaneous wounds. Melanoma tumor growth was similar in Timp4 deficient and control mice. Despite of this, lung metastasis of melanoma cells was significantly increased in Timp4 null mice. In an attempt to translate the current findings to patient material, TIMP4 expression was studied in human specimens representing different inflammatory cardiovascular pathologies, i.e. giant cell arteritis, atherosclerotic coronary arteries and heart allografts exhibiting signs of chronic rejection. The results showed that cardiovascular expression of TIMP4 is elevated particularly in areas exhibiting inflammation. The results of the present studies suggest that TIMP4 has a special role in the regulation of tissue repair processes in the heart, and also in healing wounds and metastases. Furthermore, evidence is provided suggesting the usefulness of TIMP4 as a novel systemic marker for vascular inflammation.

Identification and characterization of new genes and pathways regulating spermatogonial cell fate

Spermatogenesis, i.e sperm production in the seminiferous tubules of the testis, is a complex process that takes over one month to complete. Life-long ability of sperm production ultimately lies in a small population of undifferentiated cells, called spermatogonial stem cells (SSCs). These cells give rise to differentiating spermatogonia, which are committed to mature into spermatozoa. SSCs represent a heterogeneous population of cells and many aspects of their basic biology are still unknown. Understanding the mechanisms behind the cell fate decision of these cells is important to gain more insights into the causes of infertility and testis cancer. In addition, an interesting new aspect is the use of testis-derived stem cells in regenerative medicine. Our data demonstrated that adult mouse testis houses a population of Nanog-expressing spermatogonia. Based on mRNA and protein analysis these cells are enriched in stage XII of the mouse seminiferous epithelial cycle. The cells derived from this stage have the highest capacity to give rise to ES cell-like cells which express Oct4 and Nanog. These cells are under tight non- GDNF regulation but their fate can be dictated by activating p21 signalling. Comparative studies suggested that these cells are regulated like ES cells. Taken together these data imply that pluripotent cells are present in the adult mammalian testis. CIP2A (cancerous inhibitor of PP2A) has been associated with tumour aggressiveness and poor prognosis. In the testis it is expressed by the descendants of stem cells, i.e. the spermatogonial progenitor cells. Our data suggest that CIP2A acts upstream of PLZF and is needed for quantitatively normal spermatogenesis. Classification of CIP2A as a cancer/testis gene makes it an attractive target for cancer therapy. Study on the CIP2A deficient mouse model demonstrates that systemic inhibition of CIP2A does not severely interfere with growth and development or tissue or organ function, except for the spermatogenic output. These data demonstrate that CIP2A is required for quantitatively normal spermatogenesis. Hedgehog (Hh) signalling is involved in the development and maintenance of many different tissues and organs. According to our data, Hh signalling is active at many different levels during rat spermatogenesis: in spermatogonia, spermatocytes and late elongating spermatids. Localization of Suppressor of Fused (SuFu), the negative regulator of the pathway, specifically in early elongating spermatids suggests that Hh signalling needs to be shut down in these cells. Introduction of Hh signalling inhibitor resulted in an increase in germ cell apoptosis. Follicle-stimulating hormone (FSH) and inhibition of receptor tyrosine kinases resulted in down-regulation of Hh signalling. These data show that Hh signalling is under endocrine and paracrine control and it promotes germ cell survival.

Flexible Sensor for Wearable Vital Sign Monitoring System

In modern society, the body health is a very important issue to everyone. With the development of the science and technology, the new and developed body health monitoring device and technology will play the key role in the daily medical activities. This paper focus on making progress in the design of the wearable vital sign system. A vital sign monitoring system has been proposed and designed. The whole detection system is composed of signal collecting subsystem, signal processing subsystem, short-range wireless communication subsystem and user interface subsystem. The signal collecting subsystem is composed of light source and photo diode, after emiting light of two different wavelength, the photo diode collects the light signal reflected by human body tissue. The signal processing subsystem is based on the analog front end AFE4490 and peripheral circuits, the collected analog signal would be filtered and converted into digital signal in this stage. After a series of processing, the signal would be transmitted to the short-range wireless communication subsystem through SPI, this subsystem is mainly based on Bluetooth 4.0 protocol and ultra-low power System on Chip(SoC) nRF51822. Finally, the signal would be transmitted to the user end. After proposing and building the system, this paper focus on the research of the key component in the system, that is, the photo detector. Based on the study of the perovskite materials, a low temperature processed photo detector has been proposed, designed and researched. The device is made up of light absorbing layer, electron transporting and hole blocking layer, hole transporting and electron blocking layer, conductive substrate layer and metal electrode layer. The light absorbing layer is the important part of whole device, and it is fabricated by perovskite materials. After accepting the light, the electron-hole pair would be produced in this layer, and due to the energy level difference, the electron and hole produced would be transmitted to metal electrode and conductive substrate electrode through electron transporting layer and hole transporting layer respectively. In this way the response current would be produced. Based on this structure, the specific fabrication procedure including substrate cleaning; PEDOT:PSS layer preparation; pervoskite layer preparation; PCBM layer preparation; C60, BCP, and Ag electrode layer preparation. After the device fabrication, a series of morphological characterization and performance testing has been done. The testing procedure including film-forming quality inspection, response current and light wavelength analysis, linearity and response time and other optical and electrical properties testing. The testing result shows that the membrane has been fabricated uniformly; the device can produce obvious response current to the incident light with the wavelength from 350nm to 800nm, and the response current could be changed along with the light wavelength. When the light wavelength keeps constant, there exists a good linear relationship between the intensity of the response current and the power of the incident light, based on which the device could be used as the photo detector to collect the light information. During the changing period of the light signal, the response time of the device is several microseconds, which is acceptable working as a photo detector in our system. The testing results show that the device has good electronic and optical properties, and the fabrication procedure is also repeatable, the properties of the devices has good uniformity, which illustrates the fabrication method and procedure could be used to build the photo detector in our wearable system. Based on a series of testing results, the paper has drawn the conclusion that the photo detector fabricated could be integrated on the flexible substrate and is also suitable for the monitoring system proposed, thus made some progress on the research of the wearable monitoring system and device. Finally, some future prospect in system design aspect and device design and fabrication aspect are proposed.