<p>The homeostatic hematopoietic stem cell compartment is comprised of quiescent long term self renewing stem cells and cycling short term stem cells with finite renewal potential. To study the molecular mechanisms governing self renewal of hematopoietic cells we must force them to enter the cell cycle and proliferate. One approach to accomplish this goal is to damage the hematopoietic compartment with ionizing radiation or cytotoxic chemotherapy. Such injuries ablate mature blood cells and drive the primitive stem cells into cycle. I have elected to use a simple model of hematopoietic damage and regeneration to study the molecular mechanisms controlling self renewal in hematopoietic stem cells. At the beginning of this project it was unclear whether the signaling pathways which homeostatically control self renewal are utilized during injury repair. In particular, there is very little understanding of the signals required for regeneration after radiation damage. We hypothesized extracellular signal transduction pathways provided by the microenvironment are critical mediators of the stem cell repair process. To address these topics and extend the previous work generated in our laboratory, I chose to pursue a candidate approach focusing on the Wnt and Notch developmental signaling pathways.</p><p>In order to examine the activation and requirement for each signaling cascade after radiation and chemotherapy damage we used a combination of loss of function and reporter mouse models. To this end, we have conducted the majority of experiments for the Wnt project in animals deficient in beta-catenin, the key transcription factor required in the pathway. Our investigations revealed the Wnt pathway is turned on within regenerating stem cells and loss of beta-catenin impairs regeneration of the stem cell compartment after both radiation and chemotherapy injury. </p><p>Using a Transgenic Notch Reporter mouse to investigate the role of Notch signaling following hematopoietic damage we determined the Notch pathway is also activated during regeneration. Furthermore, using a live imaging approach we discovered Notch activated cells change their fate choice during regeneration. To determine if Notch gain of function provides radio-protection we infected stem cells with an active form of Notch prior to radiation and then scored self renewal potential in vitro. This led us to the conclusion that Notch gain of function can provide a self renewal benefit to irradiated hematopoietic stem cells.</p> / Dissertation

The purpose of thesis aims to investigate the mechanical behavior and properties of a centrally notched hybrid Al alloy/Carbon-Fiber/PEEK(APC-2) laminate at elevated temperature. The high performance hybrid composite laminates of 0.5mm Aluminum alloy sheets sandwiched by APC-2 cross-ply and guasi-isotropic laminates were fabricated. The prepregs of APC-2 were stacked into cross-ply [0/90]s and quasi-isotropic [0/45/90/-45] laminates spread uniformly with nanoparticles SiO2. The sheet surface was treated by chromic acid anodic method to achieve perfectly bonding with matrix PEEK. The modified diaphragm curing process was adopted to fabricate Al/APC-2 hybrid nanocomposite laminates. The panels were cut into the specimens and then drilled an diameter hole in the center with diameters of 1,2,4,6 mm. The MTS 810 material testing machine was used to conduct the tension and fatigue tests. In addition, the MTS 651 environmental chamber was installed to control and keep the specific testing temperatures, such as ,25¢XC(RT), 75¢XC, 100¢XC, 125¢XC and 150¢XC. At first, the nominal stress(£mnom) and stress-strain diagram were obtained due to static tension tests at elevated temperature. The constant stress amplitude tension-tension cyclic tests were carried out by using load-control mode at a sinusoidal loading with frequency of 5Hz and stress ratio R=0.1. The received fatigue data were plotted in normalized S-N curves at variously elevated temperature. For the tensile tests, at the same temperature the nominal stress of cross-ply specimens was higher than that of quasi-isotropic specimens. Comparing with the notched and unnotched of cross-ply specimens, the nominal stress of notched specimens was about 60% to 80% that of unnotched specimens. Besides, as for the notched and unnotched quasi-isotropic specimens, the nominal stress of notched specimens was about 75% to 85% that of unnotched specimens. Then, the fatigue life and stress-cycles (S-N) curves of notched specimens were obtained often tension-tension fatigue tests. In the case of the same loading, notched specimens possess worse fatigue behavior, but in the same normalized stress ratio, the S-N curves of the unnotched were below the notched ones. The fatigue resistance of notched samples decrease as the temperature rising.

The aims of this thesis to investigate the two step loading of Ti/APC-2 hybrid nanocomposite laminates and their notched effect. Ti/APC-2 laminates were composed of three layers of titanium sheets and two layers of APC-2. Nanoparticles SiO2 were dispersed uniformly on the interfaces of APC-2 with the optimal amount of 1 wt %. Then, APC-2 was stacked according to cross-ply [0/90]s and quasi-isotropic [0/45/90/-45] sequences. The modified diaphragm curing process was adopted to fabricate the hybrid panels for minimal impact of production. The panels were cur into samples and drilled an diameter hole in the center with diameters of 4 or 6 mm. Both tension and fatigue tests were carried out with MTS 810 universal testing machine at room temperature. Also, two-step loading tests include high¡÷low and low¡÷high tests, were conducted. 0.9£mnom is denoted as high load and 0.7£mnom low load for two-step loading spectrum. In both high¡÷low and low¡÷high step loadings the first step is to do cyclic tests at a half life of the corresponding load, and then finish it due to last step load. From the received results, some conclusions were made. First, the ultimate load of notched cross-ply samples was reduced about 50% and the notched quasi-isotropic samples reduced about 30% compared to their unnotched counterparts. Second, the S-N curves are very close for both centrally notched samples of diameters 4 mm and 6 mm in cross-ply and quasi-isotropic samples. Third, quasi-isotropic samples had higher average values of cumulative damage than cross-ply samples. Because of notched effect centrally notched samples of diameters 4 mm had higher average values of cumulative damage than centrally notched samples of diameters 6 mm.

The inner ear is a complex sensory organ of interconnected chambers, each with a sensory epithelium comprised of hair cells and support cells for detection of sound and motion. This dissertation focuses on the development and regulation of sensory epithelia in zebrafish and utilizes loss of function, gain of function and laser ablation techniques. Hair cells and support cells develop from an equivalence group specified by proneural genes encoding bHLH transcription factors. The vertebrate Atoh1 bHLH transciption factor is a potential candidate for this role. However, data in mouse has led some researchers to conclude it does not have a proneural activity, but, rather, is involved in later stages of hair cell differentiation. In addition, the factors regulating Atoh1 are mostly unknown. We address these issues in zebrafish and show that the zebrafish homologs atoh1a and atoh1b are required during two developmental phases, first in the preotic placode and later in the otic vesicle. They interact with the Notch pathway and are necessary and sufficient for specification of sensory epithelia. Our data confirm atoh1 genes have proneural function. We also go on to show Atoh1 works in a complex network of factors, Pax2/5/8, Sox2, Fgf and Notch. Misexpression of atoh1 alters axial patterning and leads to expanded sensory epithelia, which is enhanced by misexpression of either fgf8 or sox2. Lastly, we examine the role of sox2 in sensory epithelia development and regeneration. Sox2 has been implicated in maintainence of pluripotent stem cells as well as cell differentiation. In the inner ear, Sox2 is initially expressed in the prosensory domain and is required for its formation. Eventually, Sox2 is downregulated in hair cells and maintained in support cells; however, its later role has not been determined. We show that in the zebrafish inner ear, sox2 is expressed after sensory epithelium development has begun and, like in mouse, expression is down regulated in hair cells and maintained in support cells. Our data demonstrate a role for sox2 in maintenance of hair cells and in transdifferentation of support cells into hair cells after laser ablation. Additionally, sox2 is regulated by Aoth1a/1b, Fgf, and Notch.

Because of the huge bandwidth of Ultra-Wideband (UWB) systems, in-band narrowband interference may hinder receiver performance. In this dissertation, sources of potential narrowband interference that lie within the IEEE 802.15.3a UWB bandwidth are presented, and a solution is proposed. To combat interference in Multi-Band OFDM (MB-OFDM) UWB systems, an analog notch filter is designed to be included in the UWB receive chain. The architecture of the filter is based on feed-forward subtraction of the interference, and includes a Least Means Squared (LMS) tuning scheme to maximize attenuation. The filter uses the Fast Fourier Transform (FFT) result for interference detection and discrete center frequency tuning of the filter. It was fabricated in a 0.18 µm process, and experimental results are provided. This is the first study of potential in-band interference sources for UWB. The proposed filter offers a practical means for ensuring reliable UWB communication in the presense of such interference. The Operational Transconductance Amplifier (OTA) is the predominant building block in the design of the notch filter. In many cases, OTAs must handle input signals with large common mode swings. A new scheme for achieving rail-to-rail input to an OTA is introduced. Constant gm is obtained by using tunable level shifters and a single differential pair. Feedback circuitry controls the level shifters in a manner that fixes the common mode input of the differential pair, resulting in consistent and stable operation for rail-to-rail inputs. As the new technique avoids using complimentary input differential pairs, this method overcomes problems such as Common Mode Rejection Ratio (CMRR) and Gain Bandwidth (GBW) product degradation that exist in many other designs. The circuit was fabricated in a 0.5µm process. The resulting differential pair had a constant transconductance that varied by only ±0.35% for rail-to-rail input common mode levels. The input common mode range extended well past the supply levels of ±1.5V, resulting in only ±1% fluctuation in gm for input common modes from -2V to 2V.

Thesis (M.S.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains vii, 110 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 94-107).

The protein Liquid facets (Lqf) promotes endocytosis at the plasma membrane1. Lqf activity is required for proper Notch signaling, likely through facilitating the endocytosis of Notch ligand by indirectly linking ligand to clathrin. A genetic modifier screen to identify genes that interact with lqf was performed by a previous graduate student. Genes identified in the screen might provide new insights into how Lqf promotes endocytosis or how Notch signaling is regulated. In this work, I performed genetic mapping techniques to identify the genes mutated in each complementation group of the screen. I identified the gene mutated in complementation group 6 as mitochondrial alanyl tRNA synthetase (Aats-ala-m). tRNA synthetases link a tRNA to its cognate amino acid during translation. Mitochondrial tRNA synthetases function in the mitochondria in translation. Aats-ala-m genetically interacts with lqf, suggesting the two genes function in the same pathway. In this work, I also identified chromosomal regions where the genes mutated in complementation groups 1,2, and 9 are located. / text

Breast cancer is a heterogeneous disease with vastly different tumor progression kinetics and survival outcomes depending upon the differentiation state and gene expression patterns of the tumor. Effective treatments exist for patients with endocrine therapy sensitive or HER2 overexpressing tumors, but targeted treatments are not available for other tumor types. The mechanisms governing mammary tumor phenotype generation could prove critical to finding treatments. The c-Jun N-terminal kinase (JNK) pathway has recently been implicated in the inhibition of breast tumor luminal differentiation (1, 2) and JNK2, in particular, is important in mammary tumorigenesis and tumor progression (3-8). Therefore, the involvement of JNK2 in inhibition of mammary luminal cell differentiation was investigated in normal glands and tumors. Studies found that JNK2 inhibits luminal cell populations in normal mammary ducts. Additionally, JNK2 suppresses Notch activity in stem cell niche of the developing mammary gland. In vitro assays show that control over differentiation by JNK2 is due to suppression of p53-dependent Notch1 expression. Inhibition of luminal cell populations by JNK2 is also apparent in tumor cell models regardless of p53 expression. In the p53-competent Polyoma Middle T-antigen model, Notch1 expression is suppressed by JNK2. In the absence of p53, JNK2 suppresses luminal populations independent of Notch1. In this model, decreased luminal marker expression is accompanied by increased epithelial to mesenchymal transition. It was also found that JNK2-dependent epithelial to mesenchymal transition inhibits luminal populations and is driven by JNK2-dependent suppression of Brca1. JNK2 also confers resistance to estrogen signaling inhibition, and increases the metastatic ability of tumor cells in vivo. These data establish the importance of JNK2 in mammary epithelial cell differentiation in normal glands and tumors. They also suggest that JNK2 may be an effective prognostic marker or treatment target. / text

The process of cell fate determination, which establishes the vastly diverse set of neural cell types found in the central nervous system, remains poorly understood. During retinal development, multipotent retinal progenitor cells generate seven major cell types, including photoreceptors, interneurons, and glia, in an ordered temporal sequence. The behavior of these progenitor cells is influenced by the Notch pathway, a widely utilized signal during embryogenesis which can regulate proliferation and cell fate decisions. To examine the underlying genetic changes that occur when Notch1 is removed from individual retinal cells, microarray analysis of single cells from wild type or Notch1 conditional knockout retinas was performed. Notch1 deficient cells downregulated progenitor and cell cycle marker genes, while robustly upregulating genes associated with rod genesis. Single wild type cells expressed markers of both rod photoreceptors and interneurons, suggesting that these cells were in a transitional state. In order to examine the role of Notch signaling in cell fate specification separate from its role in proliferation, Notch1 was genetically removed specifically from newly postmitotic cells. Notch1 deficient cells preferentially became cone photoreceptors at embryonic stages, and rod photoreceptors at postnatal stages. In both cases, this cell fate change occurred at the expense of the other cell types normally produced at that time. In addition, single cell profiling revealed that Inhibitor of differentiation 1 and 3 genes were robustly downregulated in Notch1 deficient cells. Ectopic expression of these genes during postnatal development in wild type retinas was sufficient to drive production of progenitor/Müller glial cells. Moreover, Id1 and 3 partially rescued the production of Müller glial cells and bipolar cells in the absence of Notch1, even in newly postmitotic cells. We propose that after cell cycle exit, retinal precursor cells transition through a period in which they express marker genes of several different cell types as they commit to a fate, likely endowed by their progenitor cell. Specifically, cells that will become bipolars or Müller glia depend on Id-mediated Notch signaling during this transitional state to take on their respective fates.

Development leads unidirectionally towards a more restricted cell fate that is usually stable. However, it has been proven that developmental systems are reversible by the success of animal cloning of a differentiated somatic genome through somatic cell nuclear transfer (SCNT). Recently, reprogramming of somatic cells to a pluripotent embryonic stem cell (ESC)-like state by introducing defined transcripton factor has been achieved, resulting in the generation of induced pluripotent stem cells (iPSCs), which resemble ESCs. iPSC reprogramming is of great medical interest, as it has the potential to generate a source of patient-specific cells. However, the dangerous delivery method, low efficiency, and slow kinetics of the reprogramming process have hampered progress with this technology.