Early events in BRCA1-associated tumorigenesis remain poorly understood. To understand the immediate consequences of BRCA1 loss of function, we modeled BRCA1 loss of function in vitro using normal primary human mammary epithelial cells (HMEC). We have found that in HMEC, loss of BRCA1 results in a novel type of senescence. Loss of BRCA1-induced senescence is not associated with DNA damage or p53 upregulation. We find that p53 protein levels are down regulated due to proteasome-mediated degradation. Although p53 levels are down regulated, we find that BRCA1 loss induced expression of a number of p53-dependent anti-oxidant genes. In particular we uncovered that SESN2, a p53 downstream target gene, inhibits loss of BRCA1 induced ROS and activates autophagy. In contrast to human fibroblasts, we found that loss of BRCA1 induced senescence is p53 independent, and can occur in the absence of ROS upregulation and autophagy induction.

Early events in BRCA1-associated tumorigenesis remain poorly understood. To understand the immediate consequences of BRCA1 loss of function, we modeled BRCA1 loss of function in vitro using normal primary human mammary epithelial cells (HMEC). We have found that in HMEC, loss of BRCA1 results in a novel type of senescence. Loss of BRCA1-induced senescence is not associated with DNA damage or p53 upregulation. We find that p53 protein levels are down regulated due to proteasome-mediated degradation. Although p53 levels are down regulated, we find that BRCA1 loss induced expression of a number of p53-dependent anti-oxidant genes. In particular we uncovered that SESN2, a p53 downstream target gene, inhibits loss of BRCA1 induced ROS and activates autophagy. In contrast to human fibroblasts, we found that loss of BRCA1 induced senescence is p53 independent, and can occur in the absence of ROS upregulation and autophagy induction.

Bacteriophage ë has been used as a model replicon system for forty years. While the basic ë replication initiation scheme has been elucidated for several decades, many aspects of the mechanisms are unclear. I wished to study two unanswered issues in ë replication initiation. </p><p>Replication initiation of E. coli and ë each depend upon a protein generally called a licensing factor, which brings the DnaB helicase protein to the origin site to begin DNA synthesis. The licensing factors are the products of host gene dnaC and ë gene P. The synthesis of P from ë DNA in an E. coli cell can competitively interfere with DnaC activity needed for E. coli replication initiation. I wished to learn more about what happens to a host cell when exposed to extended P expression. Previous studies in this laboratory suggested that i) the continuous expression of P was tolerated by a subset of exposed cells and that ii) host defects mapping to dnaB could suppress the effect of extended P expression (P-lethality). I used DNA sequencing to determine if these suppressor mutations were within dnaB. I screened known host mutations for their influence on P-lethality. In summary: E. coli strains with GrpD55 and GrpA80 defects were found to each have two point mutations within their dnaB genes. I was unable to isolate mutations within P that suppressed P-lethality and instead obtained regulatory mutations preventing wild type P expression. Two of these sequenced mutations showed that a cI[Ts] lambda repressor was reverted to cI wild type, blocking P expression at all assay temperatures. P-lethality was reversible in cells exposed to P for up to five hours, causing me to suggest that P-Interference be used in place of the term P-lethality. A non-inducible allele of lexA prevented P-mediated cellular filamentation and enhanced P-Interference. This suggests that induction of the SOS response helps cells to tolerate extended P expression. A host strain containing a defective ClpXP protease significantly enhanced cellular sensitivity to P-Interference. This suggests an important role for the ClpXP chaperone-protease complex in degradation of P and cellular resistance to P expression. I present models to explain the P-Interference Phenotype.</p><p>Recent reports have re-opened the possibility that the tO-oop-pO element influences ë DNA replication initiation. I have also been investigating this possibility. I found that a plasmid with tO-oop-pO (the terminator, nucleotide sequence and promoter for OOP RNA) and orië DNA sequence was inhibitory to the development of repë phages, and designated this the Inhibition Phenotype (IP). In pursuing the mechanism for this inhibition, I mutated the tO-oop-pO and orië elements. I found that the expression of the 77nt OOP RNA transcript and the presence of four 18 base pair repeats (iterons) within orië were required for the IP. I isolated spontaneous phage mutants, resistant to the IP. I determined that singly infected cells were sensitive to the IP but that multiply infected cells escaped the IP. I propose that the IP to repë phage development is directed to the initial or theta mode of ë replication initiation. I found that the theta-mode of ë replication initiation can be bypassed, likely via recombination between multiple phage genomes within a singe cell. I propose models to explain the IP and also suggest a role for OOP RNA in the regulation of ë DNA replication.

Initiation of DNA replication requires the action of the Dbf4/Cdc7 kinase complex (DDK) which is also a phosphorylation target of Rad53 kinase in the S-phase checkpoint. DDK is thought to trigger DNA replication by phosphorylating members of the Mcm2-7 complex present at origins of replication. While DDK phosphorylation sites have been identified on Mcm2-7, the contributions made by Dbf4 and Cdc7 to the targeting of the complex have not been established. DDK has also been implicated in the S-phase checkpoint response since it is removed from chromatin in a Rad53-dependent manner. The interaction of Dbf4 and Cdc7 with each of the Mcm2-7 subunits was assessed and showed an interaction between Dbf4 and Mcm2 and Mcm6, while interactions between Cdc7 and Mcm4 and Mcm5 were observed. Mutations in Mcm2 and Mcm4 that disrupt the interactions with Dbf4 or Cdc7 showed modest growth impairment and compromised DNA replication, while simultaneous abrogation of both interactions resulted in lethality. Strains overexpressing Mcm2 or Mcm4 were sensitive to genotoxic agents, while overexpression of Mcm2 in a Mcm4Δ175-333 strain background resulted in a severe growth impairment as well as sensitivity to genotoxic stress. ChIP analysis revealed the possibility of Dbf4/Cdc7 localization to origin flanking regions through most of S-phase, which may redistribute to origins at the time of firing. Fluorescence microscopy of Mcm2 and Dbf4 in S-phase seem to show a punctate pattern of staining, consistent with these factors’ localization to ‘replication factories.’ By using a Dbf4ΔN mutant, the N-motif was shown to be required for the Rad53-mediated removal of Dbf4 from chromatin under checkpoint conditions. Initial optimization of a DNA combing protocol was also performed, which along with Dbf4ΔN mutant and the fluorescently-epitope tagged strains, will be useful tools for evaluating a role for DDK in the S-phase checkpoint response. Altered levels of DNA replication factors have been implicated in many human cancers. The data presented in this study provide novel insight into the normal process of the initiation of DNA replication which can be applied to research involving higher eukaryotes, including humans, and can serve as a benchmark for comparison with the cancer phenotype.

Bacteriophage ë has been used as a model replicon system for forty years. While the basic ë replication initiation scheme has been elucidated for several decades, many aspects of the mechanisms are unclear. I wished to study two unanswered issues in ë replication initiation. </p><p>Replication initiation of E. coli and ë each depend upon a protein generally called a licensing factor, which brings the DnaB helicase protein to the origin site to begin DNA synthesis. The licensing factors are the products of host gene dnaC and ë gene P. The synthesis of P from ë DNA in an E. coli cell can competitively interfere with DnaC activity needed for E. coli replication initiation. I wished to learn more about what happens to a host cell when exposed to extended P expression. Previous studies in this laboratory suggested that i) the continuous expression of P was tolerated by a subset of exposed cells and that ii) host defects mapping to dnaB could suppress the effect of extended P expression (P-lethality). I used DNA sequencing to determine if these suppressor mutations were within dnaB. I screened known host mutations for their influence on P-lethality. In summary: E. coli strains with GrpD55 and GrpA80 defects were found to each have two point mutations within their dnaB genes. I was unable to isolate mutations within P that suppressed P-lethality and instead obtained regulatory mutations preventing wild type P expression. Two of these sequenced mutations showed that a cI[Ts] lambda repressor was reverted to cI wild type, blocking P expression at all assay temperatures. P-lethality was reversible in cells exposed to P for up to five hours, causing me to suggest that P-Interference be used in place of the term P-lethality. A non-inducible allele of lexA prevented P-mediated cellular filamentation and enhanced P-Interference. This suggests that induction of the SOS response helps cells to tolerate extended P expression. A host strain containing a defective ClpXP protease significantly enhanced cellular sensitivity to P-Interference. This suggests an important role for the ClpXP chaperone-protease complex in degradation of P and cellular resistance to P expression. I present models to explain the P-Interference Phenotype.</p><p>Recent reports have re-opened the possibility that the tO-oop-pO element influences ë DNA replication initiation. I have also been investigating this possibility. I found that a plasmid with tO-oop-pO (the terminator, nucleotide sequence and promoter for OOP RNA) and orië DNA sequence was inhibitory to the development of repë phages, and designated this the Inhibition Phenotype (IP). In pursuing the mechanism for this inhibition, I mutated the tO-oop-pO and orië elements. I found that the expression of the 77nt OOP RNA transcript and the presence of four 18 base pair repeats (iterons) within orië were required for the IP. I isolated spontaneous phage mutants, resistant to the IP. I determined that singly infected cells were sensitive to the IP but that multiply infected cells escaped the IP. I propose that the IP to repë phage development is directed to the initial or theta mode of ë replication initiation. I found that the theta-mode of ë replication initiation can be bypassed, likely via recombination between multiple phage genomes within a singe cell. I propose models to explain the IP and also suggest a role for OOP RNA in the regulation of ë DNA replication.

This thesis focuses on the growth of cracks which are small in relation to the material microstructure especially the situation of clusters of small cracks grown from smooth surfaces, termed micro-multi-site cracking, as is frequently the case for components in service. A proper understanding of this regime of crack growth will allow for less conservative maintenance schedules as well as the application of more sensitive health monitoring systems which are currently under development. To address the problem a significant experimental investigation of micro-multi-site cracking was conducted on 7075-T7351 aluminum alloy. Using the resulting data a micro-structurally based transition crack length is defined to determine the point which separates small and long crack growth. This definition is based upon the observed evolution of scatter in the growth rates of growing small cracks. It is shown that this scatter falls with growth until the transition point is reached where it assumes a constant value for the growth of long cracks. It is then shown that the total population of cracks within the clusters can be considered as bi-modal. One distribution consists of primary cracks which can grow and ultimately cause specimen failure. The second distribution consists of secondary cracks, the growth of which ultimately arrests. Several methods for experimentally separating the two distributions have been developed. The first method relies upon the defined transition point between small and long crack behavior. A second method based upon the second derivative of the crack length versus cycle count data has also been developed. Since the secondary cracks cannot lead to failure their data must be discarded prior to any analysis. It is then shown that failure to do so will lead to erroneous non-conservative predictions of crack growth.

This interpretive study uses hermeneutic phenomenological methodology to understand the experience of six college students in Taiwan who participate in TV talk show as studio audience. Texts were collected from in-depth interviews. The result indicated a dramatic interaction framework toward the whole experience: participants as performers must ¡¥act¡¦ like undergraduate students, though the show ¡V from script, setting to personal front ¡V must be ratified by producer. Furthermore, two transformative effects are found in participants. First, they were socialized in the studio through the performance, and learned more social-performing skills and scripts. Second, they are bothered by mixing up their drama-roles with social-roles. It was the producer¡¦s purpose to represent ¡¥a world beneath¡¦ of college students in University. However it became ¡¥a public trial¡¦ on TV after excessive entertainment manipulation.

Textile composites have a wide variety of applications in the aerospace, sports, automobile, marine and medical industries. Due to the availability of a variety of textile architectures and numerous parameters associated with each, optimal design through extensive experimental testing is not practical. Predictive tools are needed to perform virtual experiments of various options. The focus of this research is to develop a better understanding of linear elastic response, plasticity and material damage induced nonlinear behavior and mechanics of load flow in textile composites. Textile composites exhibit multiple scales of complexity. The various textile behaviors are analyzed using a two-scale finite element modeling. A framework to allow use of a wide variety of damage initiation and growth models is proposed. Plasticity induced non-linear behavior of 2x2 braided composites is investigated using a modeling approach based on Hill’s yield function for orthotropic materials. The mechanics of load flow in textile composites is demonstrated using special non-standard postprocessing techniques that not only highlight the important details, but also transform the extensive amount of output data into comprehensible modes of behavior. The investigations show that the damage models differ from each other in terms of amount of degradation as well as the properties to be degraded under a particular failure mode. When compared with experimental data, predictions of some models match well for glass/epoxy composite whereas other’s match well for carbon/epoxy composites. However, all the models predicted very similar response when damage factors were made similar, which shows that the magnitude of damage factors are very important. Full 3D as well as equivalent tape laminate predictions lie within the range of the experimental data for a wide variety of braided composites with different material systems, which validated the plasticity analysis. Conclusions about the effect of fiber type on the degree of plasticity induced non-linearity in a ±25° braid depend on the measure of non-linearity. Investigations about the mechanics of load flow in textile composites bring new insights about the textile behavior. For example, the reasons for existence of transverse shear stress under uni-axial loading and occurrence of stress concentrations at certain locations were explained.

Hydraulic fracturing of transverse isotropic reservoirs is of major interest for reservoir stimulation and in-situ stress estimation. Rock fabric anisotropy not only causes in-situ stress anisotropy, but also affects fracture initiation from the wellbore. In this study a semi-analytical method is used to investigate these effects with particular reference to shale stimulation. Using simplifying assumptions, equations are derived for stress distribution around the wellbore's walls. The model is then used to study the fracture initiation pressure variations with anisotropy. A sensitivity analysis is carried out on the impact of Young's modulus and Poisson's ration, on the fracture initiation pressure. The results are useful in designing hydraulic fractures and also can be used to develop information about in-situ rock properties using failure pressure values observed in the field. Finally, mechanical and permeability anisotropy are measured using Pulse Permeameter and triaxial tests on Pierre shale.

The Shield&rsquo / s Diagram has been the key stone for the description of initiatial motion of a particle in open channel flow. Data in Shield&rsquo / s study and further studies are collected in channels. However, the approximation of these data for the case of withdrawal of sediment or clean water through bottom outlets has not been confirmed. Furthermore, two phase models run to simulate the phenomenon so far have used brine and water combination. In this study, an experimental attempt is made to study the behavior of deposits subject to withdrawal from a bottom outlet where there are not enough parameters to calculate the bottom shear stress and consequently the dimensionless parameters generally used for the description of initiation of motion. The experimental set up used for this purpose is a 1 m long and 0.35 m wide channel such that at the downstream of the channel there is a horizontal slit representing the bottom outlet. During the experiments, fresh water and sand with D50 = 0.298 mm and D50 = 0.912 mm are used. Two different widths of the slit, namely 0.35 m and 0.0875 m are investigated. Based on the observations of the incipient motion of the sediment, the findings in the form of a relationship among the discharge through the bottom outlet, and some other relevant parameters are reported. In addition, a comparison of these data with the literature by the help of some newly defined dimensionless parameters for the description of the initiation of motion is included.