BRCA1 is a caretaker of genome integrity with various molecular functions, which are required for development and tumor suppression. These include the homology-directed repair (HDR) of DNA double strand breaks, stalled replication fork protection (SFP), transcription, chromatin remodeling and cell cycle checkpoint control. Recent studies reported that BRCA1 is required for reprogramming to pluripotency, but its specific role remains unknown. In this work, we use separation of function mutants for the roles of BRCA1 in HDR and SFP to show that BRCA1 is required to repair replication-associated DNA double strand breaks by homologous recombination during reprogramming. Deficiency in SFP proved inconsequential to induced pluripotent stem (iPS) cell generation and cells with this phenotype did not experience reduced reprogramming. Thus, the primary limiting factor for the transition to pluripotency is a specific class of DNA damage: double strand breaks, likely occurring in late replicating regions which require repair by homologous recombination. These findings identify an important role of DNA damage, linked to the progression of DNA replication, in limiting cell type transitions during reprogramming. Most studies on iPS cell generation have focused on gene expression as a limiting step, in part due to the wide availability of tools to analyze transcription. Since the progression of DNA replication and DNA damage during S-phase are cell type specific, we have started the development of a sequencing platform to map various aspects of replication progression, such as origin usage, polymerase direction,pausing and stalling. In this work, we demonstrate that nucleotide analogs, incorporated during DNA synthesis in mammalian cells, can be detected by Nanopore sequencing.

Ovaries maintain and produce functional female gametes, oocytes, for fertilisation. Oocytes develop inside cellular assemblies, the ovarian follicles, before birth and can reside there for up to 50 years in the human. Despite recent inroads, the precise mechanisms of initial follicle recruitment and growth remain unclear. Although the pituitary gonadotrophins play a role in this developmental process, locally produced factors have been implicated strongly in initiation of follicle growth. It is known that fibroblast growth factor 2 (FGF2) is a powerful mitogen for follicular granulosa cells in culture and initial studies undertaken in this project were successful in detecting FGF2 gene expression in ovarian biopsies from fertile healthy women. To further elucidate which cells were expressing FGF2, laser microdissection was employed to isolate differentially staged follicle populations. Real-time RT-PCR was used to quantify mRNA in relation to follicle development. Decreasing levels of FGF2 expression were detected as follicles developed. Non-radioactive in situ hybridisation confirmed FGF2 mRNA localisation in granulosa cells of preantral follicles. FGF2 protein localisation was assessed with immunohistochemistry; two primary antibodies raised against different fragments of human FGF2 were used. Both antibodies detected FGF2 in the oocyte cytoplasm of putative non-growing follicles, whereas only one of the antibodies showed additional reactivity to the basement membrane region of these same follicles. These results suggest different isoforms of FGF2 may localise specifically to different cellular sites. Follicle stimulating hormone receptor (FSHR) gene expression was also investigated in follicles using laser microdissection, real-time RT-PCR and in situ hybridisation. FSHR mRNA was detected in all follicle populations, including the smallest putative non-growing follicles. Disparity to other published works was attributed to the position of primer annealing, and thus the ability to detect alternatively spliced transcripts. In conclusion, the work presented here provides evidence that FGF2 and FSHR are present in small follicles and that their actions may be stimulatory or inhibitory to initial follicle recruitment.

Bibliography: leaves 300-326. / 326 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Evaluates an in vitro experimental system which was designed to assess functions of platelets and cultured endothelial cells when they were incubated either independently or in combination. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 1995

Bibliography: 31 unnumbered leaves at end of vol

Copy of author's previously published article in pocket on back end-paper. Includes bibliography (leaves 135-185). Aims to characterise the expression of PETA-3 (Platelet Endothelial Tetraspan Antigen-3), CD9, CD63 and ?gb?s1 integrins in normal human tissue ; to determine the subcellular localisation in endothilial cells and platelets ; to investigate protein-protein interactions involving PETA-3 ; and to examine the effects of anti-PETA-3 monoclonial antibodies on platelet and endothilial cell function.

Graduation date: 1994

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 109-124).

Objective: The incidence rate and mortality of breast cancer are increasing in Taiwan during recent years. The incidence rate of breast cancer is ranked number one among top ten female cancers, and the mortality of breast cancer is ranked fourth among cause of death for female cancer sufferers. The most common age group for breast cancer is between 40 and 50 years old. Breast cancer causes huge disease burdens for individual, family and society. The breast sonography and fine needle aspiration cytology (FNAC) are common screening methods for breast cancer diagnosis. Nevertheless, little study has focused on the benefits of combing these two methods in clinical application. This study aims to fill such research gap. Method: This study conducted medical chart reviews and collected 2,776 observations that were under breast sonography and FNAC examination from a regional hospital locates in southern Taiwan. The diagnosis categories for sonography include: malignant, benign, and probably benign tumor. The diagnosis categories for FNAC include: malignant, benign, and suspicious for malignant. Results: Among 2,776 observations, there were 555 observations (20%) had operation in the studied hospital. The operation results indicated that 205 (36.9%) observations were with malignant status, and 350 (63.1%) observations were with benign status. The diagnosis categories of both sonography and FNAC were significantly associated with the operation results (p<0.001). The FNAC had specificity in 100%, false positive ratio in zero, and positive predictive value in 100%. The Odds ratios for sonography diagnosis categories, age groups, and tumor sizes were OR=4.132 (95%CI: 1.5¡V11.6), OR=31.957 (95% CI: 3.7¡V272.4), OR=0.457 (95% CI: 0.1¡V1.5), respectively. When combining sonography and FNAC in parallel tests, the diagnosis accuracy was 89.2%, sensitivity was 90.2%, specificity was 88.6%, positive predictive value was 82.2%, and negative predictive value was 93.9%. When combining sonography and FNAC in serial testing, the diagnosis accuracy was 88.1%, sensitivity was 67.8%, specificity was 100%, positive predictive value was 100%, and negative predictive value was 84.1%. Conclusion: Combining sonography and FNAC in breast cancer diagnosis can increase the accuracy, decrease false positive ratio and false negative ratio. These two methods can be conducted during outpatient visit and are fast, accurate and cost-effective tools for breast cancer diagnosis. These two methods particularly appropriate for younger female patients for early screening, early intervention, and may increase the survival rates.

A fundamental yet unresolved issue in cell biology is how force regulates actin dynamics and how this biophysical regulation is modulated by biochemical signaling molecules. Here we show, by atomic force microscopy (AFM) force-clamp experiments, that tensile force regulates the kinetics of G-actin/G-actin and G-actin/F-actin interactions by decelerating dissociation at low forces (catch bonds) and accelerating dissociation at high forces (slip bonds). The catch bonds can be structurally explained by force-induced formation of new interactions between actin subunits (Steered molecular dynamics (SMD) simulations performed by Dr. Jizhong Lou, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China). K113S mutation on yeast actin suppressed the actin catch-slip bonds, supporting the structural mechanism proposed by SMD simulations. Moreover, formin controlled by a RhoA-mediated auto-inhibitory module can serve as a "molecular switch", converting the catch-slip bonds to slip-only. These results imply anisotropic stability of the actin network in cells subjected to directional forces, possibly explaining force-induced cell and actin fiber alignment controlled by RhoA and formin. Our study suggests a molecular level crosstalk mechanism bridging the actin-mediated mechanotransduction and biochemical signal transduction pathways.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences