Current sperm processing methods used in assisted reproductive technologies can cause damage to the sperm cell. New ways that mimic the natural guidance mechanisms present in the female genital tract may offer ways to sort sperm with better fertility parameters. Sperm that respond to these cues may have better over sperm health. Human sperm exhibit positive rheotaxis by orienting and swimming against the fluid released by the female genital tract. At certain flow rates sperm can actively orient and swim against the flow. Sperm retrieved that exhibit positive rheotaxis have higher motility and better morphology than the original semen sample. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

Mammalian cell culture has gained importance in biotechnology for the development of therapeutic and diagnostic agents. Among them, Chinese hamster ovary (CHO) cells are regarded as the mammalian cell "workhorse". The use of CHO cell line for the production of recombinant proteins used in human therapy has reached a level of industrial production. However, a major problem encountered in in vitro cultures is cell death via apoptosis. Since apoptosis leads to the loss of viability of mammalian cells in vitro, especially in serum-free media. This is important and necessary to prevent the activation of apoptosis cascade and increase their cell viability and enhance their cellular robustness. The overall goal of this study is to improve our understanding of the cellular and physiological determinants of apoptosis and its relationship with other cellular functions. Apoptosis is a result of a very complex network of signaling pathways triggered from both inside and outside of the cell and a highly regulated pathway by both pro-apoptotic and anti-apoptotic proteins that promote cell survival or cell death. Although many causes of apoptotic process in mammalian cell cultures had been researched in the past and have been discussed in recent years, a lot need to be explored. In order to bring novel strategies to understand apoptosis in mammalian cell cultures, our study was not only focused on the apoptotic pathway but also expand to metabolic network to set up a link between cell growth and apoptosis. In our project, we applied systems biology methods in a mammalian cell line (CHO TF 70R), to understand the relationship between cellular metabolism and apoptosis in a typical serum free culture medium. After establishing the basic culture platform, the effects of culture conditions on initiating apoptosis will be evaluated. Healthy and apoptotic cell samples were identified and isolated using Fluorescence Activated Cell Sorting (FACS) and Magnetic Activated Cell Sorting (MACS), respectively. A comprehensive study of CHO cellular metabolism was made using a metabolic flux network to compare and analyze by metabolic flux analysis (MFA) to get more information on cell metabolism and apoptotic behavior. Furthermore, 2-NBDG combined with Annexin V-PE was also successfully applied to estimate the glucose uptake rate in real early apoptotic cells. In summary, we used the integration of the data generated by MFA to understand apoptotic behavior and establish a correlation between cell regulation and apoptosis. It will help us to identify the changes during the onset of apoptosis process will be studied by using proteomics tools to analyze the protein up-regulation or down-regulation in different cell status in the future.

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Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [103]-137).

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Suppression of genes involved in DNA repair, tumor suppression and detoxification through epigenetic modifications has been implicated in the etiology of cancer. As such analysis of promoter methylation patterns in genes frequently down regulated in breast cancer in non-cancerous subjects may serve as an indicator of breast cancer risk. CpG-island hypermethylation of single genes has been detected in cells isolated from nipple aspirate and ductal lavage, yet both isolation methods yield insufficient cells to complete an extensive analysis on any one donor sample. As an alternative we have turned to magnetic separation of human mammary epithelial cells from breast milk. Initial studies with these cells, which are detailed in chapter one, show that a breast milk sample provides sufficient epithelial cells to isolate high quality RNA for gene expression analyses or genomic DNA for methylation analysis of multiple genes. Using quantitative RT-PCR of RNA collected from these samples we detected differences in the mRNA levels for six genes known to be down regulated in breast cancers: BRCA1, p16, CDH1, TMS-1, GSTPi, and SFRP1. Additionally using methyl-specific PCR (MSP) we assayed for a small panel of genes frequently methylated in cancer and found them to be unmethylated in the few breast milk samples examined. However, given the small number of CpG sites which can be assayed by the MSP technique it is not surprising that methylation was not detected in disease-free subjects. With methods for collecting breast milk samples and processing them for genetic material established we turned to a more comprehensive study of DNA methylation in larger population of donors which is detailed in chapter two. Utilizing a highly sensitive and highly quantitative methylation analysis technique known as Pyrosequencing we examined age-related methylation patterns for RASSF1A, TMS-1, CDH1, SFRP1, GSTPi, and CRBP1 in genomic DNA purified from exfoliated epithelial cells magnetically enriched from breast milk (n=111) and whether the protective effects conveyed by early pregnancy could be partly due to decreases in DNA hypermethylation. Although firm answers about early pregnancy were inconclusive based on our sample pool, this body of work lays down a solid foundation for future studies.

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