Breast cancer is a world health problem and is a leading cause of cancer-related death among women in the United States. However, breast cancer risks were reported to be reduced through exposure to Vitamin D through its Receptors identified as the p53 target gene. The purpose of this study was to assess the associations between VDR gene polymorphisms knowledge/awareness and decisions to reduce breast cancer risks and likelihood of mammogram screening among women in Texas. Data from survey were used. Roy adaptation model was the theoretical framework that guided this quasi- experimental, quantitative research. The dependent variables were decisions to reduce breast cancer risks and likelihood of mammogram screening. The independent variables were knowledge about VDR gene polymorphisms and exposure to vitamin D. The covariates were level of education, awareness, lifestyle, breast self-exams, mammograms, age, early menarche, late menopause, and family history of breast cancer. The chi-square test and regression analysis were used to test the stated research hypotheses and to answer the research questions. Knowledge of VDR gene polymorphisms and exposure to vitamin D were not significantly associated with breast cancer risk, ï?£2 (3, N= 250) =3.84, p > 0.05. Also, awareness of the risk factors for breast cancer was not significantly associated with decisions to go for mammogram screenings or to enroll in breast cancer risk-reduction programs, ï?£2 (3, N= 250) =1.58, p > 0.05. To advocate for the promotion of awareness of the importance of pharmacogenetic testing for VDR gene polymorphisms for early detection of breast cancer, which would help to undertake appropriate therapeutic measures in a timely manner to prevent cancer metastasis, further research is warranted.

This thesis describes bioanalytical methods for generating fingerprints of biological systems for extracting relevant information with (protein) drugs in focus. Similarities and differences between samples can reveal the hidden relevant information, which can be used to optimize the production and facilitate the quality control of such protein drugs during their development and manufacture. Metabolic fingerprinting and multivariate data analysis (MVDA) can also facilitate early diagnosis of diseases and the effects and toxicity of drugs. Currently, several protein drugs are available on the global market. Nevertheless, despite, the success of such biotherapeutics significant challenges remain to be overcome in maintaining their stability and efficacity throughout their production cycle and long-term storage. The native structure and functional activity of therapeutic proteins is affected by many variables from production to delivery, incl. variables assoc. with conditions in bioreactors, purification, storage and delivery. Thus, part of the work underlying this thesis focused on structural analysis of a protein drug using chemical labeling, peptide mapping, and evaluation of the charge state distributions of the whole protein generated by ESI. The other part focuses on non-targeted metabolomics with a view to optimizing the cell cultivation process and assessment of the drug’s toxicity. A combination of appropriate analytical methods and MVDA is needed to find markers that can facilitate optimization of the cultivation system and expression of the target proteins in early stages of process development. Rapid methods for characterizing the protein drugs in different stages of the process are also required for quality control. In order to obtain high quality fingerprints analytical separation techniques with high resolution (such as HPLC or UHPLC) and sensitive analytical detection techniques (such as ESI, quadrupole or TOF MS) have been used, singly or in combination. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript.

Monoclonal antibodies are well-established as a therapeutic in the biopharmaceutical market, targeting a variety of diseases and with 79 approved products by the United States Food and Drug Administration in December 2019. Therapeutic monoclonal antibodies are commonly produced as recombinant proteins in mammalian cell lines, due to their capacity of post-translational modifications, most notably glycosylation. Furthermore, an identified bottleneck within the production of recombinant proteins is the translocation of nascent proteins from the cytosol into the lumen of the endoplasmic reticulum. The signal peptide, which is located at the N-terminal of nascent proteins, plays a central role in the process of protein secretion. Several studies have shown that optimization of signal peptides is a crucial step for attempting to achieve increased expression of recombinant antibodies in mammalian systems. The aim of this study was to evaluate the expression of three human recombinant antibodies in Human Embryonic Kidney 293 (HEK293) cells by evaluating 16 different signal peptide combinations, consisting of eight heavy chain (HC) and two light chain (LC) signal peptides. The impact goal was an efficient secretion of recombinant antibodies, and thus lower production cost of recombinant antibodies in HEK293 cells. First, 16 HC and LC signal peptide plasmid constructs were generated for each of the three recombinant antibodies. Thereafter, transient gene expression in HEK293 cells were performed at three independent experiments. Finally, the antibody titers were quantified using Biacore concentration analysis. The produced antibody titers for the three studied recombinant antibodies were highly dependent on the used signal peptides. Interestingly, the evaluated HC and LC signal peptide combinations resulted in 3 times higher and 2 times higher antibody titers compared to the original signal peptides used by the Drug Discovery and Development platform at Science for Life Laboratory, for two of the studied antibodies respectively. The results presented in this report further demonstrates the necessity to evaluate signal peptides in order to achieve increased expression of recombinant antibodies in mammalian systems.

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Tato diplomová práce se zabývá popisem principů kultivace embryonálních fibroblastových buněk myší (3T3), lidských endoteliálních buněk odebraných z pupečníkové žily (HUVEC) a epiteliálních buněk vaječníku čínského křečka (CHO) v mikrofluidních systémech simulujících kapiláry. Byly provedeny literární rešerše v oblasti realizací experimentu “Scratch Wound Healing Assay” v mikrofluidních systémech s použitím fibroblastů a endotheliálních buněk. V práci jsou dále popsány principy konfokální a fluorescenční mikroskopie a metody zpracování obrazů pro sledování buněčné migrace. Experimentální nastavení pro mikrofluidní realizaci “Scratch Wound Healing Assay” s použitím trypsinu – EDTA pro vytvoření rýhy, a konfokálního mikroskopu Leica TCS SP8 X pro následující snímání pořízených dat bylo navrženo a otestováno s dostatečným počtem opakování. Vhodný algoritmus pro analýzu buněčné migrace byl napsán v programovacím prostředí Matlab. Závěrem této práce je diskuze získaných výsledků.

Damage to the central nervous system (CNS) leads to the generation of an immune response which culminates with the encapsulation of the damaged area. The encapsulation, known as a glial scar, essentially breaks neural signal pathways and blocks signal transmissions to and from the CNS. The effect is the loss of motor and sensory control for the damaged individual. One method that has been used successfully to treat this problem is the use of a brain-machine interface (BMI) which can intercept signals from the brain and use these signals to control a machine. Although there are many types of BMI devices, implantable devices show the greatest promise with the ability to target specific areas of the CNS, with reduced noise levels and faster signal interception, and the fact that they can also be used to send signals to neurons. The largest problem that has plagued this type of BMI device is that the materials that have been used for their construction are not chemically resilient, elicit a negative biological response, or have difficulty functioning for extended periods of time in the harsh body environment. Many of these implantable devices experience catastrophic failure within weeks to months because of these negative factors. New materials must be examined to advance the future utilization of BMI devices to assist people with CNS damage or disease. We have proposed that two semiconductor materials, cubic silicon carbide (3C-SiC) and nanocrystalline diamond (NCD), which should provide solutions to the material biocompatibility problems experienced by implantable BMI devices. We have shown in this study that these two materials show chemical resilience to neuronal cellular processes, and we show evidence which indicates that these materials possess good biocompatibility with neural cell lines that, in the worst case, is comparable to celltreated polystyrene and, in most cases, even surpasses polystyrene. We have utilized 3C-SiC within an electrode device and activated the action potential of differentiated PC12 cells. This work details our initial efforts to modify the surfaces of these materials in order to improve cellular interaction and biocompatibility, and we examine our current and future work on improving our implantable BMI devices.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19759号 / 農博第2155号 / 新制||農||1039(附属図書館) / 学位論文||H28||N4975(農学部図書室) / 32795 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 宮川 恒, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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