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The thesis introduces two developed methods to quantify extracellular vesicles (EVs) isolated from cancer cell lines and healthy human saliva by using capillary zone electrophoresis. In the first chapter, the importance of EVs, as well as the existing EV isolation, characterization, and quantification methods, are described. The general principle of capillary electrophoresis (CE) is explained for a better understanding of these two methods. Chapter II describes the idea and concepts of Extracellular Vesicles quantitative Capillary Electrophoresis (EVqCE). The method evolved from the previous study carried out in our research group for the quantification of viruses. After the isolation of EVs from different cell lines, the characterization and quantification of EVs were performed using nanoparticle tracking analysis (NTA) and flow cytometry. EVqCE consists of four steps for EV quantification. In this study, EVqCE was employed to know the concentrations of EVs in unknown samples, followed by calculation of the average mass of the RNA present in EVs. In the next chapter, one of the human body fluids, i.e., saliva, was chosen for the quantification of EVs. Salivary EVqCE was developed in a similar way as EVqCE for cell lines since the general theories and procedures are practically the same. However, human saliva contains an abundant amount of viscous proteins and ribonuclease (RNase), that were the major obstacles for salivary EVs detection and quantification. The method for the isolation of EVs from the saliva was optimized, and the quantification was performed successfully. The average mass of RNA in saliva EVs was also calculated and analyzed. The concentration of saliva EVs in unknown samples were compared with the results from NTA and flow cytometry to validate the salivary EVqCE. In the last chapter, I described the application of EVqCE to study the quality control of EVs. The method calculates the degradation level of EVs samples under different conditions, providing a potential way for real-time monitoring of the EVs status in the body fluid sample.

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<p>  </p> <p>Non-small cell lung cancer (NSCLC), the most common type of lung cancer, is essentially the leading cause of cancer related deaths in United States. Only 24% of NSCLC patients survive 5-years post diagnosis, largely attributed to the lack of efficient treatment strategies at the metastatic stage. Thus, understanding the biological mechanisms that promote NSCLC metastasis is critical for the development of effective cancer-specific therapeutic agents. The development of cancer metastasis is greatly driven and influenced by intercellular communication. Key mediators of cell-to-cell communication are extracellular vesicles (EVs). For the past years, the study of EVs released by tumor cells have gained attention, given their impact in modulating the tumor immunity, supporting tumorigenesis, and contributing to the development of metastasis. However, the mechanisms though which tumor EVs contribute to tumor development are still understudied. In this study, we isolated and characterized small EVs, also referred as exosomes, from NSCLC cell lines (H358, Calu6, H460, SKMES-1) and investigated their release, uptake, and impact in non-tumorigenic lung epithelial cells recipient cells (BEAS-2B and HBEC). Our results demonstrated that EVs from NSCLC can induce migration and invasion of non-tumorigenic epithelial cells, and impair epithelial barrier permeability, suggesting their role in supporting tumorigenesis and metastasis. Furthermore, we assessed the immunomodulatory effects of NSCLC EVs on anti-tumor immune cells, particularly T cells. Our findings revealed a suppressive effect of EVs derived from mutant KRASG12C NSCLC (H358) on T-cell proliferation, suggesting their contribution to immune evasion mechanisms in mutant KRAS tumors. To dissect the underlying mechanisms, we employed a dual approach utilizing genetic manipulation (shRNA knockdown) and a small molecule inhibitor (ARS-1620) targeting the oncogenic KRASG12C. Our data demonstrated that targeting KRASG12C impaired the EV-driven cancer phenotypes, highlighting the pivotal role of KRAS oncogenic signaling in tumorigenesis and immune suppression mediated by EVs. Overall, our study sheds light on the crucial involvement of tumor derived EVs in NSCLC progression, both in terms of promoting cellular migration and invasion, as well as dampening anti-tumor immune responses. By elucidating the mechanisms underlying EV-driven tumorigenesis and highlighting the therapeutic potential of targeting KRAS signaling, our findings pave the way for the development of novel and effective therapeutic agents for NSCLC.</p>

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Yes / A series of ruthenium and iridium complexes have been synthesized and characterized with 20 novel crystal structures discussed. The library of β-ketoiminato complexes has been shown to be active against MCF-7 (human breast carcinoma), HT-29 (human colon carcinoma), A2780 (human ovarian carcinoma), and A2780cis (cisplatin-resistant human ovarian carcinoma) cell lines, with selected complexes’ being more than three times as active as cisplatin against the A2780cis cell line. Selected complexes were also tested against the noncancerous ARPE-19 (retinal pigment epithelial cells) cell line, in order to evaluate the complexes selectivity for cancer cells. Complexes have also been shown to be highly active under hypoxic conditions, with the activities of some complexes increasing with a decrease in O2 concentration. The enzyme thioredoxin reductase is overexpressed in cancer cells, and complexes reported herein have the advantage of inhibiting this enzyme, with IC50 values measured in the nanomolar range. The anticancer activity of these complexes was further investigated to determine whether activity is due to effects on cellular growth or cell survival. The complexes were found to induce significant levels of cancer cell death by apoptosis with levels induced correlating closely with activity in chemosensitivity studies. As a possible cause of cell death, the ability of the complexes to induce damage to cellular DNA was also assessed. The complexes failed to induce double-strand DNA breaks or DNA cross-linking but induced significant levels of single-strand DNA breaks, indicating a mechanism of action different from that of cisplatin. / Lord RM, Hebden AJ, Pask CM, Henderson IR, Allison SJ, Shepherd SL, Phillips RM, McGowan PC

Mathematical models, that depict the dynamics of a cancer cell population growing out of the human body (in vitro) in unconstrained microenvironment conditions, are considered in this thesis. Cancer cells in vitro grow and divide much faster than cancer cells in the human body, therefore, the effects of various cancer treatments applied to them can be identified much faster. These cell populations, when not exposed to any cancer treatment, exhibit exponential growth that we refer to as the balanced exponential growth (BEG) state. This observation has led to several effective methods of estimating parameters that thereafter are not required to be determined experimentally. We present derivation of the age-structured model and its theoretical analysis of the existence of the solution. Furthermore, we have obtained the condition for BEG existence using the Perron-Frobenius theorem. A mathematical description of the cell-cycle control is shown for one-compartment and two-compartment populations, where a compartment refers to a cell population consisting of cells that exhibit similar kinetic properties. We have incorporated into our mathematical model the required growing/aging times in each phase of the cell cycle for the biological viability. Moreover, we have derived analytical formulae for vital parameters in cancer research, such as population doubling time, the average cell-cycle age, and the average removal age from all phases, which we argue is the average cell-cycle time of the population. An estimate of the average cell-cycle time is of a particular interest for biologists and clinicians, and for patient survival prognoses as it is considered that short cell-cycle times correlate with poor survival prognoses for patients. Applications of our mathematical model to experimental data have been shown. First, we have derived algebraic expressions to determine the population doubling time from single experimental observation as an alternative to empirically constructed growth curve. This result is applicable to various types of cancer cell lines. One option to extend this model would be to derive the cell cycle time from a single experimental measurement. Second, we have applied our mathematical model to interpret and derive dynamic-depicting parameters of five melanoma cell lines exposed to radiotherapy. The mathematical result suggests there are shortcomings in the experimental methods and provides an insight into the cancer cell population dynamics during post radiotherapy. Finally, a mathematical model depicting a theoretical cancer cell population that comprises two sub-populations with different kinetic properties is presented to describe the transition of a primary culture to a cell line cell population.

Mitochondrial mutations and heteroplasmy have been associated with disease states that result from inadequate cellular energy production. As mitochondrial DNA (mtDNA) encodes many of the polypeptides involved in oxidative phosphorylation (OXPHOS), mtDNA mutations may lower energy production which is required for cell division and sustained ATP synthesis. In order to test the relationship between mtDNA mutations and the rate of cell division, a mammary epithelial cancer cell line, MCF-7, is used as a model. Nine proliferate single cell clones have been isolated from MCF-7. Population doubling times of six single cell clones and the MCF-7 stock have been determined. Clones with distinctly different growth rates were selected for mutational analysis. Growth rates of these clones appeared to be different from each other. Using polymerase chain reaction (PCR) and DNA sequencing, three cases of heteroplasmy have been identified in the mitochondrial genes of the MCF-7 stock and four single cell clones (ATPase C9119T, ND6 T14300G, Cytb G15807A). Heteroplasmy present in the Cytb gene is differs between single cell clones. Differences between the growth rates may be indicative of metabolic variations in these single cell clones. The OXPHOS enzymes encoded by the mutated genes were quantified by standard enzymatic assays. The assays demonstrated significant differences in specific activity between the clones, but were not correlated with mitochondrial heteroplasmy. This thesis determines that the differences in specific activity observed between clones is of nuclear origin.