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A hallmark of oncogenic transformation is disruption of the actin cytoskeleton, which is partly attributed to altered expression and/or activity of proteins that bind to and regulate the state of polymerization of actin in cells. Profilin-1 (Pfn1) is a key regulator of actin polymerization that is downregulated in human breast cancer. Previous studies have also shown Pfn1 has a tumor-suppressive effect on triple-negative breast cancer (TNBC) cells with mesenchymal features, and Pfn1-induced growth suppression of TNBC cells is partly mediated by upregulation of cell-cycle inhibitor p27kip1 (p27). The aim of the first part of this dissertation was to elucidate the molecular mechanism of how Pfn1 elevation causes p27 accumulation in TNBC cells. It is demonstrated that Pfn1 overexpression leads to accumulation of p27 through promoting activation of AMP-activated kinase (AMPK) and AMPK-dependent phosphorylation of p27 on T198 residue (a post-translational modification that leads to increased protein stabilization of p27) secondary to a cadherin-mediated epithelial morphological reversion. These findings not only elucidate a potential mechanism of how Pfn1 may inhibit proliferation of mesenchymal TNBC cells, but also highlight a novel pathway of cadherin-mediated p27 induction and therefore cell-cycle control in cells. The aim of the second part of this dissertation was to investigate whether Pfn1 is a classic tumor-suppressive protein. Matrigel-based 3D-outgrowth assays that measure tumor-initiating capacity of breast cancer cells demonstrated that either depletion or elevation of Pfn1 expression impairs formation of filopodia-like structures (a feature that determines dormant vs proliferative phenotype) and outgrowth of TNBC cells suggesting that a balanced Pfn1 expression is most conducive for tumor-initiating capacity of TNBC cells. Furthermore, perturbing Pfn1 expression affects expression of stem cell-associated genes thereby impacting the overall stem-like phenotype of TNBC cells. Finally, the outgrowth-deficient phenotype of TNBC upon Pfn1-depletion can be rescued by modulating ECM component which suggests that alteration in cell-matrix adhesion and downstream signaling underlies the growth-related phenotypic changes induced by loss of Pfn1. Based on these results, we conclude that Pfn1 expression level is an important determinant for stemness and outgrowth potential of TNBC cells.

Mechanical forces and torques play an important role in many biological processes ranging from gene transcription to protein conformational dynamics, and intracellular transportation. A range of spectroscopy techniques have been developed for direct measurements of force and torque applied to biomolecules, and fluorescence microscopy have been used extensively to study the dynamics of biomolecules and their interactions. However, to apply both force and torque simultaneously in electromagnetic tweezers, with two components decoupled and controlled independently, has not been accomplished. Also, the combination of torque spectroscopy with fluorescence microscopy has not been explored. The goal of this dissertation was to develop a novel magnetic trapping platform that is capable of simultaneously exerting and monitoring controlled forces and torques on single biomolecules via micrometer-sized magnetic particles while recording fluorescence images with high spatial and temporal resolution. Towards this goal, a system using electromagnets composed of a monopole that generates force, and a set of quadrupoles that generate torque, was integrated into an optical microscope. With this system, it is possible to apply tension in the piconewton force range to single molecules, and to independently modulate the trap torsional stiffness to twist molecules. Secondly, a novel method has been developed to directly track the angular displacement of optically anisotropic particles undergoing rotational motion using optoelectronic detectors. This method makes image acquisition and processing procedures commonly used in previous studies for detecting rotational motion unnecessary, and, when combined with the electromagnetic tweezers, can extend the bandwidth of control and detection of mechanical states of single molecules to the kilohertz regime. Finally, a dual-color, bifocal imaging system was implemented to allow detection of spectrally and spatially distinct fluorescent particles using a single camera with sub-pixel registration between two separate channels, and low mechanical drift. In conclusion, the advancement in the force/torque spectroscopy and fluorescence imaging techniques presented here holds significant potential in biological investigations that require fast control and detection of mechanical states of molecules and their dynamic properties. This system can be applied to studies of DNA-protein interactions, DNA conformation dynamics, and their dependence on the mechanical states of the DNA molecules.

With the ability to disentangle electronic transitions that occur on different elements and local electronic structures, time-resolved extreme ultraviolet (XUV) spectroscopy has emerged as a powerful tool for studying ultrafast dynamics in condensed phase systems. In this dissertation, a visible-pump/XUV-probe transient absorption apparatus with femtosecond resolution was constructed to investigate the carrier relaxation dynamics in semiconductors after photo-excitation. This includes timescales for carrier thermalization by carrier-carrier and carrier-phonon scattering. The 30 – 72 eV photon energy coverage (17 – 40 nm wavelength) generated by a table-top XUV light source is suitable for probing the 3p-to-3d core level absorptions of various transition metal oxides (TMOs) with specificities to elements and oxidation states.In Chapter 1, a brief introduction to charge carrier dynamics in semiconductor-based materials is given. In addition, fundamentals of core-level spectroscopy and the high harmonic generation (HHG) process are also addressed in this introductory chapter. Specifications of the experimental apparatus that was constructed are summarized in Chapter 2, including the design concepts and characterization of performance. Chapter 3 presents the spectral tunability of the XUV pulses generated from a semi-infinite gas cell (SIGC), as well as the data acquisition procedures. Charge carrier relaxation dynamics in Co3O4 following the charge transfer excitation pathway at 400 nm are documented in Chapter 4. In Chapter 5, various visible pump wavelengths are used to excite Co3O4 and the differences in the carrier dynamics versus excitation wavelength are considered. After selectively photoexciting a Si/TiO2 heterojunction, the resulted electron transfer process is observed and reported in Chapter 6. The concluding remarks of the dissertation are made in Chapter 7, while several ongoing time-resolved experiments are addressed in the Appendix sections.