abstract: The bleomycins are a family of glycopeptide-derived antibiotics isolated from various Streptomyces species and have been the subject of much attention from the scientific community as a consequence of their antitumor activity. Bleomycin clinically and is an integral part of a number of combination chemotherapy regimens. It has previously been shown that bleomycin has the ability to selectively target tumor cells over their non-malignant counterparts. Pyrimidoblamic acid, the N-terminal metal ion binding domain of bleomycin is known to be the moiety that is responsible for O2 activation and the subsequent chemistry leading to DNA strand scission and overall antitumor activity. Chapter 1 describes bleomycin and related DNA targeting antitumor agents as well as the specific structural domains of bleomycin. Various structural analogues of pyrimidoblamic acid were synthesized and subsequently incorporated into their corresponding full deglycoBLM A6 derivatives by utilizing a solid support. Their activity was measured using a pSP64 DNA plasmid relaxation assay and is summarized in Chapter 2. The specifics of bleomycin—DNA interaction and kinetics were studied via surface plasmon resonance and are presented in Chapter 3. By utilizing carefully selected 64-nucleotide DNA hairpins with variable 16-mer regions whose sequences showed strong binding in past selection studies, a kinetic profile was obtained for several BLMs for the first time since bleomycin was discovered in 1966. The disaccharide moiety of bleomycin has been previously shown to be a specific tumor cell targeting element comprised of L-gulose-D-mannose, especially between MCF-7 (breast cancer cells) and MCF-10A ("normal" breast cells). This phenomenon was further investigated via fluorescence microscopy using multiple cancerous cell lines with matched "normal" counterparts and is fully described in Chapter 4. / Dissertation/Thesis / Ph.D. Chemistry 2013

No description available.

This thesis presents the development and application of nanoscale techniques to trap and / Chemistry and Chemical Biology

Disruptions in the response of endothelial progenitor cells to changes in oxygen environment may present a possible mechanism behind multiple pediatric pulmonary disease models, such as bronchopulmonary dysplasia. Using high-throughput fixed single-cell protein and RNA imaging, we have created "stop-motion" movies of Thymosin. 4 (T beta 4) and Hypoxia Inducible Factor 1 alpha (HIF-1 alpha) protein expression and vascular endothelial growth factor (vegf) and endothelial nitric oxide synthase (eNOS) mRNA in human umbilical cord-derived endothelial colony-forming cells (ECFC). ECFC were grown in vitro under both room air and hyperoxia (50% O-2). We find elevated basal T beta 4 protein expression in ECFC derived from prematurely born infants versus full term infants. T beta 4 is a potent growth hormone that additionally acts as an actin sequestration protein and regulates the stability of HIF-1 alpha. This basal level increase of T beta 4 is associated with lower HIF1 alpha nuclear localization in preterm versus term ECFC upon exposure to hyperoxia. We find altered expression in the pro-angiogenic genes vegf and eNOS, two genes that HIF-1 alpha acts as a transcription factor for. This provides a potential link between a developmentally regulated protein and previously observed impaired function of preterm ECFC in response to hyperoxia.

No description available.

Nanopore sensors are emerging as a promising technology for single molecule analysis and polymer sequencing. Traditionally, measurements are taken by monitoring the ionic current through the nanopore, which gives information (e.g. size, shape, charge) about a molecule of interest while it is in the confined geometry of the nanopore. The dynamics of the molecule before the arrival to the nanopore, such as the capture dynamics, or molecular conformation prior to translocation, as well as clogging mechanisms and features of anomalous translocation events, are not assessed by the electrical measurements alone. To study the whole process of nanopore diffusion, capture and passage it is necessary to complement the electrical signal with another detection mode. Particularly, optical visualization of the molecules as they translocate through the nanopore has great potential. In this Thesis I present the design, construction, optimization and testing of a nanopore--‐based optofluidic instrument, which uses fluorescence microscopy to visualize individual fluorescently stained DNA molecules as they translocate a solid--‐state nanopore, while in parallel record the ionic current signal through the pore. The following challenges were overcome to achieve the integration of the optical and electrical systems: (i) the electrical detection system must account for the physical constrains of a wide field fluorescence microscope, and the optical system should in turn not affect the low--‐noise electrical detection of individual DNA molecules. The design of the instrument included a microfluidic device, so to position the nanopore within the working distance (<170--‐μm) of the microscope objective (Chapter 2). (ii) Electrical noise was optimized to a level that is indistinguishable from a standard (with no optics) nanopore system (Chapter 3). The custom instrument was used to demonstrate: 1) Electrical detection of DNA translocations with a laser light illuminating the nanopore; 2) Optical tracking of DNA capture and translocation dynamics; 3) Synchronization of the optical and electrical signals in preparation for simultaneous detection. In the process of noise optimization, a strong noise coupling between the illumination source and the ionic current was found, characterized and eliminated. Consequently, the noise performance of the custom instrument is the lowest of any other nanopore--‐based optofluidic systems described in the literature to date. This opens up the way to many new and exciting investigations of polymer translocation dynamics through nanoconfined geometries. Lastly, during the development of this custom instrument, a method to localize the fabrication of a nanopore by controlled dielectric breakdown on a membrane, with a focused laser beam, was discovered.

Nanoscopic spatial reorganization of selectin ligands, CD44 and PSGL-1, during the initial step of hematopoietic stem/progenitor cell (HSPC) homing, tethering and rolling of migrating cells over E-selectins, has been recently reported. However, the exact spatial distribution of these ligands and their spatial reorganization during the cell rolling on E-selectins are still an open question. The spatiotemporal characterization at the nanoscale level requires high resolution imaging methods. In this study, I quantitatively characterize nanoscopic spatiotemporal behavior of the selectin ligands on the migrating cells to understanding the molecular mechanism of the cell rolling at the nanoscale level by means of a microfluidics-based 3D super-resolution fluorescence microscopy technique. The obtained results suggest that PSGL-1 on the cell shows significant change in the axial distribution on the cell during the cell rolling on E-selectin whereas the spatial distribution of CD44 along the axial direction is not affected significantly by the cell rolling. These findings indicate that each selectin ligand has a distinct contribution to the initial step of the HSPC homing because of their distinct spatial localizations on the cells that regulate at least partly the accessibility of these ligands to the surface E-selectin.

Spatial and temporal dynamics of polymer chains play critical roles in their rheological properties, which have a significant influence on polymer processing and fabrication of polymer-based (nano) materials. Many theoretical and experimental studies have aimed at understanding polymer dynamics at the molecular level that give rise to its bulk phase properties. While much progress has been made in the field over the past ~60 years, many aspects of polymers are still not understood, especially in complicated systems such as entangled fluids and polymers of different topologies. In addition, the physical properties of biological macromolecules, i.e. DNA, are expected to affect the spatial organization of chromosome in a cell, which has the potential impact on a broad epigenetics research. Here, we propose new methods for simultaneous visualization of diffusive motion and conformational dynamics of individual polymer chains, two most important factors that characterize polymer dynamics, based on a new single-molecule tracking technique, cumulative-area (CA) tracking method. We demonstrate the applicability of the CA tracking to the quantitative characterization of the motion and relaxation of individual topological polymer molecules under entangled conditions, which is possible only by using the newly-developed CA tracking, using fluorescently-labeled linear and cyclic dsDNA as model systems. We further extend the technique to multi-color CA tracking that allows for the direct visualization and characterization of motion and conformation of interacting molecules. We also develop a new imaging method based on recently developed 3D super-resolution fluorescence microscopy technique, which allows direct visualization of nanoscale motion and conformation of the single molecules that is not possible by any other methods. Using these techniques, we investigate spatial and temporal dynamics of polymers at the single-molecule level, with special emphasis on the effect of topological forms of the molecules and the confined geometry on their spatiotemporal dynamics. Our results demonstrate that the new methods developed in this thesis provide an experimental platform to address key questions in the entangled topological polymer dynamics. The research will provide a platform for developing new polymer-based materials and open the possibility of studying spatial organization of DNA in a confined geometry from physics point of view.

This thesis focuses on autofluorescence of flavins in gram-negative bacteria Cupriavidus necator H16 and its mutant strain PHB-4. The main methods used were fluorescence microscopy and flow cytometry. To confirm the presence of flavins, excitation and emission spectra of the bacterial suspension were measured, which were compared with flavin standards. In the part of testing cells without stress response, the autofluorescence of bacteria in PBS buffer and cell suspensions stained with fluorescence probe BODIPY 493/503 was measured. The ratio of short fluorescence lifetime to long autofluorescence lifetime, and its dependence on fluorescence probe was compared with previous conditions. Autofluorescence of the supernatant was measured; it was found that the relative amplitude of long lifetime was multiple times higher than in the cell. In the part devoted to the stress response, this thesis was focused on the amount of dissolved oxygen in the production medium and the effect on bacterial autofluorescence. Then differently concentrated hydrogen peroxide was used, the best results were obtained from the concentration of 100 mM in media. For comparison a combination of hydrogen peroxide with ferro-ammonium sulphate was used, but there was no big difference. Sodium azide and antimycin A were selected as substances that directly influence on bacterial respiratory chain. Both compounds affected change in the ratio of the relative amplitudes, but the distribution of these lifetimes and the autofluorescence change over time was affected only by sodium azide.

Práca je zameraná na navrhnutie vhodnej metódy analýzy smerovosti neuritov. Využité boli snímky neurónov z fluorescenčnej mikroskopie. Pred samotnou segmentáciou bolo potrebné snímky predspracovať, pričom sa postupne využila úprava kontrastu, ostrenie a adaptívna filtrácia pomocou Weinerovského filtru. Jednotlivé návrhy metód segmentácie pozostávali z prostého prahovania, narastaním oblastí a využitím morfologických operácií. Následná analýza smerovosti využívala smer gradientov v obraze. Navrhnutá metóda bola využitá aj ako klasifikátor, ktorý dokázal rozdeliť jednotlivé snímky do skupín podľa smeru rastu.