The purpose of this research was to study the hybridization of molecular beacons under different conditions and designs. Data collected suggest that the inconsistency found in the emission intensity of several of these probes may be caused by 3 important factors: length of the probe, nucleotide sequence and, the formation of an alternative complex structure such as a dimer. Of all three factors, dimer formation is the most troublesome, since it reduces the emission of the reporter molecules. A new probe design was used to reduce dimer formation. The emission signal of the improved probe was several folds stronger than those probes with the early design. In this research, dimer formation is detected, furthermore a new probe with a different design was tested. If dimer formation can be reduced molecular beacons can be integrated into more complex hybridization systems providing an important tool in research and diagnosis of genetic disorders.

Thesis advisor: Torsten Fiebig / Thesis advisor: Mary Roberts / Understanding the electronic nature of DNA is profound and has been attempted for decades. Photoexcitation of DNA with UV light deposits electronic energy in the base stack and prepares highly reactive excited states. These states are precursors for photoinduced damage reactions which can lead to mutations and ultimately to cell death. While many DNA photo products have been isolated and characterized, the primary events immediately after photon absorption are not yet understood. Recent studies with ultrafast lasers have revealed that the majority of excess energy gained by DNA with light absorbance is dissipated on the femtosecond and picosecond time scales. In this study double-stranded oligonucleotides with different base sequences, content and lengths were systematically examined using femtosecond pump-probe spectroscopy. The results indicate that excitations in DNA are delocalized over more than two bases and the extent of the delocalization depends strongly on the structure of the investigated systems. Exciton delocalization domains in the longer duplexes are larger than in the shorter ones. Also, single-stranded oligonucleotides show smaller extent of exciton delocalization than duplexes with the same length. In addition to the fundamental studies on DNA photophysics, the properties and the structure of new molecular beacons based on thiazole orange dimers were studied. A full account of the optical and structural properties of the dimers in different base environments and orientations is presented here. Currently, the development of efficient ways to utilizing solar energy is at the forefront of the scientific community due to the ever rising demand for energy. Both, colloidal semiconductor nanocrystals and single-walled carbon nanotubes are potential alternatives to conventional inorganic and organic materials in photovoltaic devices Thorough understanding of the charge transfer and related photophysical phenomena in these systems will answer the question whether these nanomaterials can be applied in future generations of solar cells. The photoinduced electron transfer in donor-acceptor CdSe/CdTe heterostructured nanorods, in which CdTe is grown on top of CdSe in a single rod structure, was studied. The electron transfer between the two nanocrystals occurs on the subpicosecond time scale, competing with the ultrafast relaxation mechanisms in the quantum confined nanocrystals. Furthermore, investigations on how quantum confinement influences the phonon wavepackets in semiconductor nanocrystals were carried out. Quantum beats corresponding to longitudinal optical phonon modes were observed in the femtosecond pump-probe spectra of colloidal CdTe nanocrystals. Size-dependent experiments revealed that the optical phonon frequencies and the exciton-phonon coupling strength do not depend on the crystal's size. Only the wavepacket dephasing time was influenced by the diameter of the particles which was correlated with the hole relaxation to the exciton band edge. Electron donor-acceptor constructs, based on single-walled carbon nanotubes (SWNT), can be attained by noncovalent functionalization of the nanotubes with pyrene derivatives. However, charge transfer does not take place in the simplest pyrene-SWNT constructs. For the first time the pure SWNT-pyrene construct was isolated and investigated. Our results revealed that the optical properties of pyrene are drastically altered due to strong electronic interactions with the SWNT surface. In other words, aromatic molecules lose their electronic (and chemical) signature when non-covalently attached to carbon nanotubes. / Thesis (PhD) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Human pluripotent stem cells (hPSCs) hold the potential to revolutionize cardiac tissue engineering. Because of their ability to proliferate and differentiate into all cardiomyocyte subtypes they represent an opportunity to regenerate virtually any tissue lost from the over 1 million cardiac disease patients in the United States alone. Studies have shown, however, that hPSCs which are not terminally differentiated pose a variety of risks including teratoma formation and lack of appropriate cell engraftment. It is therefore important to ensure that only well characterized cardiac subtypes are implanted into patients or used for research purposes. Current differentiation protocols generate a mixture of cardiac subtypes, and research on cardiac subtype specification is hampered by the lack of a high throughput method to distinguish cardiac subtypes. This thesis establishes the ability to identify, enrich and characterize cardiac subtypes using MBs. This will provide a robust tool for clinical use of hPSCs in cardiac cell therapy and for analysis of differentiation protocol effects on cardiac subtype formation.

Philosophiae Doctor - PhD / Breast cancer is one of the major causes of death in South Africa. About 1 in 29 South African women are at risk of developing this type of cancer in their lifetime. The global incidence of breast cancer also increases annually with over 1 million new cases diagnosed every year. Molecular diagnostic techniques such as qRT-PCR, Fluorescent In Situ Hybridization (FISH), Immunohistochemistry (IHC) and ELISA are used to diagnose breast cancer. Some of these diagnostic techniques make use organic fluorophores as fluorescent reporter molecules. The principle of all these diagnostic techniques is reliant on the detection of molecular biomarkers that are associated with the disease. In most cases these molecular biomarkers are DNA, RNA or proteins that are up-regulated in response to or as a result of the disease. The first aim of this study was therefore to identify membrane proteins that are up-regulated in cancers that can potentially be used as biomarkers for the detection of breast cancer. The second aim of this study was to investigate the application of quantum dots in the development of a molecular diagnostic test that can detect a breast cancer biomarker. The most commonly used method to identify molecular biomarkers for diseases have traditionally been gene expression analysis using technologies such as DNA microarray. These technologies have certain limitations and have therefore not been very successful in identifying useful disease biomarkers. Biomarker II discovery by proteomics can overcome some of these limitations and is potentially a more suitable method to identify molecular biomarkers for breast cancer. In this study proteomics in combination with Stable Isotope Labelling with Amino Acids in Cell Culture SILAC was used to do a comparative analysis of the expression levels of membrane proteins present in a human breast cancer cell line (MCF-7) derived from a breast cancer patient and a human breast cell line (MCF- 12A) derived from a healthy individual. This led to the identification of the transmembrane protein, GFRA1 as potential new biomarker for breast cancer. This study showed that this protein is over expressed in MCF-7 cells as compared to MCF-12A cells and that it is also highly expressed in the myoepthelial cells of the milk ducts of breast cancer patients. This study also demonstrates the use of molecular beacon technology to develop a DNA probe for the detection of cDNA encoding the CK19 gene, which is a known biomarker for breast cancer. In the development of this probe, quantum dots were used as the fluorescence reporter. This molecular beacon probe was able to demonstrate the over expression of CK19 in MCF-7 cells. This study shows that this technology can potentially be used as a diagnostic test for breast cancer and since quantum dots are used in the development of these molecular beacon probes, this diagnostic test can potentially facilitate the development of multiplex detection systems for the diagnosis of breast cancer. Molecular beacon technology can potentially also be used to detect novel biomarkers such as GFRA1.

Terminaison de la transcription. Rho se fixe aux transcrits naissants au niveau d’un site Rut (Rhoutilization) libre à partir duquel il transloque le long de l’ARN (5’→3’) de façon ATP-dépendante pour rattraper le complexe d’élongation de la transcription et induire la dissociation de celui-ci. Il est généralement admis que les sites de fixation de Rho présentent une richesse en Cytosines et une pauvreté en Guanines, ainsi qu’une relative pauvreté en structures secondaires. Les études génomiques ou transcriptomiques n’ont pas dégagé d’éléments consensus ou de règles permettant de prédire les sites de terminaison Rho-dépendants. En combinant approches biochimiques et bioinformatiques, j’ai tenté de comprendre les mécanismes par lesquels Rho reconnait les transcrits.J’ai identifié un ensemble de déterminants de séquence qui, pris ensemble, possèdent un bon pouvoir prédictif et que j’ai utilisé pour construire le premier modèle computationnel capable de prédire la terminaison Rho-dépendante à l’échelle des génomes d’E. coli et Salmonella. J’ai caractérisé in vitro certains de ces terminateurs, en particulier dans les régions 5’UTR, avec l’espoir qu’ils soient impliqués dans des mécanismes de régulation conditionnelle. J’ai identifié des candidats dont l’activité pourrait être sous le contrôle de facteurs comme des petits ARN non codants (sRNA) ou latempérature. J’ai également développé une méthode fluorogénique pour détecter facilement la terminaison Rho-dépendante in vitro et ai commencé à adapter l’approche CLIP-seq à l’étude du transcriptome Rho-dépendant chez Salmonella. Collectivement, mes travaux offrent de nouveaux outils d’analyse et de prédiction de la terminaison Rho-dépendante, une meilleure cartographie des sites d’action de Rho chez E. coli et Salmonella, ainsi que de nouvelles pistes d’étude du rôle de Rhodans l’expression conditionnelle du génome. / Transcripts at a free Rut (Rho-utilization) site from which Rho moves along the RNA in an ATP dependentfashion to catch up with and dissociate the transcription elongation complex. It is generally believed that the Rut sites are, respectively, rich and poor in Cytosines and Guanines as well as relatively poor in secondary structures. Studies at the genomic or transcriptomic scale have notrevealed any stronger consensus features or rules for predicting potential Rho-dependent termination sites. By combining biochemical and bioinformatics approaches, I have explored the mechanisms by which Rho recognizes transcripts to induce transcription termination. I have identified a complex set of sequence determinants which, taken together, have good predictive power and which I used to build the first computational model able to predict Rho-dependent termination at the scale of Escherichiacoli and Salmonella genomes. I have characterized in vitro some of these terminators, particularly in 5'UTRs, with the hope that they will be involved in conditional regulatory mechanisms. I have identified several candidates whose activity may be under the control of factors such as small non-coding RNAs(sRNA) or temperature. I have also developed a fluorogenic method to easily detect Rho-dependent termination in vitro and have begun to adapt the CLIP-seq approach to the study of the Rhodependent transcriptome in Salmonella. Collectively, my work offers new tools for the analysis and prediction of Rho-dependent termination, a better mapping of the sites of probable Rho action in E.coli and Salmonella, as well as several lines of investigation of the role of Rho in the conditional expression of bacterial genomes.

This thesis discusses developing new tools to probe DNA damage resulting from photoinduced chemical processes and probing the initial excited-state structural dynamics of nucleic acids and sunscreen agents. The results of this thesis show that UV resonance Raman spectroscopy (UVRRS) is an information-rich probe of chemical compositions in in situ analysis of sunscreen formulations. The results indicate that 244-nm excited UV resonance Raman spectra can provide a limit of detection of 0.23% w/w of sunscreen active ingredients (AIs), far below typical active ingredient (AI) concentrations in sunscreen formulations. UVRRS was used to probe the photostability of sunscreens, by determining the initial excited-state structural dynamics of Benzophenone-3 (BZ3), a common sunscreen active ingredient. Only minor excited-state structural distortions were obtained, based on the low displacement values, suggesting an inherently stable molecule. The largest structural change occurs along the carbonyl stretch, suggesting a strong interaction with the methanol solvent and a dissipative decay path for the molecule. Similarly, to gain an insight into how the structure of nucleic acids determine their photochemistry, UVRRS was used to probe the initial excited-state structural dynamics of 9-methyladenine (9-MeA). As for BZ3, the initial excited-state structural dynamics obtained for 9-MeA, are low, and much lower than for pyrimidine bases, providing a strong evidence for the photochemical stability of this purine base analog, compared to pyrimidines. Since DNA does not exist in isolation in cells, the role of a UV-absorbing amino acid, tryptophan, in UV-induced DNA photodamage was explored using molecular beacons (MBs) to detect the damage. The results indicate that tryptophan protective effects on DNA far outweighs their photodamage potential. However, MBs are expensive and they have some technical limitations, despite their exquisite sensitivity. Therefore, a new DNA damage detection method was developed based on smart probes (SPs). These probes are shown to be sensitive and sequence specific for detecting DNA photodamage.

Osseointegration, or the direct integration of an implant into bone tissue, is necessary for implant success. Titanium is commonly used clinically in dental and orthopaedic implants because of its passivating oxide layer, which facilitates osseointegration, and its mechanical properties such as a modulus of elasticity similar to bone. Diverse studies have shown that surface microtopography, chemistry, and surface energy affect osteoblast behavior. The problem with these studies is that they access the average behavior of a culture in response to a substrate and not the behavior of individual cells. The objective of this study was to develop a method for correlating the behavior of individual cells with the characteristics of the surface underneath them. More specifically, this work developed a method to correlate integrin beta-1 (β1) expression with the surface characteristics under individual cells. Integrins are cell surface receptors that bind to specific proteins in the extracellular matrix adsorbed on the implant surface. Previous work has shown that expression of certain integrins is increased when osteoblasts on titanium substrates develop a more differentiated phenotype, and that integrin β1 is necessary for osteoblast response to roughness on titanium substrates. This study used molecular beacons specific to integrin β1 to quantify integrin β1 expression of MG63 cells cultured on titanium disks. A template was designed to coordinate the location of cells using fluorescence microscopy and scanning electron microscopy (SEM) in reference to laser etchings on the disks. After live cell imaging, cells were fixed, dried, and critical point dried for focused ion beam (FIB) milling. Transmission electron microscopy (TEM) sections of cells identified with high and low integrin β1 molecular beacon intensity were milled, and cells with high and low integrin β1 molecular beacon intensity were also serial sectioned. While our TEM results were inconclusive, SEM images from serial sectioning showed contact points between the cell body and the substrate, consistent with previous results. Cells cultured on pretreatment (PT) or sandblasted acid etched (SLA) titanium surfaces were also serial sectioned, showing that cells on SLA surfaces have more regions of contact between the cells and the substrate than cells on PT surfaces. This work is significant as it is the first study to develop a method to correlate individual cell behavior with the substrate surface characteristics under the individual cells. Previous studies have reported the average cell behavior in response to their substrates, while this work allows for the study of substrate surface characteristics that positively affect integrin β1 expression in individual cells. Further optimization of the fluorescence imaging process and FIB milling process could be done, and the method developed in this study could be used in future studies to investigate surface characteristics after using other fluorescent analyses of cell behavior, such as immunocytochemistry.

No description available.

Messenger RNA (mRNA) posttranscriptional regulation has been implicated in the development and/or progression of several diseases including many types of cancer, rheumatoid arthritis, vascular disease, and Alzheimer's disease. Differential regulation of Androgen Receptor (AR) mRNA has been associated specifically with prostate cancer progression. In this thesis, molecular beacons were developed to allow for the detection of the expression and localization of AR mRNA in live prostate cancer cells. These beacons were then applied as a tool for studying how AR mRNA regulation is involved in prostate cancer growth and advancement. Two AR mRNA targeted beacons were designed and tested in solution and in live cells to determine their functionality. The beacon-based approach for AR mRNA detection was then optimized through the use of the two beacons in tandem and alteration of their backbone chemistry. A series of validation tests were performed on these beacons, including testing their abilities to: 1) produce a feasible localization pattern, 2) discriminate between AR positive (AR+) and AR negative (AR-) prostate cancer cell lines and 3) follow stimulus-induced changes in AR mRNA expression. Based on these results, a dual chimeric beacon approach was selected to determine the role of AR mRNA regulation in two systems that represent important stages in prostate cancer growth and progression: 1) hormone stimulation of androgen-dependent prostate cancer cells and 2) progression of androgen-dependent prostate cancer cells to the androgen-independent state. Our results suggest that changes in AR mRNA expression, organization, and localization may be indicative of molecular mechanisms involved in these critical transitions associated with prostate cancer progression. Taken together, this work provides a feasibility study for visualizing changes in AR mRNA state as a diagnostic measure for evaluating the aggressiveness of the disease and demonstrates the possible utility of therapeutically targeting AR mRNA regulation in order to prevent prostate cancer advancement.

Numerous troponin T (TnT) isoforms are generated by alternative RNA splicing primarily in its NH2-terminal hypervariable region, but the functions of these isoforms are not completely understood. In this dissertation work, calcium and terbium binding behavior of several forms of TnT were investigated by spectroscopic and radioactive techniques. Chicken breast muscle TnT binds calcium and terbium through its NH2-terminal Tx motif (HEEAH)n with high affinity (10-6 mM) and fast on-rate (106 - 107 M-1 s-1). Chicken leg muscle TnT and a human cardiac TnT NH2-terminal fragment, which both lack the Tx motif on their NH2-terminal regions, do not have affinities for calcium in the physiological range. Computational predictions on TnT N47 suggest that the TnT NH2-terminal region might fold into an elongated structure with at least one high affinity metal ion binding pocket comprised primarily of the Tx motif sequence and several lower affinity binding sites. In addition, calcium binding to TnT N47 might alter its conformation and flexibility. Luminescence resonance energy transfer measurements and other experimental observations are consistent with the computational predictions suggesting the computational simulated atomic model is reasonable. TnT mutations are responsible for 15% of familiar hypertrophic cardiomyopathy (FHC) cases with a phenotype of relatively mild hypertrophy, but a high incidence of sudden death. Detection of those genetic mutations would facilitate the clinical diagnosis and initiation of treatment at an early stage. This dissertation also investigated a novel hybridization proximity assay (HYPA) combining molecular beacon and luminescence resonance energy transfer (LRET) technologies. Experimental results suggest that a shared stem probe design produces a more consistent response upon hybridization, whereas the internally labeled probe was less consistent, but can yield the highest responses. Using the optimally designed molecular probes, the HYPA provides a detection of alterations in nucleic acid structure of as little as a single nucleotide. This novel HYPA is expected to expand its applications in the analysis and screening of genetic diseases.