PTEN (phosphatase and tensin homolog deleted on chromosome ten) is a potent tumor suppressor. PTEN’s tumor suppressor action is rooted in its phosphatase function on the lipid substrate phosphatidylinositol-(3,4,5)-trisphosphate (PI(3,4,5)P3). PTEN’s enzymatic activity is specific for the third position of the inositol headgroup. PI(3,4,5)P3 is a second messenger that is a part of the PI3K-Akt pathway, and its dysregulation leads to constitutively activated AKT. The result of AKT activation is cell cycle progression, motility, cell growth, and proliferation, and consequently, overaction leads to neoplastic growth and tumorigenesis. PTEN antagonizes this pathway by regulating PI(3,4,5)P3 population through its phosphatase activity which produces the lipid PI(4,5)P2 (phosphatidylinositol-(4,5)-bisphosphate). A result of PTEN’s function is that its activity must be localized at the PM (plasma membrane) since this is where its substrate resides. Additionally, the mole percent of the phosphoinositide family of lipids is small. From highest percent composition to lowest the phosphoinositide species in the PM rank as PI(4,5)P2 (~2%), PI(4)P (~1%), and PI(3,4,5)P3 (~0.02%). For PTEN to turn over its substrate, it must first translocate from the cytosol to the PM and then search through the plasma membrane for this rare but high in demand lipid. This is at the center of the scarcity paradox. This work explores how PTEN may overcome this paradox by using its multiple lipid binding domains to interact with multiple lipid partners to efficiently localize it toward a region with a high probability of having PI(3,4,5)P3. This hypothesis is tested using two kinetic methodologies. First, we use pre- steady state stopped-flow spectrometry to determine the rates that govern PTEN-lipid binding. Second, we use single-molecule total internal reflectance fluorescence (smTIRF) microscopy to resolve the diffusion coefficients and dwell times of bound PTEN on SLBs supported lipid bilayers (SLBs). We test PTEN against various lipid compositions to determine how the bilayer structure in addition to the chemistry of the lipid influences the enzyme’s binding. These compositions include PI(4,5)P2, PI phosphatidylinositol (PI), phosphatidylserine (PS), PI(4,5)P2/PI and PI(4,5)P2/PS. In addition to this kinetic work, we will also present a novel model membrane platform that takes advantage of a microfluidic device to develop lateral lipid gradients in SLBs. This microfluidic platform, in the future, will allow for the investigation of the dynamic behavior of proteins interacting with lipids but with a bilayer that has a structure recapitulating polarized membranes like in chemotaxing cells.

Heparan sulfate (HS) is a cell-surface sulfated polysaccharide that binds to multiple proteins and has been implicated in cancer, viral infection and Alzheimer's disease. Due to the heterogeneity of HS, the structural requirements for protein binding are ill- defined. Chemical synthesis of structurally-defined HS oligosaccharides, which are tunable in terms of length, order of monosaccharides and sulfation pattern, is required for the investigation of HS-protein binding. Single molecule methods have been utilised in biophysics to study dynamic processes and can allow observation of rare events which would be 'averaged out' in ensemble measurements. Access to fluorescently labelled HS oligosaccharides should allow investigation of interactions with proteins at the single molecule level using methods such as single molecule FRET, providing a method complementary to NMR studies (ensemble) and X-ray crystallography (non-dynamic).This thesis presents the development of a method for the fluorescent labelling of a chemically synthesised HS disaccharide utilising a reducing-end amine tag. Analysis of the fluorescence properties of the labelled disaccharide at ensemble and single molecule level indicated no perturbation of the fluorophore when attached to the sugar. Fluorescence correlation spectroscopy measurements of the fluorescent HS disaccharide with the protein FGF-1 showed no binding, which is attributed to the low concentration (1 nM) of disaccharide required in the experiment. Additional work is presented in this thesis on the development of a method for atom-specific 13C labelling of HS oligosaccharides, which has been initiated by synthesis of a 13C labelled L-iduronate monosaccharide and a 13C labelled disaccharide. New strategies for the synthesis of HS oligosaccharides based on orthogonal thioglycoside-based glycosylations employing S-benzoxazolyl and S-thiazolyl donors have been investigated. Development of a chemoselective glycosylation strategy for HS oligosaccharide synthesis utilising a 'super-disarmed' [2.2.2] L-iduronic lactone is presented.

This dissertation will focus on the volatile compounds released upon the burning of incense which are numerous and varied. The first part of this dissertation is the gas chromatography-mass spectral (GC-MS) analysis of burning incense collected via solid phase microextraction (SPME) with the aim of developing a library of compounds found in incense as used in the Orthodox church. The second part of this dissertation has the aim of developing a method for forming oleic acid bilayer vesicle membranes and a fluorescence spectroscopy method by which the reactivities of these vesicles can be analyzed. These reactivities include permeability, fluidity, aggregation, and fusion of the membranes. One family of the volatile compounds found in incense are the terpenes and terpenoids. The reactivity of the terpenes and terpenoids found in incense will be analyzed using the oleic acid vesicles with the hypothesis that terpenes of the same structural groups will act similarly on oleic acid vesicle membranes and these reactivities can be related to mechanistic interactions.

Molecular assemblies capable of harvesting light and using the absorbed energy have attracted great interest in recent years because of their applicability in such domains as light emitting diodes, fluorescent labelling of biological molecules, and photonic devices. Nature has also developed in plants and photosynthetic bacteria several examples of photonic nanostructures which guide light over small distances and harvest light energy, using resonance energy transfer (RET). For some time, researchers have tried to mimic the spatial arrangements of high energy transfer efficiency found in Nature. Recent progress in the application, creation and manipulation of individual or small groups of molecules are opening new perspectives for further developments in this field. These recent advances are commonly considered to lie at the root of what is being called "Nanotechnology". Although the definitions of nanotechnology are diverse, it is commonly admitted that this new domain of Science draws ideas and concepts from disciplines including engineering, physics, chemistry, biology, mathematics and computer science. The central dogma of the “bottom up” version of nanotechnology is the notion of self-assembly, which is the spontaneous assembly of materials into predetermined ordered structures or complexes. Presented here is an example from a field of nanotechnology that utilizes self-assembly onto nano-patterned surfaces to generate nano-structured systems and devices. More precisely, in the present case we target photo-active devices based on Fluorescence Resonance Energy Transfer (FRET), taking inspiration from photosynthetic light harvesting systems, where concentric nanometric rings of chromophores funnel light energy to a reaction center. Here, we synthesize nano-patterned chromophore surfaces which are able to collect light energy over a large surface and funnel it in regions of ~100 nm size. Our results indicate that an efficient collection and transfer of light energy can be performed by properly nano-designed surfaces, which may have practical consequences for the fabrication of light-powered active nano-devices.

This thesis describes a fundamental study of the miscibility and the nature of the interface between components of core-shell polymer blends using the technique of Fluorescence Resonance Energy Transfer (FRET) coupled with data analysis that involves Monte-Carlo simulations. Our aim in this study was to develop a fundamental methodology to quantitatively determine the width of the interface between the two components in binary polymer blends. At the current state of the art, data analysis of FRET experiments requires translational symmetry. In the system under study, uniform core-shell structures satisfy this criterion. Thus, in this work our focus was directed toward the study of a blend system with a core-shell structure. For this FRET study, I have identified a number of potential donor and acceptor dye pairs that fluoresce in the visible range of the spectrum and can be chemically attached to polymers. Among them, I selected, as the donor and the acceptor, a pair of naphthalimide dyes that have not previously been used for FRET experiments. Model experiments showed that while the fluorescence decay profile of the donor chromophore was exponential in solution, it was not exponential in polystyrene (PS) or poly(methyl methacrylate) (PMMA) films. Thus, I carried out refinements to existing FRET theory to interpret the data generated by using these dyes. Also, I derived a new model to predict the fluorescence intensity of non-exponential decaying donor dyes in core-shell systems. I selected a model system composed of a PS core surrounded by a PMMA shell. The PS core particles were prepared by miniemulsion polymerization to obtain cross-linked PS particles with a narrow size distribution. Seeded emulsion polymerization under starved-fed condition was employed to synthesize monodisperse dye-labeled core-shell particles. The extent of miscibility and the nature of interface between the core and the shell polymers were retrieved from a combined study by Monte-Carlo simulations and analysis of the donor fluorescence intensity decays. Agreement between the retrieved interface thickness and the literature data on PS-PMMA validates the methodology developed here for the use of such donor dyes in FRET studies on polymer blends.

This thesis describes a fundamental study of the miscibility and the nature of the interface between components of core-shell polymer blends using the technique of Fluorescence Resonance Energy Transfer (FRET) coupled with data analysis that involves Monte-Carlo simulations. Our aim in this study was to develop a fundamental methodology to quantitatively determine the width of the interface between the two components in binary polymer blends. At the current state of the art, data analysis of FRET experiments requires translational symmetry. In the system under study, uniform core-shell structures satisfy this criterion. Thus, in this work our focus was directed toward the study of a blend system with a core-shell structure. For this FRET study, I have identified a number of potential donor and acceptor dye pairs that fluoresce in the visible range of the spectrum and can be chemically attached to polymers. Among them, I selected, as the donor and the acceptor, a pair of naphthalimide dyes that have not previously been used for FRET experiments. Model experiments showed that while the fluorescence decay profile of the donor chromophore was exponential in solution, it was not exponential in polystyrene (PS) or poly(methyl methacrylate) (PMMA) films. Thus, I carried out refinements to existing FRET theory to interpret the data generated by using these dyes. Also, I derived a new model to predict the fluorescence intensity of non-exponential decaying donor dyes in core-shell systems. I selected a model system composed of a PS core surrounded by a PMMA shell. The PS core particles were prepared by miniemulsion polymerization to obtain cross-linked PS particles with a narrow size distribution. Seeded emulsion polymerization under starved-fed condition was employed to synthesize monodisperse dye-labeled core-shell particles. The extent of miscibility and the nature of interface between the core and the shell polymers were retrieved from a combined study by Monte-Carlo simulations and analysis of the donor fluorescence intensity decays. Agreement between the retrieved interface thickness and the literature data on PS-PMMA validates the methodology developed here for the use of such donor dyes in FRET studies on polymer blends.

<p>Alzheimer's disease (AD) is a progressive neurodegenerative disease that affects over 5 million people in the United States alone. This number is predicted to triple to by the year 2050 due to both increasing life expectancies and the absence of disease-attenuating drugs. The etiology of AD remains unclear, and although there are multiple theories implicating everything from oxidative stress to protein misfolding, misregulated metal ions appear as a common thread in disease pathology. </p><p>Chelation therapy has shown some effectiveness in clinical trials, but to date, there are no FDA-approved metal chelators for the treatment of AD. One of the biggest problems with general chelators is their inability to differentiate between the metal ions involved in disease progression verses those involved in normal metabolic function. To address this problem, we have developed a prochelator approach whereby the prochelator (SWH) does not bind metals with significant biological affinity. However, once activated to the chelator (CP) via enzymatic hydrolysis, the molecule is able to bind copper and reduce its toxicity both in vitro and in a cellular model of Alzheimer's Disease. </p><p>Central to this strategy is the site-specificity provided by enzymatic activation of the prochelator. In our system, SWH to CP conversion is mediated by beta-secretase, an enzyme involved in A-beta generation. However, in order to render SWH capable of hydrolysis in cells, we modified the prochelator to contain a dihydrocholesterol membrane anchor attached via a polyethylene glycol linker. From this construct, we created beta-MAP, which is an SWH-based FRET probe to demonstrate beta-secretase-mediated conversion of SWH to CP. beta-MAP was also used to confirm the efficacy of a known beta-secretase inhibitor without the need to for mutated cells lines or expensive antibodies. beta;-MAP and the associated microscopy method represent a significant advancement to the currently available ELISA assays for beta-secretase activity.</p><p>While activation of the prochelator by an enzyme in cells is encouraging, non-specific hydrolysis of the peptide prevents significant accumulation of the chelator on the cell membrane. Furthermore, attachment of the polyethylene glycol and sterol units induce cell toxicity not seen with the native CP peptide. These drawbacks prevent the current prochelator from effectively protecting cells from AD conditions. Structural modifications to overcome these problems, including implementation of a new peptide sequence are planned for future experiments.</p> / Dissertation

Cell division requires accurate DNA replication and cells develop checkpoint mechanisms toensure the correct passage of the genetic material. Cells arrest by a checkpoint when DNAdamage is found. After the checkpoint is silenced, the cell cycle can be resumed. Polo-likekinase 1 (Plk1) and Aurora A kinase (AurA) are both important regulators for checkpointrecovery. The question how AurA is activated was studied by many researchers, but the exactmechanism stays unclear.We developed a new setup to study AurA activation during checkpoint recovery. Quantitativeimmunofluorescence of fixed cells as well as a FRET probe that monitors Plk1 activity intime-lapse filming were applied in this study as indirect readouts of Aurora A activation. Theresult suggests that a Plk1-AurA feedback loop exists during checkpoint recovery. It can alsobe concluded that the inhibition of Cdk1 reduces Plk1 and AurA activity during checkpointrecovery. We also investigated the effect of calcium interfering drugs on AurA activation butno conclusive result was obtained.

Fluorescence resonance energy transfer (FRET)-based biosensors have been designed to fluorometrically detect everything from proteolytic activity to receptor-ligand interactions and structural changes in proteins. While a wide variety of fluorophores have demonstrated effectiveness in FRET probes, several potential sensor components are particularly notable. Semiconductor quantum dots (QDs) are attractive FRET donors because they are rather bright, exhibit high quantum yields, and their nanoparticulate structure enables the attachment of multiple acceptor molecules. Fluorescent proteins (FPs) are also of particular interest for fluorescent biosensors because design elements necessary for signal transduction, probe assembly, and device delivery and localization for intracellular applications can all be genetically incorporated into the FP polypeptide. The studies described in this thesis elucidate the important parameters for concerted QD-FP FRET probe design. Experimental results clarify issues of FRET pair selection, probe assembly, and donor-acceptor distance for the multivalent systems. Various analysis approaches are compared and guidelines asserted based on the results. To demonstrate the effectiveness of the QD-FP FRET probe platform, a ratiometric pH sensor is presented. The sensor, which uses the intrinsic pH-sensitivity of the FP mOrange to modulate the FP/QD emission ratio, exhibits a 20-fold change in its ratiometric measurement over a physiologically interesting pH range, making it a prime candidate for intracellular imaging applications.

This thesis presents a modeling program to obtain equilibrium information for a fluorescent system. Determining accurate dissociation constants for equilibrium processes involving a fluorescent mechanism can prove to be quite challenging. Typically, titration curves and non-linear least squares fitting of the data using computer programs are employed to obtain such constants. However, these approaches only consider the total fluorescence signal and often ignore other energy transfer processes within the system. The current model considers the impact on fluorescence from equilibrium binding (viz., metal-ligand, ligand-substrate, etc.), quenching and resonance energy transfer. This model should provide more accurate binding constants as well as insights into other photonic processes. The equations developed for this model are discussed and are fit to experimental data from titrimetric experiments. Since the experimental data are generally in excess of the number of parameters that are needed to define the system, fitting is operated in an overdetermined mode and employs error minimization (either absolute or relative) to define goodness of fit. Examples of how changes in certain parameters affect the shape of the titrimetric curve are also presented. The detection of metal ions is very important, causing a need for the development of a metal ion sensor that provides selectivity, sensitivity, real-time in situ monitoring, and a flexible design. In order to be able to perform in situ monitoring of trace metal ions, FRET-pair labeled peptides were attached to a Tentagel[trademark] resin surface. After soaking in nonmetal and metal solutions (pH = 7.5), the resin beads gave an enhanced response in the presence of Hg²⁺ and Zn²⁺. Using a t-test, the signals of the beads that were soaked in a solution of each of these metal ions (and that of Cd²⁺) were determined to be significantly different from beads soaked in a solution without metal. However, the standard deviation between a set the beads was too large in order to differentiate a bead that was soaked in nonmetal solution versus one soaked in a metal containing solution. / text