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.

L’ADN et l’ARN double brin (ADNdb, ARNdb) subissent des transitions de surétirement avec des forces d’environ 60 pN. Nous effectuons des mesures de force à l'aide d'un piège optique à double faisceau contenant deux billes reliées par une seule molécule. Un brin du duplex est attaché aux deux extrémités aux deux billes, tandis que l’autre brin n’est attaché qu’à une seule extrémité. Quatre cas différents sont comparés: ADNdb, ARNdb, hybride ARN- ADN avec ADN sous tension et hybride ADN- ARN avec ARN sous tension.Un surétirement se produit pour les quatre duplex. La différence la plus remarquable est que les ARNdb présentent un plateau lisse, alors que les autres duplex présentent des motifs en dents de scie. Nous constatons que les ARNdb s'étirent par un mécanisme différent et expliquons pourquoi cette propriété pourrait aider les structures d'ARN à s'assembler et à jouer leurs rôles biologiques.Un surétirement de l'hybride ARN-ADN libère progressivement un brin d'ARN. Les structures formées au sein de cet ARN naissant sont visibles dans le signal de force lors du re- recuit. Pour la première fois à notre connaissance, nous imitons donc et étudions le repliement de l'ARN co-transcriptionnel dans un test in vitro. En se concentrant sur le stade précoce de l’assemblage des grandes sous- unités ribosomales de E. coli (domaines I-II de l’ARNr 23S et des protéines r L4, L13, L20, L22, L24), on observe plus souvent un recuit partiel avec les protéines r. Nos résultats indiquent que les cinq protéines r de liaison précoce agissent comme des auxiliaires de repliement bien avant que l’ARN 23S complet ne soit transcrit. / Double-stranded DNA and RNA (dsDNA, dsRNA) undergo overstretching transitions at forces around 60 pN. We perform force measurements using a dual-beam optical trap that holds two beads linked by a single molecule. One strand of the duplex is attached at both extremities to the beads, while the other strand is attached only at one extremity. Four different cases are compared: dsDNA, dsRNA, RNA-DNA hybrid with DNA under tension, and DNA-RNA hybrid with RNA under tension. Overstretching occurs for all four duplexes. The most remarkable difference is that dsRNA exhibits a smooth plateau, while the other duplexes show saw-tooth patterns. We find that dsRNA overstretches by a different mechanism and explain why this property could help RNA structures to assemble and play their biological roles.Overstretching the RNA-DNA hybrid progressively liberates an RNA strand. Structures formed within this nascent RNA are seen in the force signal upon re-annealing. For the first time to our knowledge, we thus mimic and study co-transcriptional RNA folding in an in-vitro assay. Focusing on the early stage of E.coli large ribosomal subunit assembly (domains I-II of 23S rRNA and r-proteins L4, L13, L20, L22, L24), partial re-annealing is observed more frequently with r-proteins than without. Our results indicate that the five early- binding r-proteins act as folding helpers well before the entire 23S RNA is transcribed.

With more states revealed, and more reliable rates inferred, mechanistic schemes for ion channels have increased in complexity over the history of single-channel studies. At the forefront of single-channel studies we are faced with a temporal barrier delimiting the briefest event which can be detected in single-channel data. Despite improvements in single-channel data analysis, the use of existing methods remains sub-optimal. As existing methods in single-channel data analysis are unquantified, optimal conditions for data analysis are unknown. Here we present a modular single-channel data simulator with two engines; a Hidden Markov Model (HMM) engine, and a sampling engine. The simulator is a tool which provides the necessary a priori information to be able to quantify and compare existing methods in order to optimize analytic conditions. We demonstrate the utility of our simulator by providing a preliminary comparison of two event detection methods in single-channel data analysis; Threshold Crossing and Segmental k-means with Hidden Markov Modelling (SKM-HMM).

Many highly effective drugs display serious side-effects. To limit them, one can contain the drug in tiny containers, which are subsequently delivered toward targets inside the body. The entrapment of drug molecules prevents them from coming in contact with and thus causing damage to normal cells. Inherently, it is difficult to reach 100% efficiency of drug trapping and release when employing physical caps to seal the vehicles. Instead, we propose drug trapping inside the nanopores of hydrophobic silica particles by "hydrophobic trapping". This phenomenon is associated with the repulsive "force field" generated inside nanometer-sized hydrophobic channels that completely prevents aqueous solutions from entering the channels. We demonstrate the excellent trapping efficiency using C18-modified silica particles with 10 nm pores and the anticancer drug doxorubicin. The major challenge in using hydrophobic particles in biological applications is their tendency to cluster in aqueous media. To overcome it, we use surfactants as solubilization means. We have developed protocols that effectively solubilize the outer surface of the particles while preventing surfactant micelles from entering nanopores. Consequently, particles become well-dispersible in aqueous solutions, with the pre-loaded drug safely contained inside nanopores. Nanomaterials exhibit heterogeneity on their surfaces that impact their functional applications. Although techniques such as atomic force microscopy are great tools for studying nanomaterials with their excellent spatial resolution, they cannot probe the inner surface of porous structures. We have established a method of single-molecule ratiometric imaging that is currently the only technique able to provide the nanopolarity of adsorption sites located on the pore surface. We analyze the polarity distribution of adsorptions events for the solvatochromic probe Nile Red at the C18/acetonitrile interface and discover at least two different populations of adsorption sites. One of them corresponds to the polarity of surface silanol groups while the other sites have a polarity consistent with the environment inside the C18 organic layer. We also discover an additional adsorption mode situated at a polarity higher than exposed silanol surface that could presumably be linked to a different ionization state of the silanol groups. We are developing a method for resolving spectra of complex samples using two-dimensional hetero-correlation spectroscopy by correlating the intensity fluctuations in optical spectra to those of completely separated peaks in analytical separations. We demonstrate this methodology for fluorescence spectra and electrophoregrams of mixtures anthracene-pyrene. All the individual vibronic features that overlap in mixtures are cleanly extracted in cross-sections of the two-dimensional asynchronous spectrum.

Riboswitches are gene regulatory elements found in messenger RNA that function by changing structure upon the binding of a ligand to an aptamer domain. Single adenine-binding pbuE riboswitch aptamer RNAs were unfolded and refolded co-transcriptionally using optical tweezers for single molecule force spectroscopy. The kinetic and energetic properties of distinct folding intermediates were characterised with and without the binding of adenine. These observed intermediates were related to structural elements of the aptamer, which were found to fold sequentially, in a transcriptionally independent manner. The mechanical switch underlying the regulatory action of the riboswitch was observed directly (adenine stabilisation of the weakest helix), and the energy landscape for the folding was reconstructed. The construction of a dual-beam optical trap with separate detection and trapping laser beams manipulated and focused into a rigid, modified inverted microscope is also described. This instrument aims to achieve ngstrm-level resolution through careful design to reduce noise.

Clusters of metal ions that possess large numbers of magnetically coupled unpaired electrons have attracted much interest in recent years due to their fascinating magnetic behavior. With an appreciable component of magnetic anisotropy, these large-spin paramagnetic molecules can exhibit an energy barrier to inversion of their magnetic dipole, leading to spontaneous magnetization and magnetic hysteresis below a critical temperature. Since this behavior is a property of an individual clusters rather than a collection of molecules, this phenomenon has been dubbed ??Single Molecule Magnetism??. Our approach to the study of new high-spin systems has been to exert a measure of synthetic control in the preparation of clusters. Specifically we are employing highly anisotropic metal ions with the anticipation that these ions would engender large overall magnetic anisotropy in the resulting clusters. The first step in this process was the development of the chemistry of two new d5 ReII (S = ??) complexes, namely [ReII(triphos)(CH3CN)3][PF6]2 and [Et4N][ReII(triphos)(CN)3]. The magnetic, optical and electrochemical properties were studied and theoretical models were developed to describe the origin of the large temperature independent paramagnetism that was observed. Next, we successfully employed transition metal cyanide chemistry using the ReII building blocks to prepare a family of isostructural, cubic clusters of the general formula {[MCl]4[Re(triphos)(CN)3]4} M = Mn, Fe, Co, Ni, Cu, Zn whose 3d ions adopt local tetrahedral geometries. Within the clusters, magnetic exchange is observed between the paramagnetic ions, which has been modeled using an Ising exchange model to account for the dominating anisotropy of the ReII ion. Despite the high pseudo-symmetry of the clusters (Td), this work has yielded a rare example of a metal-cyanide single molecule magnet, {[MCl]4[Re(triphos)(CN)3]4} with an S = 8 ground state, D = -0.39 cm-1 and an effective energy barrier for magnetization reversal of Ueff = 8.8 cm-1. The elucidation of this family of isostructural clusters has also allowed us to pursue fundamental work on the structure/property relationships of the exotic, paramagnetic ReII ion. As the clusters are soluble, stable compounds, the future of this chemistry lies in the development of a true building-block approach to ??super-clusters?? that exhibit very high ground state spin values.

Metalloporphyrin aggregation is critical for triplet-triplet annihilation (TTA) to occur. In order to maximize the efficiency of TTA, to use as a mechanism of photon upconversion in dye-sensitized solar cells, it is important to understand the phenomenon of absorber aggregation. The aggregation of ZnTPP in polymer films was investigated by fluorescence anisotropy and total internal reflection fluorescence microscopy (TIRFM). Single molecule spectroscopy (SMS) and spectromicroscopy were the techniques used to study single molecules and multimolecular aggregates of ZnTPP/ZnTPPS in polymer films/adsorbed on glass substrates.<p> Fluorescence anisotropy measurements consistently showed depolarized emission from films most concentrated with ZnTPP. This observation was likely a result of energy transfer in and among porphyrin aggregates. Fluorescence intensity decays were also obtained and they illustrated a pattern of decreased fluorescence lifetime (i.e. faster decays) as the concentration of porphyrin in the film increased. These results are consistent with the formation of aggregates, and their increased presence in more concentrated films. The formation of these aggregates quenches the fluorophores fluorescence, resulting in the observed shorter fluorescence lifetimes. <p> TIRFM was performed to study the structure of these polymer films doped with ZnTPP. It was determined that these films consisted of discrete domains and thus lacked homogeneity, and the presence of aggregates was clearly visible. Time-resolved TIRFM measurements were also performed but no interesting results were collected.<p> SMS and spectromicroscopy were the final techniques employed to study porphyrin aggregation. Preliminary measurements were performed with polymer films doped with ZnTPP, and the single step decay time trajectories collected indicated that single molecules were being studied. Furthermore, emission spectra of these molecules were collected and they were similar to those obtained for a bulk measurement, but the bands were slightly shifted in comparison. These measurements were repeated with ZnTPPS adsorbed to glass substrates. Two different patterns of decay trajectories were measured: (i) single step decays corresponding to single ZnTPPS molecules and (ii) multi step/complex decays representative of multimolecular aggregates. Emission spectra were also collected for the multimolecular aggregates, and they were consistent with those of an ensemble measurement but slightly blue-shifted. Such a shift is common when studying aggregates on such a highly polar surface. Thus, these results demonstrate that ZnTPPS aggregates form even at concentrations as low as 10-8 M, and can be studied using SMS despite their weak fluorescence emission.

Metalloporphyrin aggregation is critical for triplet-triplet annihilation (TTA) to occur. In order to maximize the efficiency of TTA, to use as a mechanism of photon upconversion in dye-sensitized solar cells, it is important to understand the phenomenon of absorber aggregation. The aggregation of ZnTPP in polymer films was investigated by fluorescence anisotropy and total internal reflection fluorescence microscopy (TIRFM). Single molecule spectroscopy (SMS) and spectromicroscopy were the techniques used to study single molecules and multimolecular aggregates of ZnTPP/ZnTPPS in polymer films/adsorbed on glass substrates.<p> Fluorescence anisotropy measurements consistently showed depolarized emission from films most concentrated with ZnTPP. This observation was likely a result of energy transfer in and among porphyrin aggregates. Fluorescence intensity decays were also obtained and they illustrated a pattern of decreased fluorescence lifetime (i.e. faster decays) as the concentration of porphyrin in the film increased. These results are consistent with the formation of aggregates, and their increased presence in more concentrated films. The formation of these aggregates quenches the fluorophores fluorescence, resulting in the observed shorter fluorescence lifetimes. <p> TIRFM was performed to study the structure of these polymer films doped with ZnTPP. It was determined that these films consisted of discrete domains and thus lacked homogeneity, and the presence of aggregates was clearly visible. Time-resolved TIRFM measurements were also performed but no interesting results were collected.<p> SMS and spectromicroscopy were the final techniques employed to study porphyrin aggregation. Preliminary measurements were performed with polymer films doped with ZnTPP, and the single step decay time trajectories collected indicated that single molecules were being studied. Furthermore, emission spectra of these molecules were collected and they were similar to those obtained for a bulk measurement, but the bands were slightly shifted in comparison. These measurements were repeated with ZnTPPS adsorbed to glass substrates. Two different patterns of decay trajectories were measured: (i) single step decays corresponding to single ZnTPPS molecules and (ii) multi step/complex decays representative of multimolecular aggregates. Emission spectra were also collected for the multimolecular aggregates, and they were consistent with those of an ensemble measurement but slightly blue-shifted. Such a shift is common when studying aggregates on such a highly polar surface. Thus, these results demonstrate that ZnTPPS aggregates form even at concentrations as low as 10-8 M, and can be studied using SMS despite their weak fluorescence emission.

Water-soluble fluorescent metallic clusters at sizes comparable to the Fermi wavelength of an electron (~0.5 nm for gold and silver) were created and their photophysical properties were investigated at the bulk and single molecule levels. We employed biocompatible dendrimer and peptide to prepare a series of strong fluorescent gold and silver clusters with chemical or photo reduction methods. Facilitated by the well-defined dendrimer size, electrospray ionization mass spectrometry indicates that the fluorescent silver nanocluster size ranges from 2 to 8 Ag atoms. The correlation of emission energy with the number of atoms, N, in each gold nanocluster is quantitatively fit for the smallest nanoclusters with no adjustable parameters by the simple scaling relation of EFermi/N1/3, in which EFermi is the Fermi energy of bulk gold. The transition energy scaling inversely with cluster radius indicates that electronic structure can be well described with the spherical jellium model and further demonstrates that these nanomaterials are multi-electron artificial atoms. Fluorescence from these small metal clusters can be considered protoplasmonic, molecular transitions of the free conduction electrons before the onset of collective dipole oscillations occurring when a continuous density of states is reached. In addition, very strong single molecular Stokes and Antistokes Raman enhancement by fluorescent silver clusters was observed. Pushing to larger sizes, we also created ~ 2nm diameter glutathione encapsulated luminescent gold nanoparticles. Distinct from similarly sized but nonluminescent gold nanoparticles, these 2 nm gold nanoparticles show bright, long lifetime emission but no plasmon absorption. The emission might arise from charge transfer between gold atoms and the thiol ligand. Providing the missing link between atomic and nanoparticle behavior in noble metals, these highly fluorescent, water-soluble gold and silver nanoclusters offer complementary transition energy size scalings at smaller dimensions than do semiconductor quantum dots. The unique discrete excitation and emission and strong Stokes and antistokes Raman enhancement coupled with facile creation in aqueous solution open new opportunities for noble metal nanoclusters as biological labels, energy transfer pairs, and other light emitters in nanoscale electronics.