Characterizing nanoparticles to be able to understand how they functions in the body is important for development of drugs. Furthermore with increasing number of nanoparticle product the nanotoxicity of nanoparticles is important to understand. This report is a part of the EU-project Nanoclassifier which purpose is to “develop a cost effective, high throughput screening platform for characterization of the bionanointerface and its cell-binding partners”. Single-cycle kinetic was used to determine the number of binding epitopes on polystyrene nanoparticle with transferrin corona. The number of available epitopes describes how active the Nanoparticle will be in the body. For this purpose Single-cycle kinetic methodology was successfully used on nanoparticles. Single-cycle kinetic methodology has great potential to become the standard method for high throughput nanoparticle epitope characterization.

Doctor of Philosophy / Department of Chemistry / Viktor Chikan / Colloidal semiconductor nanoparticles will be important and practical next generation materials that can be cheaply manufactured. The objective of this project is to gain more inside into chemistry is used to control the formation and assembly of semiconductor nanoparticles (NPs). As a model system CdSe and CdTe nanoparticles are used in this work. The growth kinetics, aggregation dynamics, and heterogeneous growth of NPs by using novel tools such as; in-situ monitored fluorescence and absorption techniques, time-resolved and static fluorescence spectroscopy, TEM (transmission electron microscopy), and numerical simulations are studied. This study can be divided into the following four parts. The first part presents experimental observation of the quantized growth of CdTe quantum dots (QD). The high-temperature absorption spectra indicate the evolution of multiple peaks corresponding to various sizes of QDs. The observed aggregation is driven by dipole-dipole interaction of NPs. The second part is an investigation of the aggregation dynamics of magic-sized CdTe quantum dots and how this process can be controlled. It is shown that the growth kinetics of the QDs is very sensitive to the Cd/Te ratio. Cd-rich conditions form very different aggregation pattern due to the lack of formation of magic-sized nanoparticles. Simulations also suggest that the formation mechanism is mainly coalescence of the particles rather than the ‘neck formation’ within the CdTe aggregates. The next part investigates the growth of NPs in the presence of two distinctly sized NPs in the bimodal growth regime via numerical simulations. The bimodal distribution (or quantized Ostwald ripening) technique is found to be a slower process than the repeated injection technique to focus the size distribution of NPs. Slower growth will reduce inhomogeneity in a scaled-up production of NPs. The last part focuses on the effect of addition of doping on vii heterogeneous growth and the growth kinetics. The low temperature synthesis lacks the heterogeneous growth regime. However, as the temperature is increased to 120 0C, two different sizes emerge. Addition of In dopants seems to accelerate the growth kinetics and the magic sized NPs in the solution possess a negative anisotropy that is most likely due to supperlatice formation of magic-sized NPs.

Ribosomal incorporation of unnatural amino acids (AAs) into peptides or proteins has found broad applications in studying translation mechanism, discovering potential therapeutics, and probing protein structure and function. However, such applications are generally limited by the low incorporation efficiencies of the unnatural AAs. With in vitro kinetics studies using a purified E. coli translation system, we found that the natural N-alkyl AA carrier, tRNAPro, could hasten the incorporation of N-methyl AAs. Also, the incorporation rate increased remarkably with increasing pH in the range of 7 to 8.5, suggesting the rate was limited by peptidyl transfer, not accommodation. Competition experiments revealed that several futile cycles of delivery and rejection of the A site N-methyl AA-tRNA were required per peptide bond formation, and the incorporation yield could be increased by using a higher Mg2+ concentration. Kinetics of ribosomal polymerization, using AA-tRNA substrates prepared from the standard N-NVOC-AA-pdCpA chemoenzymatic ligation method, clarified that the inefficiency of incorporation was due to the penultimate dC. This dC prompted faster peptidyl-tRNA drop-off, leading to loss of processivities along consecutive incorporations. Circumventing the penultimate dC by using our N-NVOC-AA-pCpA chemoenzymatic ligation or the flexizyme charging method to prepare the AA-tRNA substrates was able to improve the efficiencies of ribosomal consecutive incorporations of unnatural AAs. By studying the translation steps after aminoacylation of tRNAPyl, the favored carrier for unnatural AAs in vivo, we demonstrated surprisingly slow biphasic kinetics of tRNAPyl-mediated amber suppression in vitro. The fast phase amplitude increased with increasing EF-Tu concentration, allowing measurement of Kd of EF-Tu binding. Results revealed ~25-fold weaker EF-Tu binding affinity of the tRNAPyl body than that of E. coli tRNAPhe. The fast phase rate was ~30-fold slower than that of native substrates, and this rate was limited by the ~10-fold less efficient AA-tRNAPyl:EF-Tu:GTP ternary complex binding to the ribosome. The incorporation was so slow that termination by RF2 mis-reading of the amber codon became a significant competing reaction. The processivity was unexpectedly impaired as ~40% of the dipeptidyl-tRNAPyl could not be elongated to tripeptide. This new overall understanding opens a window of improving unnatural AA incorporation both in vitro and in vivo.

Reaction progress kinetic analysis (RPKA) is a powerful tool for determining kinetic parameters of catalytic reactions. Many of the published articles that have used RPKA have employed reaction calorimetry for obtaining sufficient data to be reliable. The use of gas uptake measurements, in place of calorimetry is explored in this Thesis. Chapter 2 details the use of gas uptake measurements in establishing the order with respect to substrate and gas for the rhodium catalysed hydrogenation of 1-octene. Previous studies have used initial rate measurements to establish these orders and the reaction cycle is well known. The use of RPKA allows the same information to be established in two reactions. Chapter 3 focuses on the rhodium catalysed hydroformylation of 1-octene as it involves the reaction of one substrate with two gases. Using RPKA it is possible to determine the order in substrate and the overall order in gas, but it was found difficult to determine the order with respect to the individual gases using RPKA alone. Chapter 4 shows the palladium catalysed methoxycarbonylation of vinyl acetate. The reaction has two substrate concentrations changing simultaneously as well as a gas. This chapter shows that by careful design of experiments the orders with respect to each of these substrates and CO can be determined in minimal numbers of experiments. Chapter 5 focuses on the methoxycarbonylation of alkynes, which uses RPKA in complex multistep reactions, to establish if RPKA can be used to determine the kinetics with respect to the individual reacting components for each step. This study focuses on the methoxycarbonylation of phenylacetylene to produce methyl cinnamate as well as the methoxycarbonylation of both terminal and internal linear alkynes. These linear alkynes carbonylate to produce an α,β-unsaturated ester. The double bond is isomerised from its conjugated position along the chain to the terminal position where it is trapped and carbonylated to produce an α,ω-dieter product.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Triathlon involves three different modes of endurance events, swim, bike and run, consecutively. Transitions between events are critical to be successful in the sport; however, many triathletes report impaired running performance due to adverse residual effects from cycling. One of the strategies that triathletes use to manage the adverse effects is to use a bicycle with a more vertical seat post angle. There is limited evidence that support the effectiveness of such bicycle geometry, but many of these studies lacks ecological validity. Twelve triathletes and cyclists completed a 20-km simulated course with instrumentations for 3D motion, kinetic, and electromyographic analyses under two different seat post angle settings: shallow (ROAD) and steep (TRI). Series of paired-t tests were used for statistical analysis. Results indicated cycling mechanics between two seat post angle conditions were similar; however, the steep condition resulted in time-delay in muscle activation and pedal force application. There was no significant difference in cycling performance. The athletes were able to retain relatively consistent pedaling techniques with modification of seat post angle.

Type 2 diabetes or adult onset diabetes, has been a global epidemic for the past two decades, and the number of new cases accelerates every year. Insulin resistance is one of the major factors behind this, wherein the insulin receptor, which signals to regulate glucose levels, based on the hormone insulin, loses its sensitivity. Obesity is one other major concern which is caused due to the improper balance between the caloric intake and the energy utilized. Gastric bypass surgeries (GBP) are performed to avert obesity. However, a beneficial side-effect is that the state of insulin resistance is reset to near baseline levels within a few days after the procedure. The reason behind this remains unexplained, with possible humoral effects, hypothesized to occur after the bariatric procedure. In this work, high-five insect cell line was utilized to recombinantly produce full length insulin receptors (IR). However commercially sourced IR ectodomains (eIR – soluble version of the full length IR with the completely extracellular α subunits along with extracellular and transmembrane regions of the β subunit), were used to study the interaction. Measuring the kinetics of IR-insulin interactions is critical to improving our understanding of this disease. In this study, a multiplex surface plasmon resonance (SPR) assay was developed for studying the interaction between insulin and the eIR. A scaffold approach was used in which anti-insulin receptor monoclonal antibody 83–7 (Abcam, Cambridge, UK) was first immobilized on the SPR sensorchip by amine coupling, followed by eIR capture. The multiplex SPR system (ProteOn XPR36TM, Bio-Rad Laboratories, Hercules, CA) enabled measurement of replicate interactions with a single, parallel set of analyte injections, whereas repeated regeneration of the scaffold between measurements caused variable loss of antibody activity. The main approach was to replicate the physiological IR-insulin interaction using this assay. It was also observed that insulin at higher concentrations tend to form dimers and hexamers in solution. This was tested using size exclusion chromatography analysis and proved to be true. Therefore an alternative analyte with the similar binding properties and affinity of insulin and at the same time with reduced self- association characteristics was explored. Lispro, the analogue of insulin with reduced self-association properties (generated by swapping of residue 28 and 29 with Lys and Pro respectively) was finally used to study the interaction with eIR. Interactions between recombinant human insulin with eIR-A (A isoform of the insulin receptor ectodomain) followed a two-site binding pattern (consistent with the literature), with a high-affinity site (dissociation constant KD1 = 38.1 ± 0.9 nM) and a low-affinity site (KD2 = 166.3 ± 7.3 nM). The predominantly monomeric insulin analogue Lispro had corresponding dissociation constants KD1 =73.2 ± 1.8 nM and KD2 =148.9 ± 6.1 nM, but the fit to kinetic data was improved when conformational change factor was included in which the high-affinity site was converted to the low-affinity site. Kinetics of interaction of insulin with eIR-A and eIR-B isoforms were then compared. eIR-A bound insulin with apparently higher affinity (with both the binding sites) when compared with eIR-B. This was again consistent with literature that IR-A had two-fold higher affinity for binding insulin than IR-B. The assay was further extended to study the effect of external factors such as glucose, visfatin on this interaction. Glucose (the main biomolecule which is regulated by the IR-insulin interaction) was tested, if it had any direct effect on the interaction. It was observed that glucose did not have any effect on eIR-insulin interactions. Visfatin, an adipocytokine which has been highly debated in literature for its insulin mimetic effects and IR binding properties, was then tested. The standard assay did not provide much insights as the reference channel immobilized with 83-7 monoclonal antibody to the receptor had much affinity for visfatin, leading to non-specific binding and negative responses. Therefore, in an alternative methodology was used - visfatin, Lispro and insulin were immobilized on separate channels along with bovine serum albumin immobilized on reference channel and eIR isoforms used as analyte to study the effect of visfatin on IR. This study showed that visfatin, a higher molecular weight protein compared to insulin, bound both the eIR isoforms. This is consistent with literature that visfatin binds IR at a site distinct from insulin, but the assay described here could not confirm the fact that it mimicked the signalling carried out by IR-insulin binding. Further studies are required to interpret the kinetics of visfatin-eIR interaction. To my knowledge, this is the first SPR assay developed to study eIR-insulin interactions in real-time. This could potentially be extended to study the interaction of insulin with full length insulin receptors and the effect of humoral and other external factors on the interaction, without the need for insulin labelling.

Radical polymerisation (RP) has for many years been an industrially important process, and the kinetics of the process remains an active area of research. As polymerisation proceeds, converting monomer (small molecules) into polymer (long chain molecules), chemical species of a variety of chain lengths are produced. Recent work has pointed toward the fact that rate coefficients for polymerisation reactions (specifically, termination and propagation) are often dependent on the chain-length of the reacting species. The focus of this thesis is to study the effects of chain-length-dependent reactions on the kinetics of RP, by using computer-based modeling and comparing the results of such modeling with experimental data. This enables the understanding of otherwise inexplicable trends and the building of more mechanistically detailed and accurate models for RP kinetics. In Chapter 2, a new model for termination is developed, connecting observations and analyses of termination kinetics at short chain lengths (particularly small molecule studies) with other observations and analyses at long chain lengths (conventional RP kinetics studies) in order to construct a model for termination that is shown to be capable of coherently describing termination kinetics at any chain length. In Chapter 3, this new model for termination is tested at short chain lengths on polymerisations with large quantities of added chain transfer agent. With the inclusion of chain-length-dependent propagation in the model, the model for termination is validated. Chapter 4 is aimed at extending an existing reduced-variable, compact, 'universal' description of steady-state RP kinetics by incorporating all known chain-length dependent reactivities. This both increases computational efficiency over other approaches and provides easily evaluated, approximate analytical expressions for RP kinetics. This foundational theory is applied in Chapter 5 to reach a deeper understanding of the behaviour of the model, and show how experimental data may readily be analysed to extract information about chain-length-dependent termination kinetics. In Chapter 6, the effect of chain-length dependent reactivities on the important technique of single-pulse pulsed-laser polymerisation is investigated, and this technique is validated as the best experimental method for investigation of termination kinetics. In general, a central result of this thesis is that chain-length-dependent reactivities, when acknowledged and properly incorporated into models, can explain many phenomena in RP kinetics which otherwise seem difficult to account for. No exceptions to this principle have been found.

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