Novel glass tracers, organic and inorganic polymers based on narrow band atomic fluorescence, have been developed for deployment as environmental tracers. The use of discrete fluorescent species in an environmentally stable host has been investigated to replace existing toxic, broad band molecular dye tracers. The narrow band emission signals offer the potential for the tracing of a large numbers of signals in the same environment; this has been investigated by examining multiple doped tracers which have the potential for coding to specific effluent sources or particulates. The concept of using lanthanide doped glasses as environmental tracers has been demonstrated. The spectral characterisation and concentration studies of the lanthanide doped tracer allow the selection of parameters to produce future tracers and detection systems for particular applications. Therefore by altering the chosen lanthanide dopant, number of dopants, dopant concentration and using selective excitation and emission wavelengths there are a huge number of possible unique tracer combinations. The significantly narrower bandwidth emission peaks of the lanthanide based tracers achieve more selective detection of multiple tracers without overlap interference and gives the potential to selectively and simultaneously monitor many different tracers in the same location. The spectral lifetime characteristics of the lanthanide tracers are very different from the lifetime of background fluorescence which is typically molecular in origin. This is an extra discrimination against background interference and is an important additional advantage of using lanthanide based tracers. Overall this work shows that a very large number of unique environmental tracers can be obtained by varying the concentration, the number of lanthanide ions in a glass and also the possibility of using organic and inorganic lanthanide chelate doped tracers.

The immobilisation of uranium is an important issue within the nuclear industry due to contaminated land from accidental spillage, weapons testing or mining activities. Within the environment uranium is most commonly found in the +VI oxidation state as the mobile uranyl cation [UO2]2+. Alternatively, the +IV oxidation state can also be found in the environment, forming either an insoluble crystalline uraninite phase, or a more soluble molecular uranium(IV) species. Many endogenous subsurface bacteria can bind and accumulate actinide ions through biosorption and can reduce mobile uranyl(VI) species down to immobile uranium(IV) compounds and mineral phases. This work presents an investigation into the bioreduction process by two anaerobic Gram-negative bacteria, Geobacter sulfurreducens and Shewanella oneidensis MR-1. Luminescence spectroscopy is used to monitor the intensity of uranyl(VI) emission in situ over the course of a 24 hour bioreduction experiment with uranyl(VI) acetate as the electron acceptor and either acetate or lactate as the electron donor. An increase in intensity of the emission around hour three or four during the reduction, followed by an overall decrease, is attributed as the disproportionation of an unstable uranyl(V) intermediate. The role of inner and outer membrane c-typecytochromes as well as flavin secretion is also investigated using three deletion mutants of the S. oneidensis bacteria, which shows that in their absence, the reduction of uranyl(VI) does not occur over the course of 24 hours. The emission of uranium(IV) is also investigated during bioreduction in phosphate media and results show that emission can be observed in aqueous solutions at pH 7 pointing to the presence of a molecular product. One photon confocal and two photon fluorescence microscopy has been utilised for the very first time to directly optically image the bioreduction of uranyl(VI) in combination with luminescence lifetime mapping. The sorption of uranyl(VI) onto the surface of the bacteria with differing lifetimes indicates a direct interaction between uranyl(VI) and surface bound c-type cytochromes, since this variation was not observed in mutant S. oneidensis strains where the cytochromes were not present. Combined, these results have established the applicability of optical spectroscopy and microscopyin tracking the bioreduction of uranium in situ.

Two-photon microscopy is often used in biological imaging due to its optical sectioning and depth penetration capabilities. These characteristics have made two-photon microscopy especially useful for neurobiological studies where imaging a volume at single cell resolution is typically required. This dissertation focuses on the optimization of two-photon excited fluorescence for volumetric imaging of biological samples, with special attention to imaging the mouse brain. Chapter 2 studies wavefront manipulation as a way of optimizing two-photon excited fluorescence. We show, through numerical simulations and experiments, that the magnitude of the two-photon fluorescence signal originating from cell-sized objects can be used as a metric of beam quality. We also show that the cranial window used in mouse experiment is a major source of aberrations, which can readily be represented in the Zernike basis. Finally, we implement a modal wavefront optimization scheme that optimizes the wavefront based entirely on the magnitude of the fluorescence. Along with this scheme, Zernike functions are found to be a useful basis for correcting aberrations encountered in mouse brain imaging while the Hadamard basis is found to be useful for scattering compensation. Corrections performed in mouse brain using Zernike functions are found to be valid over hundreds of microns, allowing a single correction to be applied to a whole volume. Finally, we show that the wavefront correction system can double as a wavefront encoding system for experiments that require custom point-spread-functions. Chapter 3 aims to significantly improve the volume imaging rate of two-photon microscopy. The imaging speed is improved by combining two-photon excitation with scanning confocally-aligned planar excitation microscopy (SCAPE). Numerical simulations, analytical arguments, and experiments reveal that the standard method of combining nano-joule pulses with 80 MHz repetition rates is inadequate for two-photon light-sheet excitation. We use numerical simulations and experiments to explore the possibility of achieving fast volumetric imaging using line and sheet excitation and find that the sheet excitation scheme is more promising. Given that two-photon excitation requires high photon-flux-densities near the focus, achieving high enough fluorescence has to be balanced with restrictions placed by saturation, photodamage, photobleaching and sample heating effects. Finally, we experimentally study light sheet excitation at various pulse repetition rates with femtosecond pulses and find that repetition rates near 100 kHz allow imaging of nonbiological samples of ~200x300x300 μm^3 volume at 20 volumes per second while balancing the above constraints. This work paves the way for achieving fast, volumetric two-photon imaging of the mouse brain.

Dans cette étude, nous avons développé un composé dérivé du NADPH, nommé Nanoshutter (NS). NS a été conçu pour inhiber l'activité catalytique de la NOS, c'est à dire la synthèse de NO, en occupant la place du NADPH dans le domaine réductase du NOS. La voie de synthèse de NO chez les mammifère correspond à l'oxydation de la L-arginine catalysée par la NOS, qui se produit dans son domaine oxygénase. Basée sur des données de modélisation moléculaire, la structure de NS est composée deux sous-unités: (i) le motif nucléotidique de reconnaissance du NADPH a été retenu, permettant au composé NS un ciblage approprié du site de liaison au NADPH de la NOS, (ii) le motif nicotinamide de NADPH a été remplacé par un groupe stilbène lié à un groupement terminal accepteur d'électrons. De plus, ce fragment est caractérisé par une très bonne section efficace d'absorption à deux photons (130 GM à 840 nm). NS1, le composé prototype de la famille NS, contient un groupe terminal NO2 en tant que groupe accepteur d'électrons. La valeur de Kd (~ 4,2 µM) a été estimée dans des expériences de titrage sous excitation un- ou deux-photons, et suggère une bonne affinité de liaison de NS1 à la NOS. De façon inattendue, NS1 présente une bonne sélectivité, en terme de rendement quantique de fluorescence, pour les isoformes de NOS par rapport à d'autres protéines qui contiennent ou non un site de liaison NADPH. En outre, il a été montré que NS1 inhibait de façon compétitive NOS par rapport au NADPH. Dans les expériences d'imagerie de fluorescence réalisées sur des cellules endothéliales (HUVEC), NS1 a démontré une internalisation rapide et efficace, avec un signal de fluorescence mis en évidence principalement dans la région périnucléaire, accompagné d’un signal plus sporadique à la membrane plasmique. Cette observation est en parfait accord avec la colocalisation de NS1 et eNOS mesurée par immunomarquage, démontrant ainsi que NS1 cible eNOS dans les cellules endothéliales. La vasoconstriction NO-dépendante attendue dans les anneaux aortiques isolés de souris a été montrée, mais uniquement en présence de catalase qui convertit H2O2 en H2O et O2. En revanche, en l'absence de catalase, la vasorelaxation a plutôt été observée. Ce résultat indique que la NOS n’est très certainement pas l’unique cible de NS1 dans le système endothélial, et que d’autres cibles en rapport avec la modulation de ROS (Reactive Oxygen Species) sont impliquées. En accord avec ce résultat, NS1 provoque une réponse biphasique de la production de ROS dans les cellules HUVEC : Une phase d'augmentation est observée aux faibles concentrations de NS1 (en dessous de 2 µM), suivie d'une diminution (inhibition de ROS) pour des concentrations de NS1 plus élevées. En outre, NS1 inhibe la production de O2- dans les macrophages de souris et les productions de H2O2 et de O2- survenant dans des conditions de découplage de nNOS in vitro. Des explications possibles pour interpréter ces données sont: NS1 probablement inhibe la production de ROS, soit produites au niveau de la NADPH oxydase ou (et) au cours du découplage de la NOS. L'origine de la phase d’augmentation reste plus difficile à interpréter, mais pourrait correspondre au ciblage de la glucose-6-phosphate deshydrogénase. Enfin, NS1 exerce un effet anti -angiogénique sur les cellules endothéliales et empêche la prolifération de cellules du mélanome. En conclusion, NS1 rempli l'objectif principal de cibler et inhiber la NOS en ciblant plus particulièrement le domaine réductase - il est aussi caractérisé par des propriétés d’absorption et de fluorescence à deux photons intéressantes permettant des applications in vitro et in vivo. L’ensemble de ces caractéristiques présentent un profil intéressant pour de futures applications d’imagerie en temps réel et non-invasive, avec également un fort potentiel pour des applications cliniques liées aux maladies NO-dépendantes. / In this study, we introduced a NADPH derivative named as Nanoshutter (NS). NS was designed to inhibit the catalytic activity of NOS, i.e. synthesis of NO, by occupying the NADPH site in the reductase domain of NOS. In mammals, NO participates in extensive physiological/pathological processes in the cardiovascular, nervous and immune systems. The pathway of mammal NO synthesis is the oxidation of L-arginine catalyzed by NOS, which occurs in its oxygenase domain. The catalysis requires three co-substrates (L-arginine, NADPH, and O2) and five cofactors groups (FAD, FMN, calmodulin, BH4 and heme). Guided by molecular modeling, the structure of NS contains two conjugated subunits: (i) the nucleotide recognition motif of NADPH was retained in NS, allowing a proper targeting to the NADPH binding site of NOS- (ii) the nicotinamide moiety of NADPH is replaced by a stilbene moiety linked with a terminal electron acceptor group, preventing electron flow from the reductase to the oxygenase domain of NOS. Furthermore, this moiety is characterized by a large two-photon absorption cross-section (130 at 840 nm). NS1, the first compound of the NS family, contains a NO2 terminal group as an electron acceptor group. NS1 displayed distinct fluorescence properties in its free and NOS-bound states. The Kd value (around 4.2 µM) was estimated in titration experiments performed under one- or two-photon excitation conditions, suggesting an effective binding of NS1 to NOS with a good affinity. Surprisingly, in terms of fluorescence quantum yield, NS1 displayed a good selectivity to the NOS isoforms over other proteins which contain or not a NADPH binding site. Furthermore, NS1 was shown to competitively inhibit nNOS in a dose-dependent manner. In fluorescence imaging experiments with endothelial cells (HUVEC), NS1 displayed a rapid and efficient internalization, with highlighted fluorescence signal at the perinucleus region and sporadic signal at the plasma membrane. This observation was in accordance with the colocalization imaging between NS1 and eNOS as shown by immunostaining, showing that NS1 actually targets eNOS in endothelial cells. The expected NO-dependent vasoconstriction in isolated mouse aortic rings was only evidenced in the presence of catalase, which converts H2O2 into H2O and O2. By contrast, in the absence of catalase, a contradictory vasorelaxation was observed. This result indicates that NS1 may target more than NOS in endothelium system, which is (are) likely related to Reactive Oxygen Species (ROS) production. Accordingly, NS1 led to a biphasic response of ROS production in HUVEC cells: An increasing phase occurred at low NS1 concentration (below 2 µM) and followed by a decreasing phase (ROS inhibition) at higher NS1 concentrations. Furthermore, NS1 was shown to inhibit O2- production in mouse macrophages and H2O2 and O2- production in uncoupled nNOS in vitro. Altogether, the possible but not exclusive explanations for current data are: in addition to its inhibition effect on NO production, NS1 probably also inhibits the ROS production either produced by NADPH Oxidase or by electron leakage from uncoupled NOS, or a combination of both. The origin of the increasing phase remains more elusive but could correspond to the targeting of glucose-6-phosphate-dehydrogenase (G6PD). Additionally, NS1 displayed anti-angiogenesis effect on endothelial cells and prevented proliferation of melanoma. In conclusion, NS1 fulfilled the goal as a new NOS inhibitor targeting the reductase domain and displayed a unique two-photon property in vitro and in vivo, these features may provide a promising future for non-invasive real-time imaging, and to potential clinical applications in the NO-dependent diseases.

The development of single-molecule and super-resolution fluorescence techniques has revolutionised biological imaging. Nano-scale cellular structures and heterogeneous dynamic processes are now able to be visualised with unprecedented resolution in both time and space. The achievable localisation precision and therefore the resolution is fundamentally limited by the number of photons a single-fluorophore can emit. The ideal super-resolution dye would emit a large number of photons over a short period of time. On the contrary, an optimal single-molecule tracking probe would be highly photostable and undergo no transient dark-state transitions. Single-molecule instrument development is beginning to reach technological saturation and as the frontiers of bioimaging expand, exorbitant demands are placed on the gamut of available probes that often cannot be met. Thus, the next key challenge in the field is the development of the better fluorophores that underlie these techniques; this includes both the synthesis of new chemical derivatives and alternative novel strategies to augment existing technologies. The results of this thesis are divided into two distinct parts; Project One details the development of new synthetic fluorescent probes for the study of amyloid protein aggregates implicated in neurodegenerative diseases. This includes a study of the photophysical and binding properties of a novel fluorophore library based on the amyloid dye Thioflavin-T. Following on from this, is the presentation of novel bifunctional dyes capable of simultaneously identifying hydrogen peroxide and amyloid aggregates by combining existing tools for the independent detection of these species. The sensing capabilities of these dyes are explored at the bulk and single-molecule levels. Project Two describes a new photo-modulatable fluorescent-protein fusion construct that can undergo Förster resonance energy transfer (FRET) to an organic dye molecule. This FRET cassette is comprised of a photoconvertible fluorescent protein donor, mEos3.2 and acceptor fluorophore, JF646. This strategy imparts a strong photostabilising effect on the fluorescent protein and a resistance to photobleaching. The functionality of this approach is demonstrated with in vitro single-molecule fluorescence studies and its biological applicability shown by tracking single proteins in the nuclei of live embryonic stem cells. Furthermore, initial characterisations of the excited state dynamics in effect are presented through the systematic modification of parameters.

L’étude du comportement des éléments métalliques est primordiale compte tenu leur effet souvent toxique dansde nombreux écosystèmes. Ces derniers lorsqu’ils interagissent avec la Matiere Organique (MO), peuvent formerdes complexes plus ou moins stables. Ainsi, la MO joue un rôle important dans leur spéciation chimique et leurtransport. Dans ce travail, l’analyse de cette complexation est réalisée par Quenching de Fluorescence (QF).Cette technique permet de modéliser la fixation des sites de complexation à l’aide d’une constante thermodynamiquedéterminée à partir d’un modèle 1 : 1. Le quenching de fluorescence a été mesuré par spectroscopie defluorescence en mode stationnaire et en mode résolue en temps. Les mesures de fluorescence en mode stationnairefournissent des Matrices d’Excitation et d’Émission de Fluorescence (MEEFs). L’extraction des différents composantsde ces MEEFs est effectué par séparation de sources : la décomposition multilinéaire CP/PARAFAC,qui permet de caractériser spectralement les composants. Les mesures par Spectroscopie Laser Résolue en Temps(SLRT) permettent une caractérisation spectrale et temporelle des composants fluorescents. L’étude des lois dedécroissance de la fluorescence induite par impulsion laser nanoseconde en l’occurrence a permis de déterminerle type d’interaction entre la MO et les quencheurs. Pour se faire, un algorithme de déconvolution temporellea été appliqué à chaque décroissance de fluorescence mesurée. L’interprétation des données temporelles a étéaccomplie en utilisant le graphique de Stern–Volmer. Les résultats des interactions du cuivre, de l’europiumet de l’uranium avec les Acides Humiques (AH) et les Acides Fulviques (AF) montrent des décroissances defluorescences importantes et des constantes de stabilité entre 2,04 et 4,52. Le cuivre a permis de valider notremodèle d’étude et l’interaction de l’europium et l’uranium avec les AH et AF étudiés a révélé des constantesde stabilité en général en bonne corrélation avec la littérature. Les résultats de la SLRT ont souvent révélé desdécroissances bi–exponentielles et des temps de vie entre 0,40 et 14 ns et montrent que les interactions étudiéesont principalement engendrer un quenching statique et donc la formation d’un complexe moléculaire à l’étatfondamental. Cette étude a donc permis par caractérisation spectrale et temporelle, de déterminer l’interactionde la matière organique avec les métaux plus ou moins toxiques. / The study of metal elements behavior, considering their impact in various ecosystems, is of paramount importance.The latter, upon contact with Organic Matter (OM) can form weak or strong stable complexes. Therefore,OM plays an important role in their chemical speciation and transport. The analysis of these properties is carriedout by Fluorescence Quenching (QF). This technique allows introspecting ligands–metal interactions andthe 1 : 1 modeling (one ligand site, one metal) gives information about the conditional thermodynamic constant.Fluorescence quenching was measured using steady–state and time–resolved fluorescence spectroscopy.The steady–state measurements provides Excitation and Emission Matrices of fluorescence (EEMs). The extractionof the different components from these matrices is carried out by a multi–mode factor analysis such asCP/PARAFAC, which allows a spectral composition of the samples. Time-Resolved Laser Spectroscopy (TRLS)measurements allow temporal and spectral characterization of the fluorescent components. Indeed, the study ofthe fluorescence decays induced by a nanosecond pulsed laser in this case allowed to measure the interaction betweenthe OM and the quencher. For those purposes, a fluorescence lifetime deconvolution algorithm was appliedto each fluorescence decay. Analysis of the fluorescence lifetime data was accomplished using the Stern–Volmerplot which gave reliable information on the quenching process that takes place. Copper, europium and uraniuminteractions with Humic Acids (HA) and Fulvic Acids (FA) shows significant fluorescence decays and stabilityconstants between 2.04 and 4.52. Copper allowed to calibrate our model study and the interactions of europiumand uranium with the HA and FA studied reported stability constants in agreement with the literature. TRLSresults have often revealed a bi–exponential decays and fluorescence lifetimes between 0.40 and 14 ns and showsthat the studied interactions mainly lead to static quenching and thus the formation of a molecular complex inthe ground state. This study has allowed spectral and temporal characterization to determine organic matterinteraction with toxic metals.

Calcineurin (CaN), a heterodimeric Ca2+-calmodulin-dependent Ser/Thr phosphatase, regulates diverse pathways, from stress responses in yeast to T-cell activation and cardiac hypertrophy in humans. Calmodulin (CaM), an essential mediator of calcium–dependent signaling pathways, activates CaN in the presence of calcium by binding to an intrinsically disordered region of the enzyme and altering its conformation. My hydrodynamic studies have determined that CaM participates in a 1:1 complex with the CaM-binding domain of βCaN (CaNp, residues 400–423). To explore the molecular mechanism of CaM association with CaN, I have used spectroscopic methods to determine the calcium-dependent and domain–specific interactions of CaM with CaNp. These studies revealed that the affinity of CaM1–148 for CaNp was weak in the absence of calcium, and very high (Kd in the nM to pM range) in the presence of calcium. I have demonstrated that CaNp binding to CaM increases the calcium–binding affinity of each domain of CaM1–148 to a similar degree, thereby retaining the property of sequential calcium binding to the domains, with preference for sites in the C–domain. This allows the N–domain to lag in response to an increase in cellular calcium and perhaps contribute to the regulation of CaN in a manner distinct from that of the C–domain. NMR studies of calcium–saturated CaM1–148 demonstrated that the N–domain of CaM experienced a larger structural perturbation than the C–domain upon binding CaNp. Additional NMR studies revealed that CaNp adopts an anti–parallel orientation when bound to CaM, with the sole aromatic residue of CaNp contacting the N–domain of CaM. This contrasts with many CaM-target complexes in which the sole aromatic residue contacts the C–domain of CaM. Rigorous thermodynamic studies explored how mutations in the calcium-binding sites of mammalian CaM (mCaM) and mutations known to cause disruption of CaM–mediated ion channel regulation in Paramecia (PCaM) affected the allosteric interactions of the domains of CaM in the presence of CaNp. These studies demonstrated separable roles of the domains of CaM in recognition of CaNp. The consequences of a mutation depended upon its location within the complex. Collectively, research presented in this thesis provides insight into the mechanisms whereby the two domains of CaM contribute to recognition of CaN.

A series of new, octadentate, fluorescent, macrocyclic ligands have been prepared with a view to using them to study aromatic anion sequestration. The eight-coordinate Cd(II) complexes of the ligands have been shown capable of acting as receptors for a range of aromatic oxoanions. This has been demonstrated by perturbation of both 1H NMR chemical shift values and the anthracene derived fluorescence emission intensity as the potential guest anion and the host are combined. Non-linear least squares regression analysis of the resulting titration curves leads to the determination of binding constants in 20% aqueous 1,4-dioxane which lie in the range 10^2.3 M-1 (benzoate) to 10^7.5 M-1 (2,6-dihydroxybenzoate). By reference to the X-ray determined structures of related, but non-fluorescent inclusion complexes, the primary anion retention force is known to arise from hydrogen bonding between the anion and four convergent hydroxy groups that exist at the base of a cavity that develops in the complexes as their aromatic groups juxtapose upon coordination. This work reveals significant stability enhancement when hydroxy groups are positioned on the anion at points where O-H...pi hydrogen bonding to the aromatic rings that constitute the walls of the cavity becomes geometrically possible.

Bacteria are implicated in endodontic infections. They not only infect the root canal lumen but also invade the dentinal tubules where they may remain untouched by contemporary chemomechanical preparation during root canal therapy. The contentious issue is whether the bacteria within these tubules contribute to secondary infections. Many studies have shown that clinicians fail to completely eradicate them during root canal therapy. At present there are no techniques available to detect the effectiveness of the current chemomechanical treatment regime within dentinal tubules. It is difficult to detect bacteria within the dentinal tubules. Culturing techniques have been used routinely as they are versatile and easy to use. However, they are unable to show the distribution of the bacteria within the dentinal tubules. Scanning electron microscopy, on the other hand, shows detailed surface structure in association with bacteria. Histological examination of root dentine specimens under the light microscope also shows the distribution of bacteria within the specimen but not viability. The dilemma posed by these existing techniques is that the results offer limited information; either demonstrating bacterial viability or bacterial distribution within specimens. No techniques able to show both the viability and the distribution of bacteria within the dentinal tubules have been reported to date. Fluorescent stains, in particular SYTO�9 and propidium iodide (LIVE/DEAD� Baclight[TM] viability kit, Molecular Probes Inc., Eugene, Oregon), have made it possible not only to stain bacteria but to differentiate live and dead bacteria. The combination of these two stains has yet to be applied to dental hard tissue in situ and they provide the basis for this investigation. The aim of this study was to evaluate the potential of the LIVE/DEAD� Baclight[TM] stains in conjuction with confocal laser scanning microscopy in the development of a technique to evaluate the viability and distribution of bacteria within dentinal tubules. This was extended to demonstrate the application of this technique by examining three different means of root canal disinfection both qualitatively and quantitatively. An important aspect of this study was to maintain bacterial viability, as well as to get maximum bacterial invasion into dentinal tubules. Results indicated that when the root canals were instrumented with Protaper� files and then irrigated with sodium hypochlorite (NaOCl) and ethylene diaminetetraacetic acid with cetrimide (EDTAC), there was more bacterial invasion into the dentinal tubules than when the root canals were only irrigated with NaOCl and EDTAC. Daily replenishments of nutrients resulted in deeper bacterial invasion into the dentinal tubules. Bacteria colonized the dentinal tubules up to a distance of 594 � 133 [mu]m from the canal. In the untreated tubules, 96 � 4 % of bacteria remained viable (green-fluorescent), whereas the Amoxicillin-treated tubules contained 94 � 6 % dead (red-fluorescent) bacteria. The calcium hydroxide-treated tubules resulted in 92 � 7 % bacterial death while the laser-treated tubules contained 81 � 12 % dead cells, frequently displaying an inner zone of dead cells surrounded by an outer zone of viable cells. The application of the fluorescent stains combined with confocal microscopy offers a new method for assessing the in vitro efficacy of root canal disinfection regimens.

This thesis describes several aspects of cosmic ray composition studies using the Utah Fly's Eye and High Resolution Fly's Eye (HiRes) detectors. The Fly's Eye detector utilises the atmospheric fluorescence technique to measure the development of cosmic ray cascades as they pass through the atmosphere. This is complementary to the surface array technique, as used by the Akeno experiment in Japan, which measures the electromagnetic and muon content of air showers at a single observation level. For some time it was thought that Fly's Eye and Akeno gave inconsistent composition results. In Chapter 4 I show that the inconsistency is due, for the most part, to a difference in the assumptions made about hadronic interactions. In Chapter 5 I present analysis of the composition between 10^17 and 10^18 eV using the prototype High Resolution Fly's Eye (HiRes) detector in coincidence with the Michigan Muon Array (MIA). The hybrid nature of these measurements gives us more information about cosmic ray showers than either technique on its own. The consistency or otherwise of the composition measured by the two detectors is discussed. Finally, in Chapter 6, I discuss a method of extracting the total proton-proton cross section from the cosmic ray data. This information is of interest because it is derived at centre of mass energies much higher (by at least an order of magnitude) than those currently accessible by collider experiments. I present a preliminary calculation of the cross section using the HiRes/MIA hybrid data set. / Thesis (Ph.D.)--Department of Physics and Mathematical Physics, 2001.