Inner hair cells (IHCs) are the actual sensory receptors in hearing. Immature IHCs generate spontaneous calcium-dependent action potentials. Changing the characteristic of the Ca2Å signals modulates the amplitude and duration of the action potentials in these cells. These spontaneous action potential firing patterns are thought to be important for the development of the auditory system. The aim of this thesis is to gain a deeper understanding of the electrical activity and calcium signalling during development of IHCs from a mathematical point of view. A numerical bifurcation analysis is performed to delineate the relative contributions of the model parameters to the asymptotic behaviour of the model. In particular, we investigate the pattern of periodic solutions including single (normal) spiking, pseudoplateau burstings and complex solutions using two-parameter sections of the parameter space. We also demonstrate that a simplified (three-dimensional) model can generate similar dynamics as the original (four-dimensional) IHC model. This reduced model could be characterised by two fast and one slow or one fast and two slow variables depending on the parameters’ choice. Hence, the mechanisms underlying the bursting dynamics and mixed mode oscillations in the model are studied applying 1-slow/2-fast and 2-slow/1-fast analysis, respectively.

<div><div><div><div>Mass spectrometry is a widely used tool for efficient chemical characterization and identification. The development of electrospray ionization as a soft ionization method enables mass spectrometry for large biomolecule investigation. Protein as one of the most important classes of biomolecules, its structural changes including folding, unfolding, aggregation, degradation and post-translational modification all influence protein bioactivity. Protein characterization and identification are important for protein behavior mechanism understanding, which may further contribute to disease treatment development. Protein conformation changes are normally very fast, and the initial stages, which significantly influence the conformation changing pathway, normally occur in milliseconds or shorter time scale. Such a fast structural change is hard to be monitored using traditional bulk solution manipulations, and fast sample preparation methods are required. </div><div><br></div><div>In this thesis project, theta tips are applied as a microreactor and nESI-MS emitter to perform fast protein manipulation immediately before MS analysis. Theta tips can be operated in two modes. The first mode is for submillisecond time scale reactions. Proteins and reagents are loaded into different channels and directly sprayed out simultaneously. Proteins and reagents mix and react in the Taylor cone and subsequent droplets for submillisecond time scale. Through this method, pH induced protein folding is investigated and protein folding intermediates were captured. The second mode is for milliseconds or longer reactions. Differential voltages can be applied to each channel before ionization and spray. The electric field between the two channels induces in-tip electroosmosis, which lead to an in-tip mixing and reaction. In this mode, the reaction time is not limited by the droplet lifetime as in the first mode, but is controlled by electroosmosis time. By changing the electroosmosis square wave frequency and cycles, the mixing time can be elongated to milliseconds or longer, which is suitable for slower reaction study. </div><div>Joule heating is discovered during theta tip electroosmosis when samples are dissolved in buffer. The Joule heating effect is high enough to heat up the aqueous solution to at least 75 oC based on Raman thermometry measurement, while the actual peak temperature could be higher. The Joule heating effect in theta tip electroosmosis can be easily controlled by electroosmosis voltage, time, buffer concentration etc.. Proteins are thermally denatured by the Joule heating effect, and the denaturation extent correlates with Joule heating parameters. </div><div>With this results in hand, we are developing a protein melting temperature measurement method using theta tip Joule heating effect and mass spectrometry. This new melting temperature measurement method measures changes in protein mass and charge state distributions. Therefore, it could sensitively detect ligand loss and protein tertiary structural changes, which is an important compensation to current protein melting temperature measurement techniques like CD or DSC. Since the heating time is short and protein concentration for MS is low, protein aggregation and thermal fragmentation are highly avoided so a complete protein thermal unfolding process is monitored. Theta tip electroosmosis combining MS characterized protein thermal denaturation behavior from a new aspect.</div><div><br></div><div>Besides single protein folding and unfolding, protein identification and post-translational modification are important for proteomics study. The traditional bottom-up, top-down and middle-down methods are not able to both preserve intact protein mass and efficiently generate enough fragment peaks easily without performing gas phase dissociation. In this thesis, we also developed a new way to identify proteins combining limited trypsin digestion and mass spectrometry. Intact protein mass was preserved for protein size and PTM identification. Enough tryptic peptides were also generated for protein identification through database search.</div></div></div></div><div><br></div>

La criminalistique peut être définie comme l’application de procédés techniques aux investigations judiciaires permettant l’étude scientifique des traces et des indices retrouvés sur les scènes de crime.Depuis la découverte de l’empreinte génétique par Sir Alec Jeffreys en 1984, le monde judiciaire s’est profondément ancré dans l’ère de l’ADN en raison d’évolutions technologiques successives dans le domaine de la biologie moléculaire et ses applications en criminalistique. Le besoin de réponse instantanée est omniprésent dans les esprits. La mise en œuvre de techniques d’analyses simples, sensibles, fiables et permettant d’obtenir des résultats dans les plus brefs délais sont les clés du succès.Au cours des processus techniques, la collecte du matériel biologique, et donc de l’ADN au sein de la trace, constitue une étape incontournable et cruciale qui va conditionner la réussite des analyses. Ce travail de recherche a donc consisté à développer des solutions performantes de prélèvements de matériels biologiques soit en détournant de leur fonction initiale des solutions existantes soit en développant des solutions simples mais innovantes combinant les avantages des solutions existantes. Ces travaux ont permis de donner naissance au micro-écouvillon GendSAG. Les potentialités de GendSAG permettent de proposer une solution alternative aux solutions commerciales de systèmes intégrés d’analyses rapide d’ADN. Cette solution alternative d’analyse rapide et haut débit de l’ADN mise en œuvre dans un laboratoire mobile au plus près de la scène de crime répond non seulement à la grande majorité des avantages des systèmes intégrés mais également à toutes leurs limitations. / Forensic sciences can be defined as the used of technical processes to judicial investigations allowing the scientific study of traces and evidences found on crime scenes.Since the discovery of DNA fingerprinting by Sir Alec Jeffreys in 1984, the legal world has become deeply rooted in the DNA by successive technological developments in molecular biology and its applications in forensic. The need for instant response is omnipresent in the minds. The key to success is the implementation of simple, sensitive, reliable analytical techniques that enable results to be achieved in the shortest possible time.During these technical processes, the collection of biological samples, is an unavoidable and a crucial step that will condition the analysis success rate. This study consisted in developing efficient biological collecting solutions either by diverting from their original function the existing solutions or by developing simple but innovative solutions combining the advantages of the existing solutions. This allowed developing the micro-swab GendSAG. The potentialities of GendSAG make it possible to propose an alternative solution to the commercial rapid DNA analysis integrated systems. This rapid, cost effective and high-throughput DNA analysis solution performed in a dedicated mobile laboratory directly into the crime scene enables the large majority of the rapid DNA analysis integrated systems benefits and also all of their limitations.

This thesis describes two different sample preparation strategies for inductively coupled plasma atomic emission spectrometry (ICP-AES), and their ability regarding multi element quantification in complex samples. Sensitivity, repeatability, reproducibility and accuracy were investigated. The aim was to increase the over all efficiency, the speed of analysis, and/or the sensitivity of the analytical method. The intention was to measure analytes with concentrations ranging from ng/g to mg/g simultaneously. The aim was additionally to study chemical and physical processes occurring during the sample preparation, the sample transport to the plasma, and the atomization therein. In the first sample preparation strategy, a hydrophilic highly cross-linked iminodiacetate-agarose adsorbent, IDA-Novarose, was used for preconcentration of metal ions, and matrix elimination in natural water samples. The sorbent was synthesized with different binding capacities. The effect of the capacity on preconcentration, matrix elimination, and uptake capability at high flow rates was studied. For a high capacity IDA-Novarose (≥ 45 µmole/ml) quantitative uptake was seen even at high flow rates (100 ml/min) for Cu2+ with a high affinity to the adsorbent, and for Cd2+ with a moderate affinity. For lower capacities the uptake of Cd2+ was affected by the sample matrix and the flow rate. A method based on the determination of the conditional stability constant of the metal sorbent complex was suggested for the prediction of the sorbent capacity needed to obtain quantitative recovery and optimal matrix elimination. The sorbent was used in a flow system with online buffering for the analysis of a certified riverine water (SLRS-3), tap water and lake water. With few exceptions the results obtained by ICP-AES after preconcentration agreed well with the certified concentrations and results obtained by ICP-MS. The other sample preparation strategy discussed is a method for non digested biological samples from different animal organs for the multi element analysis by ICP-AES. This “mix and measure method” consists of a simple homogenization of the sample with a mixing rod in a small amount of neutral media, followed by dilution and direct measurement with ICP-AES. The total time of analysis is only a few minutes. The ability of this fast method to accurately quantify some elements of toxic, environmental, and/or physiological concern with the lowest possible sample dilution and the highest possible plasma load was evaluated. In 10 % liver slurry Cd, Co, and Sr, at concentration levels around 0.05 µg/g were quantified simultaneously with P and K around 2000 µg/g and with several other elements in between (Al, Ca, Cu, Fe, Mg, Mn, Pb, and Zn). The relative standard deviation of repeated measurements of samples was around 5 - 6 % for regardless of the concentration of the element. The method was also used for fast screening of the elemental distribution in mice organs (brain, heart, kidney, liver, lung and spleen).