Dissertations / Theses on the topic 'Single channel recording'

Single-channel recordings (SCR) and single-molecule Fluorescence (SMF) are invaluable tools for studying membrane proteins. Combining these two techniques would allow for structural dynamics to be correlated with function at the single-molecule level. However, the different requirements of SCR and SMF measurements present a long-standing challenge. Droplet interface bilayers (DIBs) are a model membrane system in which parallelisation of such measurements is possible using optical single-channel recording to resolve the conductance of multiple proteins simultaneously. In this project, two approaches were evaluated for obtaining simultaneous measurements in DIBs: the first sought to report on structural changes associated with a gating bacterial mechanosensitive channel, MscS; the second sought to resolve kinetics of the spontaneous β-barrel folding mechanism. For both approaches, functional, labelled protiens were produced and characterised using ensemble measurements to confirm that the state-transition of interest was accompanied by a change in fluorescent signal. Characterisation of MscS in DIBs revealed that control of lateral tension (a requirement to stimulate mechanosensitive channel gating) posed a major experimental challenge, rendering further pursuit of this candidate impossible. However in the case of β-barrel folding, the single-molecule fluorescence alone revealed a rare but fast transition from a membrane-associated unfolded to a folded state. While further work is needed to fully exploit simultaneous measurements, significant progress towards this goal is presented in this work, in particular the development of reagents, experimental procedures and analytical tools.

This thesis describes studies of unitary currents flowing through two different potassium (K) channels present in sarcolemmal vesicles of the frog, Rana temporaria. The ATP-regulated K-channel is described first and the results are divided into three parts. Firstly, ATP applied to the cytoplasmic face of a membrane patch closes the channels in a dose-dependent fashion. Different nucleotides and other metabolic substances are used in order to find chemicals which can substitute for ATP or which regulate its effect. Secondly, the permeability properties of the channel are described. Ion flux is non-independent. Rubidium (Rb) is permeant, and anomalous mole-fraction behaviour is demonstrated in mixtures of K+and Rb+. The final part investigates the kinetic properties of the channel. Both voltage and ATP affect the rate constants regulating transitions between closed and open states of the channel. In particular, ATP causes the channel to occupy a very long-lived closed state. Block of the channel by tetraethylammonium (TEA) ions applied to either membrane surface is described as well. Block by external TEA+ is very fast and it is suggested that the channel cannot close when blocked. The block by internal TEA+ is slower and some evidence of voltage dependency is seen. The delayed rectifier K-channel is investigated. The first of two parts describes Rb+ permeability of the channel and its effect on open and closed times. The Hodgkin-Huxley model of the channel is questioned by the very different times of occupancy of closed states and differing voltage dependencies of the steps leading to opening of the channel. The second part describes block of the channel by externally applied TEA+ and the blocking reaction is shown to be very fast and voltage dependent.

Transient receptor potential vanilloid 2 channel (TRPV2) is a Ca2+-permeable ion channel that is highly expressed in leukocytes but is also present in skeletal and cardiac muscle and endocrine cells. The TRPV2 function is implicated in a number of physiological processes, including bacterial phagocytosis, pro-inflammatory cytokine production, cardiac hypertrophy, and cancer development. TRPV2 knockout mice exhibit a high incidence of perinatal mortality, arguing that the channel plays essential roles in physiology. Despite the importance of TRPV2 for normal homeostasis, the mechanisms that control TRPV2 gating in response to pharmacological agonists, heating, membrane stretch, bioactive lipids and reactive oxygen species (ROS) remain poorly understood. Here we demonstrate that TRPV2 is functionally expressed in microglia (i.e., ‘brain macrophages’) and the microglia-like BV-2 cell line, and demonstrate that the gating of an endogenous TRPV2-like conductance is positively modulated by the bacterial toxin lipopolysaccharide (LPS), which is known to cause pro-inflammatory (M1) activation and increase ROS production by NADPH oxidase. To determine how TRPV2 gating is modulated by ROS, we recorded single channel activity in inside-out patches excised from HEK-293 cells expressing GFP-rTRPV2. Unitary currents elicited by the TRPV2 agonist 2-aminophenyl borinate (2-APB) or cannabidiol (CBD) are linear in monovalent recording solutions and give rise to an estimated unitary conductance of ~100pS, which is similar to TRPV1 but significantly smaller than TRPV3. Intriguingly, we find that although TRPV2 is insensitive to ROS (in the form of exogenously applied H2O2) alone, apparent open probability is synergistically enhanced when H2O2 is applied together with CBD. We identify two intracellular Cys residues that are necessary for TRPV2 responses to H2O2 sensitivity and find that these residues are located close to one another, albeit in different subunits, in the TRPV2 structure, suggesting that ROS promote the formation of an inter-subunit disulfide bond that alters sensitivity to pharmacological agonists. We hypothesize that ROS-dependent modulation of TRPV2 activity may be an important contributor to pro-inflammatory activation of microglia underline central nervous system diseases and that TRPV2 antagonism could be a useful therapeutic strategy in the treatment of neuroinflammation.

Hybrid technologies, thanks to the convergence of integrated microelectronic devices and new class of microfluidic structures could open new perspectives to the way how nanoscale events are discovered, monitored and controlled. The key point of this thesis is to evaluate the impact of such an approach into applications of ion-channel High Throughput Screening (HTS)platforms. This approach offers promising opportunities for the development of new classes of sensitive, reliable and cheap sensors. There are numerous advantages of embedding microelectronic readout structures strictly coupled to sensing elements. On the one hand the signal-to-noise-ratio is increased as a result of scaling. On the other, the readout miniaturization allows organization of sensors into arrays, increasing the capability of the platform in terms of number of acquired data, as required in the HTS approach, to improve sensing accuracy and reliabiity. However, accurate interface design is required to establish efficient communication between ionic-based and electronic-based signals. The work made in this thesis will show a first example of a complete parallel readout system with single ion channel resolution, using a compact and scalable hybrid architecture suitable to be interfaced to large array of sensors, ensuring simultaneous signal recording and smart control of the signal-to-noise ratio and bandwidth trade off. More specifically, an array of microfluidic polymer structures, hosting artificial lipid bilayers blocks where single ion channel pores are embededed, is coupled with an array of ultra-low noise current amplifiers for signal amplification and data processing. As demonstrating working example, the platform was used to acquire ultra small currents derived by single non-covalent molecular binding between alpha-hemolysin pores and beta-cyclodextrin molecules in artificial lipid membranes.

Les signaux multicanaux sont des signaux captés à travers plusieurs canaux ou capteurs, portant chacun un mélange de sources, une partie desquelles est connue alors que le reste des sources reste inconnu. Les méthodes à l’aide desquelles l’isolement ou la séparation des sources est accomplie sont connues par les méthodes de séparation de sources en général, et si le degré d’inconnu est large, par la séparation aveugle des sources (SAS). Cependant, la SAS appliquée aux signaux multicanaux est en fait plus facile de point de vue mathématique que l’application de la SAS sur des signaux monocanaux, ou un seul capteur existe et tous les signaux arrivent au même point pour enfin produire un mélange de sources inconnues. Tel est le domaine de cette thèse. Nous avons développé une nouvelle technique de SAS : une combinaison de plusieurs méthodes de séparation et d’optimisation, basée sur la factorisation non-négative des matrices (NMF). Cette méthode peut être utilisée dans de nombreux domaines comme l’analyse des sons et de la parole, les variations de la bourse, et les séismographes. Néanmoins, ici, les signaux de force de réaction de terre verticaux (VGRF) monocanaux d’un groupe d’athlètes coureurs d’ultra-marathon sont analysés et séparés pour l’extraction du peak passif du peak actif d’une nouvelle manière adaptée à la nature de ces signaux. Les signaux VGRF sont des signaux cyclo-stationnaires caractérisés par des double-peaks, chacun étant très rapide et parcimonieux, indiquant les phases de course de l’athlète. L’analyse des peaks est extrêmement importante pour déterminer et prédire la condition du coureur : problème physiologique, problème anatomique, fatigue etc. De plus, un grand nombre de chercheurs ont prouvé que l’impact du pied postérieur avec la terre d’une manière brutale, l’analyse de ce phénomène peut nous ramener à une prédiction de blessure interne. Ils essayent même d’adopter une technique de course - Non-Heel-strike Running (NHS) - par laquelle ils obligent les coureurs à courir sur le pied-antérieur seulement. Afin d'étudier ce phénomène, la séparation du peak d’impact du VGRF permet d'isoler la source portant les informations patho-physiologiques et le degré de fatigue. Nous avons introduit de nouvelles méthodes de prétraitement et de traitement des signaux VGRF pour remplacer le filtrage de bruit traditionnel utilisé partout, et qui peut parfois détruire les peaks d’impact qui sont nos sources à séparer, base sur le concept de soustraction spectrale pour le filtrage, utilisée avec les signaux de parole, après l’application d’un algorithme d’échantillonnage intelligent et adaptatif qui décompose les signaux en pas isolés. Une analyse des signaux VGRF en fonction du temps a été faite pour la détection et la quantification de la fatigue des coureurs durant les 24 heures de course. Cette analyse a été accomplie au domaine fréquentiel/spectral où nous avons détecté un décalage clair du contenu fréquentiel avec la progression de la course indiquant la progression de la fatigue. Nous avons défini les signaux cyclosparse au domaine temporel, puis traduit cette définition à son équivalent au domaine temps-fréquence utilisant la transformée Fourier a court-temps (STFT). Cette représentation a été décomposée à travers une nouvelle méthode que l’on a appelé Cyclosparse Non-negative Matrix Factorisation (Cyclosparse-NMF), basée sur l’optimisation de la minimisation de la divergence Kullback-Leibler (KL) avec pénalisation liée à la périodicité et la parcimonie des sources, ayant comme but final d’extraire les sources cyclosparse du mélange monocanal appliquée aux signaux VGRF monocanaux. La méthode a été testée sur des signaux analytiques afin de prouver l’efficacité de l’algorithme. Les résultats se sont avéré satisfaisants, et le peak impact a été séparé du mélange VGRF monocanal
The purpose of the presented work is to develop a customized Single-channel Blind Source Separation technique that aims to separate cyclostationary and transient pulse-like patterns/sources from a linear instantaneous mixture of unknown sources. For that endeavor, synthetic signals of the mentioned characteristic were created to confirm the separation success, in addition to real life signals acquired throughout an experiment in which experienced athletes were asked to participate in a 24-hour ultra-marathon in a lab environment on an instrumented treadmill through which their VGRF, which carries a cyclosparse Impact Peak, is continuously recorded with very short discontinuities during which blood is drawn for in-run testing, short enough not to provide rest to the athletes. The synthetic and VGRF signals were then pre-processed, processed for Impact Pattern extraction via a customized Single-channel Blind Source Separation technique that we termed Cyclo-sparse Non-negative Matrix Factorization and analyzed for fatigue assessment. As a result, the Impact Patterns for all of the participating athletes were extracted at 10 different time intervals indicating the progression of the ultra-marathon for 24 hours, and further analysis and comparison of the resulting signals proved major significance in the field of fatigue assessment; the Impact Pattern power monotonically increased for 90% of the subjects by an average of 24.4 15% with the progression of the ultra-marathon during the 24-hour period. Upon computation of the Impact Pattern separation algorithm, fatigue progression showed to be manifested by an increase in reliance on heel-strike impact to push to the bodyweight as a compensation for the decrease in muscle power during propulsion at toe-off. This study among other presented work in the field of VGRF processing forms methods that could be implemented in wearable devices to assess and track runners’ gait as a part of sports performance analysis, rehabilitation phase tracking and classification of healthy vs. unhealthy gait

Alpha-hemolysin is a pore-forming toxin secreted by pathogenic Staphylococcus aureus. Its spontaneous oligomerization and assembly into a trans-bilayer beta-barrel pore is a model for the assembly of many other pore-forming toxins. It is studied here in vitro as a means to probe general membrane protein oligomerization and lipid bilayer insertion. This thesis details the results of experiments to develop and implement a novel in vitro lipid bilayer system, Droplet-on-Hydrogel Bilayers (DHBs) for the single-molecule imaging of alpha-hemolysin assembly. Chapter 2 describes the development of DHBs and their electrical characterization. Experiments show the detection of membrane channels in SDS-PAGE gels post-electrophoresis and DHBs use as a platform for nanopore stochastic sensing. Chapter 3 describes the engineering and characterization of fluorescently-labelled monomeric alpha-hemolysin for use in protein assembly imaging experiments described in Chapter 6. Chapter 4 describes the characterization of DHB lipid fluidity and suitability for single-molecule studies of membrane protein diffusion. In addition, a novel single-particle tracking algorithm is described. Chapter 5 describes experiments demonstrating simultaneous electrical and fluorescence measurements of alpha-hemolysin pores embedded within DHBs. The first multiple-pore stochastic sensing in a single-lipid bilayer is also described. Chapter 6 describes experiments studying the assembly of alpha-hemolysin monomers in DHBs. Results show that alpha-hemolysin assembles rapidly into its oligomeric state, with no detection of long-lived intermediate states.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1995.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 110-137).

博士
國立成功大學
資訊工程學系碩博士班
101
Sleep diseases, such as insomnia and obstructive sleep apnea, seriously affect patients’ quality of life. For diagnosis, polysomnographic (PSG) recordings are most usually taken for sleep stage scoring. However, manual scored by well-trained expert which is a time-consuming and subjective process. Many multi-channel-based automatic sleep staging methods have been developed. These approaches have many drawbacks. For example, the large amount of wires connections for conventional PSG often cause sleep interference and not self-applicable. Sleep EEG signals is the main basis of the manual sleep scoring rules in clinical diagnosis. Therefore, we develop an automatic sleep staging method based on single-channel sleep EEG signals. We utilize multiscale entropy and autoregressive model coefficients to analyze the 32 all-night sleep EEG recordings from 32 healthy adult and the linear discriminant analysis (LDA) was utilized to classify each epoch into five sleep stages. After classifying the sleep stage by LDA, some misclassified epochs can be corrected according to the temporal contextual information. We utilize the smoothing process to smooth and fine-tune the results of the classifier. The average accuracy and kappa coefficient of the proposed method applied to 16 all-night (PSG) recordings compared with the manual scorings can reach 87.15 % and 0.81, respectively. The results are also better than the range in the inter-score agreement. Compared to EEG, electrodes placement of EOG recording is around the eyes, and EOG signals are also coupling some of sleep characteristics of EEG signals. Therefore, EOG has the ease of use in the home and can be applied to sleep scoring. For the applicability of home care, we also develop an automatic sleep staging method based on single-channel sleep EOG signals. In addition to multiscale entropy and autoregressive model coefficients, we also propose a new feature, multiscale line length, to enhance the sensitivity of rapid eye movement (REM) stage. The average accuracy and kappa coefficient of the proposed method compared with the manual scorings can reach 83.33 % and 0.75, respectively. The performance of our proposed method is the best in currently automatic sleep staging methods based on single-channel sleep EOG signals. So we extend the automatic sleep staging method to the application of sleep environmental control at home. In the appendix, we also show the applicability of our automatic sleep scoring method. Recently, many systems or products to support healthy sleep by monitoring the sleep environment or users’ activities and sleep quality have been developed. However, actively on-line adjusting conditions of the sleep environment according to sleep stages of the user are rare. Light is the main factor in sleep environment. We use our proposed sleep scoring method combined with a light-control algorithm based on sleep stage to develop a closed-loop human–computer interaction (HCI) system. With the experimental results of three subjects, the feasibility of controlling the light in sleep environments is demonstrated. In the future, we will consider more sleep environmental factors for the healthy, comfortable and safe sleep quality and environment. In addition, many previous studies had pointed out that sleep and memory consolidation, learning, or performance improvement is very relevant. However, these related experiments often require experts and laboratory personnel to monitor all night, it takes a lot of manpower and time-consuming. To save more time and manpower costs, we will apply our method to related studies and experiments for automation in the future.