Calcium entry through voltage-gated calcium (CaV) channels is important in diverse cellular processes including neurotransmitter release, gene expression and cardiac pacemaker activity. CaV channels auxiliary CaVβ subunits enhance plasma membrane expression and modify the biophysical properties of CaVα1 subunits. Due to their multi-domain structures - including a conserved SH3-GK 'core' and hypervariable N- and C- terminal domains - CaVβs are also considered to be members of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, and may therefore act as molecular scaffolds both within and outside the CaV channel complex. This project studied the roles of CaVβ N- and C-terminal hypervariable domains in contributing to isoform-specific differences in CaVβ functions both in a) CaV channel complex expression and distribution, and b) interactions with non channel proteins. To analyse such contributions a series of molecular tools were developed to assess the distributions of CaVβs (both within and outside the CaV channel complex) and their interactions with novel potential partner proteins. This involved systematically testing fluorophore- and epitope-tagged CaVβs for co-localisation with both fluorophore-tagged CaV2.2 and a range of myc-tagged potential interaction partners (as quantified either by a 'Membrane Localisation Index' developed during this project or Intensity Correlation Analysis). This approach uncovered much detail about relative isoform specificities of CaVβ non-channel complex protein-protein interactions, however one particularly striking interaction was discovered between CaVβ1b/CaVβ4 and the nuclear protein Heterochromatin 1 γ (HP1γ), where nuclear translocation of CaVβ1b or CaVβ4 was induced upon association with HP1γ. Given the similarity of CaVβ1b and CaVβ4 N termini, a series of CaVβ1b N-terminal chimeras were then created, where the N terminus was exchanged with that of CaVβ3 (which did not interact with HP1γ). Subsequent imaging studies using these chimeras then confirmed that the CaVβ1b N terminus is necessary for co-localisation with HP1γ and subsequent HP1γ mediated CaVβ nuclear uptake. Given that an interaction between the CaVβ3 isoform and Pax6(S) - another nuclear protein - have been reported, where the CaVβ3-Pax6(S) interaction also induces nuclear translocation of both proteins, the CaVβ1b/CaVβ4-HP1γ interaction may represent one of a range of as-yet undiscovered CaVβ1b/gene regulatory protein interactions. As interaction with CaVβ3 suppresses the transcriptional activity of Pax6(S), nuclear targeting may be an important means by which CaVβs modulate gene expression - which in the case of HP1γ interactions may occur via de-repression.

The low power regime has attracted various researchers in the information theory and communication communities to understand the performance limits of wireless systems. Indeed, the energy consumption is becoming one of the major limiting factors in wireless systems. As such, energy-efficient wireless systems are of major importance to the next generation wireless systems designers. The capacity is a metric that measures the performance limit of a wireless system. The study of the ergodic capacity of some fading channels in the low power regime is the main subject of this thesis. In our study, we consider that the receiver has always a full knowledge of the channel state information. However, we assume that the transmitter has possibly imperfect knowledge of the channel state information, i.e. he knows either perfectly the channel or only an estimated version of the channel. Both radio frequency and free space optical communication channel models are considered. The main contribution of this work is the explicit characterization of how the capacity scales as function of the signal-to-noise ratio in the low power regime. This allows us to characterize the gain due to the perfect knowledge compared to no knowledge of the channel state information at the transmitter. In particular, we show that the gain increases logarithmically for radio frequency communication. However, the gain increases as log2(Pavg) or log4(Pavg) for free-space optical communication, where Pavg is the average power constraint imposed to the input. Furthermore, we characterize the capacity of cascaded fading channels and we applied the result to Rayleigh-product fading channel and to a free-space optical link over gamma-gamma atmospheric turbulence in the presence of pointing errors. Finally, we study the capacity of Nakagami-m fading channel under quality of service constraints, namely the effective capacity. We have shown that the effective capacity converges to Shannon capacity in the very low power regime.

Opioids are gold-standard analgesics for pain relief in chronic pain conditions. Paradoxically, chronic opioid use causes an enhanced pain sensitivity termed ‘Opioid-induced hyperalgesia’ (OIH). OIH is a clinically relevant problem associated with the use of opioids. In addition to decreasing quality of life, increased pain from OIH necessitates increasing dosages of analgesics to effectively control the pain, resulting in an increased risk of opioid epidemics, addiction, and overdose. To prevent this clinically important effect, it is necessary to understand how chronic opioid use causes hyperalgesia. Our preliminary studies revealed that synaptic plasticity in the spinal dorsal horn (SDH) is dependent on neuron type in the OIH model and occurs concurrently with hyperalgesia, suggesting central sensitization as a mechanism of OIH. We found that astrocyte ablation blocked mechanical hyperalgesia and neuron type-dependent synaptic plasticity, indicating that astrocytes are critically involved in OIH. Additionally, morphine treatment upregulated IL-1β expression in the SDH in our preliminary experiments. Inhibition of IL-1β prevented OIH and blocked the repeated morphine-induced synaptic plasticity in the SDH, suggesting IL-1β is a key player in the pathogenesis of OIH. Astrocytes and other glial cells are critical in the development and maintenance of neuroinflammatory conditions, such as OIH, through the release of proinflammatory cytokines (PICs), including IL-1β. The mechanosensitive ion channel, Piezo1, was recently found to be upregulated in astrocytes and microglia under LPS-induced inflammatory conditions, and activation of Piezo1 was found to reduce IL-1β expression in LPS-inflamed primary mouse astrocytes. The goal of this study was to investigate the function of Piezo1 as a potential treatment for neuroinflammatory diseases of the CNS in a model of LPS-induced inflammation. In this study, we created a culture cell model of LPS-induced astrocytic neuroinflammation using the C8-S type II astrocyte culture cell line. We used a multi-disciplinary approach of electrophysiology and imaging to assess changes in calcium flux induced by the selective Piezo1 agonist, Yoda1, and mechanosensitive ion channel activity in the LPS-stimulated C8-S culture astrocytes. We found that calcium flux is increased in LPS stimulation and augmented by additional Yoda1 treatment. We also found that LPS stimulation increases mechanosensitive ion currents and stiffens cell membranes using patch-clamp electrophysiology techniques. These results indicate that Piezo1 is likely upregulated in the LPS model of cultured astrocytes, thus mechanosensitive responses are increased. Results from these experiments reveal key information about the mechanical properties of Piezo1 and poise Piezo1 as a promising therapeutic for OIH and other neuroinflammatory diseases caused by astrocytic IL-1β release.

No description available.

Communication systems have remained almost unchanged since the invention of the superheterodyne receiver in 1918 by the US engineer Edwin Armstrong. With the introduction of multiple-input-multiple-output (MIMO) technologies, Index Modulation appears to be the promising technology to revolutionize the traditional radio-frequency (RF) chain. Index modulation is a high-spectrum, energy-efficient, simple digital communication technique that uses the states of the building blocks of a communication system. In this study, we have focused on the use of radiation patterns scattered by antenna arrays or a metasurface as indices that are encoded as data bits. Initially, we explore sets of 𝑁tx transmitting point source antennas located on the XY plane; we assume that every antenna has phase tunability capability. The phase, the position in space, and the size of the array determine the shape of the far-field radiation pattern. Following the antenna excitation, a set of 𝑁rx receiver antennas spread at specific locations of the spherical space measures the incoming power signal, allowing the sampling of the radiation pattern that is demodulated into information bits.This work is focused on the characterization of the measured radiation patterns under different system and channel variables and their direct effect on the Bit Error Rate.

Dans cette thèse, nous nous intéressons aux schémas de codage et de décodage source-canal conjoint des contenus multimédia. Nous montrons comment la redondance laissée par le codeur vidéo peut être exploitée pour réaliser un décodage robuste des séquences transmises sur un lien radio-mobile bruité. grâce au schéma de décodage conjoint proposé, le nombre de paquets corrompus est significativement réduit au prix d'une très légère augmentation du débit. Nous appliquons ensuite ce schéma de décodage robuste à latransmission par descriptions multiples sur une architecture mixte Internet et radio-mobile. Le décodage source-canl conjoint des paquets reçus permet de corriger les erreurs de transmission et d'augmenter ainsi le nombre de paquets utilisés par le décodeur pour compenser les paquets perdus. L'efficacité de ce schéma est étudiée par rapport à un schéma classique basé sur les décisions dures du canal et sur un code correcteur d'erreurs introduisant le même niveau de redondance. Une deuxième partie de la thèse est consacrée à l'étude de schémas de codage source-canal conjoint basés sur une transformation redondante. Deux schémas d'estimation ont été proposés. Dans le premier, nous exploitons la redondance structurée introduite et le caractère borné du bruit de quantification pour construire un estimateur cohérent corrigeant les erreurs de transmission. Dans le deuxième schéma, nous appliquons l'algorithme de propagation de croyances pour évaluer les distributions a posteriori des composantes du signal d'entrée, à partir de sorties bruitées du canal. Nous appliquons alors ces deux schémas pour estimer l'entrée d'un banc de filtres suréchantillonnés. / This thesis aims at proposing and implementing efficient joint source-channel coding and decoding schemes in order to enhance the robustness of multimedia contents transmitted over unreliable networks. In a first time, we propose to identify and exploit the residual redundancy left by wavelet video coders in the compressed bit streams. an efficient joint-source channel decoding scheme is proposed to detect and correct some of the transmission errors occurring during a noisy transmission. This technique is further applied to multiple description video streams transmitted over a mixed architecture consisting of a wired lossy part and a wireless noisy part. In a second time, we propose to use the structured redundancy deliberately introduced by multirate coding systems, such as oversampled filter banks, in order to perform a robust estimation of the input signals transmitted over noisy channels. Two efficient estimation approaches are proposed and compared. The first one exploits the linear dependencies between the output variables, jointly to the bounded quantization noise, in order to perform a consistent estmiation of the source outcome. The second approach uses the belief propagation algorithm to estimate the input signal via a message passing procedure along the graph representing the linear dependencies between the variables. These schemes ares then applied to estimate the input of an oversampled filter bank and their performance are compared.

A number of studies have demonstrated the ability of free fatty acids to activate taste cells and elicit behavioral responses consistent with there being a taste of fat. Here I show for the first time that long chain unsaturated free fatty acid, linoleic acid, depolarizes taste cells and elicits a robust intracellular calcium rise via the activation of transient receptor potential channel type M5. The linoleic acid-induced responses depend on G protein-phospholipase C pathway indicative of the involvement of G protein-coupled receptors in the transduction of fatty acids. Mice lacking transient receptor potential channel type M5 exhibit no preference for and show reduced sensitivity to linoleic acid. Together, these studies show that transient receptor potential channel type M5 plays an essential role in fatty acid transduction and suggest that fat may reflect a bona fide sixth primary taste. Studies to identify the types of taste cells that respond to fatty acids show that both type II and type III taste cells express fatty acid-activated receptors. Fatty acids elicit robust intracellular calcium rise primarily in type II taste cells and a subset of type III taste cells. However, a significant subset of type II taste cells respond to high potassium chloride, which has been broadly used as the indicator for type III taste cells as well, suggesting the expression of voltage-gated calcium channels in these cells. This finding conflicts with previous studies that type II taste cells lack voltage-gated calcium channels. To explore if voltage-gated calcium channels are expressed in subsets of type II taste cells, transgenic mice with type II or III taste cells marked by green fluorescent proteins are used. Results show that a subset of type II taste cells exhibit voltage-gated calcium currents, verifying the expression of voltage-gated calcium channels in these cells. These results question the utility of being able to use high potassium chloride solution to identify unequivocally type III taste cells within the taste buds. A model for the transduction of fatty acids in taste cells consistent with these findings and our previous data is presented.

At the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) convert chemical stimuli into electrical signals. They are heteropentameric membrane protein complexes assembled from four evolutionary related subunits (two α subunits, and one each of the β-, δ-, and ε-subunits), arranged around a central ion-conducting pore, which is regulated by the neurotransmitter acetylcholine. Understanding how the binding of acetylcholine leads to channel opening is of fundamental importance. While it is known that channel opening results from a global conformational change involving the cooperative action of all five subunits, how the subunits achieve this cooperativity is unclear. Our hypothesis is that this subunit cooperation is maintained through coevolution of the subunits, and thus studies of subunit coevolution can provide insight into subunit cooperativity. Using an ancestral reconstruction approach, combined with single-molecule patch clamp electrophysiology, we have begun dissecting the mechanistic consequences of preventing coevolution of the acetylcholine receptor β-subunit. This approach has allowed us to identify new amino acid determinants of acetylcholine receptor function.

Large conductance Ca2+-activated K+ channels (BK channels) are expressed ubiquitously in both excitable and non-excitable cells and are important for a range of physiological functions. BK channels gate K+ efflux in response to concurrent depolarized membrane voltage and increased intracellular Ca2+ to modulate action potential shape and duration in neurons, regulate contractility in smooth muscle, and control fluid secretion by epithelial cells in the airway and gut. In addition, mutations in the human BK channel gene (KCNMA1) are linked to neurological disease, such as epilepsy and paroxysmal dyskinesia. Thus, BK channel modulators may provide treatment avenues for BK channelopathies. It will be important to expand our arsenal of BK channel-selective activators and inhibitors and to grow our understanding of their molecular mechanisms of action. Discovery of new channel modulators will further lead to a greater understanding of BK channel structure and function. To better understand the basic structure-function relationship of BK channel gating in response to increased intracellular Ca2+ concentration, in this work I initially investigate structural determinants of BK channel activation in response to conformational changes following Ca2+ binding. I analyze crystal structures of the BK channel cytosolic Ca2+-sensing domain (CSD), also known as the “gating ring”, formed by the C-terminal domains of each of the four identical pore-forming subunits. In the Ca2+-bound state, N449 from the adjacent subunit contacts the bound Ca2+ ion, forming a “Ca2+ bridge.” Mutating N449 to alanine eliminates this coordinate interaction, and using electrophysiology, I found that BK channels with the N449A mutation exhibit a shift in the voltage required for half maximal activation (V1/2) towards more positive voltages. Using size-exclusion chromatography, I observed that the purified BK channel CSD with the N449A mutation shows reduced gating ring oligomerization in response to Ca2+ compared to the wild-type CSD. To further probe molecular determinants of BK channel gating and increase our arsenal of BK channel gating modulators, I optimized a fluorescence-based high throughput screening approach to discover compounds with BK channel inhibitor activity with 99.73% confidence. Through this approach I discovered that the -opioid receptor agonist, loperamide, is a potent BK channel inhibitor. Loperamide (LOP) reduced the open probability of channels at depolarized voltages, but not at very negative voltages when the voltage-sensor is at rest. I observed a weak voltage dependence of loperamide inhibition, consistent with loperamide binding shallow within the inner cavity to block the channel pore. I quantified the inhibitory effect of LOP using an allosteric model in which LOP blocks conduction through open channels and binds with 45-fold higher affinity to the open state over the closed state. These data suggest that loperamide may represent a new class of “use-dependent,” open channel blockers. Together this work describes an approach to understanding BK channel structure and function with the goal of identifying and developing novel therapeutics for the treatment of BK-related diseases. / Biochemistry

Stress during fish culture can impact growth, physiology and fillet quality. Maintenance of high quality seafood is important to ensure the production of a highly marketable product. The present study assessed how sequential stressors affect the physical, physiological, sensory and quality characteristics of channel catfish (Ictalurus punctatus) fillets. Temperature (25°C or 33°C) and dissolved oxygen (DO, ~2 mg/L or >5 mg/L) were maintained for four weeks, followed by socking and transport stress for a total of 12 treatments. After each stage of stress (environment, socking and transport), physical (length, weight and feed consumption), physiological (hematocrit, plasma cortisol, pH, glucose, lactate, total protein, osmolality and ionic composition) and fillet quality (color, texture, sensory and pathogen load) attributes of the fish and fillets were evaluated. Fillet yield decreased with increasing severity of environmental stress. Overall, increasing stress resulted in decreased feed consumption, growth and fillet yields. A cumulative stress response was identified with regard to circulating cortisol and glucose, which increased with each sequential stress event. Under low oxygen conditions there was a suppression of the stress response. Handling imposed a more pronounced physiological response than environmental conditions. The sequential stressors resulted in a less intensely colored fillet that was less red, which suggests the quality changes imposed are beneficial to the marketability of the fillets. However, increased redness in fish reared under high temperatures and oxygen levels suggest that an increased prevalence of red fillets can be expected. Sensory analysis revealed that fillet flavor was acceptable in all treatments with the severe stress treatment preferred due to lower intensity of less favorable flavor attributes. The changes in flavor were presumably due to fasting of the stressed fish and a subsequent purging of fat stores. As fish progressed through the harvest event, cook loss decreased, tenderness increased and pH increased, indicating that stress induced positive textural changes. Proteomic analysis revealed mainly down-regulation of structural and metabolic proteins, which indicates higher proteolytic activity and an adjustment in energy metabolism in response to stress. The overall effects of chronic environmental conditions and handling highlight the importance of managing for stress in cultured channel catfish.