AlGaN/GaN sensors for direct monitoring of fluids and bioreactions

AlGaN/GaN based pH-sensors have been characterized and further developed for the in situ monitoring of cell reactions. Generally, good proliferation of different cell lines was observed on AlGaN and GaN surfaces without using any kind of thin films of organic material for improving of the cellular adhesion and biocompatibility. NG108-15 nerve cells were chosen for the investigation of the sensor response on cell activity. In an open setup with contact to normal atmosphere, the monitoring of the spontaneous cell activity (“breathing”) was recorded. By titration in complex electrolytes, it was demonstrated that these sensors are able to monitor complex cell reactions on different neuroinhibitors. Numerical simulations as well as simplified analytical calculations of ion fluxes give strong evidence that the signal in the cell-transistor coupling experiments is primarily generated by the Na+ flux. In conclusion, the AlGaN/GaN-ISFETs show stable operation under physiological conditions, exhibit a very good signal resolution and are suitable for long-time monitoring of cell reactions on different stimuli.In dieser Arbeit wurden AlGaN/GaN-Heterostrukturen, die ein hohes Potenzial für pH-Sensoren aufweisen, charakterisiert und weiterentwickelt für die elektronische Erfassung von Zellreaktionen. Dazu wurden NG108-15 Nervenzellen auf den Sensoroberflächen kultiviert und deren Antwort auf Stimulierung mit verschiedenen Neuroinhibitoren aufgezeichnet. Zunächst wurde ein Messaufbau für das Erfassen extrazellularer Potenzialänderungen entworfen und das bestehende Chipdesign sowie die Herstellungstechnologie weiterentwickelt. Für die Auswahl optimaler Sensoren für die Transistor-Zell-Kopplung wurden sowohl mittels PIMBE und MOCVD gewachsene Heterostrukturen charakterisiert bezüglich ihrer elektronischen Transporteigenschaften und ihres Verhaltens als pH-Sensor. Auf AlGaN- und GaN-Oberflächen konnte eine sehr gute Kultivierung verschiedener Zelllinien erzielt werden ohne die sie sonst übliche Verwendung organischer Zwischenschichten zur Erhöhung von Adhäsion (z.B. Fibroplasten). Der Einfluss verschiedener Technologie- und sensorrelevanter Behandlungsschritte auf die Oberflächeneigenschaften der AlGaN/GaN-Sensoren wurde untersucht und die Medienstabilität bzw. Wechselwirkungen wurden analysiert. In einem offenen Setup mit Gasaustausch zur Umgebung wurde eine spontane Zellaktivität erfasst ("Zellatmung"), die in einem abgeschlossenen Setup aufgrund des reduzierten Gasaustausches nicht auftrat. Weiterhin wurde die Empfindlichkeit des Sensors auf Potenzialänderungen durch Na+ and K+ Ionen und deren Reaktionen mit Neurotoxinen bestätigt. Durch Titration in komplexe Elektrolyte und durch Kultivierung von NG108-15 Nervenzellen auf der Sensoroberfläche wurde demonstriert, dass die Sensoren in der Lage sind, komplexe Zellreaktionen zu erfassen. Berechnungen mit Hilfe von Simulationen und vereinfachten analytischen Beschreibungen für die Ionenflüsse belegten, dass bei der Zell-Transistor-Kopplung das Sensorsignal im Wesentlichen durch die Na+ Flüsse erzeugt wird. Die experimentellen Beobachtungen und die theoretischen Modellierung zeigte dafür eine gute Übereinstimmung. Zusammenfassend wurde in dieser Arbeit gezeigt, dass AlGaN/GaN-ISFETs stabil unter physiologischen Bedingungen arbeiten, sehr gute Signalauflösung ermöglichen und für Langzeitmessungen mit lebenden Zellen geeignet sind

11th European Headache Federation Congress jointly with 31st Congress of the Italian Society for the Study of Headaches : Rome, Italy. 01-03 December 2017

. Aims of the study were explore the relationship between peripheral chromatic and central visual dysfunction evaluating also the presence of functional receptor impairment in patients with migraine, with and without aura examined interictally

Characterization of mechanisms of resistance to common insecticides in noctuid pest species and resistance risk assessment for the new lepidopteran specific compound flubendiamide

Noctuid species, such as the beet armyworm Spodoptera exigua, the cotton bollworm Helicoverpa armigera and the tobacco budworm Heliothis virescens, are well-known pests in many agricultural cropping systems worldwide. The extensive and widespread use of insecticides against these species has led to the development of resistance against almost all commercially used compounds. The focus of this thesis was on the novel lepidopteran specific compound flubendiamde, to get a detailed overview about the efficacy using different kinds of bioassays for a resistance risk assessment. On the other hand, the underlying possible resistance mechanisms of a S. exigua strain from southern Spain was investigated on the toxicological, biochemical, pharmacokinetic and molecular level. The basic of all further experiments was two bioassay techniques, a leaf-disc spray application and an artificial diet bioassay, on 2nd instar larvae. Flubendiamide and 11 competitors with different mode of actions were tested on three susceptible laboratory strains (HELI-AR, HELI-VI, SPOD-EX S) and one field strain from Spain (SPOD-EX E-98). The aim was to get baseline susceptibilities of the three noctuid species and additionally the resistance profile of strain SPOD-EX E-98 against the different insecticides. The results of the two bioassay techniques were comparable with each other, emamectin shown the highest efficacy followed by low EC50-values for flubendiamide, deltamethrin and indoxacarb. The robust artificial diet bioassay was used for a worldwide resistance monitoring for flubendiamide, and resulting in a mean efficacy of 93% in 18 test populations. An interesting cross-resistance pattern against insecticides with different mode of actions demonstrated the S. exigua strain SPOD-EX E-98, and can be called as ?multi-resistant?. High resistance to three classes of insecticides, pyrethroids, benzoylphenyl ureas and carbamates/organophosphates, was detected in this strain, also moderate resistance levels to endosulfan and indoxacarb. The in vivo application study with the synergist PBO (monooxygenase inhibitor) has shown an additionally participation of this enzyme system towards the deltamethrin resistance in this strain. Additionally, PBO and two esterase inhibitors had no effect to the resistance level of triflumuron (benzoylphenyl urea) and so target-site resistance is likely in this case. Using molecular biological methods, a point mutation (kdr) in the voltage-gated sodium channel of the resistant strain SPOD-EX E-98 was detected, this channel is the target of the pyrethroids. This target-site resistance is particularly responsible for the extremely high resistance factor (~900) of the pyrethroid deltamethrin detected in the bioassays. In order to investigate the pharmacokinetic profiles of deltamethrin, triflumuron and flubendiamide in S. exigua larvae radiolabelled compounds were used. No differences in penetration or excretion of the compounds were observed between the two strains. This suggests that physiological changes have not influence as a possible mechanism of deltamethrin and triflumuron resistance. Four biochemical markers, carboxylesterases (CEs), cytochrome P450-dependent monooxygenases (monooxygenases), glutathione S-transferases (GSTs) and acetylcholinesterases (AChEs), were investigated in both S. exigua strains. These enzyme systems are known to be linked with metabolic detoxification/resistance to diverse insecticides. With the exception of GST, in the multi-resistant SPOD-EX E-98 strain was found significantly higher enzyme activities to the other three marker enzymes. The higher CE activity in the multi-resistant strain SPOD-EX E-98 was further investigated, using an nPAGE to obtaining the iso-enzyme banding patterns of both strains. In comparison, the strain SPOD-EX E-98 exhibited an additional thick band and it was not possible to inhibiting this band in vitro through the esterase inhibitor DEF. It is possible that this result is involved in resistance, but only further investigations could be clarify the exact role of the additional band in this resistant strain. The AChE of both S. exigua strains was used for in vitro inhibition studies. The resistant strain SPOD-EX E-98 was insensitive against several organophosphates and carbamates compared with strain SPOD-EX S. Only one exception was detected, the AChE of the resistant strain was more sensible to carbofuran than the enzyme from strain SPOD-EX S. This phenomenon of ?negative cross-insensitivity? is generally correlated with an altered substrate binding site of the AChE. Further investigations of kinetic parameters exhibited a higher turnover of the substrate in strain SPOD-EX E-98. The bioassays confirmed these results and so the resistance mechanism of the two chemical classes is an altered AChE in the resistant strain.Die phytophagen Larven der Familie der Noctuidae, wie z.B. Spodoptera exigua, Helicoverpa armigera und Heliothis virescens sind weltweit als wirtschaftlich bedeutende Schadinsekten bekannt und befinden sich in einer Resistenzproblematik gegenüber fast allen kommerziell erhältlichen Insektiziden. Der Fokus dieser Arbeit lag auf einem neuen insektiziden Wirkstoff, Flubendiamide, und die Einschätzung über das Risiko einer möglichen Resistenzentwicklung. Verschiedene Arten von toxikologischen Untersuchungen wurden eingesetzt um einen Überblick zu bekommen. Des Weiteren lag das Interesse auf einem S. exigua Stamm aus Südspanien, dessen mögliche Resistenzmechanismen auf biochemischer, pharmakokinetischer und molekularer Ebene untersucht wurden. Die Grundlage für alle Versuche bildeten zwei Biotestsysteme, eine Sprühapplikation auf Blattmaterial und ein Kunstfutter-Biotest. Flubendiamide und 11 Konkurrenzinsektizide mit unterschiedlichen Wirkmechanismen wurden an drei sensiblen Laborstämmen (HELI-AR, HELI-VI, SPOD-EX S) und einem Freilandstamm aus Spanien (SPOD-EX E-98) getestet. Ziel war es Basisdaten und ein mögliches Resistenzprofil für den Stamm SPOD-EX E-98 herauszuarbeiten, wobei beide Testsysteme vergleichbare Ergebnisse erzielten. Emamectin zeigte die beste Wirkung dicht gefolgt von niedrigen EC50-Werten für Flubendiamide, Deltamethrin und Indoxacarb für die drei Laborstämme. Der robuste Kunstfutter-Biotest kam zusätzlich in einem weltweiten Resistenz-Monitoring mit Flubendiamide zum Einsatz, wobei ein durchschnittlicher Wirkungsgrad von 93% bei 18 Testpopulationen erreicht wurde. Der S. exigua Stamm SPOD-EX E-98 zeigte sich interessanterweise kreuzresistent gegenüber verschiedenen Insektiziden mit unterschiedlichen Wirkmechanismen und kann somit als "multiresistent" bezeichnet werden. Eine starke Resistenz gegenüber den drei Insektizidklassen, Pyrethroide, Benzoylharnstoffe und Carbamate/Organophosphate wurde für diesen Stamm festgestellt, zusätzlich moderate Resistenzfaktoren für die Wirkstoffe Endosulfan und Indoxacarb. Die in vivo Versuche mit dem Synergisten PBO (Monooxygenase-Inhibitor) zeigten eine zusätzliche Beteiligung dieses Enzymsystems an der vorhandenen Deltamethrin-Resistenz dieses Stammes. Zusätzlich konnte kein Effekt, sowohl mit PBO als auch mit Esterase-Inhibitoren, auf den Resistenzlevel von Triflumuron (Benzoylharnstoff)erzielt werden. Daher muss in diesem Fall eine Target-Site-Resistenz angenommen werden. Mit molekularbiologischen Methoden konnte eine Punktmutation (kdr) im spannungsabhängigen Natriumkanal, dem Wirkort der Pyrethroide, des Stammes SPOD-EX E-98 ermittelt werden. Diese Target-Site-Resistenz ist teilweise fürden extrem hohen Resistenzfaktor(~900)des Pyrethroids Deltamethrin verantwortlich. In weiteren Experimenten wurde das pharmakokinetische Profil von radioaktiv markierten Wirkstoffen (Deltamethrin, Triflumuron, Flubendiamide) in S. exigua Larven nachvollzogen. Zwischen den beiden S. exigua Stämmen ergab sich keine veränderte Aufnahme oder Exkretion der Wirkstoffe, sodass gegenüber Deltamethrin und Triflumuron eine Veränderung auf physiologischer Ebene als Resistenzmechanismus auszuschließen ist. In den zwei S.exigua Stämmen wurden vier biochemische Marker, Carboxylesterasen (CEs), Monooxygenasen, Glutathion S-Transferasen (GSTs) und Acetylcholinesterasen (AChEs), untersucht, von denen bekannt ist, dass sie als Resistenzfaktoren in Frage kommen. Das Ergebnis zeigte, außer bei GSTs, in allen anderen drei Enzymsystemen signifikant erhöhte Aktivitäten des multi-resistenten Stammes SPOD-EX E-98. Zusätzlich zu der erhöhten CE-Aktivität des Stammes SPOD-EX E-98 konnten mittels nPAGE die Isoenzym-Bandenmuster für beide S. exigua Stämme bestimmt werden. Im Vergleich fiel eine zusätzliche starke Bande im Stamm SPOD-EX E-98 auf, die auch nicht durch den Esterase-Inhibitor DEF inhibiert werden konnte. Diese zusätzliche Bande kann möglicherweise als Resistenzfaktor in dem resistenten Stamm eine Rolle spielen, hierzu sind aber noch weitere Untersuchungen nötig. In vitro Inhibierungsstudien von AChE aus beiden S. exigua Stämmen zeigten das der Stamm SPOD-EX E-98 unempfindlicher gegenüber Organophosphaten und Carbamaten ist, nur in Bezug auf Carbofuran zeigte sich die AChE aus dem Stamm SPOD-EX E-98 sensibler als das Enzym aus dem Stamm SPOD-EX S. Dieses Phänomen der "negativen Kreuzinsensitivität" wird generell mit einer veränderten Substratbindestelle der AChE in Verbindung gebracht, dieses konnte bei der Untersuchung der kinetischen Parameter bestätigt werden, in Form einer erhöhten Substratumsetzung in dem Stamm SPOD-EX E-98. Dieses Ergebnis spiegelt sich auf Biotestebene wieder und kann für beide Wirkstoffgruppen als Resistenzmechanismus angesehen werden

Advanced Signal Processing in Wearable Sensors for Health Monitoring

Smart, wearables devices on a miniature scale are becoming increasingly widely available, typically in the form of smart watches and other connected devices. Consequently, devices to assist in measurements such as electroencephalography (EEG), electrocardiogram (ECG), electromyography (EMG), blood pressure (BP), photoplethysmography (PPG), heart rhythm, respiration rate, apnoea, and motion detection are becoming more available, and play a significant role in healthcare monitoring. The industry is placing great emphasis on making these devices and technologies available on smart devices such as phones and watches. Such measurements are clinically and scientifically useful for real-time monitoring, long-term care, and diagnosis and therapeutic techniques. However, a pertaining issue is that recorded data are usually noisy, contain many artefacts, and are affected by external factors such as movements and physical conditions. In order to obtain accurate and meaningful indicators, the signal has to be processed and conditioned such that the measurements are accurate and free from noise and disturbances. In this context, many researchers have utilized recent technological advances in wearable sensors and signal processing to develop smart and accurate wearable devices for clinical applications. The processing and analysis of physiological signals is a key issue for these smart wearable devices. Consequently, ongoing work in this field of study includes research on filtration, quality checking, signal transformation and decomposition, feature extraction and, most recently, machine learning-based methods

Pesticide Impact on Honeybees Declines and Emerging Food Security Crisis

Bee crisis is threatening worldwide food security. Pesticides are extensively used in the agricultural zone. Unfortunately, these pesticides cause severe toxicity toward pollinators than the target pests such as honeybees. This review summarizes the different studies related to pesticide hazards of bees. This paper reported risks of pesticides neurological and physiological poisoning toward honeybees. Pesticides act as poison and ruin vital functions involved in leaning and cognition, behavior and, the body physiological mechanisms. Many laboratory and field research data evaluated the lethal and sub-lethal poisoning on bee foraging dance, learning, and memory abilities of honeybees. Insecticide residues are detected in bee bodies and LD50 and LC50 values evaluated. It is also studied that in honeybees systemic insecticide residues and, its metabolite adulterated in their body during foraging activities. Similarly, pesticide-contaminated food stored in a hive consumed continuously by honeybees may cause sub-lethal toxicity effects. Which causes anomalous bee social behavior and ultimately leads to colony collapse disorder. If population of pollinator decline it will disturb the food chain and leads to food crisis. This review emphasized causes of bee decline with the emergence of pesticides in agricultural domains

Multimodal Wearable Sensors for Human-Machine Interfaces

Certain areas of the body, such as the hands, eyes and organs of speech production, provide high-bandwidth information channels from the conscious mind to the outside world. The objective of this research was to develop an innovative wearable sensor device that records signals from these areas more conveniently than has previously been possible, so that they can be harnessed for communication. A novel bioelectrical and biomechanical sensing device, the wearable endogenous biosignal sensor (WEBS), was developed and tested in various communication and clinical measurement applications. One ground-breaking feature of the WEBS system is that it digitises biopotentials almost at the point of measurement. Its electrode connects directly to a high-resolution analog-to-digital converter. A second major advance is that, unlike previous active biopotential electrodes, the WEBS electrode connects to a shared data bus, allowing a large or small number of them to work together with relatively few physical interconnections. Another unique feature is its ability to switch dynamically between recording and signal source modes. An accelerometer within the device captures real-time information about its physical movement, not only facilitating the measurement of biomechanical signals of interest, but also allowing motion artefacts in the bioelectrical signal to be detected. Each of these innovative features has potentially far-reaching implications in biopotential measurement, both in clinical recording and in other applications. Weighing under 0.45 g and being remarkably low-cost, the WEBS is ideally suited for integration into disposable electrodes. Several such devices can be combined to form an inexpensive digital body sensor network, with shorter set-up time than conventional equipment, more flexible topology, and fewer physical interconnections. One phase of this study evaluated areas of the body as communication channels. The throat was selected for detailed study since it yields a range of voluntarily controllable signals, including laryngeal vibrations and gross movements associated with vocal tract articulation. A WEBS device recorded these signals and several novel methods of human-to-machine communication were demonstrated. To evaluate the performance of the WEBS system, recordings were validated against a high-end biopotential recording system for a number of biopotential signal types. To demonstrate an application for use by a clinician, the WEBS system was used to record 12‑lead electrocardiogram with augmented mechanical movement information

Acetylcholine in Central Cardiorespiratory Regulation in Health and Depression

Circulation and breathing movements that are essential for life are regulated by neurons in the hypothalamus and lower brainstem. Activity of these neurons is regulated by peripheral afferent and higher order inputs that release a diverse array of amino acids, amines and peptides. In this thesis we investigated the role of the neurotransmitter acetylcholine (ACh) and its receptors in regulation of cardiorespiratory homeostasis. Secondly, we determined whether or not genetic disturbances in regulation of acetylcholine receptor sensitivity affect central control of circulation, body temperature or respiration. The findings presented in Chapter 3 reveal a novel functional role of ACh and G-protein coupled muscarinic receptor (mAChR) activation in the rostral ventrolateral medulla (RVLM). We showed for the first time that some non-C1 RVLM neurons express mRNA for the M2 or M3 receptor; however, both C1 and enkephalinergic RVLM neurons were closely apposed by c holinergic terminals positive for the vesicular acetylcholine transporter (vAChT). Physiological studies demonstrated that activation of mAChR within the RVLM in anaesthetised rats increases arterial pressure and sympathetic nerve activity and has differential effects on major cardiorespiratory reflexes: RVLM mAChR activation resets the sympathetic baroreflex to higher arterial pressures and increases its gain and, concomitantly, attenuates excitatory reflexes evoked by peripheral chemoreceptor or somatic afferent stimulation. Retrograde tracing from the RVLM combined with vAChT immunoreactivity showed that neurons in the pedunculopontine tegmental nucleus (PPT) are the sole source of cholinergic input to the RVLM. The PPT-RVLM pathway appears to be part of a central command circuit concerned with adjusting circulatory function appropriate to increased muscle activity. These data support the notion that activation of specific neurotransmitter receptors in the RVLM encodes fu nctional specificity in control of sympathetic outflow and r! eflex fu nction. The extent to which genetic variations in central mAChR sensitivity influence autonomic function is unknown. Flinders Sensitive Line (FSL) rats were bred from Sprague Dawley (SD) rats for exaggerated behavioural and hypothermic responses to cholinesterase inhibitors and direct-acting mAChR agonists. A control genetic counterpart, the Flinders Resistant Line (FRL), was also bred in parallel for reduced responses to cholinergic agonists. The findings of Chapter 5 showed for the first time that FSL rats exhibit an increase in M2 and reduction in M3 receptor expression in the rostral medulla, suggesting that cholinergic signalling in this region may be altered. However, alterations of mAChR expression specific to FSL rats were restricted to this area and there were no changes in cerebellar expression of mAChR in any strain. Physiological studies showed that conscious or anaesthetised FSL rats were more sensitive to thermoregulatory responses to central mAChR a ctivation (ie hypothermia and increase in cutaneous blood flow); whereas pressor responses were reduced compared to SD and FRL rats. The increase in sympathetic activity and depression of respiration evoked by central mAChR activation was unchanged and attenuated, respectively, in FSL rats compared to control strains. These findings indicate that mAChR involved in control of different autonomic functions are regulated independently at the genetic and / or post-transcriptional level. The findings of Chapters 4 and 6 reveal a novel effect of breeding for cholinergic hypersensitivity in FSL rats on control of vagal and sympathetic outflow. Spectral analysis of blood pressure recordings in conscious FSL rats showed a reduction in total and high frequency power of heart rate variability (HRV), an increase in the LF/HF ratio and reduction in baroreflex sensitivity (BRS) compared to controls. These changes reflect a reduction in reflex vagal input and relative predominan ce of sympathetic input to the sinus node in FSL rats. Under! urethan e anaesthesia, FSL rats had a higher heart rate and exhibited lower gain of baroreflex control of splanchnic sympathetic nerve activity (SNA). Moreover, FSL rats were more susceptible to ventricular arrhythmias during infusion of the cardiac glycoside ouabain under anaesthesia compared to controls. These data indicate that FSL rats exhibit impaired reflex regulation of vagal and sympathetic outflow that could underlie increased vulnerability to arrhythmia seen in this strain. The precise brain regions and neurotransmitters that underlie autonomic disturbances seen in FSL rats are unclear. As well as muscarinic hypersensitivity, FSL rats also exhibit increased sensitivity to nicotine, serotonin and dopamine. Multiple chemical sensitivities in FSL rats may arise from functional interactions with mAChR or changes in common intracellular regulatory or signalling pathways. FSL rats exhibit a number of behavioural and somatic abnormalities consistent with clinical depre ssion, including reduced motivated behaviour and sleep and psychomotor disturbances. These symptoms are also alleviated by treatment with antidepressants, suggesting that similar neurochemical abnormalities may underlie behavioural disturbances seen in FSL rats and human depression. Symptoms of depression are an emerging risk factor in the development of cardiovascular disease and are associated with increased risk of dying from a cardiac-related event. A reduction in HRV and BRS in depressed patients has been widely reported and is considered to be a key substrate predisposing to arrhythmia in this patient group. In this thesis we demonstrate for the first time that FSL rats exhibit similar autonomic abnormalities to those reported in human depression and are more vulnerable to ouabain-induced ventricular arrhythmias. These findings suggest that biological factors predisposing to autonomic dysfunction and arrhythmia in FSL rats could also operate in human depression. This m ay involve altered neurotransmission in cardiovascular brain! regions , or inappropriate regulation of cardiovascular function by arousal or motor control pathways. Overall, this thesis provides novel insights into cholinergic mechanisms that regulate cardiorespiratory homeostasis. ACh is important in physiological regulation of circulation via activation of G-protein coupled mAChR in the RVLM. Selective breeding for cholinergic hypersensitivity in FSL and FRL rats results in region- and subtype-specific changes in mAChR expression in the lower brainstem and differentially influences muscarinic control of circulation and breathing. Variations in central mAChR sensitivity may contribute to impaired reflex control of vagal and sympathetic outflow and could hence predispose to cardiac complications including arrhythmias. Future studies may aim to further understand the relationship between endogenous sensitivity of metabotropic neurotransmitter receptors in the CNS and cardiovascular disturbances associated with depression

Response properties of the inferior colliculus following unilateral noise induced hearing loss : the effects of cholinergic enhancement and auditory training

Tinnitus is a common and potentially debilitating condition that is often associated with hearing loss. Noise induced hearing loss is a frequently utilised animal model, in studies investigating the underlying mechanisms of tinnitus. In the central auditory system, the inferior colliculus (IC) is an obligatory nucleus for the ascending processing of auditory signals. It has been comprehensively investigated in animal models of unilateral noise induced hearing loss (UNIHL). Previous studies have mostly investigated the dominant, contralateral pathway with little attention being paid to the ipsilateral path. In normal hearing animals, contralaterally driven IC neurons are primarily excitatory while ipsilaterally driven IC neurons are primarily inhibitory. However, there are also ipsilaterally driven IC neurons that are excitatory. Literature that have investigated the response properties of these neurons, and their consequential response properties within an animal model of UNHIL is very limited. The first study presented in this thesis investigated the consequences of UNIHL of both dominant contralaterally excitatory and non-dominant ipsilaterally excitatory neurons of the IC. The findings in this thesis may have implications for the development of therapies for tinnitus. By using a human equivalent dose of an approved drug, paired with a well-known neuro-rehabilitation intervention, together these interventions can significantly affect the consequential response properties of IC neurons that are observed following acoustic trauma. In a condition where currently there are no reliable cures, further development of these findings may provide insights that could lead to the generation of novel therapeutic approaches

Investigation of antigen derived peptides and nanoparticles as a novel immunotherapy for Myasthenia gravis

Η Βαριά Μυασθένεια είναι μια αυτοάνοση νόσος που προκαλείται από αντισώματα, αλλά εξαρτάται από την ενεργοποίηση των Τ λεμφοκυττάρων. Στη βαριά Μυασθένεια τα αυτοαντισώματα στοχεύουν κυρίως τον μυϊκό νικοτινικό υποδοχέα της ακετυλοχολίνης στη νευρομυϊκή σύναψη. Η παρεμπόδιση της μετάδοσης του σήματος λόγω καταστροφής του υποδοχέα της ακετυλοχολίνης οδηγεί σε μυϊκή αδυναμία και κόπωση. Οι παρούσες θεραπείες για τη Μυασθένεια δεν είναι ειδικές και για αυτό είναι πιθανό να έχουν πολλές παρενέργειες. Για το λόγο αυτό, είναι αναγκαίο να αναπτυχθούν καινούριες θεραπείες, που έχουν μεγαλύτερη ειδικότητα. Τέτοιου είδους αντιγονοειδικές θεραπείες θα πρέπει να στοχεύουν στον επαναπρογραμματισμό συγκεκριμένων αυτοδραστικών κυττάρων του ανοσοποιητικού καθώς και στην επαναφορά της ανοσολογικής ανοχής εναντίον του αυτοαντιγόνου. Μελέτες σε πειραματικά ζωικά μοντέλα για τη Μυασθένεια έχουν δείξει πως η χορήγηση του σχετικού με την ασθένεια αντιγόνου, μέσω της ρινικής ή στοματικής οδού, έχει την δυνατότητα να επάγει την ανοσολογικής ανοχή. Παρά τα πρώιμα αυτά ενθαρρυντικά αποτελέσματα, δεν έχει ακόμα αναπτυχθεί μια σχετική θεραπεία. Αναπτύξαμε ένα πειραματικό ζωικό μοντέλο για τη Βαριά Μυασθένεια, ανοσοποιώντας Lewis αρουραίους, με την εξωκυττάρια περιοχή της α1 υπομονάδας του ανθρώπινου υποδοχέα της ακετυλοχολίνης, καθιστώντας το κατάλληλο για τον έλεγχο αντιγονο-ειδικών θεραπειών. Χρησιμοποιώντας αυτό το μοντέλο διερευνήσαμε την θεραπευτική ικανότητα της εξωκυττάριας περιοχής της α1 υπομονάδας του ανθρώπινου υποδοχέα της ακετυλοχολίνης, μέσω της χορήγησής της από διαφορετικές οδούς, σε διαφορετικές δόσεις και συχνότητα σε διαφορετικά στάδια της νόσου. Τα αποτελέσματά μας έδειξαν πως επαναλαμβανόμενες ενδοφλέβιες χορηγήσεις, έχουν τη δυνατότητα να βελτιώσουν σημαντικά τις εκδηλώσεις της πειραματικής Μυασθένειας. Αυτή η βελτίωση συνοδευόταν από τα μειωμένη απώλεια υποδοχέων ακετυλοχολίνης και χαμηλότερα επίπεδα αντισωμάτων στον ορό. Παρόλο που είναι αναγκαία η περαιτέρω διερεύνηση των εμπλεκόμενων μηχανισμών, η επαγωγή ανοσολογικής ανοχής μέσω του αυτό-αντιγόνου αποτελεί μια πολλά υποσχόμενη ανοσοθεραπεία για τη Βαριά Μυασθένεια. Επιπλέον, καθώς η Βαριά Μυασθένεια έχει χαρακτηριστεί ως μοντέλο, τυχόν καινούριες θεραπείες είναι πιθανό να μπορούν να εφαρμοστούν και σε άλλες παρόμοιες αυτοάνοσες νόσους.Myasthenia gravis (MG) is an antibody-mediated, T-cell dependent, autoimmune disease, in which autoantibodies are mainly directed against the muscle nicotinic acetylcholine receptor (nAChR) at the neuromuscular junction. Impaired signal transduction due to AChR destruction by the autoantibodies leads to muscle weakness and fatigability. MG is currently treated with non-specific immunomodulatory agents, which may have several side effects. Hence, there is a great need to develop novel, more specific therapies. Such targeted treatments should aim on re-programming the specific autoreactive immune cells and restoring the lost tolerance towards the autoantigens. Early studies with experimental autoimmune MG (EAMG) animal models showed that mucosal administration of disease-relevant antigens could induce such tolerance, providing the proof of principle for the therapeutic application of the approach. Despite these promising findings, a novel therapy has not yet been established. We have developed an EAMG model based on immunization of Lewis rats with the human nAChR α1 subunit extracellular domain (hα1 ECD), well suited for testing antigen-specific treatments. We used this model to investigate the therapeutic potency of the ha1-ECD, by different administration routes, doses and time points during disease progression. Our data showed that when given intravenously and in repeated doses, hα1 ECD dramatically ameliorated EAMG symptoms. This was accompanied by reduced loss of nAChRs and lower levels of serum autoantibodies. Although, further investigation of the underlying mechanisms involved is needed, autoantigen-induced tolerization is a promising immunotherapy for MG. Moreover, since MG is a model antibody-mediated autoimmune disease, discovery of novel therapies could be applicable to other similar autoimmune diseases

Inhibition studies of serine hydrolases by cyclic phosphates and phosphonates

The serine hydrolase superfamily is one of the largest known enzyme families comprising approximately 1% of the predicted protein product in human genome. This family of enzymes contains a catalytic triad that is mainly consists of serine, aspartic acid/glutamic acid and histidine residues in their active sites. It has been proposed that the potential drug targets for Alzheimer’s disease and diabetes type 2 are enzymes that belong to this enzyme family. Acetylcholinesterase is an enzyme that catalyses the breakdown of acetylcholine, a neurotransmitter that helps transport information from one nerve cell to another. Breakdown of acetylcholine in Alzheimer’s disease patients enhances memory loss, which could be reduced if AChE is inhibited. Cyclophostin, a bicyclic phosphate, is a natural product inhibitor of AChE having an IC50, of 8 e-4 μM. The laboratory synthesized mono- and bicyclic analogs of phosphonate analog of cyclophostin exhibited low μM potency against human AChE. It is established that these analogs covalently modify the active site of AChE and do not dissociate from the active site upon treatment with oximes. From a comparative analysis of kinetic data it is revealed that these compounds are less toxic and milder than the existing AChE inhibitors and can be used as potential chemotherapeutic agent against Alzheimer’s disease. Hormone-sensitive lipase (HSL) is another serine hydrolase enzyme that hydrolyzes lipids in the form of triglycerides. It is a homodimer of 84 kDa subunits and is mostly found in adipose tissues. HSL is a potential drug target for diabetes type 2. The activity of HSL must be inhibited in insulin deficient patients to lower the risk of associated cardiovascular disease. Cyclipostin is a natural product inhibitor of HSL. Laboratory synthesized monocyclic phosphonate analogs of cyclipostin having varying C-chain length exhibited μM potency against rat HSL. The potency of these analogs improved upon introducing longer C-chain like C16. This class of compounds showed an aggregation property that affected their potency against the enzyme. The attachment of the C-chain at the P-center of the monocyclic phosphonate analog considerably improved the potency (almost 10 fold)