Local Dosing in a 3-Mercaptopropionic Acid Chemically-Induced Epileptic Seizure Model with Microdialysis Sampling

The focus of this research was the development of an animal model for local administration of 3-mercaptopropionic acid (3-MPA) in a chemically-induced epileptic seizure model using microdialysis sampling with simultaneous electrocorticography recording (ECoG). Local administration of 3-MPA through the microdialysis probe was employed to elicit seizures in a localized brain region. Delivery of 3-MPA to the brain and changes in amino acid and catecholamine neurotransmitters were monitored. Simultaneous ECoG recordings were made using a microdialysis probe with an internal Ag/AgCl electrode. Local administration of a convulsant is important, as many clinical cases present with focal seizures. Neurochemical and electrical activity were monitored in three separate brain regions: the striatum, hippocampus, and locus coeruleus. 3-MPA was administered through the microdialysis probe in one region, while control samples were collected in the other two. These results demonstrated that unless two brain regions were connected via efferent or afferent pathways, administration of 3-MPA in one region had no neurochemical effect in the others. In the region where 3-MPA was administered, an increase in both glutamate, the main excitatory amino acid, and GABA, the main inhibitory amino acid, was seen. In addition, an increase in both dopamine and norepinephrine was seen. A multiple dosing regimen of 3-MPA was developed where 3-MPA was administered twice. These results showed that there was an attenuation in the increase of glutamate and GABA during the second administration of 3-MPA, indicating a neuronal protective mechanism taking place to decrease the effect of the second 3-MPA administration. Seizures were not detected using during local administration of 3-MPA using the microdialysis probes with an internal Ag/AgCl electrode. This was not due to the ineffectiveness of the electrodes, as they detected seizures during systemic dosing of 3-MPA. It is possible that the number of neurons excited from the local administration of 3-MPA were so limited that the signal was too small to be detected

Fast whole-brain imaging of seizures in zebrafish larvae by two-photon light-sheet microscopy

Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics. However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited by the signal-to-noise-ratio. Here, we describe a 2P LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish brain (5 Hz) while keeping low the laser intensity on the specimen. We applied our system to the study of pharmacologically-induced acute seizures, characterizing the spatial-temporal dynamics of pathological activity and describing for the first time the appearance of caudo-rostral ictal waves (CRIWs).Comment: Replacement: accepted version of the manuscript, to be published in Biomedical Optics Express. 36 pages, 15 figure

Fast whole-brain imaging of seizures in zebrafish larvae by two-photon light-sheet microscopy

Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one-photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics. However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited by the signal-to-noise-ratio. Here, we describe a 2P LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish brain (5 Hz) while keeping low the laser intensity on the specimen. We applied our system to the study of pharmacologically-induced acute seizures, characterizing the spatial-temporal dynamics of pathological activity and describing for the first time the appearance of caudo-rostral ictal waves (CRIWs)

A novel chemo-optogenetic nanomachine sensitive to intracellular pH shifts

Epilepsy is a neurological disorder characterized by repeated seizures. Nowadays drugs and other approaches to reduce them are available but, unfortunately, around 30% of patients do not respond to medical therapies. In the last decade, optogenetics has emerged as a tool to both explore neuronal networks dynamics and to treat neurological conditions such as epilepsy. The optogenetics strategy involves the expression, in precise brain areas, of light sensitive proteins called opsins that are able to change the membrane potential upon wavelength-specific illumination. This last aspect is, usually, achieved using LED-based hardware. Despite the many advantages of this technique, it still faces practical and translational challenges because of the difficulties of illuminating multiple and deep areas of the brain. In this scenario, the search of alternative light sources is a goal to achieve. Luciferases are enzymes able to emit light upon addiction of their own substrate coelenterazine, and can be used to deliver light to opsins and modulate their action. In this work, the probe called pHIL (pH sensitive inhibitory luminopsin) was developed with the purpose to modulate the epileptic phenotype. pHIL is composed by a bioluminescent protein, RLuc8, coupled to the inhibitory opsin eNpHR3.0. Moreover, the control of seizures will occur only under the intracellular acidic conditions observed in epileptic neurons. The pH sensitivity of the probe is given by the presence of a pH sensor, a pH-sensitive variant of EGFP, called E2GFP. The functioning of the probe is based on the BRET mechanism. The UV light that comes from the luciferase is transferred to the E2GFP that under acidic conditions will emit light and activate the eNpHR3.0, promoting membrane hyperpolarization of epileptic neurons. pHIL is expressed and localizes at the plasma membrane in both HEK293T cells and primary hippocampal neurons. Moreover, pHIL hyperpolarizes HEK293T under acidic conditions and upon addiction of CTZ 400a, the specific luciferase substrate able to induce the UV light emission. On the basis of our data, therefore, we propose pHIL as a potential therapeutic tool to counteract neuronal hyperexcitability

Evaluation of Fog Security Devices

The purpose of this report is to investigate and advise on the safety, regulation and procedures relevant to the use of fog security systems in greater Melbourne, Victoria. The project evaluates existing literature pertaining to these devices such as building and fire codes, incident reports, and manufacturer information, as well as interviews with emergency services personnel, system installers, and users of these systems. From these sources, this report is written as a comprehensive outlook on the safety of these systems as well as recommendations in the best interests of the fire brigade, community and manufacturers

Etude et réalisation d'un système de communications par lumière visible (VLC/LiFi). Application au domaine automobile.

The scientific problematic of this PhD is centered on the usage of Visible LightCommunications (VLC) in automotive applications. By enabling wireless communication amongvehicles and also with the traffic infrastructure, the safety and efficiency of the transportation canbe substantially increased. Considering the numerous advantages of the VLC technologyencouraged the study of its appropriateness for the envisioned automotive applications, as analternative and/or a complement for the traditional radio frequency based communications.In order to conduct this research, a low-cost VLC system for automotive application wasdeveloped. The proposed system aims to ensure a highly robust communication between a LEDbasedVLC emitter and an on-vehicle VLC receiver. For the study of vehicle to vehicle (V2V)communication, the emitter was developed based on a vehicle backlight whereas for the study ofinfrastructure to vehicle (I2V) communication, the emitter was developed based on a traffic light.Considering the VLC receiver, a central problem in this area is the design of a suitable sensorable to enhance the conditioning of the signal and to avoid disturbances due to the environmentalconditions, issues that are addressed in the thesis. The performances of a cooperative drivingsystem integrating the two components were evaluated as well.The experimental validation of the VLC system was performed in various conditions andscenarios. The results confirmed the performances of the proposed system and demonstrated thatVLC can be a viable technology for the considered applications. Furthermore, the results areencouraging towards the continuations of the work in this domain.La problématique scientifique de cette thèse est centrée sur le développement decommunications par lumière visible (Visible Light Communications - VLC) dans lesapplications automobiles. En permettant la communication sans fil entre les véhicules, ou entreles véhicules et l’infrastructure routière, la sécurité et l'efficacité du transport peuvent êtreconsidérablement améliorées. Compte tenu des nombreux avantages de la technologie VLC,cette solution se présente comme une excellente alternative ou un complément pour lescommunications actuelles plutôt basées sur les technologies radio-fréquences traditionnelles.Pour réaliser ces travaux de recherche, un système VLC à faible coût pour applicationautomobile a été développé. Le système proposé vise à assurer une communication très robusteentre un émetteur VLC à base de LED et un récepteur VLC monté sur un véhicule. Pour l'étudedes communications véhicule à véhicule (V2V), l'émetteur a été développé sur la base d’un pharearrière rouge de voiture, tandis que pour l'étude des communications de l'infrastructure auvéhicule (I2V), l'émetteur a été développé sur la base d'un feu de circulation. Considérant lerécepteur VLC, le problème principal réside autour d’un capteur approprié, en mesured'améliorer le conditionnement du signal et de limiter les perturbations dues des conditionsenvironnementales. Ces différents points sont abordés dans la thèse, d’un point de vue simulationmais également réalisation du prototype.La validation expérimentale du système VLC a été réalisée dans différentes conditions etscénarii. Les résultats démontrent que la VLC peut être une technologie viable pour lesapplications envisagées

Neuroimaging of functional and structural alterations in Juvenile Myoclonic Epilepsy and Frontal Lobe Epilepsy

Epilepsy is the commonest neurological disorder and has profound effects on patients, who suffer from epileptic seizures and also from cognitive impairment. The exact mechanisms of cognitive impairment remain unclear. Aim of this study was to analyse in more detail the functional and structural alterations in two different patient groups, juvenile myoclonic epilepsy (JME) and frontal lobe epilepsy (FLE). We recruited and investigated 26 healthy controls, 30 patients with JME and 67 patients with FLE. All participants underwent magnetic resonance imaging (MRI), including structural imaging, five functional MRI paradigms and diffusion tensor imaging (DTI) as well as neuropsychological assessment. In patients with JME we could show motor cortex hyperactivity and an increased functional connectivity between the pre-frontal cognitive cortex and the motor system. This correlated with increased structural connectivity, measured by DTI and also with disease severity: patients with more active epilepsy showed a stronger hyperconnectivity. In FLE, we could show extensive reorganization of cognitive functions, and we could show, that functional MRI can be used as a new diagnostic method, to identify dysfunctional areas, indicative of the seizure onset zone. This is particularly important in patients with nonlesional FLE, where epilepsy surgery may be advisable but is challenged by the absence of a visible surgical target. The study has provided new insights into pathophysiological mechanisms in JME, specifically explaining the characteristic effect of motor seizures triggered by cognitive effort. It has contributed strong evidence that the observed imaging alterations are the cause and not a consequence of JME, by documenting marked structural changes in seizure free patients. For patients with FLE the study showed highly individual effects of chronic epilepsy on cognitive processing in the frontal lobe. These alterations are clinically relevant for both, avoiding complications from surgery, but also to identify pathological alterations not visible in conventional MRI

NASA Tech Briefs, March 2005

Topics covered include: Scheme for Entering Binary Data Into a Quantum Computer; Encryption for Remote Control via Internet or Intranet; Coupled Receiver/Decoders for Low-Rate Turbo Codes; Processing GPS Occultation Data To Characterize Atmosphere; Displacing Unpredictable Nulls in Antenna Radiation Patterns; Integrated Pointing and Signal Detector for Optical Receiver; Adaptive Thresholding and Parameter Estimation for PPM; Data-Driven Software Framework for Web-Based ISS Telescience; Software for Secondary-School Learning About Robotics; Fuzzy Logic Engine; Telephone-Directory Program; Simulating a Direction-Finder Search for an ELT; Formulating Precursors for Coating Metals and Ceramics; Making Macroscopic Assemblies of Aligned Carbon Nanotubes; Ball Bearings Equipped for In Situ Lubrication on Demand; Synthetic Bursae for Robots; Robot Forearm and Dexterous Hand; Making a Metal-Lined Composite-Overwrapped Pressure Vessel; Ex Vivo Growth of Bioengineered Ligaments and Other Tissues; Stroboscopic Goggles for Reduction of Motion Sickness; Articulating Support for Horizontal Resistive Exercise; Modified Penning-Malmberg Trap for Storing Antiprotons; Tumbleweed Rovers; Two-Photon Fluorescence Microscope for Microgravity Research; Biased Randomized Algorithm for Fast Model-Based Diagnosis; Fast Algorithms for Model-Based Diagnosis; Simulations of Evaporating Multicomponent Fuel Drops; Formation Flying of Tethered and Nontethered Spacecraft; and Two Methods for Efficient Solution of the Hitting- Set Problem

Systematic Analysis of Molecular and Cellular Dysfunction in Accelerated Aging Phenotypes

Zur Gruppe segmentaler progeroider Erkrankungen gehören genetische Erkrankungen, denen beschleunigte Alterungsprozesse in mehreren Geweben zugrunde liegen. Klinische Merkmale dieser Patienten sind altersassoziierte Pathologien in jungen Jahren wie graue Haare, Lipodystrophie, Osteoporose, grauer Star, Schwerhörigkeit, Arteriosklerose, Diabetes mellitus und Tumore. In den vergangenen Jahren wurden viele krankheitsverursachende Gendefekte für segmentale progeroide Erkrankungen identifiziert. Die betroffenen Proteine sind an Genomstabilität und der Funktion der Mitochondrien beteiligt. Die Identifizierung der zugrundeliegenden genetischen Defekte deckte molekulare und zelluläre Mechanismen auf, die unser Verständnis von Alterungspozessen und altersassoziierten Erkankungen im Allgemeinen erweiterte. Das Ziel meiner Arbeit war, unser Wissen über diese zellulären und molekularen Mechanismen der Alterungsprozesse zu erweitern, indem ich eine einzigartige Sammlung von Fibroblasten und DNA-Proben von Patienten mit verschiedenen progeroiden Erkrankungen untersucht habe. Dazu habe ich eine Methode zur Sequenzierung der mitochondrialen DNA mit sehr hoher Abdeckung etabliert, um niederfrequente mtDNA-Varianten in Patientenproben zu quantifizieren. Durch die Behandlung von Kontrollfibroblasten mit genotoxischen Substanzen konnte ich zeigen, dass dieser Ansatz die Detektion von niederfrequenten Varianten im mitochondrialen Genom möglich macht. Die Analyse einer DNA-Probe eines von Cutis Laxa Typ IC betroffen Patienten, der eine homozygote Mutation im LTBP4 Gen trägt, deckte eine signifikant erhöhte Anzahl an mtDNA-Varianten auf. LTBP4 kodiert ein sekretiertes Protein, das den TGF-beta Signalweg reguliert und das bisher noch nicht mit mitochondrialer Dysfunktion assoziiert war. Dieses Ergebnis weist auf eine Verbindung von LTBP4 und der Integrität des mitochondrialen Genoms hin. VII Weiterhin habe ich eine Real-Time PCR-basierte Methode zur Analyse der Telomerlänge etabliert und damit die Telomerlänge in DNA-Proben von Patienten gemessen, die an segmentalen progeroiden Erkrankungen leiden. So habe ich die erwartete sigmoidale Verteilung der Telomerlänge mit dem Alter und eine hohe Varianz der Telomerlänge in Kontrollproben zeigen können. Die Verkürzung der Telomerlänge in einer Probe eines Bloom-Syndrom-Patienten und einer Probe eines Patienten mit Cutis Laxa Typ 1C kommt der Signifikanz nahe. Um die Verringerung der Telomerlänge detaillierter charakterisieren zu können, habe ich bei der Optimierung einer Telomer-qFISH-Methode kollaboriert und diese Methode dann eingesetzt, um die Telomerlänge in drei Fibroblasten von Bloom-Syndrom-Patienten zu messen. Erstaunlicherweise war in einer Patientenprobe die Signalintensität, die der Telomerlänge entspricht, und die Anzahl der Telomersignale erhöht. In dieser Patientenprobe konnte eine Chromosomenaberration und ein verzögerter Zellzyklus mit einer erhöhten Anzahl an Zellen in der G2/M-Phase nachgewiesen werden. Dies deutet darauf hin, dass der doppelte Chromosomensatz die Messung beeinträchtigte. Mit einem Southernblot konnte ich bestätigen, dass die Telomerlänge in diesen Patientenproben nicht verändert war. Des Weiteren habe ich die Anreicherung von DNA-Schäden und die DNA-Reparatur in Patientenfibroblasten mittels der Quantifizierung der γH2AX Foci nach Bestrahlung etabliert und analysiert. So konnte ich eine erhöhte Anzal von γH2AX Foci in zwei unbehandelten Fibroblastenproben mit Mutationen in den Genen GORAB und SLC25A24 identifizieren, was auf eine höhrere Anfälligkeit für DNA-Schäden oder eine Schwäche der DNA-Reparatur hinweist. Des Weiteren hat die Bestrahlung signifikant mehr γH2AX Foci in zwei Patientenfibroblasten hervorgerufen, die Mutationen in den Genen PYCR1 und GORAB tragen, was ebenfalls auf eine Beeinträchtigung der DNA-Reparatur in diesen Zellen hindeutet. Zusammengefasst habe ich in meiner Arbeit neue Methoden zur Quantifizierung von altersassoziierten zellulären Prozessen etabliert und diese Methoden genutzt, um Fibroblasten und DNA-Proben von Patienten mit segmentalen progeroiden Erkrankungen zu analysieren. So konnte ich neue Erkenntnisse zu den beteiligten Pathomechanismen gewinnen, indem ich eine Verbindung von LTBP4 mit der Integrität des mitochondrialen Genoms und einen potentiellen Einfluss von BLM und PYCR1 auf die Telomerlänge identifiziert habe.Accelerated aging in multiple tissues is the connecting characteristic of the group of genetic disorders called segmental progeroid syndromes. At young ages, affected patients show many clinical features of aging-associated pathologies such as hair graying, lipodystrophy, osteoporosis, cataracts, hearing loss, arteriosclerosis, diabetes mellitus, and malignancies. Several disease-causing genes for segmental progeroid syndromes have been identified within the last years, and the affected proteins are often involved in genome maintenance or mitochondrial function. Identifying these underlying genetic alterations revealed molecular and cellular mechanisms involved in the pathology of these diseases and furthered our understanding of aging processes and aging-associated diseases in general. The aim of my thesis was to expand our knowledge of the cellular and molecular mechanisms of aging using a unique collection of fibroblast and DNA samples of patients suffering from a large variety of different progeroid syndromes. I established an ultra-high coverage mtDNA sequencing approach to detect and quantify low-frequency mtDNA variants in patient samples. By treatment of control fibroblasts with genotoxic agents, I could show that this approach allows the detection of low-frequency variants in the mitochondrial genome. Analysis of a DNA sample of a patient suffering from Cutis laxa type IC and carrying a homozygous mutation in LTBP4 revealed a significantly increased number of mtDNA variants. LTBP4 encodes a secreted protein that regulates TGF-beta signaling and has previously not been associated with mitochondrial dysfunction. Therefore, these results indicate for the first time a link between LTBP4 and the integrity of the mitochondrial genome. Additionally, I established a real-time PCR-based method in order to analyze telomere length in DNA samples of patients suffering from progeroid syndromes. I found the anticipated sigmoidal distribution of telomere length by age as well as a high variance of telomere length in the control samples. The decrease of telomere length in two patient samples, a Bloom syndrome patient and a patient suffering from Cutis laxa type IIB, approached significance. V To characterize telomere attrition in more detail, I collaborated on the optimization of a telomere qFISH method, which I then used to measure telomere length in three fibroblast samples of Bloom syndrome patients. Strikingly, the telomere signal intensity corresponding to telomere length as well as the number of telomere signals were increased in one patient sample. A chromosomal aberration and a delayed cell cycle progression with an increased amount of cells in the G2/M phase were then detected in these fibroblasts, indicating that to the double set of chromosomes interfered with the measurement. A Southern blot confirmed that the telomere length in these patient samples was not different from the controls. Further, I analyzed the accumulation of DNA damage and induction of the DNA damage response in patient fibroblasts using the quantification of γH2AX foci upon treatment with genotoxic reagents. I found an elevated level of γH2AX foci in two untreated fibroblast samples carrying mutations in GORAB and SLC25A24 indicative of either higher susceptibility of these cells to DNA damage or deficiencies in DNA damage repair processes. Further irradiation treatment caused a significantly elevated level of γH2AX in two fibroblast samples carrying mutations in the PYCR1 and GORAB genes suggestive of DNA damage repair impairment in these cells. In summary, the results of my Ph.D. thesis help to establish new methods for the analysis and quantification of aging-associated cellular processes. Using these methods on cells and DNA samples of patients with segmental progeroid syndromes, I could provide new insights into the involved pathomechanisms by identifying a link between LTBP4 and mitochondrial DNA as well as a possible influence of BLM and PYCR1 on telomere length.2021-09-3