440 research outputs found

    Functional Human Cell-Based Cardiac Tissue Model with Contraction Force Measurements

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    SydÀmeen kohdistuvat lÀÀkkeiden haittavaikutukset ovat yksi suurimpia syitÀ lÀÀkeainekandidaattien hylkÀÀmiselle sekÀ jo markkinoille vietyjen lÀÀkkeiden poisvedoille. Nykyiset menetelmÀt lÀÀkkeiden turvallisuuden ja tehokkuuden testaamiseen eivÀt ennusta lÀÀkkeiden vaikutuksia ihmiselle riittÀvÀn tarkasti. Koska sydÀnten toiminnassa on lajikohtaisia eroja, elÀinkokeissa ei vÀlttÀmÀttÀ tunnisteta kaikkia ihmisen sydÀmelle haitallisia aineita. TÀtÀ varten tarvitaan toiminnaltaan mahdollisimman hyvin ihmisen sydÀnkudosta vastaavia ihmissolupohjaisia sydÀnkudosmalleja. TÀmÀn vÀitöskirjan tavoitteena oli kehittÀÀ toiminnallinen sydÀnkudosmalli, joka mallintaa aikuisen ihmisen sydÀntÀ, sekÀ yhdistÀÀ tÀhÀn malliin sykintÀvoiman mittaus. TÀssÀ työssÀ kehitetty sydÀnkudosmalli koostuu ihmisen rasvan kantasoluista (hASC) ja ihmisen napanuoran laskimon endoteelisoluista (HUVEC) muodostuvasta verisuoniverkostosta, jonka pÀÀlle kasvatetaan ihmisen indusoiduista kantasoluist (hiPSC) erilaistetut sydÀnlihassolut. TÀmÀ sydÀnkudosmalli karakterisoitiin rakenteellisesti, geenien ilmentymisen tasolla ja toiminnallisesti. SydÀnkudosmallien sykintÀvoimanmittaukseen kehitettiin yksi- ja kaksisuuntaisia pietsosÀhköisiÀ sensoreita. Tulosten perusteella yhteisviljelmÀ verisuonipohjan kanssa parantaa sydÀnlihassolujen kypsymistÀ edistÀmÀllÀ niiden jÀrjestÀytymistÀ ja morfologiaa, sarkomeerirakennetta ja solu-solu-liitoksia. Myös sydÀnlihassolujen geenien ilmentymisessÀ oli vastaavuuksia aikuisen sydÀmeen. Toiminnallinen karakterisointi tunnetuilla sydÀmeen vaikuttavilla lÀÀkeaineilla osoitti mallin tunnistavan tarkasti aineiden vaikutuksia. Kehitetyt pietsosÀhköiset voima-anturit soveltuivat sydÀnmallien sykintÀvoiman mittaamiseen. Antureiden todettiin pystyvÀn mittaamaan sekÀ voimaa eri mekanismein lisÀÀvien ettÀ sitÀ vÀhentÀvien aineiden vaikutuksia. Yhteenvetona voidaan todeta, ettÀ työssÀ kehitetty sydÀnkudosmalli jÀljittelee sydÀnlihaskudoksen rakennetta ja toimintaa. Malli sopii testaamaan ihmisen sydÀmeen kohdistuvia akuutteja lÀÀkeaineiden haittavaikutuksia ja sydÀnlÀÀkkeiden tehoa. Kehitettyyn sydÀnkudosmalliin liitetyllÀ pietsosÀhköisellÀ voima-anturilla on potentiaalia voimaankohdistuvien lÀÀkeainevaikutusten testaamiseen.Adverse cardiac effects are a major reason for drug attrition during drug development and for post-approval market withdrawals. Therefore, drug development would greatly benefit from tests that better predict human cardiac function. Due to intrinsic species-to-species differences in the functionality of the heart, nonclinical animal testing does not fully represent the effects of the drugs on human. Thus, there is a need for reliable, human cell -based standardised in vitro models for cardiotoxicity and drug efficacy testing. The aim of this thesis was to develop a functional human cell -based cardiac tissue model that mimics the adult human heart and to develop a contraction force measurement system for measuring the cardiac contractility of the cardiac tissue model. The cardiac tissue model that was optimised in this thesis consisted of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes that are cocultured with preformed human adipose stromal cells (hASC) and human umbilical vein endothelial cells (HUVEC) vascular-like networks. The model was characterized structurally, in gene expression levels, and functionally. For measuring the cardiac contraction force, piezoelectric cantilever sensors with single axis and dual axis sensor designs were developed. The cell culture method was adjusted according to the sensor designs. The functionality of the cardiac tissue model and contraction force measurement technology were confirmed by known inotropic drug exposures. The results show that the coculture with the vascular-like networks improved the cardiomyocyte maturity and the tissue like structure of the model. The cardiomyocytes in the model showed improved organization and morphology, well- developed sarcomeres, and cell-cell connections. The gene expression of the cardiac tissue model also showed characteristics of the adult human heart. The functional characterization with known reference compounds showed that the model had good predictivity with high correlation to human data. The developed piezoelectric contraction force sensors were suitable for measuring the contraction force of cardiac tissue constructs. Both positive and negative inotropic effects including different mechanisms were measurable in the model with the system. In conclusion, the developed cardiac tissue model mimics the myocardium structure and functionality. The model is suitable for testing cardiotoxicity and efficacy of acute drug-induced effects on human heart. Moreover, the functionality of the cardiac tissue model with the developed contraction force measurement system was shown on a proof-of-concept level

    Palmitoylation and regulation of the funny current HCN4 channel

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    The sinoatrial node (SAN) acts as the primary pacemaker of the heart as it spontaneously generates electrical activity that propagate through the cardiac conduction system, underpinning automaticity of the heart. A network of surface membrane ion currents (“membrane clock”) and the rhythmic oscillation of local CaÂČâș release from the sarcoplasmic reticulum (“calcium clock”) work interdependently to form a coupled-clock system that drives pacemaker automaticity and its regulation on a beat-to-beat basis. The “funny current” (If) is a key component of the membrane clock contributing to the diastolic depolarisation of the SAN. Hyperpolarisation-activated cyclic nucleotide-gated channel HCN4 is the predominant isoform responsible for almost 70% of the sinoatrial If. HCN4 channels localise to lipid rafts in the SAN and disorganisation of these raft membrane microdomains result in channel redistribution, thus altering its kinetic properties. Ion channels are an integral component of the complex sinoatrial pacemaking network, and their regulation is therefore central to controlling the heart rate. S-palmitoylation is a form of lipidation that involves the covalent addition of a 16-carbon palmitate to a thiol group of a cysteine residue in a protein. Unlike most lipid modifications, palmitoylation is unique due to its reversible nature, allowing the dynamic regulation of both soluble and integral proteins. In recent years, palmitoylation has emerged as an important regulator of cardiac electrophysiology as it influences the function and membrane microdomain localisation of key cardiac Naâș and CaÂČâș handling proteins. The present in-vitro study was adopted to characterise palmitoylation of HCN4 channels and to establish its functional consequences. Site-specific resin assisted capture (acyl-RAC) was used to assess palmitoylation of HCN4 in human embryonic kidney (HEK) cells as well as endogenous HCN4 in isolated neonatal rat whole heart and atrial myocytes. HCN4 was sub-stoichiometrically palmitoylated in all experimental systems examined. Truncated HCN4 intracellular amino and carboxyl termini fused to YFP and cysteine-to-alanine mutations of the palmitoylation sites in HEK-293 cells mapped HCN4 palmitoylation sites to a pair of cysteines (C93 and C179) in the HCN4 N-terminus domain. A double cysteine-to-alanine mutation C93/179AA of both palmitoylation sites reduced palmitoylation of full-length HCN4 by ~67% in comparison to wild type HCN4. Membrane impermeable biotinylation of cell surface HCN4 revealed that palmitoylation did not influence its trafficking to the cell surface or cell surface turnover rate. Standard discontinuous sucrose gradient indicated that HCN4 channels did not require palmitoylation to localise to lipid rafts in HEK-293 cells. Whole-cell patch clamp was used to investigate IHCN4 in HEK-293 cells engineered to stably express wild type and mutant HCN4. Loss of palmitoylation at the N-terminus significantly reduced HCN4 current magnitude by ~5 to 8-fold across a range of voltages. However, it did not alter its half-maximal activation voltage (V₀.₅: -90.4 ± 2.5 mV for WT vs -90.4 ± 1.6 mV for C93/179AA), nor its activation slope factor (k: 7.1 ± 0.5 mV for WT vs 6.0 ± 0.2 mV for C93/179AA). Phylogenetic analysis was used to evaluate the evolutionary acquisition of HCN4 palmitoylation within the pre-metazoan and metazoan lineage. While cysteine 93 was broadly conserved within all classes of HCN4 vertebrate orthologs, conservation of cysteine 179 was confined to placental mammals. Together, this study demonstrated the importance of palmitoylation as a regulator of HCN4 channel function by enhancing HCN4-mediated currents. Palmitoylation of the HCN4 amino terminus is likely to significantly enhance If in the SAN, accelerating diastolic depolarisation, and increasing heart rate

    Occupational exposure to electromagnetic fields: risk assessment of operators performing Transcranial Magnetic Stimulation (TMS) treatments

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    The assessment of the risk from occupational exposure to electromagnetic fields (EMF) has attracted the attention of those involved in safety in the workplace, in particular after the updating of European legislation, with the publication for EMFs, of Directive 2013/35/EU1 of the European Parliament and of the Council, which made the risk assessment mandatory for this type of physical agents. The issue is made even more relevant by the proliferation of industrial and health applications using EMF even of considerable intensity. However, the rapid technological development has not always been accompanied by adequate growth in the culture of prevention and safety. Many devices expose both operators and persons of the general public to significant risks, but often, these risks are not adequately reported by the manufacturer, nor mentioned in the instruction manual, as would be expressly required by the harmonized standards. In this general framework is placed this Ph.D. research project, whose aim is to analyze possible conditions of risk in the workplace, considering only the environment where the EMF sources potentially expose the operator to risk. The research project involves a joint collaboration between two Institutions: the National Institute for Insurance against Accidents at Work - INAIL and of course Sapienza University of Rome. The project is developed in a multidisciplinary manner, providing experimental and numerical investigations to achieve the required goals, also considering the literature review and comparison for a more realistic analysis of the risk, in terms of human exposure to EMF. The work is based on a multiphysics approach to obtain a complete evaluation of the risk in the workplace, with the prospective to improve the current approach in the assessment of the risk and eventually suggest some indications to the operator for better use of the device under test. Therefore, the starting point has been a review of the workplaces to identify any gaps and critical issues in relation to the risk assessment and therefore for which it is considered necessary to deepen the protectionist issues. A literature analysis of the state of the art on the risk in the workplace is first carried out. This has been followed by numerical and accurate modeling of the device under test as well as the workers in a real reproduced work condition of exposure. Of paramount importance is the understanding of all the parameters that can affect the distribution of the induced EM quantities, which are essential for the risk assessment and the verification of compliance with the regulations system. To do this, it was necessary to study human exposure in-depth, also using different human body models available for dosimetric analysis on dedicated software. All the research has traveled on two parallel tracks, on the one hand, the need to fill the scientific gaps in the research area of exposure assessment of workers and on the other one to take into account the regulatory aspects, essential for a correct evaluation of professional exposure. Therefore, as a last step of the overall work, a possible new protocol of risk assessment analysis is proposed to move forward on the improvement of safety and security in the workplace

    when channels cooperate or capacitance varies

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    Die elektrische Signalverarbeitung in Nervenzellen basiert auf deren erregbarer Zellmembran. Üblicherweise wird angenommen, dass die in der Membran eingebetteten leitfĂ€higen IonenkanĂ€le nicht auf direkte Art gekoppelt sind und dass die KapazitĂ€t des von der Membran gebildeten Kondensators konstant ist. Allerdings scheinen diese Annahmen nicht fĂŒr alle Nervenzellen zu gelten. Im Gegenteil, verschiedene IonenkanĂ€le “kooperieren” und auch die Vorstellung von einer konstanten spezifischen MembrankapazitĂ€t wurde kĂŒrzlich in Frage gestellt. Die Auswirkungen dieser Abweichungen auf die elektrischen Eigenschaften von Nervenzellen ist das Thema der folgenden kumulativen Dissertationsschrift. Im ersten Projekt wird gezeigt, auf welche Weise stark kooperative spannungsabhĂ€ngige IonenkanĂ€le eine Form von zellulĂ€rem Kurzzeitspeicher fĂŒr elektrische AktivitĂ€t bilden könnten. Solche kooperativen KanĂ€le treten in der Membran hĂ€ufig in kleinen rĂ€umlich getrennte Clustern auf. Basierend auf einem mathematischen Modell wird nachgewiesen, dass solche Kanalcluster als eine bistabile LeitfĂ€higkeit agieren. Die dadurch entstehende große SpeicherkapazitĂ€t eines Ensembles dieser Kanalcluster könnte von Nervenzellen fĂŒr stufenloses persistentes Feuern genutzt werden -- ein Feuerverhalten von Nutzen fĂŒr das KurzzeichgedĂ€chtnis. Im zweiten Projekt wird ein neues Dynamic Clamp Protokoll entwickelt, der Capacitance Clamp, das erlaubt, Änderungen der MembrankapazitĂ€t in biologischen Nervenzellen zu emulieren. Eine solche experimentelle Möglichkeit, um systematisch die Rolle der KapazitĂ€t zu untersuchen, gab es bisher nicht. Nach einer Reihe von Tests in Simulationen und Experimenten wurde die Technik mit Körnerzellen des *Gyrus dentatus* genutzt, um den Einfluss von KapazitĂ€t auf deren Feuerverhalten zu studieren. Die Kombination beider Projekte zeigt die Relevanz dieser oft vernachlĂ€ssigten Facetten von neuronalen Membranen fĂŒr die Signalverarbeitung in Nervenzellen.Electrical signaling in neurons is shaped by their specialized excitable cell membranes. Commonly, it is assumed that the ion channels embedded in the membrane gate independently and that the electrical capacitance of neurons is constant. However, not all excitable membranes appear to adhere to these assumptions. On the contrary, ion channels are observed to gate cooperatively in several circumstances and also the notion of one fixed value for the specific membrane capacitance (per unit area) across neuronal membranes has been challenged recently. How these deviations from the original form of conductance-based neuron models affect their electrical properties has not been extensively explored and is the focus of this cumulative thesis. In the first project, strongly cooperative voltage-gated ion channels are proposed to provide a membrane potential-based mechanism for cellular short-term memory. Based on a mathematical model of cooperative gating, it is shown that coupled channels assembled into small clusters act as an ensemble of bistable conductances. The correspondingly large memory capacity of such an ensemble yields an alternative explanation for graded forms of cell-autonomous persistent firing – an observed firing mode implicated in working memory. In the second project, a novel dynamic clamp protocol -- the capacitance clamp -- is developed to artificially modify capacitance in biological neurons. Experimental means to systematically investigate capacitance, a basic parameter shared by all excitable cells, had previously been missing. The technique, thoroughly tested in simulations and experiments, is used to monitor how capacitance affects temporal integration and energetic costs of spiking in dentate gyrus granule cells. Combined, the projects identify computationally relevant consequences of these often neglected facets of neuronal membranes and extend the modeling and experimental techniques to further study them

    Characterization of mouse preoptic area cellular populations involved in thermoregulation

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    Thermoregulation is a dynamic homeostatic process, tightly regulated by the autonomic nervous system. How the brain coordinates maintenance of the body temperature within a narrow range of 37˚C, a condition that is needed for the survival of most of the species, remains unclear. Among the brain regions implicated in thermoregulation is the anterior portion of the hypothalamus, the preoptic area (POA). In this region neurons that respond to direct temperature stimuli and to the skin and spinal cord warming were found, suggesting that these warm-sensitive neurons (WSNs), are the cells that detect deep brain temperature and integrate it with temperature information from the periphery. The limiting factor in studying the WSNs of the POA and their role in thermoregulation is the lack of specific molecular markers that identify them. Therefore, the goal of this thesis work was to characterize WSNs of the POA at the molecular level and to find their genetic marker(s). To achieve this goal I used a primary POA cell culture and performed calcium imaging while applying a temperature stimulus of 45˚C in the presence and the absence of the TRPM2 antagonist, 2-Aminoethoxydiphenyl borate (2-APB). I identified and hand-picked temperature responding cells and temperature non-responding cells for the single-cell RNA-sequencing (scRNA-seq). Analysis of the scRNA-seq data pointed to the limitations of the P9 cell culture used. The majority of the temperature non-responding cells expressed glial marker genes together with neuronal markers, a combination not found in vivo. I concluded that the genetic heterogeneity of the sequenced cells was too large and putative WSNs’ molecular markers identified from cultured neurons would be ambiguous. In addition, to find markers of WNSs one has to take another approach, such as Patch-seq to analyze these neurons in more natural conditions. One of the POA neuronal populations activated by a change in ambient temperature is expressing leptin receptor (POALepR). These neurons also exhibit an increase in action potential firing frequency (AP FF) during the process of chronic heat exposure to 36˚C that also leads to an increased heat endurance (at 39˚C). in mice. This intrinsic property of neurons (not affected by synaptic blockers), seems to be needed for a mouse to endure heat, as the animals in which the firing of POALepR is abolished fail to do so. To learn more about the role of the POALepR neurons in the heat acclimation process I used FACS (Fluorescence Activated Cell Sorting) to isolate the POALepR neurons from POA of non-acclimated and acclimated ( 5 and 30 days at 36 ˚C) LeprCreHTB mice and performed RNA sequencing. I identified three genes Kcnq2, Kcnn2, and Kcnh2, all three coding for potassium ion channels, whose expression level changed with the course of heat acclimation. I have tested the functionality of these ion channels in AP viii firing of the POALepR neurons, by employing electrophysiology and pharmacology in acute POA slices. Ion channels Kv7.2 and Kv11.1, coded by Kcnq2, and Kcnh2, respectively, exhibited a role in shaping the AP firing of POALepR neurons. Applying the antagonist of Kv7.2 disrupted harmonious AP firing of POALepR neurons coming from acclimated mice, rendering their membrane potential unsteady and their firing bursty. In addition, the application of the Kv11.1 antagonist increased the AP FF of POALepR neurons even further in the long-term acclimated condition. Heat acclimation is a naturally occurring process happening across mammalian species, including humans. It is important for enduring physical burdens in hotter climates as it leads to the improved function of the thermoregulatory system. It is forthright to hypothesize that the POA, the central regulator of temperature homeostasis, plays a role in heat acclimation. However, knowledge about it is scarce. Knowing which molecules change in POALepR neurons transcriptome to increase firing, and to which other thermoregulatory relays these neurons project will help us understand their role and the role of POA in heat acclimation
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