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Multi-user equipment approved in grant 2017/24615-5: qPCR Thermocycler Quantum S5 for 384-well plates

Abstract

Based on preliminary data and on the amount of evidence produced by our group, this research project will investigate the following hypothesis: melanopsin (OPN4) and/or transient receptor potencial channels (TRPs), expressed by cardiomyocytes, monitor among other putative signals, the variation of the core temperature and/or the local matbolic variation.We suggest that temperature variation would be brought about by the oxygenated blood entering the heart from the respiratory system, and the blood coming from the periphery, with lower pressure and poor in oxygen, in addition to the circadian variation of internal temperature. The temperature variation, or the metabolic alteration induced by the temperature change, would be translated by the heart, for instance, in the expression of its major hormones, natriuretic peptides (NPs) among others, to regulate local clocks and the metabolism of target organs such as white adipose tissue (WAT) and brown adipose tissue (BAT), and of the heart itself. Once we assumed a link (through NPs) between cardiac function and metabolism, and between metabolic disfunction and clock disruption (Tables 1, 2 and 3), we may speculate that the unbalance of this machinery would be responsible for pathologies not only in the heart itself, but in the whole organism. To understand how this circuitry is affected in metabolic extremes such as obesity and cachexia will open the possibility of intervention in the pathological syndromes, or in the prevention or mitigation of those alterations. We will evaluate how temperature, or metabolic alteration, is sensed, the local clocks and the canonical tissue-specific processes in vivo, and in tissue explants or cell culture of the suprachiasmatic nucleus (SCN), médio-basal nucleus, heart, WAT and BAT of WT and ²1 adrenoceptor, Opn4, TrpV1, TrpM8 and TrpA1 KO mice, in physiological conditions and in the metabolic extremes of obesity and cachexia, associated with assays of temperature variation. Initially, we will obtain the transcriptome of the mentioned organs from WT mice subject to 22oC (cold challenge) and to 30oC (thermo-neutrality), what will allow us to focus the investigation in the pathways and signaling of sensation altered by the temperature challenge. The in vivo assays will be performed at 22oC (or 8oC below the thermoneutral temperature determined in the other genotypes by indirect calorimetry) and 30oC (or other temperature for thermo-neutrality found for the other genotypes) and the in vitro assays at 34 and 37oC for WT mice (or other range of maximal circadian temperature, and 3 degrees below, to be determined for the altered genotypes); some assays will be conducted in the presence of agonists and antagonists of OPN4 and TRP channels, or in CRISPER KO cells. In the in vivo experiments, we will perform telemetry of locomotor activity and core temperature, and calorimetry. We will analyze clock genes and the tissue-specific genes identified by the comparative transcriptoma, using among others the following techniques: immunocytochemistry, quantitative PCR, Western blot, flow cytometry with image, CRISPER. Along the duration of the project we intend to establish the strain of organ-specific Opn4 KO with Cre-lox system. (AU)

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