Autofluorescence Across Scales: An Integrated Understanding Of Redox Cofactors As Intrinsic Probes Of Metabolic State

跨尺度的自发荧光：对氧化还原辅因子作为代谢状态内在探针的综合理解

• 批准号: BB/W009242/1
• 负责人: Thomas Blacker
• 金额: $ 51.65万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FW009242%2F1
• 关键词: Autofluorescence; Across; Scales; Integrated; Understanding

Living cells require the constant input of energy to carry out their defined roles. This is released from the molecular constituents of food by a set of chemical reactions known as metabolism. Different sets of metabolic reactions, known as metabolic pathways, can be utilised depending on the type of cell, its environment and the type of fuel available, such as sugars, fats or proteins. The incorrect functioning of metabolism is known to be both a cause and effect of a wide range of diseases. For example, it has long been known that many types of cancer will metabolise vast amounts of sugar, releasing its energy inefficiently by avoiding pathways that require oxygen. This is known as the Warburg effect. Metabolic dysfunction also plays a major role in diabetes, heart disease and neurodegenerative diseases, as well as the fundamental processes of ageing.Our understanding of metabolism and its role in disease has largely resulted from experiments whereupon cells are broken down and the contents are extracted for analysis. Destroying the cell makes it difficult to investigate how the metabolism of a tissue changes with time, and the metabolites may degrade outside of the cellular environment. Furthermore, when more than one cell type is present in a complex tissue, these methods are insensitive to cell-specific differences. To continue advancing our understanding of metabolism, we require tools that allow us to visualise metabolic processes inside the different cell types of an intact, living tissue. I will achieve this by exploiting the intrinsic fluorescence of key molecules involved in metabolism, known as redox cofactors. These transfer electrons between different metabolic reactions, either as small, mobile carriers such as nicotinamide adenine dinucleotide (NADH), or as a functional group within a protein, such as flavin. The fluorescence characteristics of these molecules change depending on the specific enzyme they are bound to or whether they are carrying an electron. Images of this autofluorescence across a tissue will be taken and its properties analysed to extract information on the metabolic state of each cell.As a scientist whose expertise spans the use of lasers to study the dynamic behaviour of molecules and the application of these methods to investigate metabolic processes in living tissues, I am well equipped to investigate the use of autofluorescence for studying metabolic state. I will perform experiments and carry out computer simulations to analyse how cellular-scale metabolic processes impact the molecular-scale fluorescence of redox cofactors. I will then work out the most accurate and user-friendly ways to extract metabolic information from autofluorescence measurements to establish a novel experimental method for use by the wider biomedical research community.

活细胞需要持续不断的能量输入来执行其特定的角色。这是从食物的分子成分通过一系列化学反应被称为新陈代谢释放。不同的代谢反应，称为代谢途径，可以根据细胞的类型，其环境和可用燃料的类型（如糖，脂肪或蛋白质）来使用。已知代谢的不正确功能是多种疾病的原因和结果。例如，人们早就知道，许多类型的癌症会代谢大量的糖，通过避开需要氧气的途径来有效地释放能量。这被称为瓦尔堡效应。代谢功能障碍在糖尿病、心脏病和神经退行性疾病以及衰老的基本过程中也起着重要作用。我们对代谢及其在疾病中的作用的理解在很大程度上来自于将细胞分解并提取内容物进行分析的实验。破坏细胞使得难以研究组织的代谢如何随时间变化，并且代谢物可能在细胞环境之外降解。此外，当复杂组织中存在多于一种细胞类型时，这些方法对细胞特异性差异不敏感。为了继续推进我们对代谢的理解，我们需要能够使我们可视化完整活组织的不同细胞类型内的代谢过程的工具。我将通过利用参与新陈代谢的关键分子的内在荧光来实现这一点，这些分子被称为氧化还原辅因子。这些电子在不同的代谢反应之间转移，或者作为小的、移动的载体，如烟酰胺腺嘌呤二核苷酸（NADH），或者作为蛋白质内的功能基团，如黄素。这些分子的荧光特性会根据它们所结合的特定酶或它们是否携带电子而变化。将拍摄组织中这种自发荧光的图像，并分析其特性，以提取每个细胞代谢状态的信息。作为一名科学家，他的专业知识包括使用激光研究分子的动态行为，以及将这些方法应用于研究由于我对活体组织中的代谢过程有深入的了解，我有能力研究利用自体荧光来研究代谢状态。我将进行实验并进行计算机模拟，以分析细胞尺度的代谢过程如何影响氧化还原辅因子的分子尺度荧光。然后，我将研究出最准确和用户友好的方法来从自体荧光测量中提取代谢信息，以建立一种新的实验方法，供更广泛的生物医学研究界使用。

项目成果

Gradients of glucose metabolism regulate morphogen signalling required for specifying tonotopic organisation in the chicken cochlea.

• DOI: 10.7554/elife.86233
• 发表时间: 2023-08-04
• 期刊: eLife
• 影响因子: 7.7
• 作者: O'Sullivan JDB;Blacker TS;Scott C;Chang W;Ahmed M;Yianni V;Mann ZF
• 通讯作者: Mann ZF

NAD(P)H binding configurations revealed by time-resolved fluorescence and two-photon absorption.

通过时间分辨荧光和双光子吸收揭示 NAD(P)H 结合构型。

• DOI: 10.1016/j.bpj.2023.02.014
• 发表时间: 2023
• 期刊: Biophysical journal
• 影响因子: 3.4
• 作者: Blacker TS

Nrf2 regulates glucose uptake and metabolism in neurons and astrocytes.

• DOI: 10.1016/j.redox.2023.102672
• 发表时间: 2023-06
• 期刊: REDOX BIOLOGY
• 影响因子: 11.4
• 作者: Esteras, Noemi;Blacker, Thomas S.;Zherebtsov, Evgeny A.;Stelmashuk, Olga A.;Zhang, Ying;Wigley, W. Christian;Duchen, Michael R.;Dinkova-Kostova, Albena T.;Abramov, Andrey Y.
• 通讯作者: Abramov, Andrey Y.