Metabolism is the ensemble of biochemical processes allowing a cell to live and build all of its constituents, exert its functions and cooperate to determine, in multicellular organisms, the functions of tissues and organs. Different tissues are characterized by metabolic processes that can differ both qualitatively (for example the type of substrates degraded to obtain chemical energy) and quantitatively (depending on the state of activity). Thus, metabolism is modulated during development and, in adult life, as a function of the level of activity of the whole animal and of each organ. Alterations of the regulation of metabolism can lead to pathologies and a remarkable change in the metabolic asset is characteristic also of cancer cells. At the whole animal level, the measurement of metabolism is largely based on the consumption of oxygen and the production of carbon dioxide; these measurements provide a course indication on the rate of metabolism and allow correlation with physiological or pathological conditions. A more detailed measurement of metabolism, at the cellular level, would provide key insights in the processes of differentiation during embryo development, and possibly clarify the mechanisms of several pathologies. Thus, methods enabling a real time in vivo measurement of metabolism in the whole animal are highly desirable. A fundamental organelle involved in the metabolic activities of the cell is the mitochondrion. Here, ATP is produced with consumption of oxygen and the involvement of coenzymes which are among the most important components contributing to a cell’s autofluorescence. The signal of these molecules (NADH and FAD), as it will be shown in this thesis, represents a great tool to measure metabolism in vivo with cellular resolution. Most of inhaled oxygen is used by mitochondria. Through oxidative phosphorylation, mitochondria oxidize the substrates of the Krebs cycle and produce adenosine triphosphate (ATP) which is the molecule that powers most of the cell's activities that require a source of energy. An increasing number of studies demonstrated that a wide number of human diseases are due to mitochondrial dysfunctions. Mitochondrial diseases are a large group of pathologies associated with defects in mitochondrial energy metabolism, mainly due to anomalies in the mitochondrial respiratory chain and/or in oxidative phosphorylation (OXPHOS)[1]. Oxidative phosphorylation deficiency can cause dysfunctions in the respiratory chain, a heteromultimeric structure embedded in the inner mitochondrial membrane, or it can be associated to a single or multiple defects of the five complexes forming the respiratory chain itself [2]. During my PhD work I tested if it was possible to perform in vivo morpho-functional imaging of zebrafish larvae tissues and organs through nonlinear optical (NLO) microscopy that offers the advantage to obtain images from tissue and organs in vivo without requiring exogenous stains or tissue excision. Moreover, a goal of the thesis has been to setup methods to employ NLO microscopy for characterizing not only the morphology of a tissue but also its metabolism and functionality. NLO microscopy is a high resolution laser scanning imaging technique that allows obtaining images with a good penetration depth into tissues. Cells and extracellular matrix of biological tissues contain a variety of intrinsically fluorescent molecules (NADH, FAD, tryptophan, keratin, melanin, elastin, cholecalciferol and others). These molecules, and their interactions with the surrounding environment, can provide a tool for measuring variations of metabolism and of the cellular and extracellular environmental conditions. One of the parameters commonly utilized to study the mitochondrial health and functions is the redox ratio [1], i.e. the NADH/FAD ratio, which allows obtaining information of the cell’s metabolic activity. In fact, Nicotinamide Adenine Dinucleotide (NADH) transfers electrons to the electron transport chain (ETC) while Flavin Adenine Dinucleotide (FAD) acts as an intermediary acceptor of electrons in the ETC. For this reason, it was decided to take advantage of the endogenous signal produced by these molecules to obtain information on metabolic state using nonlinear microscopy without having to add exogenous probes. The redox ratio can be obtained through the measurement of NADH fluorescence intensity, divided by the FAD fluorescence intensity. As stated above, this ratio is sensitive to changes in the cellular metabolic rate and vascular oxygen supply [1, 3]. An increase in the NADH/FAD redox ratio usually indicates increased cellular metabolic activity [4] and an increase of glycolytic state. The NADH and FAD fluorescence are reliable biomarkers reflecting the mitochondria functions. A complementary method to optically assess cellular metabolism based on NADH fluorescence relies fluorescence lifetime measurements. Combining TPEF and fluorescence-lifetime imaging microscopy (FLIM), it is possible to obtain more information about the molecular microenvironment of a fluorescent molecule. In the case of NADH, this method provides a tool to discriminate free and protein-bound components of NADH. In fact, NADH not bound to proteins has a short lifetime (around 0.3ns), whereas when it is bound to proteins it has long lifetime (2ns – 4.5ns). Therefore, in this work I also developed methods for the in vivo measurement of NADH fluorescence lifetime in zebrafish larvae and compared these measurements with those bases on intensity NADH/FAD ratios for different experimental conditions. To characterize the spectroscopic techniques developed, the zebrafish larvae were treated with compounds known for their effects on metabolism (Rotenone) and metabolic signaling pathways (DMOG). Another test of the method was performed with a transient know-down of the mitochondrial DNA Polymerase by use of morpholinos antisense oligos; in this study it has been used two kinds of morpholinos: splicing morpholinos that is zygotic-specific interfering with the maturation of a primary transcript of the mitochondrial DNA Polymerase; ATG morpholinos that not only interfering with the maturation of a primary transcript but also with mature maternal mRNAs of the mitochondrial DNA Polymerase; (thus inducing a decrease if the density of mitochondria in the developing embryo) In the following sections, the thesis will provide: - a discussion on mitochondrial diseases and different methods that reduce the activity of oxidative phosphorylation or activating the HIF signaling, that mimics the lack of oxygen, and the interference of MOpolG (morpholino against the transcription of mitochondrial DNA polymerase) on mitochondria replication in zebrabrafish larvae; moreover, the physical theories of nonlinear optics, in particular Two-Photon Excited Fluorescence (TPEF) and Fluorescence Lifetime Imaging Microscopy (FLIM) (chapter 1); - a description of the housing and care of zebrafish, the procedures adopted to characterize and calibrate both experimental setups, and the methods applied for data analysis (chapter 2); - a report on the results obtained from each study (chapter 3) - Conclusions (chapter 4).

Morpho-functional imaging of tissues by time- resolved fluorescence microscopy / Cristina Giubani. - (2018).

Morpho-functional imaging of tissues by time- resolved fluorescence microscopy

Cristina Giubani
2018

Abstract

Metabolism is the ensemble of biochemical processes allowing a cell to live and build all of its constituents, exert its functions and cooperate to determine, in multicellular organisms, the functions of tissues and organs. Different tissues are characterized by metabolic processes that can differ both qualitatively (for example the type of substrates degraded to obtain chemical energy) and quantitatively (depending on the state of activity). Thus, metabolism is modulated during development and, in adult life, as a function of the level of activity of the whole animal and of each organ. Alterations of the regulation of metabolism can lead to pathologies and a remarkable change in the metabolic asset is characteristic also of cancer cells. At the whole animal level, the measurement of metabolism is largely based on the consumption of oxygen and the production of carbon dioxide; these measurements provide a course indication on the rate of metabolism and allow correlation with physiological or pathological conditions. A more detailed measurement of metabolism, at the cellular level, would provide key insights in the processes of differentiation during embryo development, and possibly clarify the mechanisms of several pathologies. Thus, methods enabling a real time in vivo measurement of metabolism in the whole animal are highly desirable. A fundamental organelle involved in the metabolic activities of the cell is the mitochondrion. Here, ATP is produced with consumption of oxygen and the involvement of coenzymes which are among the most important components contributing to a cell’s autofluorescence. The signal of these molecules (NADH and FAD), as it will be shown in this thesis, represents a great tool to measure metabolism in vivo with cellular resolution. Most of inhaled oxygen is used by mitochondria. Through oxidative phosphorylation, mitochondria oxidize the substrates of the Krebs cycle and produce adenosine triphosphate (ATP) which is the molecule that powers most of the cell's activities that require a source of energy. An increasing number of studies demonstrated that a wide number of human diseases are due to mitochondrial dysfunctions. Mitochondrial diseases are a large group of pathologies associated with defects in mitochondrial energy metabolism, mainly due to anomalies in the mitochondrial respiratory chain and/or in oxidative phosphorylation (OXPHOS)[1]. Oxidative phosphorylation deficiency can cause dysfunctions in the respiratory chain, a heteromultimeric structure embedded in the inner mitochondrial membrane, or it can be associated to a single or multiple defects of the five complexes forming the respiratory chain itself [2]. During my PhD work I tested if it was possible to perform in vivo morpho-functional imaging of zebrafish larvae tissues and organs through nonlinear optical (NLO) microscopy that offers the advantage to obtain images from tissue and organs in vivo without requiring exogenous stains or tissue excision. Moreover, a goal of the thesis has been to setup methods to employ NLO microscopy for characterizing not only the morphology of a tissue but also its metabolism and functionality. NLO microscopy is a high resolution laser scanning imaging technique that allows obtaining images with a good penetration depth into tissues. Cells and extracellular matrix of biological tissues contain a variety of intrinsically fluorescent molecules (NADH, FAD, tryptophan, keratin, melanin, elastin, cholecalciferol and others). These molecules, and their interactions with the surrounding environment, can provide a tool for measuring variations of metabolism and of the cellular and extracellular environmental conditions. One of the parameters commonly utilized to study the mitochondrial health and functions is the redox ratio [1], i.e. the NADH/FAD ratio, which allows obtaining information of the cell’s metabolic activity. In fact, Nicotinamide Adenine Dinucleotide (NADH) transfers electrons to the electron transport chain (ETC) while Flavin Adenine Dinucleotide (FAD) acts as an intermediary acceptor of electrons in the ETC. For this reason, it was decided to take advantage of the endogenous signal produced by these molecules to obtain information on metabolic state using nonlinear microscopy without having to add exogenous probes. The redox ratio can be obtained through the measurement of NADH fluorescence intensity, divided by the FAD fluorescence intensity. As stated above, this ratio is sensitive to changes in the cellular metabolic rate and vascular oxygen supply [1, 3]. An increase in the NADH/FAD redox ratio usually indicates increased cellular metabolic activity [4] and an increase of glycolytic state. The NADH and FAD fluorescence are reliable biomarkers reflecting the mitochondria functions. A complementary method to optically assess cellular metabolism based on NADH fluorescence relies fluorescence lifetime measurements. Combining TPEF and fluorescence-lifetime imaging microscopy (FLIM), it is possible to obtain more information about the molecular microenvironment of a fluorescent molecule. In the case of NADH, this method provides a tool to discriminate free and protein-bound components of NADH. In fact, NADH not bound to proteins has a short lifetime (around 0.3ns), whereas when it is bound to proteins it has long lifetime (2ns – 4.5ns). Therefore, in this work I also developed methods for the in vivo measurement of NADH fluorescence lifetime in zebrafish larvae and compared these measurements with those bases on intensity NADH/FAD ratios for different experimental conditions. To characterize the spectroscopic techniques developed, the zebrafish larvae were treated with compounds known for their effects on metabolism (Rotenone) and metabolic signaling pathways (DMOG). Another test of the method was performed with a transient know-down of the mitochondrial DNA Polymerase by use of morpholinos antisense oligos; in this study it has been used two kinds of morpholinos: splicing morpholinos that is zygotic-specific interfering with the maturation of a primary transcript of the mitochondrial DNA Polymerase; ATG morpholinos that not only interfering with the maturation of a primary transcript but also with mature maternal mRNAs of the mitochondrial DNA Polymerase; (thus inducing a decrease if the density of mitochondria in the developing embryo) In the following sections, the thesis will provide: - a discussion on mitochondrial diseases and different methods that reduce the activity of oxidative phosphorylation or activating the HIF signaling, that mimics the lack of oxygen, and the interference of MOpolG (morpholino against the transcription of mitochondrial DNA polymerase) on mitochondria replication in zebrabrafish larvae; moreover, the physical theories of nonlinear optics, in particular Two-Photon Excited Fluorescence (TPEF) and Fluorescence Lifetime Imaging Microscopy (FLIM) (chapter 1); - a description of the housing and care of zebrafish, the procedures adopted to characterize and calibrate both experimental setups, and the methods applied for data analysis (chapter 2); - a report on the results obtained from each study (chapter 3) - Conclusions (chapter 4).
2018
Francesco Vanzi
ITALIA
Cristina Giubani
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