| 1. |
- Akkuratov, Evgeny E.
(author)
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The Biophysics of Na+,K+-ATPase in neuronal health and disease
- 2020
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Doctoral thesis (other academic/artistic)abstract
- Na+,K+-ATPase is one of the most important proteins in the mammalian cell. It creates sodium and potassium gradients which are fundamental for the membrane potential and sodium-dependent secondary active transport. It has a second role in the cell as a receptor that by binding chemicals from the cardiotonic steroids family, the most knowledgeable of them is ouabain, triggers various signaling pathways in the cell which regulate gene activation, proliferation, apoptosis, etc. It has been shown that several severe neurological diseases are associated with mutations in the Na+,K+-ATPase encoding genes. Although Na+,K+-ATPase was discovered already in 1957 by the Danish scientist Jens Skou, the knowledge about the function of this enzyme is still not complete. In the studies included in the thesis, we have learned more about the function of Na+,K+-ATPase in different aspects of health and disease. In study I we showed a mechanism of ouabain-dependent regulation of the NMDA receptor, one of the most important receptors in the nervous system, via binding with Na+,K+-ATPase. This allows us to look at the Na+,K+-ATPase as regulator via protein-protein interaction. In study II we investigated a different aspect of Na+,K+-ATPase functioning – to look at how binding of ouabain to Na+,K+-ATPase activates a number of signaling cascades by looking at the phosphoproteome status of the cells. This allows us to see the whole picture of ouabain-mediated cascades and further characterize them. In study III we focused on the role of Na+,K+-ATPase in severe epileptic encephalopathy caused by a mutation in the ATP1A1 gene. We performed a molecular and cellular study to describe how mutations affects protein structure and function and found that this mutation converts the ion pump to a nonspecific leak channel. In study IV we performed a translational study of the most common mutation for rapid-onset dystonia-parkinsonism. We studied how this mutation affects the nervous system on the protein-, cellular-, and organism level and found that the complete absence of ultraslow afterhyperpolarization (usAHP) could explain gait disturbances found in patients. In the on-going study we showed that Na+,K+-ATPase can oligomerize and that this effect is triggered by ouabain binding to the Na+,K+-ATPase. In this study, we utilized a novel fluorescence labelling approach and used biophysical techniques with single molecule sensitivity to track Na+,K+-ATPase interactions. In summary, we applied biophysical and molecular methods to study different aspects of the function of Na+,K+-ATPase, and gained insights that could be helpful not only for answering fundamental questions about Na+,K+-ATPase but also to find a treatment for patients with diseases associated with mutations in this protein.
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| 2. |
- Bernhem, Kristoffer
(author)
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Quantitative bioimaging in single cell signaling
- 2017
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Doctoral thesis (other academic/artistic)abstract
- Imaging of cellular samples has for several hundred years been a way for scientists to investigate biological systems. With the discovery of immunofluorescence labeling in the 1940’s and later genetic fluorescent protein labeling in the 1980’s the most important part in imaging, contrast and specificity, was drastically improved. Eversince, we have seen a increased use of fluorescence imaging in biological research, and the application and tools are constantly being developed further.Specific ion imaging has long been a way to discern signaling events in cell systems. Through use of fluorescent ion reporters, ionic concentrations can be measured inliving cells as result of applied stimuli. Using Ca2+ imaging we have demonstrated that there is a inverse influence by plasma membrane voltage gated calcium channels on angiotensin II type 1 receptor (a protein involved in blood pressure regulation). This has direct implications in treatment of hypertension (high blood pressure),one of the most common serious diseases in the western civilization today with approximately one billion afflicted adults world wide in 2016.Extending from this more lower resolution live cell bioimaging I have moved into super resolution imaging. This thesis includes works on the interpretation of super resolution imaging data of the neuronal Na+, K+ - ATPase α3, a receptor responsible for maintaining cell homeostasis during brain activity. The imaging data is correlated with electrophysiological measurements and computer models to point towards possible artefacts in super resolution imaging that needs to be taken into account when interpreting imaging data. Moreover, I proceeded to develop a software for single-molecule localization microscopy analysis aimed for the wider research community and employ this software to identify expression artifacts in transiently transfected cell systems.In the concluding work super-resultion imaging was used to map out the early steps of the intrinsic apoptotic signaling cascade in space and time. Using superresoultion imaging, I mapped out in intact cells at which time points and at which locations the various proteins involved in apoptotic regulation are activated and interact.
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| 3. |
- Edwards, Steven, 1992-
(author)
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Advancing tissue clearing and expansion methods for high-resolution volumetric imaging of biological samples
- 2022
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Doctoral thesis (other academic/artistic)abstract
- The development of advanced light microscopes, capable of imaging samples at ever-higher spatial resolution and increasing speeds is an ongoing endeavour. The sample itself is an integral part of the microscope and, unlike the intricately positioned and highly polished lenses, it is an optically unpredictable component. Composed of a mixture of biological polymers, lipids, inorganic ions, the sample is a hindrance to the otherwise predictable path of light and frequently degrades the microscope’s performance. The optical properties of the sample are therefore of equal importance to those of the microscope hardware. Preparing a sample for microscopy involves tuning these optical properties to maintain or in some cases, enhance the microscope’s performance.Optical tissue clearing includes a wide range of protocols aiming at making large, opaque biological samples optically transparent. This in turn facilitates volumetric imaging of whole organ systems and negates the requirement for physical sectioning of the sample. Expansion microscopy is a technique in which biological samples can be physically magnified. This method not only clears the sample but improves the effective resolution that can be achieved in a microscope. Optical tissue clearing and expansion microscopy protocols must be further adapted and developed to address the variety of biological samples, ranging from single cells to complex tissues and model organisms.In Paper I, we developed a clearing protocol, termed CUBIC-f, which was optimised for fragile samples. We used this method to quantify neuronal cell density and trace neuronal projections in the salamander brain. In Paper II, we explored the use of expansion microscopy on 3D cell cultures to perform high-resolution imaging with improved labelling and signal-to-background ratio, resulting in more accurate image segmentation. In paper III, expansion microscopy was used in combination with light-sheet and STED microscopy to reveal the role of cerebrospinal fluid-contacting neurons in the central canal of the lamprey spinal cord. Finally, in Paper IV we combined non-canonical amino acid fluorescent labelling with expansion microscopy, demonstrating two colour super-resolution imaging of the alpha and beta subunit of the sodium pump with minimal fluorophore linkage error.
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| 4. |
- Guala, Dimitri, 1979-
(author)
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Functional association networks for disease gene prediction
- 2017
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Doctoral thesis (other academic/artistic)abstract
- Mapping of the human genome has been instrumental in understanding diseasescaused by changes in single genes. However, disease mechanisms involvingmultiple genes have proven to be much more elusive. Their complexityemerges from interactions of intracellular molecules and makes them immuneto the traditional reductionist approach. Only by modelling this complexinteraction pattern using networks is it possible to understand the emergentproperties that give rise to diseases.The overarching term used to describe both physical and indirect interactionsinvolved in the same functions is functional association. FunCoup is oneof the most comprehensive networks of functional association. It uses a naïveBayesian approach to integrate high-throughput experimental evidence of intracellularinteractions in humans and multiple model organisms. In the firstupdate, both the coverage and the quality of the interactions, were increasedand a feature for comparing interactions across species was added. The latestupdate involved a complete overhaul of all data sources, including a refinementof the training data and addition of new class and sources of interactionsas well as six new species.Disease-specific changes in genes can be identified using high-throughputgenome-wide studies of patients and healthy individuals. To understand theunderlying mechanisms that produce these changes, they can be mapped tocollections of genes with known functions, such as pathways. BinoX wasdeveloped to map altered genes to pathways using the topology of FunCoup.This approach combined with a new random model for comparison enables BinoXto outperform traditional gene-overlap-based methods and other networkbasedtechniques.Results from high-throughput experiments are challenged by noise and biases,resulting in many false positives. Statistical attempts to correct for thesechallenges have led to a reduction in coverage. Both limitations can be remediedusing prioritisation tools such as MaxLink, which ranks genes using guiltby association in the context of a functional association network. MaxLink’salgorithm was generalised to work with any disease phenotype and its statisticalfoundation was strengthened. MaxLink’s predictions were validatedexperimentally using FRET.The availability of prioritisation tools without an appropriate way to comparethem makes it difficult to select the correct tool for a problem domain.A benchmark to assess performance of prioritisation tools in terms of theirability to generalise to new data was developed. FunCoup was used for prioritisationwhile testing was done using cross-validation of terms derived fromGene Ontology. This resulted in a robust and unbiased benchmark for evaluationof current and future prioritisation tools. Surprisingly, previously superiortools based on global network structure were shown to be inferior to a localnetwork-based tool when performance was analysed on the most relevant partof the output, i.e. the top ranked genes.This thesis demonstrates how a network that models the intricate biologyof the cell can contribute with valuable insights for researchers that study diseaseswith complex genetic origins. The developed tools will help the researchcommunity to understand the underlying causes of such diseases and discovernew treatment targets. The robust way to benchmark such tools will help researchersto select the proper tool for their problem domain.
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| 5. |
- Guldevall, Karolin, 1981-
(author)
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Single Cell Investigations of the Functional Heterogeneity Within Immune Cell Populations : a Microchip-based Study
- 2014
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Doctoral thesis (other academic/artistic)abstract
- Immune cell populations are constantly divided into smaller and smaller subsets defined by newly emerging cellular markers. However, there is a growing awareness of the functional heterogeneities in between cells even within small populations, in addition to the heterogeneity over time. One may ask whether a population is correctly defined only by cellular markers or if the functionality should be regarded as well? Many of today’s techniques only measure at the population level, giving an average estimate of the behavior of that pool of cells, but failing to detect rare possibly important events. Thus, high-throughput experimental approaches to analyze single cells over time are required to address cellular heterogeneity.Progress in the fields of microfabrication, microscopy and computing have paved the way for increasingly efficient tools for studies on the single cell level, and a variety of devices have been described by others. However, few of them are suitable for long-term imaging of dynamic events such as cell-cell interactions or migration. In addition, for efficient recording of many individual events it is desirable to scale down the cells’ interaction volume; not only to shorten the time to interaction, but also to increase the number of individual events in a given area; thereby pushing a screening approach.To address these questions, a complete microwell array system for imaging of immune cell responses with single-cell resolution was designed. The platform consists of a range of silicon-glass microchips with arrays of miniature wells for incubation of cells and a custom made holder that fits conventional microscopes. The device has been designed to allow cells to be kept viable for several days in the wells, to be easy to use and to allow high-resolution imaging. Five different designs were fabricated; all with a specific type of assay in mind, and were evaluated regarding biocompatibility and functionality. Here, the design aimed for screening applications is the main focus. In this approach a large amount, tens of thousands, of small wells are imaged two to three times: first directly post-seeding of effector and target cells to register the well’s content, and second after some time has passed to allow for cell-cell interactions. The final read-out is the number of killed target cells in each well, making an automatic cell counting protocol necessary in order to analyze the massive amount of data generated.We here show that our silicon microwell platform allows long-term studies with the possibility of both time-lapse and high-resolution imaging of a variety of immune cell behavior. Using both time-lapse imaging and the screening approach we confirmed and investigated immune cell heterogeneity within NK cell populations in regards to both cytotoxicity and migrational behavior. In addition, two different types of cytolytic behavior in NK cells, termed fast and slow killing, were described and evaluated in regards to dynamic parameters; like conjugation and attachment time. We could also quantify the type of cytolytic response in relation to serial killing NK cells, and saw that serial killing NK cells more often induced fast target cell death. Further investigations using the screening approach have shown that serial killing NK cells also differ from other NK cells in their morphology, being both larger and with a more elongated shape. So far the platform has been used to gain better understanding of some aspects of NK cell biology, but there is still much left to explore. With the addition of an automatic counting program, the large numbers of wells that can be simultaneously imaged will provide new statistical information and enable higher throughput.Altogether, our family of techniques enables novel types of cellular imaging assays allowing data collection at a level of resolution not previously obtained – this was shown to be important for performing basic cell biological studies, but may also prove valuable in the proposed future medical applications such as adoptive cell therapy and stem cell transplantation.
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| 6. |
- Hueting, David A., 1993-
(author)
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In silico protein design for the enhancement of protein stability and function
- 2023
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Doctoral thesis (other academic/artistic)abstract
- Enzymes are natures catalysts that increase the rate of a chemical reaction. The increased rate of a reaction is required to be able to sustain life. Despite the huge impact of enzymes, they are not perfect catalysts. Enzyme and protein engineering is the discipline in which proteins are characterized and engineered to have improved inherent properties. Interesting properties of an enzyme to improve include stability and activity. The aim of this work is to understand how proteins and enzymes function and use a variety of different protein engineering techniques to enhance the properties of different proteins. In this work proteins and enzymes are engineered to increase our knowledge of the target proteins for downstream biomedical applications. A mix between rational and semi-rational engineering is applied in this work. In paper I and paper II, the method used is ancestral sequence reconstruction. A method that utilizes the evolutionary relationship between homologous sequences. In paper I the method was applied to a terpene cyclase, which cyclizes a precursor terpene into potential interesting drug leads. The result was a hyperstable enzyme variant. In paper II the technique was applied to the SARS-CoV-2 Spike protein. The protein is responsible for the virus SARS-CoV-2 to enter human cells. The work yielded a stable spike protein that readily expresses and can be utilized as a vaccine lead. In paper III, the aim was to understand human oxidosqualene cyclase (hOSC). A terpene cyclase essential in cholesterol synthesis. The enzyme hOSC was rationally engineered to change the driving force of the reaction. Through targeted mutations the reaction changed from entropy driven to enthalpy driven. Finally, in paper IV, a rationally engineered PETase, which is capable of degrading PET polymers into monomers, was proven to be active in human serum and verifies the proof-of-concept of degrading plastic in human blood. To summarize, the results in this thesis show the applicability of different enzyme engineering techniques to stabilize or change the function of proteins and the potential of engineered proteins in medical applications.
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| 7. |
- Jess, David Unnersjö
(author)
-
High-resolution Imaging of Cleared and Expanded Kidney Tissue Samples
- 2019
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Doctoral thesis (other academic/artistic)abstract
- The kidney is one of the most important and complex organs in the humanbody with the task of filtering hundreds of litres of blood daily. It is responsiblefor the salt and acid/base balance in the body, as well as secretinghormones important for red blood cell production and blood pressure regulation. Kidney disease is one of the fastest growing causes of death in the modern world, and this motivates extensive research for better understandingthe function of the kidney in both health and disease. Kidney failure or end stage renal disease (ESRD) is irreversible and requires treatment with dialysisor transplantation. Some of the most important cellular structures for blood filtration in the kidney are of very small dimensions (below 200 nanometers), and thus electron microscopy has previously been the only method with high enough resolution to study the morphology and topology of these minute structures. In three studies included in this thesis, we show that the finest elements of the kidney can now be resolved using different light microscopy techniques. In study 1, we show that by combining optical clearing with STED microscopy, protein localizations in the slit diaphragm of the kidney can be resolved, with widths around 75 nanometers. In study 3, a novel sample preparation method, expansion microscopy, is utilized to isotropically expand kidney tissue samples in space. Expansion improves the effective resolution by a factor of 5, making it possible to resolve podocyte foot processes and the slit diaphragmusing diffraction-limited confocal microscopy. We also show that by combining expansion microscopy and STED microscopy, the effective resolution can be improved even further (<20 nm). In our most recent work, study 5, we apply a simplified, moderate tissue swelling protocol which together with optimization of the confocal imaging provides sufficient resolution to resolve foot processes and parts of the filtration barrier. This new protocol is fast and technically simple, making it ideal for routine use, such as for future clinical pathology. In collaboration with kidney researchers, we have applied both STED microscopy and expansion microscopy to various disease models, showing that these tools can be used to both visualize and quantify pathologies occurring in different parts of the glomerular filtration barrier (GFB). In study 2, STED microscopy in combination with optical clearing is used to study the localization of Coro2b in secondary foot processes in both mouse and human tissue. In two ongoing studies with preliminary results presented in the thesis, we use STED microscopy and optical clearing to study the pathogenesis of focal segmental glomerulosclerosis (FSGS) by the use of genetic mouse models. Based on STED images, we extract different morphological parameters from foot processes and the glomerular filtration barrier (GFB) at different stages of the disease. In study 4, we apply a tissue expansion protocol to answer questions about the phenotype seen in podocytes where the mediator complex subunit 22 (Med22) is inactivated. By inactivating Med22 in a transgenic mouse line with cytosolic expression of tdTomato in podocytes, we saw strong indications that the vesicle-like structures seen in EM micrographs were indeed intracellular vesicles and not dilated sub-podocyte space. In summary, the work presented in this thesis has contributed to the development of a new toolbox for imaging renal ultra-structure using light microscopy, a field previously reserved for electron microscopy.
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| 8. |
- Kamali-Zare, Padideh
(author)
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Modeling Biophysical Mechanisms underlying Cellular Homeostasis
- 2010
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Doctoral thesis (other academic/artistic)abstract
- Cellular homeostasis is the effort of all living cells to maintain their intracellular content when facing physiological change(s) in the extracellular environment. To date, cellular homeostasis is known to be regulated mainly by time-consuming active mechanisms and via multiple signaling pathways within the cells. The aim of this thesis is to show that time-efficient passive (physical) mechanisms also, under the control and regulation of bio-physical factors such as cell morphology and distribution and co-localization of transport proteins in the cell membrane, can regulate cellular homeostasis. This thesis has been developed in an interface between physics and biology and focuses on critical cases in which cells face physiologically unstable environments at their steady state and therefore may need a constituent effort to maintain their homeostasis. The main hypothesis here is that the cell geometry is oriented in such a way that cellular homeostasis is preserved in a given environment. For exploring these cases, comparative spatial models have been developed that combine transporting function of membrane proteins with simple versus complex geometries of cells. Models confirm the hypothesis and show that cell morphology, size of extracellular space and intercellular distances are important for a dynamic regulation of water and ion homeostasis at steady state. The main clue is the existence of diffusion limited space (DLS) in the bulk extracellular space (ECS). DLS can, despite being ECS, maintain its ionic content and water balance due a controlled function of transport proteins in the membrane facing part of DLS. This can significantly regulate cellular water and ion homeostasis and play an important role in cell physiology. In paper I, the role of DLS is explored in the kidney whereas paper II addresses the brain. The response of cells to change in osmolarity is of critical importance for water homeostasis. Cells primarily respond to osmotic challenge by transport of water via their membranes. As water moves into or out of cells, the volumes of intra- and extracellular compartments consequently change. Water transport across the cell membrane is enhanced by a family of water channel proteins (aquaporins) which play important roles in regulation of both cell and the extracellular space dimensions. Paper III explores a role for aquaporins in renal K+ transport. Experimentally this role is suggested to be different from bulk water transport. In a geometrical model of a kidney principal cell with several DLS in the basolateral membrane, a biophysical role for DLS-aquaporins is suggested that also provides physiological relevance for this study. The biophysical function of water channels is then extensively explored in paper IV where the main focus has been the dynamics of the brain extracellular space following water transport. Both modeling and experimental data in this paper confirmed the importance of aquaporin-4 expressed in astrocytes for potassium kinetics in the brain extracellular space. Finally, geometrically controlled transport mechanisms are studied on a molecular level, using silicon particles as a simplified model system for cell studies (paper V and VI). In paper V the role of electrostatic forces (around the nano-pores and in between the loaded material and the silicon surface) is studied with regard to transport processes. In paper VI the roles of pore size and molecular weight of loaded material are studied. All together this thesis presents various modeling approaches that employ biophysical aspects of transport mechanisms combined with cell geometry to explain cell homeostasis and address cell physiology-based questions.
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| 9. |
- Kowalewski, Jacob, 1978-
(author)
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Modeling and Data Analysis in Cellular Biophysics
- 2009
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Doctoral thesis (other academic/artistic)abstract
- Cellular biophysics deals with the physical aspects of cell biology. This thesis presents a number of studies where mathematical models and data analysis can increase our understanding of this field. During recent years development in experimental methods and mathematical modeling have driven the amount of data and complexity in our understanding of cellular biology to a new level. This development has made it possible to describe cellular systems quantitatively where only qualitative descriptions were previously possible. To deal with the complex data and models that arise in this kind of research a combination of tools from physics and cell biology has to be applied; this constitutes a field we call cellular biophysics. The aim of this doctoral thesis is to develop novel approaches in this field. I present eight studies where quantitative modeling and analysis are involved. The first two studies concern cells interacting with their surrounding environment in the kidney. These cells sense fluid flow and respond with calcium (Ca2+) signals. The interaction between fluid and cells in renal tubular epithelium can be described by biomechanical models. This thesis describes a mathematical model of flow sensing by cilia with focus on the flow frequency response and time delay between the mechanical stress and the Ca2+ signaling response. Intracellular Ca2+ is kept at a very low level compared to the extracellular environment, while several intracellular compartments have higher Ca2+ concentration than the cytoplasm. This makes Ca2+ an efficient messenger for intracellular signaling, the process whereby signals are transduced from an extracellular stimulus to an intracellular activity such as gene expression. An important type of Ca2+ signaling is oscillations in intracellular Ca2+ concentration which occur due to the concerted interplay between different transport mechanisms within a cell. A study in this thesis examines ways to explain these mechanisms in terms of a mathematical model. Another study in the thesis reports that erythropoietin can regulate the water permeability of astrocytes and that it alters the pattern of Ca2+ oscillations in astrocytes. In this thesis the analysis of this Ca2+ signaling is described. Simulations described in one of the studies show how different geometries can affect the fluorescence recovery and that geometrically constrained reactions can trap diffusing receptors in dendritic spines. When separate time scales are present in a fluorescence revovery after photobleaching (FRAP) experiment the reaction and diffusion components can be studied separately. Applying single particle tracking methods to the migration trajectories of natural killer cells shows that there is a correlation between the formation of conjugates and transient confinement zones (TCZs) in these trajectories in vitro. TCZs are also present in in vivo experiments where they show strong similarities with the in vitro situation. This approach is a novel concept in data analysis methods for tracking immune cells.
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| 10. |
- Krishnan, Kalaiselvan, 1988-
(author)
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Studies on molecular mechanisms in calcium signaling and cellular energy consumption
- 2017
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Doctoral thesis (other academic/artistic)abstract
- Ion signaling plays fundamental role in cell survival. Na+ and Ca2+ are critical players in ion signaling. Cells spend the major amount of energy to maintain and regulate Na+ and Ca2+ gradients across the cell membrane. Any disruption in cellular energy consumption by plasma membrane ATPases affects ion signaling and vice versa. This thesis is a combination of four separate research studies. In the first study, We measured ATP consumption dynamics of Na+/K+-ATPase using a genetically encoded fluorescent indicator called Perceval HR. we demonstrate that PercevalHR is an excellent tool to visualize ATP:ADP in mammalian cells.In the second study, We studied the role of calcium signaling and TRP channels in angiotensin II type 1 receptor signaling cascade. We prove that low inhibition of CaV1.2 with physiological and therapeutically relevant concentration of Angiotensin II up regulate AT1R signaling.In the third study, We studied the role of the TRPM5 channel in regulating insulin secretion, and cytoplasmic free calcium concentration in the rat β-cells by usingtriphenyl phosphine oxide, a selective inhibitor of the channel.In the fourth study, We tested whether, the genetically engineered human β-cell line (EndoC-BH1) could be used as models for studying Ca2+signaling in the context of Type II Diabetes. We found that the EndoC-BH1 cells could be a relevant model to study stimulus-secretion coupling and Ca2+ signaling in the human β-cells.
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