Shedding light on living cells and mineralised tissues using Raman spectroscopy

Raman micro-spectroscopy presents a highly sensitive, non-invasive, and rapid way to collect biochemical information from cells and tissues. The resulting Raman spectrum is a chemical ‘fingerprint’ containing a wealth of molecular level information which has been used to characterize, monitor, compare and confirm biological processes from the cellular to tissue levels. The work presented in this thesis utilizes Raman spectroscopy to test live in vitro cellular models, classify human tissues of interest, and determine the biomolecular differences in tissue samples which are diseased or undergoing therapeutic treatment. Additionally new ways of visualizing and interpreting multivariate analytical results are proposed and demonstrated to ease the determination of the biomolecular features which are most important when comparing sample groups. A persistent challenge in the interpretation of information rich biological Raman spectra includes the multitude of signals from lipids, proteins, carbohydrates, nucleic acids, and minerals found in a limited spectral range and in some instances overlapping significantly. Partial Least Squares – Discriminant Analysis (PLS-DA) Variable Importance Projection (VIP) scores were presented as heat maps overlaying difference spectra to ease the visualization of significant biochemical bond changes between sample groups and their trends. The advantages of applying PLS-DA VIP scores in this way are demonstrated in well studied and known system including a cultured cellular model incorporating fixation methods and a human tissue comparison between healthy and osteoporotic bone. PLS-DA VIP score plots were additionally utilized to characterize and compare the biolomecular environments surrounding the recently described microscopic mineral inclusions in human aortic valves and aortae. The PLS-DA VIP score plots exposed the chemical differences in these systems through highlighting the corresponding spectral bands in an easy to read and interpret way. Raman micro-spectroscopy was also applied to investigate an in vitro ‘calcified’ porcine aortic valvular interstitial cell model. This model system was probed for the first time using the combination of Raman micro-spectroscopy and complimentary gold standard biological techniques to determine the protein and potential mineral content within these nodular, cellular systems. The ‘calcified’ porcine aortic valvular cell nodules showed no evidence of mineral inclusion. These nodules did exhibit a heavy extracellular matrix production including the production of collagen I. The porcine aortic valvular cell nodules acting as a model system for diseased aortic valve tissue requires not only the characterization of the cell nodule in vitro but also the characterization of the human disease spectrum which the model is suggested to replicate. The discovery and characterisation of microscopic mineral spherical inclusions (50nm-200µm) located in both valvular and vascular tissues leads to an interesting question on the introduction and role of microscopic mineral deposits in cardiovascular disease. Here Raman micro-spectroscopy was utilized to investigate the organic matrix surrounding these microscopic mineral deposits to determine if any colocalised protein changes exist. Protein and specifically collagen changes are demonstrated between tissues with and without the spherical mineral deposits despite being macroscopically indistinguishable. Raman spectroscopy was also utilized to provide direct insights into tissue constituent and structural changes on the molecular level in heat-induced tissue fusion via radio-frequency (RF) energy. This type of tissue fusion has gained wide acceptance clinically and is presented here as the first optical-Raman-spectroscopy study on tissue fusion samples in vitro. This study exposed spectroscopic evidence for the loss of distinct collagen fibres rich tissue layers as well as the denaturing and restructuring of collagen crosslinks post RF fusion. Raman spectroscopy is a demonstrated, powerful, biomolecular imaging technique which benefits from advancements in mathematical analytical techniques as well as its own application in biological investigations. This thesis explores the application of Raman spectroscopy in combination with powerful analytical techniques to further characterize and compare biological systems of interest.Open Acces

UVR8 mediated spatial differences as a prerequisite for UV-B induced inflorescence phototropism

In Arabidopsis hypocotyls, phototropins are the dominant photoreceptors for the positive phototropism response towards unilateral ultraviolet-B (UV-B) radiation. We report a stark contrast of response mechanism with inflorescence stems with a central role for UV RESISTANCE LOCUS 8 (UVR8). The perception of UV-B occurs mainly in the epidermis and cortex with a lesser contribution of the endodermis. Unilateral UV-B exposure does not lead to a spatial difference in UVR8 protein levels but does cause differential UVR8 signal throughout the stem with at the irradiated side 1) increase of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), 2) an associated strong activation of flavonoid biosynthesis genes and flavonoid accumulation, 3) increased GA2oxidase expression, diminished gibberellin1 levels and accumulation of DELLA protein REPRESSOR OF GA1 (RGA) and, 4) increased expression of the auxin transport regulator, PINOID, contributing to local diminished auxin signalling. Our molecular findings are in support of the Blaauw theory (1919), suggesting that differential growth occurs trough unilateral photomorphogenic growth inhibition. Together the data indicate phototropin independent inflorescence phototropism through multiple locally UVR8-regulated hormone pathways

Elucidating the Role of Endogenous Electric Fields in Regulating the Astrocytic Response to Injury in the Mammalian Central Nervous System

Endogenous bioelectric fields guide morphogenesis during embryonic development and regeneration by directly regulating the cellular functions responsible for these phenomena. Although this role has been extensively explored in many peripheral tissues, the ability of electric fields to regulate wound repair and stimulate regeneration in the mammalian central nervous system (CNS) has not been convincingly established. This dissertation explores the role of electric fields in regulating the injury response and controlling the regenerative potential of the mammalian CNS. We place particular emphasis on their influence on astrocytes, as specific differences in their injury-induced behaviors have been associated with differences in the regenerative potential demonstrated between mammalian and non-mammalian vertebrates. For example, astrocytes in both mammalian and non- mammalian vertebrates begin migrating towards the lesion within hours and begin to proliferate after an initial delay of two days; subsequently, astrocytes in non-mammalian vertebrates support neurogenesis and assume a bipolar radial glia-like morphology that guides regenerating axons, whereas astrocytes in mammals do not demonstrate robust neurogenesis and undergo a hypertrophic response that inhibits axon sprouting. To test whether injury-induced electric fields drive the astrocytic response to injury, we exposed separate populations of purified astrocytes from the rat cortex and cerebellum to electric field intensities associated with intact and injured mammalian tissues, as well as to those electric field intensities measured in regenerating non-mammalian vertebrate tissues. Upon exposure to electric field intensities associated with uninjured tissue, astrocytes showed little change in their cellular behavior. However, cortical astrocytes responded to electric field intensities associated with injured mammalian tissues by demonstrating dramatic increases in migration and proliferation, behaviors that are associated with their formation of a glial scar in vivo; in contrast, cerebellar astrocytes, which do not organize into a demarcated glial scar, did not respond to these electric fields. At electric field intensities associated with regenerating tissues, both cerebellar and cortical astrocytes demonstrated robust and sustained responses that included morphological changes consistent with a regenerative phenotype. These results support the hypothesis that physiologic electric fields drive the astrocytic response to injury, and that elevated electric fields may induce a more regenerative response among mammalian astrocytes

Tree Peony Species Are a Novel Resource for Production of α-Linolenic Acid

Tree peony is known worldwide for its excellent ornamental and medical values, but recent reports that their seeds contain over 40% α-linolenic acid (ALA), an essential fatty acid for humans drew additional interest of biochemists. To understand the key factors that contribute to this rich accumulation of ALA, we carried out a comprehensive study of oil accumulation in developing seeds of nine wild tree peony species. The fatty acid content and composition was highly variable among the nine species; however, we selected a high- (P. rockii) and low-oil (P. lutea) accumulating species for a comparative transcriptome analysis. Similar to other oilseed transcriptomic studies, upregulation of select genes involved in plastidial fatty acid synthesis, and acyl editing, desaturation and triacylglycerol assembly in the endoplasmic reticulum was noted in seeds of P. rockii relative to P. lutea. Also, in association with the ALA content, transcript levels for fatty acid desaturases (SAD, FAD2 and FAD3), which encode for enzymes necessary for polyunsaturated fatty acid synthesis were higher in P. rockii compared to P. lutea. We further showed that the overexpression of PrFAD2 and PrFAD3 in Arabidopsis increased linoleic and α-linolenic acid content, respectively and modulated their final ratio in the seed oil. In conclusion, we identified the key steps that contribute to efficient ALA synthesis and validated the necessary desaturases in P. rockii that are responsible for not only increasing oil content but also modulating 18:2/18:3 ratio in seeds. Together, these results will aid to improve essential fatty acid content in seeds of tree peonies and other crops of agronomic interest

Measurement and mathematical modeling of hyperthermia induced bioeffects in pancreatic cancer cells

Doctor of PhilosophyDepartment of Electrical and Computer EngineeringPunit PrakashSurgical resection is the standard of care for pancreatic cancer, although treatment outcomes remain poor, and a large fraction of the patient population are not surgical candidates. Minimally invasive interventions employing non-ionizing energy, such as image-guided thermal ablation, are under investigation for treatment of unresectable tumors and potentially for debulking and downstaging tumors. Tissue regions at the periphery of an ablation zone are exposed to sub-ablative thermal profiles (referred to as “mild hyperthermia”), which may induce a range of bioeffects including change in perfusion, immune modulation, and others. Bioeffects induced by heating are a function of intensity of heating and duration of thermal exposure. This dissertation presents a suite of tools for integrated in vitro experimental studies and modeling for characterizing bioeffects following thermal exposure to pancreatic cancer cells. An instrumentation platform was developed for exposing monolayer cell cultures to temperatures in the range 42–50°C for 3–60 minutes. The platform was employed to determine the Arrhenius kinetic parameters of thermal injury to pancreatic cancer cells (i.e. loss in viability) following heating. When coupled with bioheat transfer models, these parameters facilitate investigations of thermal injury profiles in pancreatic tumors following thermal exposure with practical devices. There has been growing interest in exploring the potential of thermal therapies for modulating tumor—immune system interactions, due in part to release of damage associated molecular patterns (DAMPs) from stressed tumor cells and their role in recruiting and activating antigen presenting cells. The in vitro thermal exposure platform was further expanded to allow for experimental measurement of extracellular DAMPs released from murine pancreatic cancer cells following heating to temperatures in the range 42 – 50°C for 3-60 mins. A model predicting the dynamics of heat-induced DAMPs release was developed and may inform the design of experiments investigating the role of heat in modulating the anti-tumor immune response. While in vitro experiments on monolayers are informative, 3D cell cultures (e.g., spheroid, organoids) provide an experimental platform accommodating multiple cell types in an environment that may be more representative of tumors in vivo. Furthermore, while the water-bath based in vitro platform applied for monolayers is well suited to achieving near-uniform temperature profiles, in vivo delivery of hyperthermia often yields a gradient of temperatures that is not achieved through water-bath based heating. Thus, an in vitro platform for exposing cells in 3D culture (co-culture of multiple cell populations) to 2.45 GHz microwave hyperthermia was developed. The platform includes a printed patch antenna and associated thermal management elements and was applied to study changes in gene expression profile of a 3D culture of pancreatic cancer cells and fibroblasts. This non-contact microwave heating approach may help enable additional studies for exploring the bioeffects of heat on cancer cells

Microglia: Development of a human in vitro model, analysis of motility and effects of phagocytosis in the hippocampal neurogenic niche

135 p.This PhD Thesis discusses several topics related to microglia, the resident macrophages of the brain parenchyma. They are derived from yolk sack primitive macrophages that colonize the brain early during embryonic development. Microglia contribute to the correct development and functioning of the central nervous system with their multiple functions, including their role as phagocytes clearing the brain parenchyma from apoptotic cells and protein aggregates. Microglia are also involved in most neurological and neurodegenerative diseases, especially since genome-wide association studies have found that many microglia-specific genes are significant risk factors for neurodegenerative diseases. This PhD Thesis focusses on two aspects of microglial physiology. First, on the development of human models of microglia and their importance in the use of microglia as a therapeutic target in neurodegenerative diseases. Finally, on microglial process motility and phagocytosis, two essential functions of homeostatic microglia that can become compromised in pathology. This thesis defines a method for the semi-automated analysis of microglia in 3D and how inflammation affects microglial motility. This thesis also shows how an impairment of phagocytosis in the adult neurogenic niche negatively affects the neurogenic cascade suggesting a crosstalk between microglia and neural progenitor cells