<p dir="ltr">The vagus nerve (VN) is the tenth cranial nerve that mediates most of the parasympathetic functions of the autonomic nervous system. The axons of the human VN comprise a mix of unmyelinated and myelinated axons, where ~80% of the axons are unmyelinated C fibers (Havton et al., 2021). Understanding that most VN axons are unmyelinated, there is a need to map the pathways of these axons to and from organs to understand their function(s) and whether C fiber morphology or signaling characteristics yield insights into their functions. Developing a machine learning model that detects and predicts the morphology of VN single fiber action potentials based on select fiber characteristics, e.g., diameter, myelination, and position within the VN, allows us to more readily categorize the nerve fibers with respect to their function(s). Additionally, the features of this machine learning model could help inform peripheral neuromodulation devices that aim to restore, replace, or augment one or more specific functions of the VN that have been lost due to injury, disease, or developmental abnormalities.</p><p dir="ltr">We designed and trained four types of Multi-layer Perceptron Artificial Deep Neural Networks (MLP-ANN) with 10,000 rat abdominal vagal C-fibers simulated via the peripheral neural interface model ViNERS. We analyze the accuracy of each MLP-ANN’s SFAP predictions by conducting normalized cross-correlation and morphology analyses with the ViNERS C-fiber SFAP counterparts. Our results showed that our best MLP predicted over 94% of the C-fiber SFAPs with strong normalized cross-correlation coefficients of 0.7 through 1 with the ViNERS SFAPs. Overall, this novel tool can use a C-fiber’s biophysical characteristics (i.e., fiber diameter size, fiber position on the x/y axis, etc.) to predict C-fiber SFAP morphology.</p>

Viscoelasticity and its alteration in time and space has turned out to act as a key element in fundamental biological processes in living systems, such as morphogenesis and motility. Based on experimental and theoretical findings it can be proposed that viscoelasticity of cells, spheroids and tissues seems to be a collective characteristic that demands macromolecular, intracellular component and intercellular interactions. A major challenge is to couple the alterations in the macroscopic structural or material characteristics of cells, spheroids and tissues, such as cell and tissue phase transitions, to the microscopic interferences of their elements. Therefore, the biophysical technologies need to be improved, advanced and connected to classical biological assays. In this review, the viscoelastic nature of cytoskeletal, extracellular and cellular networks is presented and discussed. Viscoelasticity is conceptualized as a major contributor to cell migration and invasion and it is discussed whether it can serve as a biomarker for the cells’ migratory capacity in several biological contexts. It can be hypothesized that the statistical mechanics of intra- and extracellular networks may be applied in the future as a powerful tool to explore quantitatively the biomechanical foundation of viscoelasticity over a broad range of time and length scales. Finally, the importance of the cellular viscoelasticity is illustrated in identifying and characterizing multiple disorders, such as cancer, tissue injuries, acute or chronic inflammations or fibrotic diseases.

No description available.

No description available.

Cancer metastasis, the spread of cancer from the primary site to distant regions in the body, is the major cause of cancer mortality, accounting for almost 90% of cancer related deaths. During metastasis, cancer cells from the primary tumor initially probe the surrounding fibrous tumor microenvironment (TME) prior to detaching and subsequently migrating towards the blood vessels for further dissemination. It has widely been acknowledged that the biophysical cues provided by the fibrous TME greatly facilitate the metastatic cascade. Consequently, there has been a tremendous wealth of work devoted towards elucidating different modes of cancer cell migration. However, our knowledge of how cancer cells at the primary tumor site initially sense their fibrous surroundings prior to making the decision to detach and migrate remains in infancy. In part, this is due to the lack of a fibrous in vitro platform that allows for precise, repeatable manipulation of fiber characteristics. In this study, we use the non-electrospinning, Spinneret based Tunable Engineered Parameters (STEP) technique to manufacture suspended nanofiber networks with exquisite control on fiber dimensions and network architecture and use these networks to investigate how single cancer cells biophysically sense fibers mimicking in vivo dimensions. Using high spatiotemporal resolution imaging (63x magnification/1-second imaging interval), we report for the first time, that cancer cells sense individual fibers by coiling (i.e. wrapping around the fiber axis) at the tip of a cell protrusion. We find that coiling dynamics are mediated by both the fiber curvature and the metastatic capacity of the cancer cells with less aggressive cancer cells showing diminished coiling. Based on these results, we explore the possibility of using coiling in conjunction with other key biophysical metrics such as cell migration dynamics and forces exerted in the development of a genetic marker independent, biophysical predictive tool for disease progression. Finally, we identify the membrane curvature sensing Insulin Receptor tyrosine kinase Substrate protein of 53 kDa (IRSp53) as a key regulator of protrusive activity with IRSp53 knockout (KO) cells exhibiting significantly slower protrusion dynamics and diminished coil width compared to their wild-type (WT) counterparts. We demonstrate that the hindered protrusive activity ultimately translates to impaired contractility, alteration in the nucleus shape and slower migration dynamics, thus highlighting the unique role of IRSp53 as a signal transducer – linking the protrusive activity at the cell membrane to changes in cytoskeletal contractility. Overall, these findings offer novel perspectives to our understanding of how cancer cells biophysically sense their fibrous surroundings. The results from this study could ultimately pave the way for elucidating the precise fiber configurations that either facilitate or hinder cancer cell invasion, allowing for the development of new therapeutics in the long term that could inhibit the metastatic cascade at a relatively nascent stage and yield a more promising prognosis in the perennial fight against cancer. / Doctor of Philosophy / Cancer is a leading cause of death worldwide. Almost ninety percent of cancer related deaths arise from the spreading of cancer cells from the primary tumor site to secondary sites in the body – a processed termed as metastasis. The environment surrounding a tumor (tumor microenvironment) is highly fibrous in nature and can assist in the metastatic process by providing biophysical cues to the cells at the tumor boundary. These cells sense the presence of the surrounding fibers by extending "arms" termed as protrusions, and then eventually detach from the primary tumor and start migrating through the fibrous microenvironment. While numerous studies have investigated the various modes of cell migration in fibrous environments, there is very little information regarding how cancer cells use protrusions to initially sense the fibers prior to detaching. In this study, we used the Spinneret based Tunable Engineered Parameters (STEP) technique to manufacture suspended nanofiber networks with robust control on fiber diameter and network architecture and use these networks to systematically investigate how single cancer cells biophysically sense fibers that mimic in vivo dimensions. We discovered that cancer cells sense individual fibers by "wrapping-around" the axis of the fiber at the tip of the protrusion – a phenomenon we refer to as coiling. We found both the fiber diameter as well as the invasive capacity of cells can influence the coiling mechanics. Based on these results, we explored the use of coiling in conjunction with other key biophysical metrics such as the cell migration speed and how much force a cell can exert to develop a biophysical predictor for cancer cell aggressiveness. Finally, given that cells sense the fiber curvature by coiling, we explored the role of a key curvature sensing protein Insulin Receptor tyrosine kinase Substrate protein of 53 kDa (IRSp53) in mediating coiling activity and found that knocking out (KO) IRSp53 results in reduced coiling and slower protrusions compared to wild-type (WT) cells. Furthermore, IRSp53 KO cells showed impaired contractility which led to an alteration in the nucleus shape and slower migration dynamics thus highlighting the role of IRSp53 in linking changes at the cell membrane to the underlying cell cytoskeleton. The results from this study could ultimately help us understand what type of fiber conditions around a primary tumor would either help or delay the emergence of the tumor boundary cells and thus allow for the development of therapeutics that could significantly slow down the metastatic process at a relatively early stage.

<p dir="ltr">CaaX proteins, comprising approximately 300 members in the human protein database, represent a diverse group implicated in fundamental cellular processes, including proliferation, differentiation, trafficking, and gene expression. To carry out such vital cellular functions, CaaX proteins need to undergo three sequential post-translational modifications (PTM) through the CaaX pathway, which consists of isoprenylation (farnesylated or geranylgeranylated), endoproteolysis, and methylation. Among the CaaX family of protein, the Ras superfamily, plays a pivotal role in cell growth and survival. Mutations in <i>Ras proteins</i> are associated with a spectrum of cancers, presenting a significant challenge for therapeutic intervention. This thesis explores the intricate landscape of PTMs of CaaX proteins, with a focus on methylation, which is carried out by membrane protein isoprenylcysteine carboxyl methyltransferase (Icmt), and its potential as a therapeutic target, particularly for Ras-driven cancers.</p><p><br></p><p dir="ltr">Icmt is unique as it is the sole methyltransferase which carries out the third PTM of methyl esterification of CaaX proteins with the aid of co-substrate SAM, which serves as the methyl donor. Additionally, how Icmt, a membrane protein localized in the endoplasmic reticulum (ER), brings these two chemically diverse molecules in close enough proximity to promote catalysis, is very intriguing and is not yet fully understood. This thesis focuses on studying the structural and functional properties of Ste14, the yeast homolog of Icmt, in order to better understand the Icmt family of proteins. Ste14 is a functional homolog of human Icmt, sharing 41% sequence identity and 62% sequence similarity. Furthermore, Ste14 can be functionally purified unlike human Icmt. Together, these attributes make Ste14 an ideal system to study.</p><p dir="ltr"><br>The first project explores Ste14 and substrate binding, focusing on residues that determine farnesylated vs geranylgeranylated substrate specificity. It is essential to note that wild-type Ste14 recognizes farnesylated and geranylgeranylated substrate equally, with no preference to one over the other. Conserved residues found in Loop 2 and Transmembrane 3 of Ste14 were mutated to alanine and assessed for their activity with AGGC, the minimal geranylgeranylated CaaX substrate. Mutants which showed nearly zero percent activity with AGGC in comparison to wild type were further analyzed to understand if this loss of mutant activity with AGGC was potentially due to the mutant's inability to bind with AGGC. A photoreactive AGGC analog was used to carry out the photolabeling experiments and residues were analyzed for their binding ability with geranylgeranylated substrate. Mutants were further analyzed to understand the effect of mutation on structural integrity, to gauge which residues are essential for catalysis and for maintaining structural integrity of Ste14. Results demonstrated that residues F80 and E98 are essential for structural stability while L81 and L82 are essential for catalysis. This project would overall help better understand the lesser studied Ste14-substrate binding.</p><p><br></p><p dir="ltr">In the second project, the focus shifts to study Ste14 and co-substrate SAM binding by using electron paramagnetic resonance spectroscopy (EPR) and site directed spin labeling (SDSL). The biophysical technique of EPR requires much less protein and serves as great tool to study conformational change Ste14 undergoes on SAM binding, 3 non conserved residues found in the SAM binding region of Ste14, were individually mutated to cysteine, and had a spin label MTSL attached to their purified active mutant forms. Through EPR the conformational changes of Ste14 during methylation specifically during SAM binding was analyzed by visualizing the movement of MTSL attached residue. Results showed of the three non-conserved residues, A223 and E227 were immobile during SAM binding while T164 residue displayed flexibility during SAM binding during SAM binding and release process. This study would help understand the protein dynamics that Icmt undergoes upon SAM binding.</p><p><br></p><p dir="ltr">The final section centers on inhibiting the third step of the CaaX pathway, which is methyl esterification, by targeting Icmt. The project involved testing a library of Icmt inhibitors and evaluating their ability to inhibit Icmt activity. Of this library of bi-substrate analog inhibitors, compounds YD 1-66, YD 1-67 and YD 1-77 emerge as promising inhibitors against human Icmt, laying the foundation for further studies to develop more potent inhibitors. This section accentuates the strategies employed to target Icmt and the potential of these inhibitors in combating Ras-driven cancers.</p><p><br></p><p dir="ltr">This thesis provides an extensive analysis of the structure and function of Ste14. The varied studies and their insights contribute to a comprehensive understanding of Icmt and pave the way for the rational design of potent chemotherapeutic inhibitors for Ras-driven cancers. The multifaceted research presented in this thesis reveals several new possibilities for targeted therapies in the field of oncology.</p>

Résumé : Au-delà de tous débats scientifiques actuels, un constat unanime est certainement la réduction du couvert de glace dans l’océan Arctique, associé au réchauffement planétaire. La réduction du couvert de glace aura sans doute des impacts encore imprévisibles sur le milieu marin. Nous avons, dans ce contexte, traité des données satellitaires et des données de mesures de réalité de terrain de campagnes océanographiques dans la portion sud-est de la mer de Beaufort afin d’étudier les variabilités spatiale et temporelle de la biomasse phytoplanctonique et tenter de les relier aux processus physiques existants dans ce milieu. La mer de Beaufort étant fortement influencée par les eaux douces du fleuve Mackenzie, il était probable que les algorithmes de couleur de l’eau opérationnels actuels ne permettaient pas une estimation juste de la concentration de la chlorophylle-a (chl-a) et, par conséquent, de la production primaire qui est à la base de la chaîne alimentaire marine. L’analyse des données bio-optiques a confirmé cette hypothèse montrant une surestimation de la chl-a in situ par un facteur variant entre 3 et 5. La forte contribution de la matière organique colorée dissoute et des particules non-algales à l’absorption de la lumière apparaît comme la source principale de cette surestimation. Nous avons donc proposé des algorithmes adaptés ainsi que de nouveaux algorithmes utilisant deux rapports de bandes spectrales permettant une estimation plus précise de la chl-a dans le sud-est de la mer de Beaufort. Une comparaison entre des données de réalité de terrain et des images satellitaires a aussi montré que la réflectance normalisée à la surface de l’eau, de même que le rapport bleu-vert, étaient plus précis à l’aide des données du capteur SeaWiFS que de celles des capteurs MODIS et MERIS. Nous avons procédé à une analyse des patrons de chl-a et de température de surface pour cinq sous-régions géographiques dans la mer de Beaufort à l’aide de sept années de données satellitaires SeaWiFS et AVHRR (1998-2004). Les résultats ont montré que les variabilités spatiale, temporelle et interannuelle de la biomasse phytoplanctonique sont régies par plusieurs facteurs environnementaux affectant la stratification de la colonne d’eau, soit le forçage du vent, la dynamique de la glace, la température de l’air, l’ensoleillement et les courants marins. Une approche statistique basée sur le concept de provinces non statiques a permis de partitionner la mer de Beaufort en quatre provinces biophysiques distinctes, apportant un nouvel éclairage sur les propriétés biophysiques de cette mer. L'analyse des données a aussi permis de détecter une tendance à l'augmentation de la chl-a dans deux secteurs de la mer de Beaufort : le plateau du Mackenzie et la partie sud du golfe d'Amundsen. Finalement, une analyse de gradients spatiaux, effectuée à partir d’images de température de surface de l’eau a permis de détecter des fronts thermiques récurrents. Ces structures spatiales jouent un rôle majeur dans l’écosystème marin, en particulier en raison de leur impact sur le développement de la biomasse phytoplanctonique. Nous avons mis en évidence des nouvelles structures frontales sur le plateau du Mackenzie et dans la région de la polynie du cap Bathurst. Les nouveaux fronts détectés sont principalement reliés à des particularités bathymétriques de la région, à la présence du panache du fleuve Mackenzie ainsi qu’à la gyre de Beaufort. En conclusion, la réalisation de cette étude a permis de générer de nouvelles informations sur les interactions entre les processus physiques et biologiques, permettant ainsi de mieux appréhender les conséquences biogéochimiques et écologiques résultant des modifications climatiques dans la mer de Beaufort. // Abstract : The Arctic Ocean ecosystem is experiencing significant changes such as a drastic reduction in seasonal sea-ice cover linked to global warming. These changes are likely to modify the physics, biogeochemistry and ecology of this unique environment in ways that are yet to be understood. In this context, we processed satellite data and in situ measurements in the southeastern Beaufort Sea to explore the spatial and temporal variability of phytoplankton biomass and link it to existing physical processes in this region. The optical properties of the Beaufort Sea being under the influence of the Mackenzie River plume, it was likely that operational ocean color algorithms did not allow an accurate estimate of chlorophyll-a concentration (Chl-a) that is a key indicator of phytoplankton biomass and marine productivity. Analysis of bio-optical data confirmed this hypothesis showing an overestimation of Chl-a in situ by a factor of three to five. High contribution of colored dissolved organic matter and non algal particles to the blue light absorption appears as the source of that poor performance. We propose regionally adapted and new algorithms using ratio of two spectral bands allowing better accuracy estimation of Chl-a in the southeastern Beaufort Sea. A match-up analysis of coincident in situ data and satellite overpass showed that the normalized water-leaving reflectance and the blue-to-green ratio retrieval were more accurate for SeaWiFS data than for MODIS and MERIS data. We investigated temporal and spatial linkages between physical and biological parameters to infer the boundaries of biophysical areas in the Canadian Beaufort Sea. Monthly sea surface temperature (AVHRR) data and chlorophyll a data from SeaWiFS were collected over seven years in five geographical sub-regions in the Beaufort Sea (1998-2004). Results showed that the spatial, temporal and inter-annual variability of phytoplankton biomass are driven by several environmental factors affecting the stratification of the water column : wind forcing, ice dynamics, air temperature, irradiance and currents. A cluster analysis based on the concept of non-static provinces was used to define four biophysical provinces in this sea. Positive temporal trends were detected for Chl-a over two regions of the Beaufort Sea : the Mackenzie Shelf and the southern portion of Amundsen Gulf. Finally, an analysis of spatial gradients, using 11 years of sea surface temperature images, allowed the detection of recurrent thermal fronts. These spatial structures play a major role in the marine ecosystem, particularly because of their impact on the development of phytoplankton biomass. We highlighted new frontal structures on the Mackenzie Shelf and in the Cape Bathurst polynya area. These identified new fronts are mainly related to bathymetric features of the region, the presence of the Mackenzie River plume and the Beaufort Gyre. In conclusion, this study has generated new information on the interactions between physical and biological processes to better understand the biogeochemical and ecological consequences of climate change in the Beaufort Sea.

Protein tertiary structure is more conserved than amino acid sequence, leading to a diverse range of functions observed in the same fold. Despite < 20 % overall sequence identity, cytochromes P450 all have the same fold. Bacterial Class I P450s receive electrons from a highly specific, often unidentified, ferredoxin, in which case the hemoprotein is termed “orphaned”. CYP199A2, a Class I P450, accepts electrons from ferredoxins Pux and HaPux. Five orientation-dependent and one orientation-independent DEER measurements on paramagnetic HaPux and spin-labelled CYP199A2 yielded vector restraints, which were applied to building a model of the CYP199A2:HaPux complex in silico. A different binding mode was observed compared to P450cam:Pdx and P450scc:Adx, both recently elucidated by X-ray crystallography. This protocol was also applied to the CYP101D1:Arx complex. The first three measurements indicate that this heterodimer does not have a similar orientation to CYP199A2:HaPux, P450cam:Pdx, or P450scc:Adx. P450cam was fused to putidatredoxin reductase (PdR) to explore the kinetic effects with a view to improving electron transfer to orphan P450s. Heme incorporation of this enzyme depends on linker length. In whole cells, the fusion was more active after longer incubations. In vitro kinetics of the fusion exhibited some co-operativity and enhanced kinetics over the unfused system under steady-state conditions. The putative iron-sulfur biosynthesis ferredoxin PuxB had been engineered by rational mutagenesis to support catalysis by CYP199A2. It was confirmed this arose from improved protein-protein recognition. Engineering of E. coli ferredoxin based on these findings was carried out, resulting in electron-transfer to CYP199A4 from a novel engineered alien ferredoxin.

Droplet Interface Bilayers (DIBs) have a number of attributes that distinguish them from conventional artificial lipid bilayers. In particular, the ability to manipulate bilayers mechanically is explored in this thesis. Directed bilayer area changes are used to make precise measurements of the specific capacitance of DIBs and to control the two dimensional concentration of a membrane protein reconstituted in the bilayer. Chapter 1 provides a general introduction to the role of the lipid membrane en- vironment in the function of biological membranes and their integral proteins. An overview of model lipid bilayer systems is given. Chapter 2 introduces work carried out in this laboratory previously and illustrates the experimental setup of DIBs. Some important bilayer biophysical concepts are covered to provide the theoretical background to experiments in this and in later chapters. Results from the characterisation of DIBs are reported, and an account of the development of methods to manipulate the bilayer by mechanical means is given. Chapter 3 describes experiments that apply bilayer area manipulation in DIBs to achieve precise measurement of specific capacitance in a range of lipid systems. Chapter 4 reports results from experiments investigating the response of bilayer specific capacitance to an applied potential. Chapter 5 covers the background and experimental setup for total internal fluo- rescence microscopy experiments in DIBs and describes the expression, purification and characterisation of the bacterial β-barrel membrane protein pore α-Hemolysin. Chapter 6 describes experiments that apply the mechanical manipulation of bilayer area in DIBs to the corralling and control of the surface density of α-Hemolysin.

This thesis describes the development and application of condensed-phase cavity-based spectroscopic techniques - namely cavity ring-down spectroscopy (CRDS); cavity enhanced absorption spectroscopy (CEAS); broadband cavity enhanced absorption spectroscopy (BBCEAS) and evanescent wave (EW) variants of all three. The recently-developed cavity technique of EW-broadband cavity enhanced absorption spectroscopy (EW-BBCEAS) has been used—in combination with a supercontinuum source (SC) and a sensitive, fast readout CCD detector—to record of the full visible spectrum (400–700 nm) of a silica-liquid interfacial layer (with an effective thickness ca. 1 µm), at rapid acquisition rates (> 600 Hz) that are sufficient to follow fast kinetics in the condensed phase, in real time. The sensitivity achieved (A_min= 3.9 x 10^-5) is comparable with previous EW-CRDS and EW-CEAS studies, but the spectral region accessed in this broadband variant is much larger. The study of liquid|air interfaces using EW cavity-based techniques is also illustrated for the first time. The first application of BBCEAS to the analysis of microfluidic samples, flowing through a microfluidic chip, is illustrated. Proof-of-principle experiments are presented, demonstrating the technique’s ability to provide full visible broadband spectral measurements of flowing microfluidic droplets, with both high detection sensitivity (α_min < 10^-2 cm^-1) and excellent spatial and temporal resolution: an SC light source and sensitive, fast readout CCD allowed measurement repetition rates of 273 Hz, whilst probing a very small sample volume (ca. 90 nL). A significant portion of this thesis is devoted to demonstrating the powerful capabilities of CEAS, CRDS and BBCEAS in monitoring radical recombination reactions and associated magnetic field effects (MFEs) in solution. The efficacy of CEAS as a high-sensitivity MFE detection method has been established in a proof-of-principle study, using narrow band CEAS in combination with phase-sensitive detection: MFE-induced absorbance changes of ca. 10^-6 could be detected using the modulated CEAS technique and the data are shown to be superior to those obtained using conventional transient absorption (TA) methods typically employed for MFE measurements. The powerful capabilities of CRDS in monitoring radical recombination reactions and associated MFEs are also demonstrated. In particular, a pump-probe CRDS variant allows not only high sensitivity (A_min on the order 10^-6), but also sub-microsecond time-resolution. Combined, these features represent significant advantages over TA. Finally, SC-BBCEAS is used to measure full visible spectra of photoinduced reactions and their MFEs. The applicability of this approach to in vitro MFE studies of Drosophila cryptochrome is demonstrated—the results mark the first in vitro observation of a magnetic field response in an animal cryptochrome, a key result supporting the hypothesis that cryptochromes are involved in the magnetic sense in animals.