A plant's response to pathogen infection is tailored dependent on infection strategy. Successful plant pathogens employ various infection strategies to avoid or reduce plant defense responses for the establishment of host compatibility. Autophagy is a non-selective degradation pathway conserved in eukaryotic organisms, which has been implicated in the regulation of cell survival or cell death, depending on cell type and stimulus. In <i>Arabidopsis thaliana</i>, an autophagic response has been reported to be activated during nutrient deprivation. Cellular contents, such as cytoplasm and organelles, are sequestered into double-membraned autophagosomes and delivered to the vacuole for degradation; degradative products, such as amino acids, are released back into the cell and reutilized to maintain cellular function. In this study, the response of the autophagy pathway was investigated in <i>A. thaliana</i> leaf tissues upon biotrophic <i>Erysiphe cichoracearum</i> and hemibiotrophic <i>Colletotrichum higginsianum</i> infections. Expression of some autophagy genes was induced in <i>A. thaliana</i> at 9 days post infection with <i>E. cichoracearum</i> and, 3 and 5 days post infection with <i>C. higginsianum</i>. Using a transgenic <i>A. thaliana</i> plant line over expressing autophagosome associated protein autophagy-8e (<i>ATG8e</i>) conjugated to green fluorescent protein (GFP) (<i>ATG8e-GFP</i>), confocal analysis revealed that autophagosomes specifically accumulated at the infection sites during <i>E. cichoracearum</i> and <i>C. higginsianum</i> invasions. These results indicate that the plant autophagic pathway responds to an interaction between <i>A. thaliana</i> and fungal pathogens. None of the defense signaling molecules including salicylic acid, jasmonic acid, ethylene, hydrogen peroxide and nitric oxide consistently triggered expression of autophagy genes. The insensitivity to defense signaling molecules and the delayed induction of autophagy genes compared to expression of pathogenesis-related genes suggest that the activation of this pathway does not contribute to host resistance responses during the infection process. In <i>A. thaliana</i> mutants, <i>atg4a/b, atg5-1, atg9-1</i> and <i>atg9-6</i> deficient for the autophagic response, virulence of <i>E. cichoracearum</i> was retarded whereas pathogenesis of <i>C. higginsianum</i> was accelerated. Taken together, these data suggest that the autophagy pathway is a potential host susceptibility factor for pathogen infection, possibly involved in establishing/facilitating biotrophy in <i>A. thaliana</i>.

Filamentous fungi, such as <i>Aspergillus nidulans</i>, are composed of tubular, highly polarized, multinucleate cells called hyphae. Polar growth involves secretion specifically at the hyphal tip. Secretion involves intracellular transport and co-ordination of the cytoskeleton and the endomembrane system. <p>Intracellular transport is likely mediated by cytoskeletal elements, which, in fungal cells consist primarily of actin and microtubules (MTs). An <i>A. nidulans</i> strain transformed with green fluorescent protein (GFP) tagged α-tubulin was utilized in the investigation of relationship between cytoplasmic MT arrays and hyphal growth rate. <i>A. nidulans</i> MTs were observed to be long and flexuous and to run roughly parallel to the long axis of hyphae. No correlation between relative MT abundance and hyphal growth rate was observed, although non-growing hyphae had a lower relative MT abundance than growing hyphae. Actin depolymerization decreased hyphal growth rate while MT depolymerization did not. MT stabilization increased hyphal growth rate. Ethanol, the solvent in which the MT and actin inhibitors were dissolved, increased both average overall growth rate and growth rate variability for individual hyphae. Taxol appeared to interact with irradiation to decreased growth rate during imaging. <p>Golgi are involved in secretion and potentially in polar growth. An <i>A. nidulans</i> α-coatomer protein (COP)I homolog (α-COPI), tagged with GFP, was used to investigate the role(s) of fungal Golgi in polar growth. α-COPI-GFP co-localized with the known Golgi marker, α-2,6-sialyltransferase (ST), tagged with red fluorescent protein (RFP), in untreated hyphae. Based on this observation, I propose that α-COPI-GFP can be used as a proxy for fungal Golgi localization. Fungal Golgi were more abundant at hyphal tips than subapically. Fungal Golgi forward (tipward) velocity correlated with hyphal growth rate. Fungal Golgi forward velocity was, on average, approximately ten times greater than average hyphal growth rate. Actin depolymerization reduced fungal Golgi forward velocity while MT depolymerization did not. However, MT stabilization increased fungal Golgi forward velocity. <p>Polymerized MTs do not appear to be essential for hyphal growth but do appear to be involved in hyphal growth rate variability. MTs also appear to play some role in the movement of fungal Golgi. The distribution and movement of fungal Golgi is clearly related to polarity.

One of the critical challenges in the Canadian oil sand industry is improving processes used to separate bitumen from oil sands and to remove clay particulates from produced oil. The fine clay particles are believed to play a significant role in the oil sands industry, from stabilizing process emulsions to fouling problems in water treatment. Addressing the problems caused by these fine clay particulates is limited by the ability to characterize the hydrocarbon-clay interactions. Scanning Transmission X-ray Microscopy (STXM) is used to study hydrocarbon-clay interactions in controlled model systems, where all components are known, and in process samples extracted from oil sands. To use STXM to study our desired systems, many experimental developments were required. Well developed sample preparation was needed to provide samples free from contaminants and experiments free of artifacts. Clean clays, free of extraneous carbon were required for model studies. A device to reduce photodeposition in the STXM chamber was also required to examine interactions of hydrocarbons on clay surfaces. <p>Using these developments, Near Edge X-ray Absorption Fine Structure (NEXAFS) spectra of model clays and model hydrocarbon mixtures were recorded using the STXM microscope on beamline 5.3.2 at the Advanced Light Source, in Berkeley CA. Using NEXAFS spectroscopy in conjunction with the STXM microscope, allowed us to explore preferential interactions between specific hydrocarbon and fine clay particles (smaller than 1 µm) in our model studies. We were also able to assess the chemistry of the hydrocarbons before association with the clay particles. <p>Process samples, consisting of a set of four bitumen froths extracted from the oil sands were investigated. The carbon chemistry of the froths was assessed and quantitatively analyzed. The findings were correlated with previous confocal microscopy results from our collaborators at CANMET Energy Technology Centre in Devon, Alberta.

Specialized cells of the innate immune system, such as macrophages, employ lysosomal enzymes, together with cationic peptides and reactive oxygen intermediates, to eliminate invading microorganisms ensnared within phagosomes. The effectiveness of this impressive armamentarium is potentiated by the acid pH generated by the vacuolar-type ATPase (V-ATPase). The determinants of the luminal pH of phagosomes and of the lysosomes they fuse with are not completely understood, but the V-ATPase is known to be electrogenic and net accumulation of protons requires charge compensation. For this reason, counter-ion pathways are thought to serve a central role in the control of acidification. It has generally been assumed that a parallel anion influx accompanies proton pumping to dissipate the voltage that tends to build up. In fact, impaired chloride channel activity in cystic fibrosis has been proposed to underlie the defective phagolysosome acidification and microbial killing reported in lung macrophages. In the first part of this thesis, I devised methods to dialyze the lumen of lysosomes in intact cells, while monitoring lysosomal pH, in order to assess the individual contribution of counter-ions to acidification. Surprisingly, anions were found to be completely dispensable for proton pumping, whereas the presence of permeant cations in the lysosomal lumen was essential. Accordingly, defects in lysosomal anion permeability cannot explain the impaired microbicidal capacity of phagocytes in cystic fibrosis. Even though counter-ion permeation pathways exist, dissipation of the electrical contribution of the V-ATPase may not be complete. If present, a transmembrane potential would alter the rate and extent of proton accumulation in phagosomes and lysosomes. However, no estimates of the voltage across the phagosomes were available. To overcome this deficiency, in the second part of this thesis, I describe a noninvasive procedure to estimate the voltage across the phagosome using fluorescence resonance energy transfer. This novel approach, in combination with organellar pH measurements, demonstrated that proton pumping is not limited by counter-ion permeability.

La progresiva miniaturización de los materiales a la escala nanométrica ha abierto en la última década nuevas expectativas en el campo de la Ciencia de Materiales. Dichos materiales nanométricos presentan propiedades únicas, difiriendo a menudo de las propiedades del propio material a la macroescala, las cuales abren un amplio abanico de nuevas fenomenologías, y en consecuencia, de aplicaciones tecnológicas. De especial interés han sido los nanomateriales magnéticos, incluyendo las nanopartículas magnéticas o sistemas moleculares, dado que emergen como sistemas clave en el desarrollo de nuevas tecnologías de interés tales como sistemas de almacenamiento de memoria, computación cuántica o dispositivos de espintrónica. El desarrollo de aplicaciones reales en base al uso de dichos nanomateriales requiere primero la búsqueda de nuevas estrategias de estructuración que permitan organizar dichos sistemas magnéticos en superficies, a la vez que estudiar cómo es su comportamiento en la transición de la macroescala a la micro- o nanoescala. En este sentido es de interés estudiar cómo sus propiedades magnéticas pueden verse afectadas por efectos de la estructuración o de la propia superficie. En este contexto, de cara a estudiar las propiedades magnéticas de estos sistemas, una de las aproximaciones que está atrayendo mayor interés es el uso de dispositivos superconductores de interferencia cuántica (SQUIDs) y sensores Hall. Estos sensores han experimentado una gran revolución al ser miniaturizados, lo que ha permitido aumentar notablemente su sensibilidad hasta la detección de la magnetización de una sola nanopartícula o molécula. En este continuo avance, una de las principales limitaciones es la necesidad de desarrollar nuevas estrategias de integración que permitan depositar los sistemas magnéticos de un modo controlado en las zonas de máxima sensibilidad de estos sensores miniaturizados. En este contexto, la presente Tesis doctoral ha sido dedicada al desarrollo de nuevas estrategias de integración de cara a mejorar el control en la integración de sistemas magnéticos. Las estrategias propuestas han permitido por primera vez realizar este proceso sin la necesidad de modificar previamente ni el material magnético ni la superficie del sensor, consiguiendo depositar en zonas definidas del sensor con control (sub)micrométrico. En concreto, se ha demostrado la viabilidad y la universalidad de la técnica de deposición directa por litografía de AFM (conocida como Dip-pen Nanolithography, DPN) para la integración de nanomateriales magnéticos, abarcando un amplio rango de materiales desde nanopartículas hasta sistemas moleculares con comportamiento de imán unimolecular, en una gran variedad de sensores con diferentes dimensiones y requisitos. / The reduction of magnetic materials to the nanometric scale has opened in the last decades new perspectives in Material Science. Nanosized materials exhibit unique properties, which can considerably differ from the properties of the corresponding bulk materials, opening a wide range of new phenomenologies and therefore of technological applications. Two of the most prospective nanomaterials over the last years have been magnetic nanoparticles and molecular materials. Such nanostructured materials have attracted much interest not only from a fundamental point of view but also because their potential use in high-density information storage devices, quantum computing applications and in spintronics. However, before these applications become a reality, there is a fundamental issue that needs to be addressed, namely, the development of strategies to evolve from bulk materials to single entities suitable to be grafted on surfaces, sensors or other systems able to act as a device. The challenge is the definition of experimental strategies to properly assemble and integrate these molecular materials into functional devices without compromising their properties. In this context, there is an interest in understanding how the structuration procedure or even the surface can modify the behavior of such nanomaterials once deposited. Nevertheless, to answer these questions one of the approximations that is attracting more attention and probably more results is that of typical magnetic characterization techniques for bulk crystalline samples such as superconducting quantum interference devices (SQUID) or Hall magnetometer sensors. These systems have been miniaturized to increase their sensitivity down to a single magnetic or NP moment, involving the need for the development of specific deposition and structuration techniques to integrate the magnetic materials into the sensors with the required control on positioning and quantity of material. In this context, the present Thesis has been devoted to the development of novel structuration strategies to improve the control on the integration of magnetic nanosystems. The proposed strategies have allowed for first time to perform this process without the need of pre-modifying either the magnetic nanomaterial or the surface of the sensor, while depositing on specific areas of the sensor with (sub)micrometric precision. In particular, we have demonstrated the viability and generality of direct-write atomic force microscopy (AFM) lithography (also known as Dip-pen Nanolithography, DPN) to overcome most of the challenges that implies such integration. For this, different experimental approaches have been explored for the integration of magnetic nanomaterials intended to be as much representative as possible, ranging from magnetic nanoparticles to molecular systems exhibiting single molecule magnet (SMM) behavior, on a wide variety of sensors displaying different dimensions and requirements.

Advances in several research areas increase the need for more sophisticated fabrication techniques and better performing materials. Tackling this problem from a bottom-up perspective is currently an active field of research. The bottom-up fabrication procedure offers sub-nanometer accurate manipulation. At this time, candidates to achieve nanomanipulation include chemical (self-assembly), biotechnology methods (DNA-based), or using controllable physical forces (e.g. electrokinetic forces, mechanical forces). In this thesis, new methods and techniques for mechanical nanomanipulation using probe force interaction are developed. The considered probes are commonly used in Atomic Force Microscopes (AFMs) for high resolution imaging. AFM-based mechanical nanomanipulation will enable arranging nanoscale entities such as nanotubes and molecules in a precise and controlled manner to assemble and produce novel devices and systems at the nanoscale. The novelty of this research stems from the development of new modeling of the physics and mechanics of the tip interaction with nanoscale entities, coupled with the development of new smart cantilevers with multiple degrees of freedom. The gained knowledge from the conducted simulations and analysis is expected to enable true precision and repeatability of nanomanipulation tasks which is not feasible with existing methods and technologies.

Alzheimer's Disease (AD) is a neurodegenerative disorder that is prevalent among the elderly population. Aß protein has been heavily implicated in the pathogenesis of AD. This protein in its fibrillar form is a major component in the senile plaques that form on neuronal cellular membranes during the course of AD. Despite substantial efforts the exact mechanism of Aß toxicity towards a cell membrane is not well-understood. The determination of this mechanism, however, is of utmost importance, since the membrane presents the first site of Aß interaction with neurons, which in turn maybe the origin of Aß neurotoxicity. The purpose of this study was to find a lipid composition that can be used as a model of neuronal membrane for subsequent studies of the role of topographical heterogeneity (domain formation) on Aß-membrane interaction as related to AD. The lipids used in the study were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), cholesterol (Chol), sphingomyelin (SM) and ganglioside GM1 (GM1). These lipids were combined in different proportions and deposited on a mica substrate to form supported monolayers. They were then imaged with an atomic force microscope (AFM) to determine if any of them exhibited domain formation. Three of the studied samples: POPC/POPG/SM 40:40:20 +5%Chol, POPC/SM/Chol 75:20:5 and POPC/SM/GM1/Chol 74:2:1:23 were found to possess interesting topography, rich in structural features: pores and domains. The average height difference between the domain features for each sample was found to be 0.58±015 nm, 0.61±0.12 nm and 0.27±0:07 nm.

Filamentous fungi, such as <i>Aspergillus nidulans</i>, are composed of tubular, highly polarized, multinucleate cells called hyphae. Polar growth involves secretion specifically at the hyphal tip. Secretion involves intracellular transport and co-ordination of the cytoskeleton and the endomembrane system. <p>Intracellular transport is likely mediated by cytoskeletal elements, which, in fungal cells consist primarily of actin and microtubules (MTs). An <i>A. nidulans</i> strain transformed with green fluorescent protein (GFP) tagged α-tubulin was utilized in the investigation of relationship between cytoplasmic MT arrays and hyphal growth rate. <i>A. nidulans</i> MTs were observed to be long and flexuous and to run roughly parallel to the long axis of hyphae. No correlation between relative MT abundance and hyphal growth rate was observed, although non-growing hyphae had a lower relative MT abundance than growing hyphae. Actin depolymerization decreased hyphal growth rate while MT depolymerization did not. MT stabilization increased hyphal growth rate. Ethanol, the solvent in which the MT and actin inhibitors were dissolved, increased both average overall growth rate and growth rate variability for individual hyphae. Taxol appeared to interact with irradiation to decreased growth rate during imaging. <p>Golgi are involved in secretion and potentially in polar growth. An <i>A. nidulans</i> α-coatomer protein (COP)I homolog (α-COPI), tagged with GFP, was used to investigate the role(s) of fungal Golgi in polar growth. α-COPI-GFP co-localized with the known Golgi marker, α-2,6-sialyltransferase (ST), tagged with red fluorescent protein (RFP), in untreated hyphae. Based on this observation, I propose that α-COPI-GFP can be used as a proxy for fungal Golgi localization. Fungal Golgi were more abundant at hyphal tips than subapically. Fungal Golgi forward (tipward) velocity correlated with hyphal growth rate. Fungal Golgi forward velocity was, on average, approximately ten times greater than average hyphal growth rate. Actin depolymerization reduced fungal Golgi forward velocity while MT depolymerization did not. However, MT stabilization increased fungal Golgi forward velocity. <p>Polymerized MTs do not appear to be essential for hyphal growth but do appear to be involved in hyphal growth rate variability. MTs also appear to play some role in the movement of fungal Golgi. The distribution and movement of fungal Golgi is clearly related to polarity.

One of the critical challenges in the Canadian oil sand industry is improving processes used to separate bitumen from oil sands and to remove clay particulates from produced oil. The fine clay particles are believed to play a significant role in the oil sands industry, from stabilizing process emulsions to fouling problems in water treatment. Addressing the problems caused by these fine clay particulates is limited by the ability to characterize the hydrocarbon-clay interactions. Scanning Transmission X-ray Microscopy (STXM) is used to study hydrocarbon-clay interactions in controlled model systems, where all components are known, and in process samples extracted from oil sands. To use STXM to study our desired systems, many experimental developments were required. Well developed sample preparation was needed to provide samples free from contaminants and experiments free of artifacts. Clean clays, free of extraneous carbon were required for model studies. A device to reduce photodeposition in the STXM chamber was also required to examine interactions of hydrocarbons on clay surfaces. <p>Using these developments, Near Edge X-ray Absorption Fine Structure (NEXAFS) spectra of model clays and model hydrocarbon mixtures were recorded using the STXM microscope on beamline 5.3.2 at the Advanced Light Source, in Berkeley CA. Using NEXAFS spectroscopy in conjunction with the STXM microscope, allowed us to explore preferential interactions between specific hydrocarbon and fine clay particles (smaller than 1 µm) in our model studies. We were also able to assess the chemistry of the hydrocarbons before association with the clay particles. <p>Process samples, consisting of a set of four bitumen froths extracted from the oil sands were investigated. The carbon chemistry of the froths was assessed and quantitatively analyzed. The findings were correlated with previous confocal microscopy results from our collaborators at CANMET Energy Technology Centre in Devon, Alberta.

A plant's response to pathogen infection is tailored dependent on infection strategy. Successful plant pathogens employ various infection strategies to avoid or reduce plant defense responses for the establishment of host compatibility. Autophagy is a non-selective degradation pathway conserved in eukaryotic organisms, which has been implicated in the regulation of cell survival or cell death, depending on cell type and stimulus. In <i>Arabidopsis thaliana</i>, an autophagic response has been reported to be activated during nutrient deprivation. Cellular contents, such as cytoplasm and organelles, are sequestered into double-membraned autophagosomes and delivered to the vacuole for degradation; degradative products, such as amino acids, are released back into the cell and reutilized to maintain cellular function. In this study, the response of the autophagy pathway was investigated in <i>A. thaliana</i> leaf tissues upon biotrophic <i>Erysiphe cichoracearum</i> and hemibiotrophic <i>Colletotrichum higginsianum</i> infections. Expression of some autophagy genes was induced in <i>A. thaliana</i> at 9 days post infection with <i>E. cichoracearum</i> and, 3 and 5 days post infection with <i>C. higginsianum</i>. Using a transgenic <i>A. thaliana</i> plant line over expressing autophagosome associated protein autophagy-8e (<i>ATG8e</i>) conjugated to green fluorescent protein (GFP) (<i>ATG8e-GFP</i>), confocal analysis revealed that autophagosomes specifically accumulated at the infection sites during <i>E. cichoracearum</i> and <i>C. higginsianum</i> invasions. These results indicate that the plant autophagic pathway responds to an interaction between <i>A. thaliana</i> and fungal pathogens. None of the defense signaling molecules including salicylic acid, jasmonic acid, ethylene, hydrogen peroxide and nitric oxide consistently triggered expression of autophagy genes. The insensitivity to defense signaling molecules and the delayed induction of autophagy genes compared to expression of pathogenesis-related genes suggest that the activation of this pathway does not contribute to host resistance responses during the infection process. In <i>A. thaliana</i> mutants, <i>atg4a/b, atg5-1, atg9-1</i> and <i>atg9-6</i> deficient for the autophagic response, virulence of <i>E. cichoracearum</i> was retarded whereas pathogenesis of <i>C. higginsianum</i> was accelerated. Taken together, these data suggest that the autophagy pathway is a potential host susceptibility factor for pathogen infection, possibly involved in establishing/facilitating biotrophy in <i>A. thaliana</i>.