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.

Traditionally, hydrogen sulfide (H2S) has been regarded as toxic. It can affect the various human systems and even cause death. However, research in the 1990s has shown that H2S can be endogenously generated by many cells and tissues in mammalian bodies, and that H2S also may play physiological roles such as those of neuromodulator and vasorelaxant in the biological system. As such, the precise measurement of the amount of H2S in a mammalian body has generated researchers strong interest. The ultimate goal of such a measurement should be conducted in-vivo and in real time.<p>The existing methods for H2S measurement require both a large quantity of tissue samples and a complex procedure, so they are not highly practicable for the purpose of achieving the aforementioned goal. In this dissertation, a new method that uses carbon nanotube as an absorbent or transducer and laser-based microscopy techniques (Raman and confocal laser scanning microscopy) as signal excitation and acquisition is proposed and developed. Experimental studies are described of using this new method for analysis of both distilled water samples and serum samples in which a group of proteins are present. <p>The study concludes that the new method (1) can measure H2S in water solutions down to a low level of concentration of 10 µM, (2) can measure H2S in sera down to a low concentration of approximately 20 µM), and (3) has a high feasibility for being used in the clinical context. Regarding (3), this is confirmed by presenting a control system that allows the laser microscopy to track carbon nanotube in a solution that has Brownian motion.<p>While not having reached the ultimate goal as mentioned above, this work advances the state-of-the-art of the measurement of low concentration and nano-quantity of H2S in water and serum samples, in particular providing a promise toward a real-time and in-vivo H2S measurement.

En sectores tan diversos como la industria alimentaria, la biotecnología y el tratamiento de aguas residuales, la filtración tangencial con membranas se viene utilizando de forma creciente en la separación, purificación y clarificación de distintas corrientes de proceso que contienen gran variedad de compuestos orgánicos. La limitación principal para el empleo industrial de las técnicas de separación por membranas es el ensuciamiento de éstas. El ensuciamiento se atribuye, de forma general, a la reducción en el diámetro de los poros, a su bloqueo y/o a la formación de un depósito en la superficie de la membrana. El avance en el desarrollo de técnicas para la caracterización, el control y la prevención del ensuciamiento de las membranas ha estado limitado por la falta de técnicas adecuadas y no invasivas para la medición del ensuciamiento. El objetivo principal del presente proyecto es desarrollar estrategias apropiadas para aplicar microscopía láser confocal de barrido (CSLM) al estudio del ensuciamiento de membranas de filtración, centrándose en el ensuciamiento causado por macromoléculas biológicas. En la tesis se han llevado a cabo experimentos de microfiltración (MF) de soluciones modelo puras y de mezclas de proteínas, polisacáridos y polifenoles. Las imágenes captadas mediante CSLM de las membranas al final de diferentes experimentos de filtración, han servido para obtener información cualitativa, sobre localización de las distintas moléculas, y cuantitativa, sobre la presencia individual de cada compuesto en el interior y la superficie de la membrana. Se han realizado también intentos de aplicación de visualización en línea mediante CSLM del proceso de microfiltración. / In fields such as the food and dairy industries, biotechnology, and the treatment of industrial effluents, pressure-driven membrane processes such as microfiltration are increasingly being used for the separation, purification and clarification of protein-containing solutions. A major limitation to the widespread use of membrane filtration, however, is fouling. Fouling is usually attributed to pore constriction, pore blocking or the deposition of cells and cell debris on the membrane surface and can lead to a reduction in the filtrate flux of more than an order of magnitude. Progress in developing a means for characterizing, controlling and preventing membrane fouling has been impeded by lack of suitable non-invasive fouling-measurement techniques. The main aim of this study is to develop suitable strategies for applying Confocal Scanning Laser Microscopy (CSLM) to characterise membrane fouling caused by biological macromolecules. Microfiltration experiments of single, binary and ternary model solutions of proteins, polysaccharides and polyphenols were carried out and CSLM images of the membranes at the end of the different filtration runs were obtained, in order to obtain quantitative and qualitative information about fouling patterns. Some trials of on-line monitoring of cross-flow microfiltration processes were also carried out.

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.

Traditionally, hydrogen sulfide (H2S) has been regarded as toxic. It can affect the various human systems and even cause death. However, research in the 1990s has shown that H2S can be endogenously generated by many cells and tissues in mammalian bodies, and that H2S also may play physiological roles such as those of neuromodulator and vasorelaxant in the biological system. As such, the precise measurement of the amount of H2S in a mammalian body has generated researchers strong interest. The ultimate goal of such a measurement should be conducted in-vivo and in real time.<p>The existing methods for H2S measurement require both a large quantity of tissue samples and a complex procedure, so they are not highly practicable for the purpose of achieving the aforementioned goal. In this dissertation, a new method that uses carbon nanotube as an absorbent or transducer and laser-based microscopy techniques (Raman and confocal laser scanning microscopy) as signal excitation and acquisition is proposed and developed. Experimental studies are described of using this new method for analysis of both distilled water samples and serum samples in which a group of proteins are present. <p>The study concludes that the new method (1) can measure H2S in water solutions down to a low level of concentration of 10 µM, (2) can measure H2S in sera down to a low concentration of approximately 20 µM), and (3) has a high feasibility for being used in the clinical context. Regarding (3), this is confirmed by presenting a control system that allows the laser microscopy to track carbon nanotube in a solution that has Brownian motion.<p>While not having reached the ultimate goal as mentioned above, this work advances the state-of-the-art of the measurement of low concentration and nano-quantity of H2S in water and serum samples, in particular providing a promise toward a real-time and in-vivo H2S measurement.

Many Americans are afflicted with inflammation of the colon. They are also at a higher risk of developing colon cancer. Confocal microscopy of bulk epithelial tissue has the potential to provide information on tissue structural properties that may be lost in the fixation and slicing procedures required for histopathology. Optical sectioning provides images in three dimensions capturing the organizational structure of cells and colon crypts throughout the entire colon. I have constructed a custom built fluorescence and reflectance confocal microscope for imaging molecular and morphological changes associated with development of inflammation in a mouse model. A confocal microscope is a point scanning system that removes out of focus light by placing a pinhole aperture in the conjugate image plane located in front of the detector. We have two sources, 488 nm and 811 nm, for fluorescence and reflectance imaging, respectively. A polygon scanning mirror and a galvanometer scanning mirror allow for a variable scan rate between 8 and 15 fps. The lateral resolution of the system is approximately 3 μm with an axial resolution of 6 μm and 4 μm for reflectance and fluorescence mode, respectively. As colon tissue becomes inflamed, there is a distinct change in the structure and architecture of the tissue. The colon crypts are no longer uniform in size or distribution throughout the tissue. Having a large field of view of 1mm2 allows for many colon crypts to be visualized within a single frame. Histology was performed on the same tissue imaged for the inflammatory study confirming the constructed confocal microscope’s ability to characterize inflamed tissue and the potential use for guided biopsy. Mosaicing, or image tiling, is an imaging technique that stitches single frames together to produce a much larger field of view. An extended frame with 1 mm x 2 cm field of view is achieved within seconds. This extended frame would allow mosaicing of the entire mouse colon much faster than conventional methods without loss of resolution. The acquired confocal images of colon tissue demonstrate the microscope’s ability to resolve cell nuclei lining the colon crypts within a relatively large field of view.

Thesis (M. S.. in Biomedical Engineering)--Vanderbilt University, Dec. 2009. / Title from title screen. Includes bibliographical references.

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