Thesis (M.S.)--Ohio University, June, 2005. / Title from PDF t.p. Includes bibliographical references (p. 77-88)

The discovery of the existence of the cell membrane has led to a search for its organization on a molecular scale. The advent of artificial lipid bilayers and the development of electron microscopy in the 1930's provided direct visual evidence for the existence of the cell membrane and drove forward models of membrane structure based its known composition of proteins, lipids and carbohydrates. The fluid mosaic model of membrane structure, based on thermo- dynamics and newly developed protein structural studies of the time, placed integral globular membrane proteins within a fluid phospholipid bilayer. This model allowed for the association of proteins into groups and the possible mobility of proteins within the lipid bilayer. At the the same time fluorescence microscopy demonstrated movement of proteins in the plane of the lipid bilayer. Since then experimental techniques have been developed that show protein complexes of varying sizes do exist and so this gives us the opportunity to ask how receptor proteins fit into the molecular organization of the cell membrane. This thesis presents an investigation into how the epidermal growth factor receptor (EGFR) organizes in the cell membrane of colorectal carcinoma cells. First a new cell line for studying the receptor by stably expressing the epidermal growth factor receptor conjugated to enhanced green fluorescent protein (EGFR-eGFP) in SW620 cells was developed. This is an interest- ing cell line because it originates from a colonic adenocarcinoma that during the process of metastasis has lost the ability to express the EGFR. It therefore provided an environment for the expression of the fluorescent form of the receptor more in keeping with its natural environment. The technique of total internal reflection fluorescence (TIRF) microscopy was used to visualize the fluorescently tagged receptor in the cell membrane. This technique uses the principles of total internal reflection to excite fluorescence in molecules located only 100 nm into the cell. Because sources of fluorescence from outside the illuminated area are minimized individual fluorescent molecules can be imaged. The spots in the images, produced by the fluorophores, were detected using a single molecule detection and tracking algorithm. The intensities of these detected spots were analysed and compared with that from a single molecule of enhanced green fluorescent protein (eGFP). This gave an estimate of the number of receptors contained within each receptor complex. Before ligand binding most of the receptors were found to be located in complexes containing up to eight molecules and most frequently they were found in complexes of two molecules. Larger complexes of receptors were found to have formed after activation of the receptor by its ligand.

The emergence of unique or enhanced physical, chemical and optical material properties at the nanoscale underlies the swift rise of nanomaterials science over recent decades. Within this interdisciplinary field, catalysis performed by nanomaterials (i.e. nanocatalysis) is one area where differences between nanoscale and bulk material properties offer particularly attractive opportunities for application. The consequent pursuit of viable nanomaterials with unprecedented catalytic activity has inevitably expanded across the periodic table, whereby a number of highly efficient precious metal, metal oxide and composite nanostructured catalysts have been developed for a wide range of synthetic organic and inorganic transformations. The lanthanide series has not been excluded from this search, but is still underrepresented in catalysis despite some rich chemistry and reactivity which sets these elements apart from many other metals. More recently however, the necessary paradigm shift away from commonly utilized but expensive, potentially toxic precious metal catalysts, and toward more sustainable alternatives, has seen an upsurge in the development of novel nanomaterials for heterogeneous catalysis: the general topic of this doctoral thesis. Heterogeneous nanocatalysis offers distinct advantages over homogeneous catalysis. Catalyst recyclability, ease of separation from reaction mixtures, and minimal product contamination all contribute to the higher overall effectiveness of heterogeneous catalysts relative to their homogeneous counterparts. The use of light as an abundant reagent, both in nanomaterial fabrication and for photocatalysis, is another attractive prospect. This dissertation addresses both points, describing the iterative development and application of photochemically-prepared samarium oxide based nanomaterials for heterogeneous catalysis and photocatalysis. Through a series of related peer-reviewed publications and associated commentary, the evolution of the application-driven design of a nanomaterial which is both efficient and effective for a diversity of heterogeneous catalytic and photocatalytic transformations is presented. Major findings include 1) both colloidal and supported samarium oxide nanoparticles can be prepared photochemically and comprise primarily Sm2O3 but may contain localized mixed valences or dynamic surface oxidation states; 2) colloidal samarium oxide nanoparticles possess high activity for Brønsted acid and oxidative catalysis, but recyclability and overall effectiveness is less than optimal due to a combination of polydispersity and size-dependent catalytic activity; 3) a similarly-prepared “second generation” samarium oxide/titanium dioxide nanocomposite presented several advantages over its predecessor, performing highly efficient and effective pure heterogeneous, dual photoredox-Lewis acid catalysis in two different types of synthetically relevant photocyclizations. Effects of different nanoparticle supports, rare insights into the catalytic mechanisms and behaviour of these nanomaterials‒obtained at the single molecule level by innovative application of Total Internal Reflection Fluorescence Microscopy (TIRFM) to catalysis research‒as well as advances in TIRFM data analysis protocols, are also discussed.

Total internal reflection fluorescence microscopy (TIRFM) has been widely applied for the study of biomolecules because of their ability to quantify biomolecules in a sample pretreatment and enrichment free manner, when compared with those costly, sample consuming and labor intensive conventional detection assay. Here, we have applied the TIRFM imaging system for the direct quantification and analysis of the biomarkers for the age-related diseases. Three research works on the quantification and study of biomarkers with the aid of TIRFM were herein described.

Background: SF processing has been firstly applied on the processing and storage of the rhizome of Dioscareapersimilis Prain et Burkill. in Wenxian country since 1900. Due to the simple, quick and low-cost characteristics of SF, it soon became a common postharvesting method for CMMs. However, recent studies showed that SF can either cause chemical changes to CMMs or affect human health. The awareness of identification of sulfur-fumigated CMMs is arisen. Comparing with chemical methods, FM is more simple and user-friendly to be established in authentication. Also, recent studies showed that different chemical profiles of CMMs can emit fluorescence differently. This research aimed to validate if FM was suitable for identification of sulfur-fumigated CMMs through using Codonopsis Radix and Ginseng Radix as examples. Method: 16 herbal samples were collected in different commercial market in different time, in which 6 of them was Codonopsis Radix and 8 of them was Ginseng Radix. Firstly, their chemical profiles of the samples were analyzed by UHPLC-QTOF-MS to make a chemical authentication. Then, their fluorescence characteristic were localized and captured on their transverse sections. Result: All the samples of Codonopsis Radix and 2 samples of Ginseng Radix were confirmed to be sulfur-fumigated as compounds sulfates or sulfites were detected. Investigated by fluorescence, herbal samples emitted blue and yellow fluorescence in different intensity under blue and green light filter. The fluorescence of groups of laticiferous tubes and resin canals were remarkable in Codonopsis Radix and Ginseng Radix respectively. Sulfur-fumigated samples showed similar characteristic to those raw samples. It was significant that samples of Codonopsis Radix emitted fluorescence differently even all of them were sulfur-fumigated. Conclusion: In the present study, samples with different growing condition, storage time and SF processing had some variation in their fluorescence characteristics. The result showed that fluorescence microscopy was not probable for identification of the sulfur-fumigated CMMs. The application of FM on the identification of sulfur-fumigated CMMs should be further investigated comprehensively. Key works: sulfur-fumigation; Codonopsis Radix; Ginseng Radix; authentication; fluorescence microscopy; UHPLC-QTOF-MS 背景：硫磺薰蒸中藥技術是在1900年由溫縣人民發明並最早記載於河南溫縣縣誌的。由於硫磺薰蒸技術擁有最高效，低成本，便於操控的優點，因此一直被藥農及中藥開發藥廠用於中藥防蟲及乾燥加工。但近年不少論文開始報道有關硫磺對中藥材的危害性，其中以硫磺薰蒸會影響中藥材內部的化學成分和藥理活性的報告最令人擔憂。礙於高效液相色譜法(HPLC)等化學檢測手段的複雜性，使用化學手段鑒別硫磺熏蒸藥材存在困難及難以普及。而近年實驗證明，中藥材裏不同的化學成分在熒光顯微鏡下會發放出不同熒光顔色。本研究以人參和黨參為例子，為應用熒光顯微鏡鑒別硫磺熏蒸中藥材，提供科學證據支持。 實驗方法：本研究採集了6個黨參及8個人參樣品進行分析。實驗首先以高效液相色譜-四極杆飛行時間串聯質譜(UHPLC-QTOF/MS)技術分析各樣品的化學成分用於鑒別樣品有否被硫磺熏蒸。然後各樣品先進行冷凍切割，並在熒光顯微鏡下進行橫切面觀察，從外到内觀察各特徵結構的熒光顔色，比較不同樣品的熒光差別。 結果：實驗結果顯示所有黨參樣品及2個白皮參樣品存在硫磺熏蒸后產生的硫酸鹽或亞硫酸鹽化合物，提示該樣品被硫磺熏蒸。所有樣品在熒光顯微鏡下都顯示不同程度的藍色及綠色熒光。儘管所有黨參樣品均被硫熏，但其顯示的熒光仍存在明顯差異，其中以樣品5和6最爲明顯。而被硫熏的人參樣品，其熒光則非常類似於非硫熏樣品。非硫熏的人參其熒光仍存在個體差異，其中2個樣品的熒光強度較其他為強及明顯。黨參内的乳管群及人參内的樹脂道，其熒光強度及顔色最爲突出。 結論：研究結果表明不同來源的樣品，其熒光特徵存在區別。熒光顯微鑑定技術不能有效鑒別市場上的黨參和人參是否經過硫磺熏蒸。熒光顯微鑑定硫磺熏蒸中藥方面的應用有待深入及系統探討。 關鍵詞：硫磺熏蒸；黨參；人參；熒光顯微鏡；中藥鑒別；高效液相色譜-四極杆飛行時間串聯質譜(UHPLC-QTOF/MS)

Cell division is a fundamental biological process that occurs in all kingdoms of life. Our understanding of cell division in bacteria stems from studies in the rod-shaped model organisms: Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. The molecular underpinnings of cell division regulation in non-rod-shaped bacteria remain to be studied in detail. Rod-shaped bacteria possess many positive and negative regulatory proteins that are essential to the proper placement of the division septa and ultimately the production of two identical daughter cells, many of which are absent in cocci. Given that essential cell division proteins are attractive antibacterial drug targets, it is imperative for us to identify key cell division factors especially in pathogens, to help counter the emergence of multi-drug resistance. In Staphylococcus aureus, a spherical Gram-positive opportunistic pathogen that causes a range of diseases from minor skin infections to life-threatening sepsis, we have identified the role of an essential protein, GpsB, in the regulation of cell division. We discovered that GpsB preferentially localizes to cell division sites and that overproduction of GpsB results in cell enlargement typical of FtsZ inhibition, while depletion of GpsB results in cell lysis and nucleoid-less minicell formation. The identification of GpsB’s interaction partners will allow us to understand the molecular mechanism by which GpsB regulates cell division.

No description available.

Poly(ADP-ribose)polymerase-1 and -2 (PARP1/2) are nuclear proteins involved in DNA repair. Tumors with defects in homologous recombination, including BRCA1- and BRCA2-deficient cancers, have been shown to be sensitive to PARP inhibition. The Weissleder group has synthesized fluorescent and radioactive derivatives of the PARP1/2 inhibitor AZD2281. We hypothesized that fluorescent and radioactive AZD2281-based imaging agents would quantify PARP1/2 expression in vitro and in vivo. To test this hypothesis, a panel of pancreatic ductal adenocarcinoma and ovarian carcinoma cell lines were characterized by immunocytochemistry for PARP1/2 expression. AZD2281-derived fluorescence signal correlated with anti-PARP antibody fluorescence signal strength in vitro. Four cell lines representing a range of PARP1/2 expression levels were then xenografted into Nu/Nu mice. Mice bearing four tumor types each were imaged with AZD2281-derived imaging agents, sacrificed, and their tumors excised for stand-alone imaging and Western blot. AZD2281-derived signal correlated with tumor PARP1/2 expression determined by Western blot, indicating that PARP1/2 expression level is a determinant of fluorescent signal strength and SUVs of AZD2281-derived agents in vivo. These data indicate that AZD2281-derived agents are useful tools for quantifying intracellular PARP1/2 both in vitro and in vivo, which could one day enable prospective identification of tumors likely to respond to PARP inhibitors.

No description available.

This thesis describes the development of novel fluorescent and photocaged lipids, and their application as tools to probe the morphological effects of ceramide (Cer)-mediated membrane reorganization in supported lipid bilayers. Cer is a sphingolipid found in eukaryotic cells that plays a key role in regulating biological processes such as apoptosis, cell-to-cell communication, differentiation and some types of pathogenesis. Sphingolipid and cholesterol-rich lipid rafts in the plasma membrane are thought to be the point of origin for many of this lipid second messenger’s effects. Cer is formed in the exoplasmic leaflet of the plasma membrane via the enzymatic hydrolysis of sphingomyelin. The compositional complexity of biological membranes has prompted the adoption of simpler model systems to study the effects of Cer generation. When it is directly incorporated into model membranes, Cer segregates into highly ordered domains with physical properties that are distinct from those of the surrounding fluid environments. However, enzymatic generation of Cer induces complex and dynamic membrane heterogeneity that is difficult to interpret and reconcile with its direct incorporation. Here I describe the synthesis of 4-nitrobenzo-2-oxa-1,3-diazol-7-yl (NBD)-labelled cholesterol (Chol) and Cer analogs, and their use as probes in model membranes exhibiting liquid-disordered (Ld) and liquid-ordered (Lo) phase coexistence. The Chol probes reproduce the modest enrichment of Chol in Lo membrane domains as well as the Cer-induced displacement of cholesterol. One of the NBD Chol probes is used to provide direct visualization of Chol redistribution during enzymatic Cer generation, and assists in identifying new features as Cer-rich regions. The NBD-labelled Cer quantifies membrane order using orientational order parameter measurements derived from polarized total internal reflection fluorescence microscopy (pTIRFM) images. The probe reports on changes in membrane order upon enzymatic generation of Cer, and indicates a significant increase in the molecular order of Ld membrane regions that is consistent with the redistribution of Chol into these areas. The probe also identifies de novo Cer-rich domains as areas of particularly high molecular order. In the final project area, 6-Bromo-7-hydroxycoumarin-4-ylmethyl (Bhc)-caged Cers are shown to release Cer rapidly and efficiently upon irradiation with near-visible UV light. The caged lipids are then incorporated into supported membranes and photolyzed to release Cer with a high degree of spatial and temporal control. Controlled Cer generation is then used to drive protein-ganglioside clustering in lipid bilayers.