abstract / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Public Health / Master / Master of Public Health

Optical molecular imaging has burgeoned into a major field within biomedicine, and technologies that incorporate surface plasmon resonance effects have become a major focus within this field. Plasmon resonance has been defined as the collective oscillation of the conduction band electrons in certain metals (such as gold) in response to an electric field, such as an impinging wave of light. We show that elastic light scattering due to the plasmon resonance of nanometer-sized gold particles makes them powerful tools for optical imaging of epidermal growth factor receptor (EGFR) expression -- a major biomarker for carcinogenesis. Optical technologies in general are poised as cheap, flexible ways to aid in diagnosis and treatment of disease. In addition to supplying a bright, stable optical scattering signal and a convenient conjugation platform for targeting molecules, these materials display a unique behavior termed "plasmon coupling". This term refers to the dramatic optical property changes brought about by the presence of other nearby nanoparticles. These changes include a dramatic red-shifting in their peak plasmon resonance wavelength, as well as a non-linear, per-particle increase in the overall scattered power. We show that such conditions exist in cells and are primarily due to intricate protein trafficking mechanisms as part of the EGFR life-cycle. The observed variations in plasmon coupling can give clues as to the nanoscale organization of these important proteins. In addition, the resulting optical property changes result in a large, molecular-specific contrast enhancement due to the shifting of the resonance closer to the near infrared region, where biological tissues tend to be most transparent. Despite this enhancement, however, many tissues contain large endogenous signals, as well as barriers to delivery of both light and the nanoparticles. As such, we also show an example of a multifaceted approach for further increasing the apparent molecular-specific optical signals in imaging of EGFR expression by using an oscillating magnetic field. This serves to encode the signal from magnetically susceptible plasmonic nanoparticles, making their extraction from the background possible. Overall, the studies presented in this dissertation should serve to stimulate further investigations into a wide variety of technologies, techniques, and applications.

Cell-cell signalling molecules play important roles in neural development. In response to extracellular signals, neuronal progenitor cells proliferate, differentiate, and form a neuronal network. In the vertebrate retina, retinal ganglion cells (RGCs) are the first neurons produced during development and are the only neurons that send projections to the brain. However, the molecular mechanisms for RGC development have not been fully understood. In this study, I have investigated the expression and functions of Nel (Neural Epidermal Growth Factor Like), an extracellular glycoprotein that contains chordin-like domains and epidermal growth factor-like domains, in the development of the chick RGCs and retinotectal projection. I found that on embryonic days (E) 2-3.5, Nel was expressed in the presumptive retinal pigment epithelium of the developing eye. Correspondingly, Nel-binding activity (Nel receptor activity) was detected in the retinal pigment epithelium and also the progenitor layer of the neural retina. At the early stages during RGC formation, Nel overexpression increased the total number of RGCs and accelerated the progression of RGC differentiation wave. Conversely, Nel expression knockdown decreased the total number of RGCs and slowed down the progression of RGC differentiation wave. At later stages (E3-E18), expression of Nel in the retina was in the retinal pigment epithelium and the RGC layer, whereas receptor activity for Nel was localized in the retinal pigment epithelium and the RGC axons. In vivo, Nel overexpression in the developing retina induced the inhibition of RGC axons and thus disrupting the intraretinal RGC axon projection. These results suggest that Nel can positively regulate the production of RGCs at the early stages during retinal development, and at the later stages, Nel can function as an inhibitory guidance cue in vivo for RGC axons.

No description available.

Tissue factor (TF) is a cell-associated receptor for coagulation factor VIIa (FVIIa) that initiates the coagulation cascade and transmits intracellular signals through protease activated receptors (PARs). This thesis documents for the first time that in human glioma cells (U373) oncogenic epidermal growth factor receptor (EGFRvIII) simultaneously upregulates the expression of several elements of the TF pathway (TF, FVIIa, PAR-1 and PAR-2). In the absence of EGFRvIII, TF triggers tumor formation, albeit with a long latency, while treatment of glioma cells with FVIIa activates MAPK phosphorylation and stimulates the expression of angiogenic factors (VEGF and IL-8). Moreover, selective targeting of the host (mouse) TF reveals its independent role in glioma tumorigenesis. We propose that TF may represent an attractive potential target to treat human brain tumors.

The problems and high medical costs associated with chronic wounds necessitate an economical bioactive wound dressing. A new strategy was investigated to inhibit MMP-9 proteases and to release epidermal growth factor (EGF) to enhance healing. Doxycycline (DOX) and EGF were encapsulated on polyacrylic acid modified polyurethane film (PAA-PU) using Layer-by-Layer (LbL) assembly. The number of bilayers tuned the concentration of DOX and EGF released over time with over 94% bioactivity of EGF retained over 4 days. A simple wound model in which MMP-9 proteases were added to cell culture containing fibroblast cells demonstrated that DOX inhibited the proteases providing a protective environment for the released EGF to stimulate cell migration and proliferation at a faster healing rate. In the presence of DOX, only small amounts of the highly bioactive EGF are sufficient to close the wound. Results show that this is new and promising bioactive dressing for effective wound management.

Sterols have been identified as major components of membrane lipids that are part of specialized membrane domains necessary for organizing events such as polar protein targeting and signal transduction in plants, fungi and animals. However a common modification of sterols is the addition of sugar moieties via glycosylation abundantly found in plants. An exact physiological role for such diversification of sterols in plants is still unknown. Using reverse genetics and transcriptomics we show that UDP-glucose: sterol glucosyltransferase encoded by UGT80B1 is necessary for correct epidermal patterning in Arabidopsis root. Patterning of hair cells (trichoblasts) and non-hair cells (atrichoblasts) in the epidermis of the Arabidopsis root involves signaling through SCRAMBLED (SCM), a plasma membrane localized LRR-RL kinase. Feedback regulation via the transcriptional regulatory complex containing R2R3-MYB transcription factor WEREWOLF (WER) represses SCM and activates the homeodomain-leucine-zipper protein GLABRA2 (GL2) in atrichoblasts. Evidence suggests symplastic connections between cells, known as plasmodesmata, establish passage ways for single-repeat R3-MYB transcription factors to activate SCM expression in trichoblasts. Mutations in UGT80B1 cause atypical localization patterns of GL2, WER, and SCM in the root epidermis. The ugt80B1 formed fewer trichoblasts in comparison to wild-type. A translational fusion of UGT80B1 to GFP localizes to the ER, plasma membrane and to sites that appear to be plasmodesmata-associated desmotubules. Ultrastructural analysis revealed abnormalities in plasmodesmata formation and morphology in ugt80B1 mutants. Steryl glucoside profiling indicated deficiencies in specific glycosylated sterol compounds in roots. This study identifies UGT80B1 as a novel membrane component that is critical for plasmodesmata morphogenesis and cell-fate determination in the root epidermis. A model is proposed in which UGT80B1 activity provides spatially discreet sterol and steryl glucoside architecture within the plasma membrane to anchor the SCM receptor and within plasmosdesmata to facilitate intercellular movement of R3-MYB regulatory proteins underlying proper differentiation of trichoblasts versus atrichoblasts. Moreover, evidence from reverse genetics, proteomics and live cell imaging point to a actin dependent localization of UGT80B1 at the vesicle rich zone of root hair tip. This localization actively supports root hair elongation via tip growth, possibly by membrane modifications required for vesicle trafficking.

The problems and high medical costs associated with chronic wounds necessitate an economical bioactive wound dressing. A new strategy was investigated to inhibit MMP-9 proteases and to release epidermal growth factor (EGF) to enhance healing. Doxycycline (DOX) and EGF were encapsulated on polyacrylic acid modified polyurethane film (PAA-PU) using Layer-by-Layer (LbL) assembly. The number of bilayers tuned the concentration of DOX and EGF released over time with over 94% bioactivity of EGF retained over 4 days. A simple wound model in which MMP-9 proteases were added to cell culture containing fibroblast cells demonstrated that DOX inhibited the proteases providing a protective environment for the released EGF to stimulate cell migration and proliferation at a faster healing rate. In the presence of DOX, only small amounts of the highly bioactive EGF are sufficient to close the wound. Results show that this is new and promising bioactive dressing for effective wound management.

The calcium binding epidermal growth factor-like (cbEGF) 12-13 domain pair from human fibrillin-1 was the focus of studies for this dissertation. Various nuclear magnetic resonance (NMR) spectroscopy techniques were employed to analyse the calcium binding, structural and dynamic properties of this pair, and to assess the effects of a disease-causing mutation. Fibrillin-1 is a mosaic protein composed mainly of 43 cbEGF domains arranged as multiple, tandem repeats, and mutations within fibrillin-1 have been linked to Marfan syndrome (MFS). 66% of MFS-causing mutations identified thus far are localised to cbEGF domains, emphasising that the native properties of these domains are critical to the functional integrity of this protein. The cbEGF 12-13 pair is found within the longest run of cbEGFs in fibrillin-1, and many mutations that cluster in this region are associated with the severe, neonatal form of MFS. It is thought that this region may be important for fibrillin-1 assembly into 10- 12nm connective tissue microfibrils. Calcium binding studies of cbEGF 12-13 demonstrated that cbEGF 13 contains the highest affinity site thus far investigated from human fibrillin-1. Comparison with previous results showed that fibrillin-1 cbEGF calcium binding affinity can be significantly modulated by the type of domain which is linked to its N-terminus, and also highlighted the high affinity of the "neonatal" region. The NMR solution structure of cbEGF 12-13 is a near-linear, rod-like arrangement of two cbEGF domains, with both exhibiting secondary structure characteristic of this domain type. The rod-like arrangement is stabilised by calcium binding by cbEGF 13 and by hydrophobic interdomain packing interactions. This observation supports the hypothesis that all Class I EGF/cbEGF-cbEGF pairs, characterised by a single linker residue, possess this rod-like structure. The structure also exhibits additional packing interactions to those previously observed for cbEGF32- 33 from fibrillin-1, which may explain the higher calcium binding affinity of cbEGF13. A model of cbEGF 11-15, created based on structural data for cbEGF 12-13 and a model of cbEGF32-36, has highlighted a potential protein binding interface, which encompasses all known neonatal MFS mutations, as well as a flexible, unstructured loop region of cbEGF 12. Backbone dynamics data confirmed the extended structure of cbEGF 12-13. These data, combined with previous data for cbEGF32-33, highlighted a potential dynamics signature for Class I cbEGF domain pairs. Comparison of data for these pairs also suggested that, in addition to the role of calcium in stabilising rigidity on the picoto millisecond time-scale, calcium affinity may play a key role in determining the anisotropy of cbEGF pairs. Possible dynamic explanations for the variation in calcium binding affinity of cbEGF domains from human fibrillin-1 were also noted. The Gl 127S mutation located in cbEGF 13 of fibrillin-1 causes a mild variant of MFS. NMR studies of the G1127S cbEGF12-13 mutant pair showed that cbEGF12 may chaperone folding of mutant cbEGF 13, an effect most likely mediated through interdomain packing interactions. These studies have also shown that the effects of this mutation are localised to cbEGF13, suggesting that a "partial" MFS phenotype is the result of altered structural, dynamic and/or calcium binding properties of this domain.