No description available.

The aim of this thesis is the study of protein resistant oligo(ethylene glycol) (OEG) self-assembled monolayers (SAMs) using in situ techniques, such as neutron reflectivity (NR), polarisation modulation infrared spectroscopy (PMIR) and small-angle x-ray scattering (SAXS). In order to elucidate the mechanisms that lead to the nonfouling properties of these SAMs, the SAM-water, protein-protein and protein-SAM interactions have been studied separately. NR measurements, focused on the solid-liquid interface between OEG SAMs and water, show clear evidence of an extended layer with reduced density water. The reduction in density is up to 10% compared to the bulk value, and extends up to 5 nm into the bulk. The effective area (density reduction x length) of this reduced density water layer did not significantly change when the temperature was reduced to 5°C. In a complementary study, the interaction of water with protein-resistant HS(CHV₂)₁₁(OCH₂CH₂)₃OMe monolayers was examined using in and ex situ PMIR. In particular, shifts in the position of the characteristic C-O-C stretching vibration were observed after the monolayers had been exposed to water. The shift in frequency increased when the SAM was observed in direct contact with a thin layer of water. It was found that the magnitude of the shift also depended on the surface coverage of the SAM. These results suggest a rather strong interaction of oligo(ethylene glycol) SAMs with water and indicate the penetration of water into the upper region of the monolayer. These findings indicate the presence of a tightly bound water layer at the SAM-water interface. Further NR studies of the interface between OEG SAMs and a highly concentrated protein solution revealed an oscillating protein density profile. A protein depleted region of about 4-5 nm close to the SAM was followed by a more densely populated region of 5-6 nm. These oscillations were then rapidly damped out until the bulk value was reached. The influence of temperature and salt concentration on the protein density profile was small, indicating a rather minor contribution of electrostatic interactions to the protein repulsive force. SAXS measurements of OEG coated gold colloids mixed with proteins in solution did also not show any pronounced salt concentration dependence of the colloid-protein interaction. The strong association of water with the SAM and the layer of tightly bound water, together with the lack of electrostatic repulsion, suggest that the adsorption of proteins is energetically hindered by the presence of a strongly bound hydration layer.

Seed formation consists of two major stages: embryo pattern formation and maturation. During seed maturation, the embryo accumulates storage material, acquires desiccation tolerance, and enters a stage of dormancy. Genetic analyses have identified several master regulators that orchestrate late embryogenesis, including the B3-domain transcription factor FUSCA3 (FUS3). In Arabidopsis, FUS3 has been shown to be a central regulator of hormonal pathways; it positively regulates late embryogenesis by increasing abscisic acid (ABA) level while repressing gibberellin (GA) synthesis. In turn, FUS3 protein level is positively and negatively regulated by ABA and GA, respectively. However, the mechanism of how this regulation occurs has not been well characterized. In this study, FUS3 has been shown to be an unstable protein rapidly degraded by the proteasome through a PEST instablility motif. To further characterize the mechanisms involved in FUS3 homeostasis, FUS3-interacting proteins were identified. The SnRK1 kinase AKIN10 was shown to interact with and phosphorylate FUS3 at its N-terminus. Furthermore, overexpression of AKIN10 delays FUS3 degradation, suggesting AKIN10 positively regulates FUS3 protein accumulation. Overexpression of AKIN10 delays developmental phase transitions, and causes defects in lateral organ development. These defects were partially rescued by the loss-of-function fus3-3 mutation, suggesting FUS3 and AKIN10 genetically interact to regulate these developmental processes. SnRK1/AMPK/Snf1 kinases are regulators of energetic stress responses. Overexpression studies suggest both FUS3 and AKIN10 positively regulate ABA signaling, but differ in sugar responses during germination; AKIN10 mediates glucose sensitivity, while FUS3 regulates osmotic stress responses. Overexpression of AKIN10 and FUS3 results in glucose and osmotic stress hypersensitivities, respectively, both of which are partially dependent on de novo ABA synthesis. Thus, FUS3 and AKIN10 act in overlapping pathways and combine different environmental signals to generate a common ABA-dependent response. In summary, novel mechanisms that regulate FUS3 homeostasis and function were identified. A model explaining the interaction between FUS3 and AKIN10 during embryonic and vegetative development, and the function of these two central developmental regulators in hormonal and stress signaling pathways is discussed.

Seed formation consists of two major stages: embryo pattern formation and maturation. During seed maturation, the embryo accumulates storage material, acquires desiccation tolerance, and enters a stage of dormancy. Genetic analyses have identified several master regulators that orchestrate late embryogenesis, including the B3-domain transcription factor FUSCA3 (FUS3). In Arabidopsis, FUS3 has been shown to be a central regulator of hormonal pathways; it positively regulates late embryogenesis by increasing abscisic acid (ABA) level while repressing gibberellin (GA) synthesis. In turn, FUS3 protein level is positively and negatively regulated by ABA and GA, respectively. However, the mechanism of how this regulation occurs has not been well characterized. In this study, FUS3 has been shown to be an unstable protein rapidly degraded by the proteasome through a PEST instablility motif. To further characterize the mechanisms involved in FUS3 homeostasis, FUS3-interacting proteins were identified. The SnRK1 kinase AKIN10 was shown to interact with and phosphorylate FUS3 at its N-terminus. Furthermore, overexpression of AKIN10 delays FUS3 degradation, suggesting AKIN10 positively regulates FUS3 protein accumulation. Overexpression of AKIN10 delays developmental phase transitions, and causes defects in lateral organ development. These defects were partially rescued by the loss-of-function fus3-3 mutation, suggesting FUS3 and AKIN10 genetically interact to regulate these developmental processes. SnRK1/AMPK/Snf1 kinases are regulators of energetic stress responses. Overexpression studies suggest both FUS3 and AKIN10 positively regulate ABA signaling, but differ in sugar responses during germination; AKIN10 mediates glucose sensitivity, while FUS3 regulates osmotic stress responses. Overexpression of AKIN10 and FUS3 results in glucose and osmotic stress hypersensitivities, respectively, both of which are partially dependent on de novo ABA synthesis. Thus, FUS3 and AKIN10 act in overlapping pathways and combine different environmental signals to generate a common ABA-dependent response. In summary, novel mechanisms that regulate FUS3 homeostasis and function were identified. A model explaining the interaction between FUS3 and AKIN10 during embryonic and vegetative development, and the function of these two central developmental regulators in hormonal and stress signaling pathways is discussed.

The focus of this thesis is to characterise the interactions between GLUT4-related SNARE proteins – syntaxin4, SNAP23 and VAMP2 – and a regulatory protein, Munc18c. GLUT4 is the primary insulin-regulated glucose transporter and is presentin fat and muscle cells. GLUT4 is held in intracellular pools of vesicles until it is transported to the cell surface upon insulin stimulation. Insulin initiates a cellular signalling cascade via the insulin receptor on the cell membrane, which in turn stimulates GLUT4 vesicles to move to the cell surface where they fuse to the plasmamembrane via SNARE proteins. SNAREs are membrane-anchored proteins present on both vesicle and target membranes that form a tight complex which brings themembranes together for fusion. Fusion of vesicles to the target membrane releases the vesicular cargo.SNARE-mediated membrane fusion is a conserved mechanism that controls many other vesicle fusion processes such as neurotransmitter release and yeast vesicular trafficking. However, the regulation of the SNARE mechanism is not fully understood. SNAREs can interact with many other proteins that could act as regulatory factors,and studies have focused primarily on a group of effector proteins called Sec1p/Munc18 (SM) proteins. SM proteins were discovered and characterised because they bind to one type of SNARE protein, syntaxin. The SM protein that interacts with the GLUT4-related SNARE, syntaxin4, is Munc18c.The aim of this thesis was to investigate Munc18c interactions with SNARE proteins, principally syntaxin4, using biochemical techniques with purified recombinant proteins. This work was carried out in several stages including: 1) development of methods to produce and purify GLUT4-related SNARE proteins, SNARE complexes and Munc18c, 2) development of an assay to quantify Munc18c interactions with binding partners using surface plasmon resonance, 3) investigation into interactions between Munc18c and SNARE ternary complex, 4) characterising Munc18c interactions with syntaxin4, and 5) developing a method to produce selenomethionine-containing Munc18c in a baculovirus system to be used in structural studies. The methods and outcomes of these experiments are described inthis thesis. There were two major outcomes from this work. Firstly, Munc18c interacts with SNARE ternary complex, and secondly, Munc18c requires only the N-terminal 29residues of syntaxin4 for an interaction to occur. These results were determined using pulldown assays with purified proteins, as well as other chromatographic methods to show that protein complexes were formed. The steps taken to develop these binding assays are also discussed. Initial crystallisation conditions forMunc18c-HIS and a peptide consisting of syntaxin4 residues 1-20 have been identified using crystallisation screens. The interactions determined for Munc18c binding to Sx4 are in direct contrast to those of neuronal SM protein, Munc18a, and its interaction with neuronal SNARE proteins - Munc18a does not bind to its ternary complex and binds to the entire cytoplasmic domain of Sx1a. Rather, the Munc18c:Sx4 interactions are similar to that for the yeast SM protein, Sly1p, which can interact with both its SNARE ternary complex and with its syntaxin via the Nterminal residues. Another interesting outcome of this research was that syntaxin4 binds to metals (cobalt and nickel). This finding represents the first reported for a syntaxin interacting with metals. Preliminary results indicate that un-tagged syntaxin4 can bind to cobalt resin, and to nickel immobilised on a chip. This interesting and novel property of syntaxin4 binding was serendipitously discovered while investigating conditions for the Munc18c assay. Overall, I have shown that Munc18c, the SM protein involved in GLUT4 trafficking, interacts with SNARE proteins in a different manner to its mammalian counterpart inneurons, Munc18a, and is more like Sly1p, a yeast ER-Golgi SM protein. Munc18c interacts with SNARE complexes and only the N-terminal residues of syntaxin4.These interactions demonstrate that the regulatory mechanism for SNARE-mediated fusion is conserved between yeast and mammals. This finding has several implications for the role of Munc18c in the exocytosis of GLUT4-containing vesicles. Munc18c could act at several stages in the fusion process via syntaxin4 binding.These interactions could involve binding to other proteins (such as synip or tomosyn), conformational switching of syntaxin4 or interaction with metal ions to induce conformational changes in the proteins. Finally, these studies of GLUT4 exocytosis contribute to our understanding of glucose transport disorders such as Type 2 diabetes and could one day pave the way for the design of therapeutic agents.

[Truncated abstract] Neuregulins (NRG’s) are pleiotropic growth factors that participate in a wide range of biological processes. The family of membrane-bound growth factors bind to and activate ErbB receptors on adjacent target cells, mediating multiple biological processes. NRG-1, NRG-2 and NRG-3 are all highly expressed in the nervous system, where it has been shown that NRG-1 is important for neuronal development, migration, synapse formation and glial cell proliferation. Little is known, however, on the specific roles of NRG-2 and NRG-3, although it is apparent that despite similar expression patterns and overlapping receptor specificity, NRG-2 and NRG-3 do not compensate for the loss of NRG-1 and mediate their own distinct activities. … Subcellular localisation experiments showed that this domain is important for trafficking of the fulllength protein to various intracellular compartments in an activity dependent manner. In addition, the ICD is required to elicit a cell death response in cultured cells and provoke an elevated α-amino-3-hydroxyl-5-methylisoxazole-4-propionate (AMPA) response in organotypic neuronal cultures following transient expression of NRG-3. A yeast two-hybrid screen identified 14-3-3ζ and PICK1 as two proteins that interacte with the human NRG-3 ICD. These interactions were confirmed both in vitro and in vivo, and were further characterised at a molecular level. This study demonstrates the ability of NRG-3 to mediate signal transduction through a biologically active ICD; a conclusion supported by identifying cytoplasmic proteins that interact with the ICD. These observations point to an additional layer of complexity where bi-directional signalling contributes to the full repertoire of NRG-3 functions.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D. in Molecular Physiology and Biophysics)--Vanderbilt University, May 2006. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2006. / Additional advisors: Vithal K. Ghanta, Denise R. Shaw, Stephen A. Watts, Bradley K. Yoder. Description based on contents viewed Feb. 20, 2009; title from PDF t.p. Includes bibliographical references.