Investigating the molecular signatures of β1-adrenergic receptor activation

In this thesis I have investigated the molecular signatures of receptor activation, using the β1-adrenergic receptor (β1AR) as a prototypical class A G-protein coupled receptor (GPCR). I have used a minimally thermostabilised turkey β1AR and expressed it functionally in insect cells using a baculovirus system. The work described here established the labelling, expression, purification and sample preparation of the receptor in LMNG detergent micelles for use in nuclear magnetic resonance (NMR) spectroscopy. GPCRs are highly dynamic molecular systems, and the use of solution NMR is highly suited to the study of fine structural changes that take place within the receptor as a consequence of receptor activation. To this end the receptor was selectively labelled with 13C at the methyl position of methionine residues. The labelling was carried out during insect cell expression, by supplementing methionine deficient media with the labelled amino acid. In this way the methionines throughout the receptor served as reporters. The radio frequency signals emitted by the nuclei of these labelled residues were monitored. NMR experiments were recorded on the receptor in the presence of ligands of various efficacies together with and without G-protein mimetic nanobodies, and changes in the signal were recorded. This allowed for a pattern of molecular signatures to be established, reporting on the effect ligands and G-protein mimetics have on the receptor. This identified two conformational equilibria, between an inactive and a ligand bound-pre-activated state and between a more and a less active ternary state when bound to a G-protein mimetic. Furthermore, it was also observed that ligand binding to the G-protein mimetic saturated basal active state elicits further changes on the receptor cytoplasmic side, demonstrating that ligand efficacy modulates the nature of receptor interaction with G-proteins, which may underpin partial agonism. It was also observed that ligand binding affects the dynamics and rigidity of the receptor, with a full agonist bound receptor exhibiting extensive µs to ms timescale dynamics, compared to a more rigid nanobody bound state. The increased dynamics suggest that full agonist binding primes the receptor for interaction with various downstream signalling partners. Once this coupling takes place, ligand efficacy determines the quality of interaction in this rigidified system. In addition to activation by ligands, certain proteins, such as antibodies can cause receptor agonism in the absence of a small molecule agonist. An example of this takes place in chronic Chagas’ heart disease, where anti-Trypanosoma cruzi antibodies inappropriately cross-react to β1AR, leading to chronic cardiac overstimulation and heart failure. In this thesis, the production of a published monoclonal antibody fragment was explored, in order to generate a tool for the study of this activation mechanism.MRC CASE, NIBR, Wolfson College, Biochemical Society, Philosophical Societ

Traffic Classification over Gbit Speed with Commodity Hardware

This paper discusses necessary components of a GPU-assisted traffic classification method, which is capable ofmulti-Gbps speeds on commodity hardware. The majority of the traffic classification is pushed to the GPU to offload the CPU, which then may serve other processing intensive tasks, e.g., traffic capture. The paper presents two massively parallelizable algorithms suitable for GPUs. The first one performs signature search using a modification of Zobrist hashing. The second algorithm supports connection pattern-based analysis and aggregation of matches using a parallel-prefix-sum algorithm adapted to GPU.The performance tests of the proposed methods showed that traffic classification is possible up to approximately 6 Gbps with a commodity PC

Polymeric alkali fullerides are stable in air

Infrared transmission, electron spin resonance, and X-ray diffraction measurements show unambiguously that RbC$_{60}$ and KC$_{60}$ are stable in air, in contrast to Rb$_{6}$C$_{60}$ which decomposes rapidly upon exposure. The specimens studied transform into pure C$_{60}$ and other byproducts when heated above 100\dd C, approximately the temperature of the orthorhombic-fcc phase transition. The stability of these compounds raises the possibility of applying them as protective layers for the superconducting fullerides.Comment: Scheduled for publication in Appl. Phys. Lett. 66, 20 Feb. 1995, typeset in REVTEX v3.0 in LaTeX. Postscript file including all figures is available on WWW http://insti.physics.sunysb.edu/~mmartin/ under my list of publications, or will be e-mailed by request

Location and type of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia

Background: Mutations of isocitrate dehydrogenase 1 and 2 are novel common genetic alterations identified in acute myeloid leukemia. Aims: To investigate the frequency, clinical associations and prognostic effect of isocitrate dehydrogenase 1 and 2 mutations together, followed by a detailed investigation of particular mutations. Methods: A consecutive cohort of 376 patients diagnosed with acute myeloid leukemia were enrolled to compare clinical characteristics. Prognostic impact was analyzed for 314 patients younger than 60 years treated with curative intention. Isocitrate dehydrogenase 1 and 2 mutations were screened using allele-specific PCR and high resolution melting, followed by a confirmatory sequencing. Results: Isocitrate dehydrogenase (IDH) 1 and 2 mutations were mutually exclusive, detected in 8.5% and 7.5% of the cases respectively. Presence of mutations was associated with older age (p=0.001), higher platelet count (p=0.001), intermediate risk karyotype (p<0.0001), nucleophosmin1 mutation (p=0.022), and with lower mRNA expression level of ABCG2 gene (p=0.006), as compared to mutation negative cases. Remission, relapse rates and overall survival were not different in IDH-mutation positive patients. Interestingly, particular mutations differred in association with nucleophosmin1 mutation: co-occurrence was observed in 14.3% of R132C vs. 70% of R132H carriers (p=0.02); and in 47.4% of R140Q vs. 0% R172K carriers (p=0.02) of IDH1 and IDH2 genes, respectively. R132H negatively influenced overall survival compared to isocitrate dehidrogenase 1 and 2 negative (p=0.02) or to R132C (p=0.019) patients. Conclusions: IDH mutations are frequent recurrent mutations in acute myeloid leukemia. Although a general common pathogenetic role is proposed, our results indicate that differences in clinical characteristics and treatment outcome may exist among disctinct mutations of both genes

Disconnection Mechanism and Regional Cortical Atrophy Contribute to Impaired Processing of Facial Expressions and Theory of Mind in Multiple Sclerosis: A Structural MRI Study

Successful socialization requires the ability of understanding of others' mental states. This ability called as mentalization (Theory of Mind) may become deficient and contribute to everyday life difficulties in multiple sclerosis. We aimed to explore the impact of brain pathology on mentalization performance in multiple sclerosis. Mentalization performance of 49 patients with multiple sclerosis was compared to 24 age- and gender matched healthy controls. T1- and T2-weighted three-dimensional brain MRI images were acquired at 3Tesla from patients with multiple sclerosis and 18 gender- and age matched healthy controls. We assessed overall brain cortical thickness in patients with multiple sclerosis and the scanned healthy controls, and measured the total and regional T1 and T2 white matter lesion volumes in patients with multiple sclerosis. Performances in tests of recognition of mental states and emotions from facial expressions and eye gazes correlated with both total T1-lesion load and regional T1-lesion load of association fiber tracts interconnecting cortical regions related to visual and emotion processing (genu and splenium of corpus callosum, right inferior longitudinal fasciculus, right inferior fronto-occipital fasciculus, uncinate fasciculus). Both of these tests showed correlations with specific cortical areas involved in emotion recognition from facial expressions (right and left fusiform face area, frontal eye filed), processing of emotions (right entorhinal cortex) and socially relevant information (left temporal pole). Thus, both disconnection mechanism due to white matter lesions and cortical thinning of specific brain areas may result in cognitive deficit in multiple sclerosis affecting emotion and mental state processing from facial expressions and contributing to everyday and social life difficulties of these patients

Development and In-Depth Characterization of Bacteria Repellent and Bacteria Adhesive Antibody-Coated Surfaces Using Optical Waveguide Biosensing

Bacteria repellent surfaces and antibody-based coatings for bacterial assays have shown a growing demand in the field of biosensors, and have crucial importance in the design of biomedical devices. However, in-depth investigations and comparisons of possible solutions are still missing. The optical waveguide lightmode spectroscopy (OWLS) technique offers label-free, non-invasive, in situ characterization of protein and bacterial adsorption. Moreover, it has excellent flexibility for testing various surface coatings. Here, we describe an OWLS-based method supporting the development of bacteria repellent surfaces and characterize the layer structures and affinities of different antibody-based coatings for bacterial assays. In order to test nonspecific binding blocking agents against bacteria, OWLS chips were coated with bovine serum albumin (BSA), I-block, PAcrAM-g-(PMOXA, NH(2), Si), (PAcrAM-P) and PLL-g-PEG (PP) (with different coating temperatures), and subsequent Escherichia coli adhesion was monitored. We found that the best performing blocking agents could inhibit bacterial adhesion from samples with bacteria concentrations of up to 10(7) cells/mL. Various immobilization methods were applied to graft a wide range of selected antibodies onto the biosensor’s surface. Simple physisorption, Mix&Go (AnteoBind) (MG) films, covalently immobilized protein A and avidin–biotin based surface chemistries were all fabricated and tested. The surface adsorbed mass densities of deposited antibodies were determined, and the biosensor;s kinetic data were evaluated to divine the possible orientations of the bacteria-capturing antibodies and determine the rate constants and footprints of the binding events. The development of affinity layers was supported by enzyme-linked immunosorbent assay (ELISA) measurements in order to test the bacteria binding capabilities of the antibodies. The best performance in the biosensor measurements was achieved by employing a polyclonal antibody in combination with protein A-based immobilization and PAcrAM-P blocking of nonspecific binding. Using this setting, a surface sensitivity of 70 cells/mm(2) was demonstrated