Spring 2013 Seminar Schedule

All seminars are held on Fridays 3:30-5:00 PM

Izabela Sokolowska
PhD Candidate,
Chemistry & Biomolecular Science Department
Clarkson University

"Investigation of Tumor Differentiation Factor (TDF) and its putative receptor (TDF-R)"

Abstract:
TDF is an under-investigated 12 kDa protein produced by the pituitary and secreted into the blood stream. TDF protein and TDF-P1 (a peptide selected from the open reading frame of TDF) have differentiation activity on breast and prostate cancer cells, suggesting a possible endocrine role. Yet, to date TDF has no definitive function and its characterization is incomplete.

Therefore to investigate the signal transduction pathways that are activated by TDF we used TDF-P1 to identify and purify TDF-R candidates from human breast and prostate cancer cell. We identified members of the Heat Shock 70 ka family of proteins as potential receptor candidates (GRP78 and HSP70). These results also suggest that TDF protein may interact with GRP78 and/or GRP78 in complex with HSP70 to promote differentiation in breast and prostate cancer cells through a steroid-independent pathway.

To gain more knowledge about TDF protein itself we over-expressed and characterized recombinant TDF (rTDF). Our experiments suggest that rTDF is expressed mostly as an insoluble monomeric form. Mass spectrometry-based analysis was used as a direct evidence of TDF existence. In addition, we assessed the potential three dimensional structure of TDF including disulfide connectivities using molecular modeling.

Lastly, we analyzed distribution of TDF expression in the body. TDF mRNA was initially detected in brain but not in other tissues such as heart, lung, liver, pancreas or skeletal muscles. However it was not known precisely which cells in the brain produce TDF. Therefore, we investigated the distribution of TDF protein in the rat tissues using fluorescent immunohistochemistry (IHC). TDF protein was expressed in select cells in breast, pituitary and select neurons in brain. Double-staining for neuronal and glial markers showed that TDF is expressed in some neuronal cells but not in astrocytes. Further experiments revealed that the GABAergic subtype of neurons seem to be TDF-positive.

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Friday, May 3, 2013
212 B.H. Snell Hall

Yuanfang Lu
Chemistry & Biomolecular Science Department
Clarkson University

"Post-Chemical Mechanical Planarization Cleaning: A NiP Substrate Study"

Abstract:
With the hard disk drive (HDD) capacity increasing by 60% per year, the surface of HDD substrate has reached atom-scale planarization after chemical mechanical polishing (CMP). Surface roughness, particle residues, metallic contaminants and corrosion spots on the disk surface may lead to head crashes, so the surface of HDD must be ultra clean. During post-CMP cleaning, cleaning solution plays a key role in cleaning efficiency. In this study, both acidic and alkaline cleaners were investigated for HDD post-CMP cleaning. Atomic force microscopy (AFM) and optical microscope analysis indicated that both acidic and alkaline cleaners facilitate the removal off silica from NiP substrates. And XPS is also used to explore the cleaning mechanism.

Professor Devon Shipp with graduate student Yuanfang Lu.

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Friday, April 26, 2013
212 B.H. Snell Hall

Professor James F. Rusling
Chemistry Department, University of Connecticut
Department of Cell Biology, University of Connecticut
School of Chemistry, National University of Ireland at Galway

"Nanoscience-based microfluidic arrays for cancer diagnostics by detection of biomarker panels"

Abstract:
Sensitive measurement of biomarker proteins overexpressed in individuals with cancer holds great promise for early detection and personalized therapies. Broad implementation of diagnostic strategies requires reliable, inexpensive devices to measure multiple proteins in patient samples. Emerging aspects of nanotechnology and microfluidics provide exciting new opportunities to design and fabricate such devices. In this talk, we discuss advances for the ultrasensitive multiplexed detection of proteins in patient serum. Approaches are based first on nanostructured sensor surfaces coupled with novel labeling strategies that achieve very large signal amplification. Microfluidic immunoarrays utilizing amperometry, electrochemiluminescence, or surface plasmon resonance are interfaced with microfluidics to achieve unprecedented sensitivities for detection of biomarker proteins into the low fg/mL range in serum. High sensitivity can also be traded for speed to achieve immunoassays in less than 10 min.

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Friday, April 19, 2013
212 B.H. Snell Hall

Dr. Daniel S. Spellman
Principal Scientist, Group Leader, Proteomics and Metabolomics, West Point Molecular Biomarkers (MBMx), PPDM, Merck Research Laboratories

"Mass Spectrometry-based Biomarkers as Tools for Decision Making in the Drug Development Process"

Abstract:
Identification of biomarkers from accessible tissues and biofluids is important to drug discovery and development, with the goal of enabling decision making at critical stage gates during the development process. A general proteomic approach for the discovery and identification of protein biomarkers, differential mass spectrometry (dMS), based on the analysis of full scan mass spectrometry data, has been previously described. Examples of how such a quantitative label-free proteomics approach can be applied to characterize changes resulting from pharmacological intervention, disease state, and observed clinical characteristics will be described.  A workflow for translating protein markers from discovery to robust, quantitative triple-quadrupole mass spectrometry assays that are more amenable to measuring large numbers of clinical samples will also be presented.

Dr. Daniel Spellman from Merck Research Laboratories (left) and Professor Costel Darie.

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Friday, April 12, 2013
212 B.H. Snell Hall

Erica Sharpe
Chemistry Department
Clarkson University

"Portable nanoparticle-based sensors for characterization of antioxidant-containing samples"

Abstract:
With increased awareness of nutrition and the advocacy for healthier food choices, there is need for a simple, easy-to-use test that can reliably measure the quality and content of food antioxidants.  We report development of the first portable nanoparticle-based sensor for the detection of antioxidants.  It offers three tiers of analysis, which sets it apart from other antioxidant assays: it is capable of analyzing samples for 1) antioxidant activity 2) constituent concentration 3) constituent identity. The platform is based on immobilized nanoparticles that show a distinct color change in the presence of antioxidants by means of redox and surface chemistry reactions.  The sensor operates in a variety of environments and food products; it does not require specialized equipment or external reagents and can be used by unskilled personnel as well as the general population. Multiple sensing-components have been found to produce unique colorimetric responses, creating opportunities for cross-validation of assay results using more than one sensor type. This presentation will discuss fabrication and performance evaluation of this assay for the detection of common food-antioxidants, and describe functionality of the assay in real samples including tea infusions, botanical extracts, and human serum. This sensing method will be compared to a commonly used USDA assay; and future work including the development of a high-throughput analysis method and the creation of a portable electronic color-reference database will be discussed.

Professor Silvana Andreescu with graduate student Erica Sharpe.

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Friday, April 5, 2013
212 B.H. Snell Hall

Yan Mu
Chemistry Department
Clarkson University

"Chemical Mechanical Polishing of Hard Disk Drive (HDD) Substrates: preliminary experimental investigation"

Abstract:
Nickel–Phosphorous (NiP) substrates have been widely used as computer hard disk drive (HDD) substrate. With rapid increase of data storage density on computer HDD, the operation distance between read/write head and disk surface has fallen into few nanometers. In order to avoid crash of read/ write head on to the disk, the NiP substrates must be perfectly flat and free of defects such as pits, scratches, and bumps. Chemical mechanical polishing (CMP) can produce high quality surface of substrates with NiP plating for HDD application. In this talk, I will discuss the effect of particle size, hydrogen peroxide as an oxidizer and BTA as a passivating agent in chemical mechanical planarization (CMP) of hard disk substrates with nickel–phosphorous plating. NiP-CMP was studied using material removal rate measurement and etching rate measurements in solutions containing the oxidizer and passivating agent. X-ray photoelectron spectroscopy was also done to understand the mechanism of NiP-oxidant interaction during polishing.

Professor Devon Shipp with graduate student Yan Mu.

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Friday, April 5, 2013
9:00 a.m.
169 B.H. Snell Hall

Lu Lu 
Chemistry Department
Clarkson University

"Preparation and Formation Mechanisms of Metallic Particles with Controlled Size, Shape, Structure and Surface Functionality"

Abstract:
Due to their excellent conductivity and chemical stability, particles of silver (Ag), gold (Au), copper (Cu) and their alloys are widely used in the electronic industry. Other unique properties extend their uses to the biomedical, optical and catalysis fields. All these applications rely on particles with well controlled size, morphology, structure, and surface properties. Chemical precipitation from homogeneous solutions was selected as the synthetic route for the investigations described in this work. Based on the evaluation of key process parameters (temperature, reactant concentrations, reactant addition rate, mixing, etc.) the general formation mechanisms of metallic particles in various selected precipitation systems were investigated and elucidated.

Five different systems for preparing particles with controlled size, morphology, structure and surface functionality are discussed. The first system involves the precipitation of Ag nanoparticles with spherical and anisotropic (platy or fiber-like) morphology. It will be shown that the formation of a stable Ag/Daxad complex has a significant impact on the reaction kinetics, and the chromonic properties of Daxad molecules are responsible for the particle anisotropy. In the second system, Au-Ag core-shell nanoparticles were prepared in aqueous solution by a two-step precipitation process. The optical properties of these particles can be tailored by varying the thickness of the Ag shell. It was also determined that the stability of the bimetallic metallic sols depends on the Cl^- ions concentration in solution. The third system discussed deals with the preparation by the polyol process of well dispersed Cu nanospheres with high crystallinity and excellent oxidation resistance. We show that the heterogeneous nucleation (seeding) approach has significant merit in controlling particle size and uniformity. The functionalization of the Au nanoparticles surface with glutathione molecules is discussed in the next section. The developed method is used to quantify the maximum adsorption load of glutathione on the gold particles and assess their potential in biomedical applications. Finally, a process capable of generating uniform Ag particles with diameters ranging from 80 nm to 60 µm and with controlled crystallinity/internal structure is described.

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Friday, March 29, 2013
212 B.H. Snell Hall

Dr. Gabriel Caruntu
Chemistry Department
University of New Orleans

"Polar Ordering in Nanoscale Ferroic Oxides and its Control via Colloidal Processing"

Abstract:
Research on nanoscale ferroic oxides has developed at a truly relentless pace due to their wide variety of outstanding properties and limited availability of similar materials to meet the increasing technological demands of present and future applications. Although of prime technological interest is the existence of a switchable polarization with direct applicability in memory devices for computer bit storage, these materials are the leading candidates for the design of novel probes in cellular imaging, due to their biocompatibility and outstanding optical properties. Although much progress has been made in understanding the proprieties of nanoscale magnetic materials, the current knowledge is far less advanced in the case of low-dimensional ferroelectrics despite their enormous technological potential.  Based upon empirical observation, it is believed that when the size of a ferroelectric is reduced, the polar ordering decreases exponentially and vanishes below a critical size. Despite years of intense research in this field, a clear understanding whether a polar ordering still exist in low-dimensional perovskite structures is still lacking and several fundamental questions should be addressed before nanoscopic perovskites become feasible for applications.

In this talk I will try to address this long-standing fundamental question by using, aggregate-free, monodisperse colloidal nanocrystals of BaTiO₃, an archetypal ferroelectric material as a model system. Selective solvent evaporation techniques enable the manipulation and isolation of individual nanoparticles allowing the examination of the local structural and electrical behavior at nanometer length scales. Although the average structure of the nanocrystals seems to be metrically cubic, at microscopic scales they retain an acentric structure down to a size of 5 nm, the smallest accessible length scale. Moreover, in-situ electron microscopy investigations revealed that nanocrystals retain a primarily linear, monodomain dielectric polarization, despite the theoretical predictions of vortice-like structures in nanoscale ferroelectrics. These findings they provide for the first time a glimpse of the structural and electrical manifestation of the polar ordering down to its ultimate size limits. Such fundamental knowledge of the nanoscale polar ordering can potentially result in the improvement of the actual design technologies in smart perovskite materials with programmable ferroelectric, dielectric and piezoelectric properties.

Professor Gabriel Caruntu from University of New Orleans (left) and Professor Egon Matijević, Victor K. LaMer Chair of Colloid and Surface Science at Clarkson University.

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Friday, March 19, 2013,
11:00 a.m
212 B.H. Snell Hall

Dr.Manfred Stamm
Leibniz Institute of Polymer Research in Dresden

"THE CHALLENGE OF POLYMER NANOSTRUCTURES AND FUNCTIONALITIES AT SURFACES: FROM SINGLE MOLECULES TO BRUSHES AND NANOTEMPLATES"

Manfred Stamm graduated in physics at the University of Frankfurt am Main/Germany in 1974, followed by his PhD on determination of polymer chain conformations at Mainz University in the group of Prof. E.W. Fischer.

He then spent three years at the CNRS and Max‐Planck Institute of Solid State Physics in Grenoble/France. After some neutron work at Forschungszentrum Jülich and interface analysis at Brookhaven National Lab/USA, he got his habilitation at University of Mainz in physical chemistry on the investigation of polymer surfaces and interfaces with nanometer resolution. He became staff scientist at the Max Planck Institute on Polymer Research in Mainz before he took a professorship on physical chemistry of polymeric materials at the Technische Universität Dresden in 1999. At the same time, he became Head of the institute of Physical Chemistry and Physics of Polymers at the Leibniz Institute of Polymer Research in Dresden. His main areas of work are connected with nanostructured polymers, polymer interfaces, advanced polymer materials and characterization techniques.

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Friday, March 15, 2013
212 B.H. Snell Hall

Katie L. Poetz
Chemistry Department
Clarkson University

"Erosion and Release Studies from Polyanhydrides"

Abstract:
Polyanhydrides are degradable polymers known to often undergo surface erosion, which is where the erosion is limited to the surface of the polymer.  This is beneficial because this allows for near zero-order release kinetics of drugs in addition to the mechanical properties of the polymer being conserved throughout most of the degradation process.  Polyanhydrides have been traditionally synthesized through polycondensation reactions, which limits their applications. In this work, we have successfully synthesized polyanhydrides through thiol-ene polymerizations.  Thiol-ene polymerizations occur via radical intermediates but molecular weight development is akin to what is seen in step-growth polymerizations.  These unique features decrease the susceptibility to undergo shrinkage, oxygen inhibition and are also able to form a more uniform crosslink density. We have shown that these polymers undergo surface erosion, in addition to exhibiting near zero-order release kinetics for the release of a drug mimic. 

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Friday, March 8, 2013
212 B.H. Snell Hall

Xing X. Liu
Chemistry Department
Clarkson University

"Templated Functionalization of Recombinant Proteins"

Abstract:
Application of synthetic organic chemistry for modification of proteins has found a common use in academic research and industry. The conventional method for production of protein conjugates has changed little in the last twenty years mostly relying on reactions of side chains of cysteine and lysine residues. Due to the presence of large numbers of similar reactive amino acid residues in proteins common synthetic methods generally produce complex mixtures of products which are difficult to separate. The lack of selectivity in these reactions constitutes the major challenge for preparation of pure protein derivatives. We explore a new approach toward functionalization of recombinant proteins that involves formation of a covalent bond with a hexahistidine tag (his-tag) present in recombinant proteins. This reaction will be specific to hexahistidine sequence and will not interfere with other amino acid residues in the molecule of protein. The approach is based on formation of a complementary complex of the hexahistidine sequence with a metal cation chelated by ligand bearing a group capable of subsequent formation of a covalent bond with one of the histidine residues of the His-tag. We will discuss reactions of alkylation of histidine residues with Baylis-Hillman esters and pathways to increase activity of these agents, design and synthesis of ligands combining Baylis-Hillman group with nitrilotriacetate function, choice of appropriate metal cations to template this reaction, and our model alkylation of N-acetylhexahistidine polypeptide by this approach. Finally, we will discuss application of this approach for chemoselective derivatization of recombinant Protein A with a fluorescent group through two step methodology using a "click" cycloaddition reaction.

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Friday, March 1, 2013
212 B.H. Snell Hall

Olivia Z Durham
Chemistry Department
Clarkson University

"Water-Borne Crosslinked Polymer Microspheres Prepared by Thiol-Ene Suspension Photopolymerization"

Abstract:
We present here the first instance of spherical, crosslinked polymer microspheres that are prepared by a water-borne thiol-ene suspension photopolymerization process.  The reaction mechanism behind thiol-ene chemistry is well understood and offers many practical advantages to polymer synthesis for water-based systems as well as for the development of multifunctional materials.  The step-growth mechanism of a thiol-ene polymerization means that the production of highly crosslinked water-borne microspheres is fundamentally different from the chain-growth polymerizations normally associated with emulsion, dispersion, and suspension polymerizations of acrylic and styrenic monomers.  We demonstrate here that thiol-ene polymerizations can be conducted in a water-borne system to yield crosslinked spherical particles that have diameters in the range of sub-micron to hundreds of microns.  Homogenization energy (mechanical shear), surfactant concentration, and surfactant species greatly influence colloidal stability, particle size, and the particle size distribution of the microspheres.  Further development of this approach offers great potential for various applications and additional research into water-borne materials.

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Friday, February 15, 2013
212 B.H. Snell Hall

Dr. Karina Ckless
Chemistry Department
SUNY Plattsburgh, Plattsburgh NY

"Redox regulation of NLRP3-inflamassome"

Abstract:
The focal point of this presentation will be on how reactive oxygen species (ROS), commonly known as “free radicals”, participate and even enhance the inflammatory response. Inflammatory diseases in general include several conditions that are associated in some extent with inappropriate production of pro-inflammatory mediators, the cytokines, such as interleukin 1β (IL-1β). The secretion of IL-1β by inflammatory cells involves activation of the muti-protein complex called NLRP3-inflammasome. In addition to inappropriate production of cytokines, an inflammatory process also can generate high amounts of ROS. These ROS can promptly attack important macromolecules in our cells, such as proteins. Attack of ROS on proteins is known as protein oxidation, or oxidative post-translational modifications (oxPTM). When proteins involved in an inflammatory complex protein network undergo oxidation they can become dysfunctional. Dysfunctional proteins can be responsible for augmentation of inflammatory processes, including those that are in the NLRP3-inflamassome/ IL-1β secretion.  In this presentation we will focus on the participation of ROS on the oxidation of NLRP3, the major protein responsible for regulating the pro-inflammatory cytokine, interleukin 1β. We will also discuss the contribution of mitochondria as the main source of ROS that are potentially causing oxidation of NLRP3 and the consequences for the augmented secretion of IL-1β by inflammatory cells.

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Friday, February 8, 2013
212 B.H. Snell Hall

Dr. Vivekananda Shetty
Progenics Pharmaceuticals, Inc
Tarrytown, NY

"Investigation of Cancer Associated Sialylation Changes in N-linked Glycopeptides by Quantitative Proteomics"

Abstract:
Over the years a great deal of attention has focused on the study of glycosylation aberration in N-linked glycopeptides due to the implication of specific glycosylation changes, in particular sialylation changes in cancer. The level of sialic acid was observed to be significantly elevated in the N-linked glycoproteome of patients with many types of cancers compared to the healthy controls. In view of this, we recently developed a novel lectin-directed tandem labeling (LTL) quantitative proteomics method to probe sialylation changes between normal and cancer serum samples. We employed the LTL method to unambiguously identify N-linked sialylation sites and accurately identified the changes in sialylation between normal and cancer serum samples based on the N-deglycosylated peptide analysis. These results were further validated by non-glycosylated peptide analysis as well as by western blot experiments to understand if these changes indeed occur at the N-linked glycosylation sites. The results of the sialylation aberration analysis of both prostate cancer and ovarian cancer N-linked glycoproteome will be discussed in this presentation.

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Friday, January 25, 2013
212 B.H. Snell Hall

Shay Mailloux
Chemistry Department
Clarkson University

"Iron-Alginate Systems for Controlled Signal-Responsive Electrochemical Release"

Abstract:
Switchable electrode interfaces, functionalized with signal-responsive materials, have become useful in a diverse range of applications, specifically for biocomputing, “smart” biosensing, and biomedical applications. Iron-alginate allows for an electrochemically responsive matrix capable of the entrapment and release of biomolecules, triggered by applied potential from en external source or from a second biochemically responsive electrode. Two systems are demonstrated.

Lysozyme is entrapped in the alginate matrix and released upon application of an electrochemical signal. The bactericidal outcome of the released lysozyme is measured using Gram-positive bacterium and shown to be comparable to the commercial product before entrapment. In the consequent project, the release system is advanced to releasing upon application of a biochemical stimulus using a second electrode with immobilized enzyme, PQQ-GDH, producing a negative potential in the presence of glucose. The alginate/PQQ-GDH system is shown to release the ‘drug’ only in the presence of glucose. Leakage is controlled using Au nanoparticle supports. Versatility of the system allows for a variety of drugs to be used, given that the Au nanoparticles can be easily functionalized.

The systems demonstrate straightforward, applicable concepts for future development in diabetes and cancer treatment, centered on ‘Sense and Act’ methodology, resulting in unparalleled rapid injury diagnosis and treatment.

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Friday, January 23, 2013
9:30 a.m.
CAMP 372

Yongqing Lan
Chemistry Department
Clarkson University

"Low Defect Chemical Mechanical Planarization of Copper"

Abstract:
Chemical mechanical planarization (CMP) has become an essential step in the fabrication of ultra-large-scale integration (ULSI) devices. Continuous miniaturization and introduction of reactive and fragile materials, such as copper and low K dielectrics, require every step of the manufacturing process including CMP process with low defects for each generation of the new devices. The key objective of this work is to explore low defect CMP process solutions at a fundamental level from a slurry formulation point of view. The use of specialty organic polymer particles to carry out the functions of abrasive, stability, and selectivity control is investigated.  In order to achieve these functions, low stiffness polymer particles are surface modified with chemical groups and tested in copper CMP. The performance of polymer particle containing slurries was found dependent on selection of functional groups. However, polymer particles carrying strong reactivity often generate heavy residues on polished copper substrate. Surface analysis indicates that the residues are mainly polymer particle films related to strong adsorption of polymers on the copper film during polishing. Solutions to eliminate the polymeric residue from copper film by adjusting reactivity of functional groups or slurry formulation were studied.  Hybrid inorganic/organic, organic/organic abrasives, and thermally sensitive polymers were also tested for low defect copper CMP. Aspects of slurry handling, a process that may cause particle agglomeration and lead to severe damage to the substrate during polishing, were also investigated

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Friday, January 18, 2013
212 B.H. Snell Hall

Izabela Sokolowska
Chemistry Department
Clarkson University

"Characterization of Tumor Differentiation Factor (TDF), novel factor expressed in select neurons"

Abstract:
Tumor Differentiation Factor (TDF) is a 17 kDa protein produced by the pituitary and secreted into the blood stream that has differentiation activity on breast and prostate cancer cells.  Yet, to date TDF has no definitive function and its characterization is incomplete. To gain more knowledge about TDF protein we over-expressed and characterized recombinant TDF (rTDF). Our experiments suggest that rTDF is expressed mostly as an insoluble monomeric and dimeric form. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified peptides that are part of the TDF protein. So far, the existence of TDF was demonstrated only through indirect methods (mRNA and Western Blotting identification), therefore our mass spectrometry identification is the first direct proof of TFD existence. In addition, we assessed the potential three dimensional structure of TDF with disulfide as its connectivity using molecular modeling, which suggests linkage between four cysteine residues within TDF (Cys17 – Cys98 and Cys70 – Cys97).

TDF was discovered indirectly in pituitary extract almost a decade ago. cDNA of tdf gene encoding a 108 amino acids was isolated from human pituitary cDNA library. TDF mRNA was detected in brain but not in other tissues such as heart, lung, placenta, liver, pancreas or skeletal muscles. However TDF has an unclear function and it is not known precisely which cells in the brain produce TDF. Therefore, we investigated the distribution of TDF protein in the rat brain using fluorescent immunohistochemistry (IHC). TDF protein was expressed in pituitary and select cells in brain. Double-staining for neuronal and glial markers showed that TDF is expressed in some neuronal cells but not in astrocytes. Further experiments revealed that the GABAergic subtype of neurons seem to be TDF-positive. These cells are inhibitory and therefore may counteract excitatory glutamate release during brain insults or disease, conferring neuroprotection. A neuroprotective role of TDF is further supported by its upregulation by oxidative stress in cultured neuroblastoma cells. In addition, we found that TDF protein is expressed in previously unreported select cells of mammary and prostate glands. It may suggest that TDF is a novel factor derived from select neurons in central nervous system (CNS) and pituitary with yet unknown functional activity on breast and prostate.

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Friday, January 11, 2013
212 B.H. Snell Hall

Dr. Steven R. Goodman
Institute of Biomedical Sciences and Technology
SUNY Upstate Medical University

"The Proteomics and Interactomics of Sickle Cell Disease"

Abstract:
My red blood cell (RBC) studies began over 30 years ago with the co-discovery of the spectrin membrane attachment protein subsequently named ankyrin. My laboratory worked out many of the protein interactions that make up the RBC membrane skeleton and then in 1981 we published an article in the Proceedings of the National Academy of Science that demonstrated for the first time that spectrin was present in diverse eukaryotic cells in addition to RBCs. This study led, over the next twenty years, to my laboratory’s complete characterization of the location, sequence, and interactions of multiple spectrin isoforms in brain and the demonstration that the axonal isoform plays an essential function as an initial docking site for small synaptic vesicles at the active zone of the presynaptic plasma membrane.

We also turned our attention to the role of the RBC membrane skeleton in hemolytic anemias including sickle cell disease. We demonstrated how oxidative stress leads to altered spectrin and actin causing the locked membrane skeleton that is the molecular basis of the irreversibly sickled cell. We also demonstrated how this altered redox status in sickled RBCs leads to a defective Gardos Channel, potassium loss, and dehydration of Sickle Cell RBCs. This work led to our determining that n-acetyl cysteine (NAC) can block dense ISC formation in vitro and in phase II clinical trials. Indeed, in the phase II clinical trial the end result was that crisis rate could be lowered by 60% in sickle cell subjects receiving 2400 mgs of NAC per day.

Over the past eight years we have performed the first complete study of the RBC proteome, demonstrated how it changes in sickle cell disease, how it is affected by drug treatments (pharmaco-proteomics) and most recently demonstrated twenty one monocyte protein biomarkers that are predictive of sickle cell crisis rate. These latter studies could transform the sickle cell field by laying the groundwork for personalized medicine for sickle cell patients that will be able to be applied within the first few months of life. A by-product of our proteomic studies on the RBC was the discovery of proteasomal subunits where the dogma had been that mature RBCs contain no proteasomal activity. Our subsequent work has demonstrated that RBCs do indeed contain functional 20S proteasomes that can degrade specific proteins.

We performed the first interactome mapping on erythrocytes. We analyzed the normal and sickle cell interactome using network centrality measures, statistical clustering methods, and most recently the Voronoi Diagram for Graphs (VDG). We were the first to use VDG to assess interactome networks and demonstrated that this technique is superior to network centrality measures and statistical clustering methods in that it tells which other proteins in a cluster are most affected by the disease altered protein; also it has a shorter computational running time.  This lecture will focus primarily, but not exclusively, on the last eight year’s research.