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Kumari, Vandana. "Structure-Based Computer Aided Drug Design and Analysis for Different Disease Targets." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1311612599.
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Dickerson, Matthew Thomas. "PROTEIN BASED BIOMIMETIC APPROACHS TO SURFACE HEMOCOMPATIBILITY AND BIOCOMPATIBILITY ENHANCEMENT." UKnowledge, 2012. http://uknowledge.uky.edu/cme_etds/6.
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T. pallidum can survive a primary immune response and continue growing in the host for an extended period of time. T. pallidum is thought to bind serum fibronectin (FN) through Tp0483 on the surface to obscure antigens. A Tp0483 fragment (rTp0483) was adsorbed onto functionalized self-assembled monolayers (SAMs) with FN. FN capture by adsorbed rTp0483 depended greatly on surface chemistry with COO- groups being best for FN binding. Hemocompatibility was determined by analysis of plasma protein adsorption, intrinsic pathway activation, and platelet activation. rTp0483+FN bound an equal or lesser amount of fibrinogen (Fg), human serum albumin (HSA), and factor XII (FXII) compared to rTp0483 or FN alone and adsorption of rTp0483 prior to FN greatly decreased platelet activation. Inhibition of protein binding and platelet activation suggested an attenuated hematological response. Biocompatibility of rTp0483 and FN coated surfaces was characterized by macrophage uptake of protein coated polystyrene microspheres (PSMs), macrophage adsorption onto protein coated surfaces, cytotoxic effects of adsorbed rTp0483 and FN, and TNF-α and NO2- release in macrophages stimulated with rTp0483 and FN adsorbed and in solution. Addition of FN to rTp0483 on plain and COO- PSMs reduced phagocytosis compared to rTp0483 alone and on plain PSMs compared to FN alone. On plain PSMs addition of FN to adsorbed rTp0483 decreased TNF-α generation. Adsorption of rTp0483 before FN on large, flat COO- surfaces decreased macrophage adsorption and TNF-α and NO2- generation. High concentrations of rTp0483 were mildly cytotoxic to macrophages. FN binding by Tp0483 on T. pallidum likely plays a role in antigenic disguise and rTp0483+FN coatings may potentially inhibit FN and rTp0483 specific interactions with macrophages. Molecularly imprinted polymer coatings were also examined for biomaterial development. Fouling resistant 2-methacryloyloxyethyl phosphorylcholine (MPC) was imprinted with bovine serum albumin (BSA) protein templates to facilitate BSA specific binding. The BSA template was constructed and verified and BSA specific binding quantified using quartz crystal microbalance (QCM) and enzyme linked immunosorbent assay (ELISA). BSA imprinted coatings were determined to bind significantly more BSA than nonfouling MPC controls demonstrating the feasibility of targeted protein capture.
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Drobin, Kimi. "Antibody-based bead arrays for high-throughput protein profiling in human plasma and serum." Licentiate thesis, KTH, Proteinvetenskap, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-225980.
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Affinity-based proteomics utilizes affinity binders to detect target proteins in a large-scale manner. This thesis describes a high-throughput method, which enables the search for biomarker candidates in human plasma and serum. A highly multiplexed antibody-based suspension bead array is created by coupling antibodies generated in the Human Protein Atlas project to color-coded beads. The beads are combined for parallel analysis of up to 384 analytes in patient and control samples. This provides data to compare protein levels from the different groups. In paper I osteoporosis patients are compared to healthy individuals to find disease-linked proteins. An untargeted discovery screening was conducted using 4608 antibodies in 16 cases and 6 controls. This revealed 72 unique proteins, which appeared differentially abundant. A validation screening of 91 cases and 89 controls confirmed that the protein autocrine motility factor receptor (AMFR) is decreased in the osteoporosis patients. Paper II investigates the risk proteome of inflammatory bowel disease (IBD). Antibodies targeting 209 proteins corresponding to 163 IBD genetic risk loci were selected. To find proteins related to IBD or its subgroups, sera from 49 patients with Crohn’s disease, 51 with ulcerative colitis and 50 matched controls were analyzed. From these targeted assays, the known inflammation-related marker serum amyloid protein A (SAA) was shown to be elevated in the IBD cases. In addition, the protein laccase (multi-copper oxidoreductase) domain containing 1 (LACC1) was found to be decreased in the IBD subjects. In conclusion, assays using affinity-based bead arrays were developed and applied to screen human plasma and serum samples in two disease contexts. Untargeted and targeted screening strategies were applied to discover disease-associated proteins. Upon further validation, these potential biomarker candidates could be valuable in future disease studies.<br><p>QC 20180412</p>
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Liu, Jiyun. "Structure based design of inhibitors toward disease related multivalent protein targets /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/8482.
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Freer, Rosie. "Molecular origins of tissue vulnerability to aberrant aggregation in protein misfolding diseases." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/275420.
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Neurodegenerative disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD), are increasingly common in our ageing society, are remain incurable. A major obstacle encountered by researchers in their attempts to find effective therapies is represented by the current lack of understanding of the molecular origins of these disorders. It is becoming clear that, although the aggregation of specific proteins, including amyloid β (Aβ) and tau in AD and α-synuclein in PD, hallmark these disorders, such behaviour is a consequence of a wider, system-level disruption of protein homeostasis. In order to identify the genetic factors contributing to such a disruption, the transcriptional changes that occur during neurodegenerative disease progression have received considerable scientific attention in recent years. In our approach, we considered another hallmark of these diseases - their characteristic patterns of spreading across the brain - to identify the nature of the transcriptional signature which underlies tissue vulnerability to protein aggregation. By understanding why tissues succumb in their characteristic sequential pattern in neurodegenerative diseases, and why some tissues remain almost completely resistant throughout, we hoped to obtain insight into the molecular origins of these disorders. Our results show that the AD progression can be predicted from a transcriptional signature in healthy brains related to the protein aggregation homeostasis of Aβ, tau, and the wider proteome. We highlight a relationship between a specific subproteome at high risk of aggregation (formed by supersaturated proteins), and the vulnerability to neurodegenerative diseases. We thus identify an AD-specific supersaturated set of proteins - termed the metastable subproteome, whose expression in normal brains recapitulates the staging of AD, with more vulnerable tissues having higher metastable subproteome expression. We find evidence of these vulnerability signatures transcending the tissue level of interrogation, with cellular and subcellular analysis also showing elevated levels of proteins known and predicted to predispose the aberrant aggregation of Aβ and tau. These results characterise the key protein homeostasis pathways in the inception and progression of AD, and establish an approach with the potential to be applied to other protein misfolding diseases, in the brain and beyond.
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Lewandowski, Eric Michael. "Structure Based Drug Design Targeting Bacterial Antibiotic Resistance and Alzheimer's Disease." Scholar Commons, 2015. http://scholarcommons.usf.edu/etd/5982.
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Structure based drug design is a rapidly advancing discipline that examines how protein targets structurally interact with small molecules, or known inhibitors, and then uses this information to lead inhibitor optimization efforts. In the case of novel inhibitors, protein structural information is first obtained via X-ray crystallography, NMR studies, or a combination of both approaches. Then, computational molecular docking is often used to screen, in silico, millions of small molecules and calculate the potential interactions they may have with the target protein’s binding pocket, in hopes of identifying novel low affinity inhibitors. By examining the interactions these small, low affinity, inhibitors have with the binding pocket, optimization efforts can be focused on maximizing interactions with “hot spots” within the pocket, thus leading to larger, high affinity inhibitors. A similar optimization technique can also be applied to known inhibitors. By examining the interactions of a known inhibitor with the binding site, new compounds can be designed to target “hot spots” in the binding pocket using the known inhibitors core structure as a starting point. The affinity of the newly designed compounds can then be compared to the affinity of the original inhibitor, and further rounds of optimization can be carried out. While simple in design, there are many challenges associated with structure based drug design studies, and there is no guarantee novel inhibitors will be found, but ultimately, it is an extremely powerful methodology that results in a much higher hit rate than other, similar, techniques. The work herein describes the use of structure based drug design to target several different proteins involved in bacterial antibiotic resistance, and a protein that has been implicated in the development of Alzheimer’s disease.
The goal of the first project was to design a new PBP inhibitor based upon an existing scaffold, and to better understand the binding mechanism and molecular interactions between penicillin binding proteins and their inhibitors. PBPs are a group of proteins that catalyze the last steps of bacterial cell wall formation, and are the targets of the β-lactam antibiotics. Two compounds were designed which conjugated a ferrocene or ruthenocene group to 6-aminopenicillinic acid, and their antibiotic properties were tested against a range of bacterial strains. To get a better understanding of how the 6-APA organometallic compounds interacted with the PBP active site, a CTX-M-14 β-lactamase model system was used for X-ray crystallographic studies. CTX-M-14 was chosen as its active site shares many key catalytic features with PBPs, and it easily, and reproducibly, yields crystals capable of diffracting to sub-atomic (< 1.0 Å) resolution.
I determined a 1.18 Å structure of 6-APA-Ru in complex with CTX-M-14 E166A β-lactamase and was able to gain unprecedented details of the interactions of the ruthenocene group with the CTX-M active site. This structure also revealed that the compound bound in the CTX-M active site was actually the decarboxylated and hydrolyzed product, which was the first time a decarboxylated product had been captured in the CTX-M active site. A second, 0.85 Å, structure of CTX-M in complex with 6-APA-Ru was determined and shed light on how the hydrogen bonding network in the CTX-M active site changes in response to the 6-APA-Ru product binding. A final, 1.30 Å, structure captured the carboxylated and hydrolyzed 6-APA-Ru product in complex with CTX-M, which was the first time the carboxylated product had been captured in the CTX-M active with the catalytic Ser70 residue intact. The results show the potential of the ruthenocene group in improving antibiotic potency, and help to better elucidate the changes that occur in the CTX-M active site upon inhibitor binding, while at the same time, telling us what changes could occur in the active site of PBPs.
The next project was focused on novel inhibitor discovery against several different PBPs. PBPs have been successfully inhibited by β-lactam antibiotics for decades, but the alarming rise of bacteria resistant to these antibiotics has placed increased urgency on the discovery of novel PBP inhibitors. A fragment based molecular docking approach was employed to virtually screen millions of small compounds for interactions with the targeted active sites, and then high scoring compounds were selected for visual inspection and inhibitory testing. Virtual screening was first done against Staphylococcus aureus monofunctional transglycosylase, a type of PBP. MTG provided a good binding pocket for virtual screening, but proved challenging to purify and crystallize. However, through great effort MTG crystals were eventually obtained. After repeated rounds of virtual screening against MTG, multiple compounds were selected for inhibition testing, and testing is currently ongoing. Virtual screening was also done against Pseudomonas aeruginosa PBP5 and PBP1a. Purification and crystallization of these proteins proved to be easier than MTG, and both yielded diffraction quality crystals.
The final project focused on virtual screening against a protein implicated in the development of Alzheimer’s disease, Slingshot Phosphatase 1. The brains of AD patients have been found to contain elevated levels of active Cofilin, and these elevated levels of active Cofilin may lead to the overproduction of amyloid β. Aβ overproduction, and its resulting accumulation, is believed to be one of the pathways that lead to AD symptoms. Cofilin is activated when it is dephosphorylated by SSH1, and inhibiting this activation may decrease the production of Aβ and the development of AD symptoms. There is no known structure of SSH1, so to perform virtual screening a SSH1 homology model was constructed using the homolog SSH2 as a starting point. Virtual screening was then performed using the SSH1 homology model and many compounds were selected for inhibition testing. Initial testing found several compounds that could prevent Cofilin dephosphorylation at levels > 10μM. However, three compounds were found to be exceptionally active, and could prevent Cofilin dephosphorylation at both 1 and 10 μM. One of these three compounds was tested directly against purified SSH1 and found to inhibit its activity, and reduce Aβ production. Crystallization of purified SSH1, and SSH2, was attempted in order to get complex structures with the three best compounds. SSH2 crystals were obtained which diffracted to 1.91 Å, and several initial hits were found for SSH1. Optimization of crystals for both proteins is currently ongoing. The SSH1 inhibitor, along with the two other highly active compounds, provides an excellent starting point for the development of highly potent SSH1 inhibitors.
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Hilbert, Brendan J. "Structure-based Targeting of Transcriptional Regulatory Complexes Implicated in Human Disease: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/681.
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Transcriptional regulatory complexes control gene expression patterns and permit cellular responses to stimuli. Deregulation of complex components upsets target gene expression and can lead to disease. This dissertation examines proteins involved in two distinct regulatory complexes: C-terminal binding protein (CtBP) 1 and 2, and Interferon Regulatory Factors (IRF) 3 and 5. Although critical in developmental processes and injury response, CtBP transcriptional repression of cell adhesion proteins, pro-apoptotic factors, and tumor suppressors has been linked to the pathogenesis of multiple forms of cancer. IRFs function in the immune system and have been implicated in autoimmune disorders.
Understanding IRF activation is critical to treating pathogens that target IRF function or for future autoimmune disease therapies. We attempted to determine crystal structures that would provide the details of IRF activation, allowing insight into mechanisms of pathogen immune evasion and autoimmune disorders. Although no new structures were solved, we have optimized expression of C-terminal IRF-3 / co-activator complexes, as well as full-length IRF3 and IRF5 constructs. Modifying the constructs coupled with new crystal screening will soon result in structures which detail IRF activation, advancing understanding of the roles of IRF family members in disease.
Through structural and biochemical characterization we sought to identify and develop inhibitors of CtBP transcriptional regulatory functions. High concentrations of CtBP substrate, 4-Methylthio 2-oxobutyric acid (MTOB), have been shown in different cancer models to interfere with CtBP transcriptional regulation. We began the process of structure based drug design by solving crystal structures of both CtBP family members bound to MTOB. The resulting models identified critical ligand contacts and unique active site features, which were utilized in inhibitor design. Potential CtBP inhibitors were identified and co-crystallized with CtBP1. One such compound binds to CtBP more than 1000 times more tightly than does MTOB, as a result of our structure-based inclusion of a phenyl ring and a novel pattern of hydrogen bonding. This molecule provides a starting point for the development of compounds that will both bind more tightly and interfere with transcriptional signaling as we progress towards pharmacologically targeting CtBP as a therapy for specific cancers.
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Hilbert, Brendan J. "Structure-based Targeting of Transcriptional Regulatory Complexes Implicated in Human Disease: A Dissertation." eScholarship@UMMS, 2007. http://escholarship.umassmed.edu/gsbs_diss/681.
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Transcriptional regulatory complexes control gene expression patterns and permit cellular responses to stimuli. Deregulation of complex components upsets target gene expression and can lead to disease. This dissertation examines proteins involved in two distinct regulatory complexes: C-terminal binding protein (CtBP) 1 and 2, and Interferon Regulatory Factors (IRF) 3 and 5. Although critical in developmental processes and injury response, CtBP transcriptional repression of cell adhesion proteins, pro-apoptotic factors, and tumor suppressors has been linked to the pathogenesis of multiple forms of cancer. IRFs function in the immune system and have been implicated in autoimmune disorders.
Understanding IRF activation is critical to treating pathogens that target IRF function or for future autoimmune disease therapies. We attempted to determine crystal structures that would provide the details of IRF activation, allowing insight into mechanisms of pathogen immune evasion and autoimmune disorders. Although no new structures were solved, we have optimized expression of C-terminal IRF-3 / co-activator complexes, as well as full-length IRF3 and IRF5 constructs. Modifying the constructs coupled with new crystal screening will soon result in structures which detail IRF activation, advancing understanding of the roles of IRF family members in disease.
Through structural and biochemical characterization we sought to identify and develop inhibitors of CtBP transcriptional regulatory functions. High concentrations of CtBP substrate, 4-Methylthio 2-oxobutyric acid (MTOB), have been shown in different cancer models to interfere with CtBP transcriptional regulation. We began the process of structure based drug design by solving crystal structures of both CtBP family members bound to MTOB. The resulting models identified critical ligand contacts and unique active site features, which were utilized in inhibitor design. Potential CtBP inhibitors were identified and co-crystallized with CtBP1. One such compound binds to CtBP more than 1000 times more tightly than does MTOB, as a result of our structure-based inclusion of a phenyl ring and a novel pattern of hydrogen bonding. This molecule provides a starting point for the development of compounds that will both bind more tightly and interfere with transcriptional signaling as we progress towards pharmacologically targeting CtBP as a therapy for specific cancers.
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Lau, Kin-chong, and 劉健莊. "Microarray-based investigations of genetic diseases." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B45894760.
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Hall, David. "An XML-based Database of Molecular Pathways." Thesis, Linköping University, Department of Computer and Information Science, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-3717.
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<p>Research of protein-protein interactions produce vast quantities of data and there exists a large number of databases with data from this research. Many of these databases offers the data for download on the web in a number of different formats, many of them XML-based.</p><p>With the arrival of these XML-based formats, and especially the standardized formats such as PSI-MI, SBML and BioPAX, there is a need for searching in data represented in XML. We wanted to investigate the capabilities of XML query tools when it comes to searching in this data. Due to the large datasets we concentrated on native XML database systems that in addition to search in XML data also offers storage and indexing specially suited for XML documents.</p><p>A number of queries were tested on data exported from the databases IntAct and Reactome using the XQuery language. There were both simple and advanced queries performed. The simpler queries consisted of queries such as listing information on a specified protein or counting the number of reactions.</p><p>One central issue with protein-protein interactions is to find pathways, i.e. series of interconnected chemical reactions between proteins. This problem involve graph searches and since we suspected that the complex queries it required would be slow we also developed a C++ program using a graph toolkit.</p><p>The simpler queries were performed relatively fast. Pathway searches in the native XML databases took long time even for short searches while the C++ program achieved much faster pathway searches.</p>
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Awan, Waqas Ahmed. "Structure-based characterisation and prediction of protein molecular function." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613923.
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Crane, Peter. "Protein based molecular probes by unnatural amino acid incorporation." Thesis, University of Oxford, 2018. http://ora.ox.ac.uk/objects/uuid:772076fc-00f2-4ca7-bfa9-3da1ce7093cb.
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The "tag & modify" strategy for protein modification relies upon the genetic incorporation of an uncommon or unnatural amino acid into a protein backbone, followed by a chemo-selective modification to yield differentially modified proteins. This thesis describes the creation of a protein-based glycoconjugate tool for interrogating biological function. In Chapter 2, the unnatural amino acid, azidohomoalanine was genetically incorporated into a library of distance defined Np276 proteins via a selective pressure incorporation. Methods to prevent the common post translational modification N-terminal gluconylation were identified, including preliminary work on a small molecule intervention. The proteins were subsequently characterised with respect to other members of the (limited) family of pentapeptide repeat protein and the key biophysical parameters (TM, stability) with relate to it being a multivalent scaffold were investigated. In Chapter 3, An initial investigation into obtaining a selective amine modification initially via N-hydroxysuccinimide esters, led to the discovery (and characterisation) of a clear selectivity for N-terminal proline Isothiocyanate modification. The dual modification of proteins via the N-term Pro & the ubiquitous (glyco) copper(I)-catalysed azide alkyne cycloaddition was subsequently used to generate homogenously dual modified Np276 scaffolds. In Chapter 4, these proteins were then used in a FACS assay against a murine sialoadhesin - chinese hamster ovary cell line, the results showing promise for the further development of multivalent glycated probes of function.
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Fortun, Jenny. "Protein aggregation in peripheral myelin protein 22 (pmp22)-associated neuropathies." [Gainesville, Fla.] : University of Florida, 2005. http://purl.fcla.edu/fcla/etd/UFE0010065.
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Thesis (Ph.D.)--University of Florida, 2005.<br>Typescript. Title from title page of source document. Document formatted into pages; contains 123 pages. Includes Vita. Includes bibliographical references.
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Shames, Igor. "Phenotypic differences between Peripheral Myelin Protein-22 (PMP-22) and Protein Zero (PO) mutations associated with Charcot-Marie-Tooth related diseases." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=79122.
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Many hereditary peripheral neuropathies are characterized by abnormal myelination in peripheral nerves. Two structural myelin proteins, PMP22, a polytopic myelin protein, and P0, an Ig-like transmembrane protein, play a major role in myelin formation. A large number of mutations have been identified in P0 and PMP22 genes. Neuropathies associated with PMP22 and P0 mutations have varying severities suggesting their effects are pleiotropic. It is of great importance to explore molecular pathogenesis of mutated P0 and PMP22 proteins in order to understand the basic process of myelination and its pathology. We hypothesize that these mutations have a variety of effects on the cell biological processes, including activation of ER quality control mechanisms, abnormal intracellular trafficking, or disruption of normal function. In order to dissect these possible effects, we studied the intracellular distribution and trafficking of mutated forms of PMP22 and P0 proteins in mammalian cells. Our studies indicate that the various P0 and PMP22 mutants may exert their pathogenic effects in different compartments and by different mechanisms in the mammalian cell.
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Raut, Nilesh G. "BIOSENSING SYSTEMS FOR THE DETECTION OF BACTERIAL QUORUM SENSING MOLECULES: A TOOL FOR INVESTIGATING BACTERIA-RELATED DISORDERS AND FOOD SPOILAGE PREVENTION." UKnowledge, 2012. http://uknowledge.uky.edu/chemistry_etds/13.
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Quorum sensing enables bacteria to communicate with bacteria of the same or different species, and to modulate their behavior in a cell-density dependent manner. Communication occurs by means of small quorum sensing signaling molecules (QSMs) whose concentration is proportional to the population size. When a QSM threshold concentration is reached, certain genes are expressed, thus allowing control of several processes, such as, virulence factor production, antibiotic production, and biofilm formation. Not only many pathogenic bacteria are known to produce QSMs, but also QSMs have been identified in some bacteria-related disorders. Therefore, quantitative detection of QSMs present in clinical samples may be a useful tool in the investigation and monitoring of bacteria-related diseases, thus prompting the use of QSMs as biomarkers of disease. Herein, we have developed and utilized whole-cell biosensing systems and protein based biosensing systems to detect QSMs in clinical samples, such as, saliva, stool, and bowel secretions. Additionally, since bacteria are responsible for food spoilage, we employed the developed biosensing systems to detect QSMs in food samples and demonstrated their applicability for early identification of food contamination. Furthermore, we have utilized these biosensing systems to screen antibacterial compounds employed for food preservation, namely, generally regarded as safe (GRAS) compounds, for their effect on quorum sensing.
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Mohamed, Nahla. "Molecular Diagnosis of Common Viral Infectious Diseases Based on Real-Time PCR." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7118.
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Xu, Jiru. "Application of PCR and DNA sequencing based molecular diagnosis in infectious diseases." Thesis, University of Ulster, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399727.
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Sorarù, Antonio. "Molecular and nanodimensional metal based systems for the therapy against neurodegenerative diseases." Doctoral thesis, Università degli studi di Padova, 2015. http://hdl.handle.net/11577/3424628.
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Reactive oxygen species (ROS) are harmful species produced during metabolic processes, such as photosynthesis and respiration, of living organism. In both case, the substrate (oxygen/water) undergoes several multi electronic reaction, during which some electrons can “escape” from the catalytic cycle and produce ROS, such as superoxide radical anion, hydrogen peroxide, hydroxyl radical and other derivatives. These species are really dangerous, since they are able to oxidize almost all cellular components. Indeed they can damage lipids, proteins, DNA, affecting cellular functions till cell death. Under oxidative stress condition, accumulation of damage due to ROS has been supposed to play a role in several pathologies, and in particular in age-related ones, such as Alzheimer’s disease (AD). This disease is characterized by an accumulation of neurotoxic senile plaques, mainly made of short peptide monomers, that tend to aggregate into fibrils, called amyloid β peptide (Aβ). Although the mechanism involved in the production of these peptides is still unknown, some hypothesis suggest that ROS are produced within the fibrils and are responsible for further Aβ production.
Nature has developed different catalytic strategies to limit ROS production, and the most important is the enzymatic pathway: superoxide dismutases (SOD) and catalase (CAT) enzymes are able to dismutate, respectively, superoxide and hydrogen peroxide. Nevertheless, in case of unpaired ROS production it is of huge interest to find new artificial systems that are able to help natural enzymes in their task. In this thesis, four different classes of synthetic enzymes (synzymes) that mimic natural anti ROS systems have been investigated:
I. Isostructural mononuclear manganese complexes, with general formula [Mn(L)X2], characterized by a pentadentate ligand L, containing different heteroatoms (N, O or S) have been used for the dismutation of hydrogen peroxide and superoxide anion. Their activity, also depending on heteroatoms, and stability were studied, first in organic solvents to have a comparison with literature similar compounds, then in aqueous solution, where only few compounds were known to work. The sulfur-containing complex [Mn(L)(OTf)2] was found to exhibit high dual SOD/CAT-like activity with excellent stability, when used in the presence of a base.
II. Isostructural dinuclear manganese complexes, with general formula [Mn2L2X], were widely studied as artificial catalases. A comparison with other dimanganese complexes, in terms of Michealis-Menten parameter, KM and kcat, was performed. Superoxide dismutase activity was also evaluated, demonstrating the unique dual SOD/CAT behavior of [Mn2L2X] with respect to other dinuclear complexes. Finally, the ligands were modified with mitochondriotropic functionalities. In particular, two fluorescent rhodamine derivatives and a triphenylphosphonium salt, were taken into account.
III. Some multimetallic manganese oxoclusters, containing 6-13 manganese atoms, were synthetized during a Short Term Scientific Mission in Dublin, in the group of Prof. Wolfgang Schmitt. The catalase-like activity of these compounds was tested for the first time. Their H2O2 dismutation capability was thus demonstrated and their stability in aqueous environment was checked. Preliminary test as SOD mimics were also performed.
IV. In the end, a completely inorganic compound, a polyoxometalate (POM) substituted with four ruthenium atoms, with formula [Ru4O4(OH)2(H2O)4(γ-SiW10O36)2]10-, able to dismutate hydrogen peroxide was studied. Its activity in different biological buffers and media was initially optimized. Its capability of interaction with Aβ peptides, coupled with its catalase activity, were exploited to control these two major events involved in Alzheimer’s disease. Preliminary test on neuronal cells were then performed (with Dr.ssa de Bartolo (ITM-CNR, Rende, CS)), confirming the interesting properties of the compound in vitro and finding a very low toxicity. Finally, encapsulation of POM was achieved in order to enables delivery and targeting in cells, using polymeric multilayer biocompatible microcapsules in which POM is deposited. The presence of POM and its catalytic activity were confirmed and analyzed.<br>Le reazioni biochimiche che coinvolgono il trasferimento di elettroni dall’ossigeno per dare acqua, durante la respirazione cellulare, e dall’acqua per dare ossigeno, durante la fotosintesi, possono portare alla formazione di specie reattive dell’ossigeno (ROS, reactive oxygen species), dovute alla “perdita” di elettroni dal ciclo catalitico. Tra queste specie troviamo inizialmente il superossido O2-•, l’acqua ossigenata e il radicale ossidrile. Queste possono reagire con altre molecole per dare origine ad altre specie reattive, per esempio dell’azoto, ma soprattutto possono danneggiare peptidi, lipidi e DNA e causare ingenti danni alle funzioni cellulari fino a portare alla morte della cellula stessa. In condizioni di stress ossidativo, l’accumulo di queste specie sembra giocare un importante ruolo nelle malattie degenerative, come ad esempio il morbo di Alzheimer (AD). In questo caso, la malattia è caratterizzata dalla presenza di aggregati proteici in forma di placche, che hanno un effetto neurotossico. Questi accumuli proteici sono costituiti principalmente da peptidi di 40-42 amminoacidi chiamati β-amiloidi (Aβ), che tendono ad aggregare, in forma di fibrille. Le cause della formazione e accumulo di questi peptidi non sono ancora del tutto chiare, ma si hanno evidenze sul coinvolgimento delle ROS nella fase di formazione dei peptidi, e sull’aumento della loro produzione, dopo la formazione delle fibre, a causa di reazioni mediate dai metalli intrappolate nelle fibre stesse.
La natura ha sviluppato dei sistemi per proteggersi da queste specie reattive, tra questi citiamo gli enzimi superossido dismutasi (SOD) e catalasi (CAT), capaci rispettivamente di eliminare superossido e acqua ossigenata, che tuttavia in certe situazioni di elevato stress ossidativo possono risultare insufficienti per prevenire i danni.
È quindi di estremo interesse lo studio di composti artificiali capaci di aiutare gli enzimi naturali nel loro compito di eliminare le ROS dall’ambiente biologico. Considerando ciò, in questa tesi sono state considerate le seguenti quattro classi di composti, utilizzati come enzimi sintetici (synzymes), per imitare le funzioni dei sistemi anti ROS naturali:
I. Complessi mononucleari ed isostrutturali di manganese, di formula generale [Mn(L)X2], caratterizzati da un legante pentadentato, L, contenente differenti eteroatomi (N, O, o S), sono stati studiati nella dismutazione dell’acqua ossigenata e del radicale anione superossido. L’attività è stata inizialmente analizzata in solvente organico (acetonitrile) per aver dei termini di paragone con altri composti di letteratura. In seguito l’attività è stata studiata anche in acqua, dove solo pochi composti di letteratura sono risultati attivi. Se utilizzati in presenza di base, i complessi [Mn(L)(OTf)2] contenenti zolfo mostrano una duplice attività SOD/CAT ed un’elevata stabilità.
II. Complessi dinucleari ed isostrutturali di manganese, di formula generale [Mn2L2X], sono stati studiati inizialmente per l’eliminazione dell’acqua ossigenata. Un confronto con simili composti di letteratura è stato effettuato tramite il calcolo dei parametri, derivati dall’ equazione di cinetica enzimatica di Michalis-Menten, KM e kcat. E’ stata anche analizzata la capacità di smaltire il superossido, dimostrando le caratteristiche uniche di [Mn2L2X] nella duplice attività CAT/SOD, in ambiente acquoso, rispetto ad altri complessi dinucleari. Infine, modificando i leganti, si è cercato di introdurre nuove funzionalità adatte alla veicolazione del composto in cellula. In particolare, sono stati utilizzati residui organici noti per la loro affinità verso i mitocondri, come i derivati della rodamina e i sali di trifenilfosfonio.
III. Sono stati studiati oxoclusters multimetallici di manganese, contenti 6-13 atomi di metallo, sintetizzati durante un Short Term Scientific Mission (STSM, COST action CM1203) a Dublino, presso il laboratorio del Prof. Wolfgang Schmitt, analizzandone per la prima volta l’attività di dismutazione dell’ acqua ossigenata e del superossido, oltre che la stabilità in soluzioni acquose.
IV. Un composto completamente inorganico, un poliossometallato (POM) contenete quattro atomi di rutenio, di formula [Ru4O4(OH)2(H2O)4(γ-SiW10O36)2]10-, è considerato per la sua solubilità in ambiente acquoso e la capacità di dismutare efficacemente l’acqua ossigenata. L’attività è stata analizzata in diversi tamponi e mezzi comunemente usati per analisi di sistemi biologici. In soluzione, il complesso è capace di ridurre la produzione di ROS e anche di interagire con peptidi amiloidei, evitandone l’aggregazione in fibrille, dimostrandosi quindi promettente nel contrastare due importanti eventi che si verificano durante la malattia di Alzheimer. In collaborazione con la Dr.ssa de Bartolo (ITM-CNR, Rende, CS) sono state quindi effettuate prove preliminari in cellule neuronali, per verificare sia la tossicità del composto (che risulta essere nulla anche a 100µM di concentrazione) che l’effettiva attività anti-ROS e anti-amiloidogenica in vitro.
Infine si è studiato l’inserimento del POM all’interno della shell di microcapsule polimeriche multistrato, con la prospettiva di controllarne la veicolazione in cellula.
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Yang, Hui. "Theoretical Studies of Molecular Recognition in Protein-Ligand and Protein-Protein Complexes." University of Toledo / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1282339026.
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Xue, Chunyi, and 薛春宜. "Molecular characterization of infectious bursal disease virus (IBDV) receptor." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B31246187.
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Peters, Theodore Walter. "Investigating the relationship between protein aggregates and cellular dysfunction in polyglutamine disease /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2008.
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Thesis (Ph.D. in Biochemistry) -- University of Colorado Denver, 2008.<br>Typescript. Includes bibliographical references (leaves 128-144). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;
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Hosseini, Azade S. "Developing Peptide-Based Receptors to Study Molecular Recognition in Water." Thesis, Boston College, 2016. http://hdl.handle.net/2345/bc-ir:107218.
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Thesis advisor: Jianmin Gao<br>My graduate research career has focused on studying the principles that underlie molecular recognition, which include protein folding, protein-membrane interactions, structural preoranization for target binding and non-covalent interactions. This thesis will present an overview of this work through three different projects. I) Synthetic receptors for target binding in water. Molecular interactions in water provide the foundation for life. More specifically, the interactions between one or more molecules, through hydrogen bonding, π-effects, hydrophobic interactions and electrostatic interactions, all play a significant role essential to biological processes. This chapter will present an overview of supramolecular chemistry in water, with a focus on small molecule receptor “warheads” that target biomolecules of interest. The discussion will then move towards the ability to preorganize these “warheads” on a scaffold to improve their potency towards a target. The fundamental principles discussed in this section will provide a foundation for the following chapter in this thesis.II) Understanding Phosphatidylserine Recognition Using the Model cLac Peptide. The plasma membrane serves as a defining feature of the cell membrane, acting as a barrier for material exchange between a cell and its local environment. More importantly, membrane lipids are involved in mediating numerous cell-signaling events and acting as receptors to recruit proteins that carry out a specific function. Due to the important role that lipids play, it is highly desirable to develop affinity ligands for the diverse range of lipid headgroup structures on a cell membrane. Although prevalent, proteins have intrinsic limitations due to their size, low stabilities and slow clearance rates. This chapter will focus on the model peptide, cLac, which was previously developed as an affinity ligand for phosphatidylserine recognition. We will focus on understanding the key properties that contribute to PS selectivity and affinity, then attempt to improve this scaffold through structural preorganization. III) A prolinomycin-based scaffold for developing functional peptides. Nature has evolved proteins to bind cell-signaling molecules with exquisite affinity and specificity, making molecular recognition an essential part of biology. It has been a highly sought after goal within the chemistry field to be able to mimic the structure and function of certain proteins with smaller molecules, such as peptides. Specifically, cyclic peptides are showing promise as therapeutic agents due to their high proteolytic stabilities, faster clearance rates and ease of synthesis compared to proteins. One challenge, however, is that peptides generally do not possess the ability to properly fold and display their side chains for target binding, as proteins do. In this chapter, I will present a prolinomycin-based scaffold, which can fold in the presence of K+ ions to preorganize its side chains for target binding. Moreover, the focus will be on the structural aspects of this cyclic peptide, along with proof-of-concept studies demonstrating its ability to recognize a target under physiological conditions. The findings in this study will be useful in developing peptide-based tools that recognize various targets. IV) Dissecting the energetic consequences of fluorinating a protein core. Proteins have emerged as a powerful class of therapeutic agents due to their superior properties over small molecules in the clinic. Some of the key advantages include their large surface areas and highly defined structures, which allow them to perform very specific functions that are generally not reproducible with traditional small molecule scaffolds. In addition, proteins possess the ability to properly fold under physiological conditions through precise, noncovalent interactions between their side chain residues. Perhaps the most relevant interactions arise from aromatic side chains, which can interact in a variety of ways to help proteins fold. In this chapter, we will focus on the model protein, VHP35, which contains a hydrophobic core of three interacting Phe residues, to study the effects of fluorination on an edge-face interaction<br>Thesis (PhD) — Boston College, 2016<br>Submitted to: Boston College. Graduate School of Arts and Sciences<br>Discipline: Chemistry
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Holler, Christopher J. "THE CELLULAR NUCLEIC ACID BINDING PROTEIN REGULATES THE ALZHEIMER’S DISEASE β-SECRETASE PROTEIN BACE1". UKnowledge, 2012. http://uknowledge.uky.edu/biochem_etds/12.
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Alzheimer’s disease (AD) is the most common neurodegenerative disease affecting the elderly population and is believed to be caused by the overproduction and accumulation of the toxic amyloid beta (Aβ) peptide in the brain. Aβ is produced by two separate enzymatic cleavage events of the larger membrane bound amyloid precursor protein, APP. The first, and rate-limiting, cleavage event is made by beta-secretase, or BACE1, and is thus an attractive therapeutic target. Our lab, as well as many others, has shown that BACE1 protein and activity are increased in late-stage sporadic AD. We have extended these findings to show that BACE1 is increased in the earliest stages of AD before the onset of significant Aβ accumulation, indicating a potential causal role in the disease. Interestingly, BACE1 mRNA levels are unchanged in AD, leading to reason that a post-transcriptional method of BACE1 regulation is altered in disease. To date, the mechanism for this aberrant post-transcriptional regulation has not been elucidated. This study has implicated the cellular nucleic acid binding protein (CNBP), a highly conserved RNA binding protein, as a positive regulator of BACE1 translation, with implications for the etiology of sporadic AD. CNBP overexpression in cultured cells or spiked into a cell-free in vitro translation system increased BACE1 protein expression without affecting BACE1 mRNA levels. Knockdown of CNBP reduced BACE1 protein and mRNA slightly. Furthermore, CNBP associated with BACE1 mRNA in cell lysates and bound directly to the BACE1 5’ UTR in vitro, which confers most of the regulatory activity. Importantly, CNBP was increased in the progression of AD and correlated with BACE1 expression. Cellular stressors (such as glucose deprivation and oxidative stress) that occur in the AD brain increase BACE1 translation and we have found that these stressors increased CNBP expression as well. Early experimental evidence suggests that CNBP may enhance BACE1 translation through a cap-independent mechanism, which is an alternative translational pathway activated by cell stress. These studies indicate that the RNA binding protein CNBP is a novel trans-acting factor important for the regulation of BACE1 protein production and may be a viable therapeutic target for AD.
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Ennen, Franka. "Protein-Glycopolymer Biohybrid Structures Based on Molecular Recognition Processes for Biomedical Applications." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-158789.
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The design of versatile biohybrid nanosized materials has revealed itself as a promising avenue towards biomedical applications in today´s life sciences. In this regard the combination of components of synthetic and natural origin facilitates an applicability which is supposed to be far beyond the sum of their single components. These biohybrid structures (BHS) can be built by a huge variety of building blocks including solid or soft nanoparticles, peptides/proteins, polynucleotides or low molecular weight drugs. Along with the latter the attachment of biologically active entities or imaging moieties, e. g. enzymes, fluorescence markers or targeting motifs display thereby a key step towards the development of carrier systems for drug delivery purposes.
Among the soft nanoparticles especially dendritic polymers such as perfectly branched dendrimers or hyperbranched polymers are considered as ideal building blocks, since they allow an easy tailoring of crucial properties such as solubility, biocompatibility or bioactivity by means of surface functionalization. Especially in the field of targeted drug delivery the crucial role of sizes and size distributions of carriers has been highlighted recently, since it critically influences important factors such as circulation time or biodistribution within the body.
The ability of avidin to form high molecular weight associates with biotinylated macromolecules as well as its inherent properties makes it a suitable candidate for passive and active targeting in combination with biotinylated (bio-)polymers. Furthermore, along with the covalent attachment of bioactive moieties, non-covalent attachment is a frequently used approach, because it is assumed to only require stoichiometric mixing. In context of the latter molecular recognition processes such as the avidin-biotin, β-cyclodextrin-adamantane or Ni(II)-NTA-histidine-tag interactions have shown to be fruitful strategies for the attachment of bioactive entities.
The overall aim of this work was to fabricate BHS based on dendritic glycopolymers with varied sizes in the nano- and micrometer range as models for biomedical applications e. g. carriers for drug delivery. Therefore the molecular recognition of avidin with biotin derivatives and β-cyclodextrin with adamantane derivatives was utilized in order to tailor final sizes, functionality or catalytic activity of those BHS.
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Degani, G. "MOLECULAR CHARACTERIZATION OF MEMBRANE-BOUND GLYCOPROTEINS INVOLVED IN HUMAN DISEASES AND POTENTIAL TARGETS FOR NEW THERAPIES." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/274187.
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The present thesis is focused on the molecular characterization of two eukaryotic membrane glycoproteins that are promising candidates for new therapeutic approaches to human diseases.
The first glycoprotein is the human Receptor for the Advanced Glycation End products (hRAGE), a member of the immunoglobulin superfamily. RAGE is a type I transmembrane glycoprotein that is beneficial in normal physiological conditions but it is also a key player in the etiology and progression of several chronic pathologies such as neurodegenerative disorders (Alzheimer), atherosclerosis, cancer and complications of metabolic diseases such as diabetes, by exacerbating the inflammatory response. A variety of ligands sharing an acidic charge, as the advanced glycation End products (AGEs), S100 proteins, HMGB1, Aβ-amyloids, bind to the extracellular V or VC1 domains of RAGE. These domains are N-glycosylated and stabilized by disulphide bonds. To overcome the tendency to aggregate of the V and VC1 domains expressed in bacteria, in this work V and VC1 domains were expressed as secreted proteins in the methylotrophic yeast Pichia pastoris. While VC1 was secreted into the culture medium and was functional, the V domain was retained intracellularly, providing the first in vivo indication that V requires C1 to fold into a structurally stable domain. The glycosylation pattern of VC1 reflects the glycosylation of RAGE isolated from mammalian sources. A simple procedure for the purification to homogeneity of VC1 from the medium was generated and the folded state of the purified protein was assessed by thermal shift assays. The protein showed a remarkable improved thermal stability compared to VC1 expressed in bacteria. The stability and full solubility of glycosylated VC1 may be beneficial for in vitro studies aimed at the identification of new ligands or inhibitors of RAGE.
The second object of this thesis was the Phr family of Candida albicans, a dimorphic fungal pathogen responsible of life-threatening invasive infections. These glycoproteins are anchored to the plasma membrane through a GPI. Phr proteins belong to family GH72 of cell wall glucan remodelling enzymes that are unique to fungi and essential for morphogenesis, cell wall integrity and virulence. For these reasons, these enzymes are targets for inhibitors of the cell wall formation to be used in therapy, similarly to what penicillins have been for bacteria. The catalytic properties of Phr proteins were studied using a new fluorescent assay. Phr1p and Phr2p are specific for β-1,3-glucan, the pH optimum was 5.8 for Phr1p and 3 for Phr2p and the temperature optimum was 30°C. Pga4p was inactive suggesting that it turned out into a structural cell wall protein. Finally, we studied the transcriptome of cells lacking β-1,3-glucan remodelling (phr1Δ cells) after induction of growth as hypha, the invasive form of this pathogen. About 310 genes were modulated and genetic analysis showed that chitin synthesis by the Chs3p isoform is essential for viability of phr1Δ cells.
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Vijayaraghavan, Jagamya. "MOLECULAR AND MACRO-MOLECULAR CYCLIZATION: STRUCTURE BASED DRUG DESIGN OPPORTUNITIES FOR TWO LYASE ENZYMES." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1485963601042409.
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Klingeborn, Mikael. "The prion protein in normal cells and disease : studies on the cellular processing of bovine PrPC and molecular characterization of the Nor98 prion /." Uppsala : Department of Molecular Biosciences, Swedish University of Agricultural Sciences, 2006. http://epsilon.slu.se/2006105.pdf.
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Cong, Xiaojing. "Molecular Simulation Studies on the Prion Protein Variants: Insights into the Intriguing Effects of Mutations." Doctoral thesis, SISSA, 2013. http://hdl.handle.net/20.500.11767/4810.
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Prion diseases, or transmissible spongiform encephalopathies (TSE), are a group of rare fatal neurodegenerative maladies that affect humans and animals. The fundamental breakthrough in TSE research was the discovery of the "prion"⎯proteinaceous infectious particle⎯ and the verification of the “protein-only” hypothesis, which states that prions could self-propagate by converting the cellular prion protein (PrPC) into the scrapie form, PrPSc (or prions), and lead to neurodegeneration without using any nucleic acids. The concept of prions may unify neurodegenerative diseases under a common pathogenic mechanism. Indeed, growing evidence shows that TSE may share similar pathogenesis with common neurodegenerative syndromes such as Alzheimer’s disease and Parkinson’s disease, for which there are currently no cure. Today, PrP is one of the most studied models for protein misfolding mechanism and TSE serve as an excellent model for studying many other neurodegenerative diseases. Understanding the molecular mechanism of the PrP misfolding process may profoundly influence the development of diagnostics and effective therapies for neurodegenerative diseases in general.
Investigating human (Hu) PrP TSE-linked mutations (more than 50 currently identified mutations, linked to ~15% of the cases) may be very instrumental in this respect, as it can provide hints on the molecular basis of the PrPC→PrPSc conversion. These mutations cause spontaneous TSE, which are likely due to modifications in the native structure of PrPC. They are located all over the structure. Polymorphisms (i.e. non-pathogenic, naturally occurring mutations) in the PrP gene have been found to influence the etiology and neuropathology of the disease in both humans and sheep. In transgenic (Tg) mice, artificial mutations can determine the susceptibility to the infection of different prion strains. Intriguingly, mouse (Mo) PrP containing artificial mutations (denoted MoPrP chimera, hereinafter) have very different effects in vitro: some MoPrP chimera were found to resist PrPSc infection, whereas some others did not; some of the resistant MoPrP chimeras even exhibited a protective effect (known as the dominant-negative effect) over the co-expressed endogenous wild-type (WT) MoPrPC. Most mutations are located in the folded globular domain (GD) while fewer are located in the intrinsically disordered N-terminal domain (N-term). The N-term of PrPC has been suggested to serve multiple functions in vivo, which likely relies on the structural flexibility of this domain. Therefore, characterizing the structural features of the N-term is central for investigating not only the mutations in this domain, but also the physiological role of the N-term.
Based on previous studies in our lab, in this thesis we first applied molecular dynamics simulations to studying the impact of all the known Hu TSE-linked mutations in HuPrPC GD. We next applied the same approach to study the GD structure of MoPrP chimeras which contain one or two residues from Hu or sheep PrP sequence. By studying these PrP variants, we aim to identify the structural determinants of the mutants that may play a role in the PrPC→PrPSc conversion. Our calculations discovered that these mutants exhibit different structural features from those of the WT PrP GD mainly in two common regions that are likely the “hot spots” in the protein misfolding process. These features can be classified into different types that are correlated to the types of mutants (i.e. pathogenic, resistant or dominant-negative), thus hinting to the molecular mechanisms of PrPSc formation and propagation. We have then predicted the structure of the entire PrP N-term and the impact of the Hu TSE-linked mutations in this domain using a novel Monte Carlo-based simulation approach, PROFASI. PROFASI has already shown to provide structural predictions in a disordered protein such as α-synuclein. Our results are consistent with available experimental data and therefore firmly allow us to provide the first overview on the structural determinants of all Hu TSE-linked mutations in PrP.
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Kravchenko, Anna. "Fragment-based modelling of protein-RNA complexes for protein design." Electronic Thesis or Diss., Université de Lorraine, 2023. http://www.theses.fr/2023LORR0370.
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Les complexes protéine-ARN jouent un rôle crucial dans la régulation cellulaire. La prédiction de leur structure 3D a des applications dans la conception de protéines et de médicaments. Le projet ITN RNAct visait à combiner des méthodes expérimentales et informatiques pour concevoir de nouveaux "motifs de reconnaissance de l'ARN" (RRM) - domaines protéiques interagissant avec l'ARN simple brin (ARNsb) - pour la biologie synthétique et la bioanalyse. La modélisation des complexes protéine-ARNsb (amarrage) est ardue car l'ARNsb n'a pas de structure propre dans sa forme libre. L'amarrage traditionnelle échantillonne les positions relatives (poses) de 2 structures moléculaires et les note pour sélectionner les plus probables. Il n'est pas directement applicable ici en raison de l'absence de structures libres d'ARNsb, pas plus que l'apprentissage profond en raison du nombre trop faible de structures connues. L'amarrage par fragments, état de l'art pour l'ARNsb, amarre toutes les conformations possibles de fragments d'ARN sur une protéine et assemble les poses les mieux notées de manière combinatoire. Notre méthode ssRNA'TTRACT utilise le logiciel d'amarrage ATTRACT et sa représentation gros grain qui remplace des groupes d'atomes par une bille. Cependant, les paramètres ARN-protéine de sa fonction de notation (ASF) ne sont pas spécifiques à l'ARNsb et peuvent être optimisés. De plus, des caractéristiques spécifiques aux RRM peuvent être apprises et guider l'amarrage. Nous avons développé un pipeline d'amarrage RRM-ssRNA basé sur les données, pour actualiser une stratégie existante. Les RRM ont 2 acides aminés aromatiques de position conservée, chacun liant par empilement un nucléotide de l'ARN. Mon collègue H. Dhondge a regroupées les structures RRM-ARNsb connues sur critère géométrique et obtenu un ensemble de prototypes de coordonnées 3D de tels empilements dans les RRM. J'ai créé un pipeline qui prend en entrée une séquence de RRM et d'ARN et l'identification des nucléotides empilés, récupère la structure du RRM dans AlphaFoldDB, identifie les positions 3D possibles des nucléotides empilés et exécute ssRNA'TTRACT avec des contraintes de distance maximales vers chaque position. En parallèle, nous avons dérivé HIPPO (HIstogram-based Pseudo-POtential), un potentiel de notation pour les poses gros-grain RRM-ARNsb basé sur la fréquence des distances bille-bille dans les poses quasi-natives versus erronées. HIPPO combine 4 ensembles de paramètres en une note consensus, afin de prendre en compte les divers modes de liaison RRM-ARNsb. Testé dans une approche "leave-one-out", il atteint un enrichissement d'un facteur 3 en quasi-natives dans les 20% de poses mieux notées pour ½ des cas contre ¼ avec ASF, et 'un facteur 4 pour ⅓ des cas contre 7% avec ASF. Surprenamment, HIPPO obtient aussi de meilleurs résultats qu'ASF sur un ensemble test de protéines sans RRM, bien que entraîné sur des RRM. Les approches par fragment rencontrent un problème intrinsèque de notation car certains fragments se lient plus spécifiquement/fortement que d'autres. Or nous avons constaté que, pour le fragment le mieux noté par complexe, HIPPO sélectionne systématiquement plus de quasi-natifs qu'ASF. Cela nous a inspiré une approche d'amarrage incrémentale: chacune des poses bien notées d'un fragment sont utilisées comme graine pour construire une chaîne d'ARN complète de manière incrémentale. Cette stratégie élimine le besoin de contacts conservés connus, jusqu'alors nécessaires pour obtenir des modèles précis, ce qui la rend généralisable aux protéines sans RRM. Nos recherches futures visent à identifier le Η le plus performant pour chaque fragment, potentiellement par apprentissage automatique (profond). Notre approche pour dériver des paramètres de notation est en principe applicable à tout type de protéine/ligand et nous prévoyons de l'étendre à d'autres domaines de protéines liant l'ARN, ainsi qu'à l'ADNsb et aux peptides longs<br>Protein-RNA complexes play crucial roles in cell regulation. Predicting their 3D structure has applications in protein design and drug development. The ITN project RNAct aimed to combine experimental and computational methods to design new "RNA recognition motifs" (RRM) - protein domains interacting with single-stranded RNA (ssRNA) - for applications in synthetic biology and bioanalysis. Modelling protein-ssRNA complexes (docking) is an arduous task due to the flexibility of ssRNA, which lacks a proper structure in its free form. Traditional docking methods sample the relative positions (poses) of 2 molecular structures and score them to select the correct (near-native) ones. It is not directly applicable here due to the absence of free ssRNA structures, nor is deep learning due to the too low number of known structures for training. Fragment-based docking (FBD), the state-of-the-art approach for ssRNA, docks all possible conformations of RNA fragments onto a protein and assembles their best-scored poses combinatorially. ssRNA'TTRACT, our FBD method, uses the well-known ATTRACT docking software, with its coarse-grained representation that replaces atom groups by one bead. Yet the RNA-protein parameters of ATTRACT scoring function (ASF) are not ssRNA-specific and require optimisation. Additionally, RRM-specific features can be learned and used to guide the docking. With my colleague H. Dhondge, we have developed a data-driven FBD pipeline for RRM-ssRNA complexes, as an updated version of an existing strategy. RRMs have two aromatic amino acids (aa) in conserved positions, each stacking with a nucleotide of the bound ssRNA. H. Dhondge collected all known RRM-ssRNA structures with such stacking and clustered them to obtain a set of prototypes for the 3D coordinates of such interactions in RRM. I then set up a docking pipeline with as input the RRM and RNA sequences and the identification of the stacked nucleotides. The pipeline retrieves the RRM structure from AlphaFoldDB, identifies possible 3D positions of the stacked nucleotides and runs ssRNA'TTRACT with maximal distance restraints toward each position. In parallel, we addressed the weakness of ASF for ssRNA by deriving HIPPO (HIstogram-based Pseudo-POtential), a new scoring potential for ATTRACT poses of ssRNA on RRM, based on the frequency of bead-bead distances in near-native versus wrong poses. It combines 4 distinct parameter sets (four Η) into a consensus scoring, to better account for the diverse RRM-ssRNA binding modes. Tested in a leave-one-out approach, HIPPO reaches a 3-fold enrichment of near-natives in 20% top-scored poses for ½ of the ssRNA fragments, versus ¼ with ASF. It even reaches a 4-fold enrichment for ⅓ of the fragments, versus 7% of the fragments with ASF. Surprisingly, HIPPO performed better than ASF also on a benchmark of non-RRM proteins, while trained only on RRMs. Most FBD approaches encounter inherent scoring issues, probably due to some fragments binding more specifically/strongly than others. To address this point, we examined the best-scored fragment per complex and found that HIPPO consistently selects more near-natives than ASF for this fragment. This inspired an incremental docking approach: the top-ranked poses of one fragment are used as a starting point to build a full RNA chain incrementally. This strategy eliminates the need for known conserved contacts, which have been required so far to obtain accurate models, making it generalizable to non-RRM proteins. Future research aims to identify the best-performing Η for each fragment, potentially using (deep) machine learning. Our workflow to derive scoring parameters is in principle applicable to any protein/ligand type and we plan to expand it to other RNA-binding protein domains, as well as ssDNA and long peptides
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Nanni, Paolo <1979>. "Mass spectrometry-based protein profiling strategies for biomarker discovery in liver and inflammatory bowel diseases." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2008. http://amsdottorato.unibo.it/1025/1/Tesi_Nanni_Paolo.pdf.
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The study of protein expression profiles for biomarker discovery in serum and in
mammalian cell populations needs the continuous improvement and combination of
proteins/peptides separation techniques, mass spectrometry, statistical and bioinformatic
approaches.
In this thesis work two different mass spectrometry-based protein profiling strategies have
been developed and applied to liver and inflammatory bowel diseases (IBDs) for the
discovery of new biomarkers.
The first of them, based on bulk solid-phase extraction combined with matrix-assisted laser
desorption/ionization - Time of Flight mass spectrometry (MALDI-TOF MS) and
chemometric analysis of serum samples, was applied to the study of serum protein
expression profiles both in IBDs (Crohn’s disease and ulcerative colitis) and in liver
diseases (cirrhosis, hepatocellular carcinoma, viral hepatitis).
The approach allowed the enrichment of serum proteins/peptides due to the high
interaction surface between analytes and solid phase and the high recovery due to the
elution step performed directly on the MALDI-target plate. Furthermore the use of
chemometric algorithm for the selection of the variables with higher discriminant power
permitted to evaluate patterns of 20-30 proteins involved in the differentiation and
classification of serum samples from healthy donors and diseased patients. These proteins
profiles permit to discriminate among the pathologies with an optimum classification and
prediction abilities. In particular in the study of inflammatory bowel diseases, after the
analysis using C18 of 129 serum samples from healthy donors and Crohn’s disease,
ulcerative colitis and inflammatory controls patients, a 90.7% of classification ability and a
72.9% prediction ability were obtained. In the study of liver diseases (hepatocellular
carcinoma, viral hepatitis and cirrhosis) a 80.6% of prediction ability was achieved using
IDA-Cu(II) as extraction procedure. The identification of the selected proteins by MALDITOF/
TOF MS analysis or by their selective enrichment followed by enzymatic digestion
and MS/MS analysis may give useful information in order to identify new biomarkers
involved in the diseases.
The second mass spectrometry-based protein profiling strategy developed was based on a
label-free liquid chromatography electrospray ionization quadrupole - time of flight
differential analysis approach (LC ESI-QTOF MS), combined with targeted MS/MS
analysis of only identified differences. The strategy was used for biomarker discovery in
IBDs, and in particular of Crohn’s disease. The enriched serum peptidome and the
subcellular fractions of intestinal epithelial cells (IECs) from healthy donors and Crohn’s
disease patients were analysed.
The combining of the low molecular weight serum proteins enrichment step and the LCMS
approach allowed to evaluate a pattern of peptides derived from specific exoprotease
activity in the coagulation and complement activation pathways. Among these peptides,
particularly interesting was the discovery of clusters of peptides from fibrinopeptide A,
Apolipoprotein E and A4, and complement C3 and C4. Further studies need to be
performed to evaluate the specificity of these clusters and validate the results, in order to
develop a rapid serum diagnostic test.
The analysis by label-free LC ESI-QTOF MS differential analysis of the subcellular
fractions of IECs from Crohn’s disease patients and healthy donors permitted to find many
proteins that could be involved in the inflammation process. Among them heat shock
protein 70, tryptase alpha-1 precursor and proteins whose upregulation can be explained
by the increased activity of IECs in Crohn’s disease were identified. Follow-up studies for
the validation of the results and the in-depth investigation of the inflammation pathways
involved in the disease will be performed.
Both the developed mass spectrometry-based protein profiling strategies have been
proved to be useful tools for the discovery of disease biomarkers that need to be validated
in further studies.
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Nanni, Paolo <1979>. "Mass spectrometry-based protein profiling strategies for biomarker discovery in liver and inflammatory bowel diseases." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2008. http://amsdottorato.unibo.it/1025/.
Full textAbstract:
The study of protein expression profiles for biomarker discovery in serum and in
mammalian cell populations needs the continuous improvement and combination of
proteins/peptides separation techniques, mass spectrometry, statistical and bioinformatic
approaches.
In this thesis work two different mass spectrometry-based protein profiling strategies have
been developed and applied to liver and inflammatory bowel diseases (IBDs) for the
discovery of new biomarkers.
The first of them, based on bulk solid-phase extraction combined with matrix-assisted laser
desorption/ionization - Time of Flight mass spectrometry (MALDI-TOF MS) and
chemometric analysis of serum samples, was applied to the study of serum protein
expression profiles both in IBDs (Crohn’s disease and ulcerative colitis) and in liver
diseases (cirrhosis, hepatocellular carcinoma, viral hepatitis).
The approach allowed the enrichment of serum proteins/peptides due to the high
interaction surface between analytes and solid phase and the high recovery due to the
elution step performed directly on the MALDI-target plate. Furthermore the use of
chemometric algorithm for the selection of the variables with higher discriminant power
permitted to evaluate patterns of 20-30 proteins involved in the differentiation and
classification of serum samples from healthy donors and diseased patients. These proteins
profiles permit to discriminate among the pathologies with an optimum classification and
prediction abilities. In particular in the study of inflammatory bowel diseases, after the
analysis using C18 of 129 serum samples from healthy donors and Crohn’s disease,
ulcerative colitis and inflammatory controls patients, a 90.7% of classification ability and a
72.9% prediction ability were obtained. In the study of liver diseases (hepatocellular
carcinoma, viral hepatitis and cirrhosis) a 80.6% of prediction ability was achieved using
IDA-Cu(II) as extraction procedure. The identification of the selected proteins by MALDITOF/
TOF MS analysis or by their selective enrichment followed by enzymatic digestion
and MS/MS analysis may give useful information in order to identify new biomarkers
involved in the diseases.
The second mass spectrometry-based protein profiling strategy developed was based on a
label-free liquid chromatography electrospray ionization quadrupole - time of flight
differential analysis approach (LC ESI-QTOF MS), combined with targeted MS/MS
analysis of only identified differences. The strategy was used for biomarker discovery in
IBDs, and in particular of Crohn’s disease. The enriched serum peptidome and the
subcellular fractions of intestinal epithelial cells (IECs) from healthy donors and Crohn’s
disease patients were analysed.
The combining of the low molecular weight serum proteins enrichment step and the LCMS
approach allowed to evaluate a pattern of peptides derived from specific exoprotease
activity in the coagulation and complement activation pathways. Among these peptides,
particularly interesting was the discovery of clusters of peptides from fibrinopeptide A,
Apolipoprotein E and A4, and complement C3 and C4. Further studies need to be
performed to evaluate the specificity of these clusters and validate the results, in order to
develop a rapid serum diagnostic test.
The analysis by label-free LC ESI-QTOF MS differential analysis of the subcellular
fractions of IECs from Crohn’s disease patients and healthy donors permitted to find many
proteins that could be involved in the inflammation process. Among them heat shock
protein 70, tryptase alpha-1 precursor and proteins whose upregulation can be explained
by the increased activity of IECs in Crohn’s disease were identified. Follow-up studies for
the validation of the results and the in-depth investigation of the inflammation pathways
involved in the disease will be performed.
Both the developed mass spectrometry-based protein profiling strategies have been
proved to be useful tools for the discovery of disease biomarkers that need to be validated
in further studies.
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Lykidis, Dimitrios Aristotle. "Development of a zinc-based fixative for DNA, RNA and protein molecular studies." Thesis, Imperial College London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.444137.
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Ledmyr, Helena. "Molecular regulation of microsomal triglyceride transfer protein, MTP : functional genetic studies in relation to cardiovascular disease /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-142-3/.
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Garousi, Javad. "Development of ADAPT-based tracers for radionuclide molecular imaging of cancer." Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-327419.
Full textAbstract:
ABD-Derived Affinity Proteins (ADAPTs) is a novel class of small engineered scaffold proteins based on albumin-binding domain (ABD) of streptococcal protein G. High affinity ADAPT binders against various therapeutic targets can be selected. In this thesis, we report a development of ADAPT-based radionuclide imaging agents providing high sensitivity and specificity of molecular imaging of HER2 expression in disseminated cancers. We investigated the feasibility of the use of ADAPTs as imaging agents and influence of molecular design and radiolabeling chemistry on in vivo targeting and biodistribution properties of the tracers. In Paper I we demonstrated the feasibility of the use of anti-HER2 ADAPT6 molecule as a high contrast imaging agent; In Paper II we evaluated the influence of composition of histidine-containing tag on in vivo biodistribution of ADAPT-based tracers labeled with 99mTc using 99mTc(CO)3 binding to histidine-containing tags and 111In using DOTA chelator at N-terminus; In Paper III we evaluated the influence of different aspects of N-terminus leading sequence on targeting including effect of sequence size on clearance rate and effect of the composition of the sequence on biodistribution profile; In Paper IV, we evaluated the influence of residualizing properties and positioning of the label on biodistribution and targeting; and In Paper V, we compared tumor-targeting properties of the ADAPT6 labeled at C-terminus with 99mTc using N3S chelator and 111In using DOTA chelator. In conclusion, ADAPTs constitute a very promising class of targeting probes for molecular imaging providing high contrast. Molecular design of the ADAPT proteins and chelators/linkers for labeling has an appreciable effect on their imaging properties.
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Morris, Viktoriya Dickstein Rebecca. "Map-based cloning of the NIP gene in model legume Medicago truncatula." [Denton, Tex.] : University of North Texas, 2007. http://digital.library.unt.edu/permalink/meta-dc-3638.
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Jang, HyeIn. "FUNCTIONAL CHARACTERIZATION OF SCAFFOLD PROTEIN SHOC2." UKnowledge, 2018. https://uknowledge.uky.edu/biochem_etds/39.
Full textAbstract:
Signaling scaffolds are critical for the correct spatial organization of enzymes within the ERK1/2 signaling pathway and proper transmission of intracellular information. However, mechanisms that control molecular dynamics within scaffolding complexes, as well as biological activities regulated by the specific assemblies, remain unclear.
The scaffold protein Shoc2 is critical for transmission of the ERK1/2 pathway signals. Shoc2 accelerates ERK1/2 signaling by integrating Ras and RAF-1 enzymes into a multi-protein complex. Germ-line mutations in shoc2 cause Noonan-like RASopathy, a disorder with a wide spectrum of developmental deficiencies. However, the physiological role of Shoc2, the nature of ERK1/2 signals transduced through this complex or mechanisms regulating the function of Shoc2 remain largely unknown. My dissertation addresses the mechanisms by which Shoc2 accelerates ERK1/2 signal transmission and the biological outputs of the Shoc2-guided signals.
To delineate Shoc2-mediated ERK1/2 signals, I have utilized a vertebrate zebrafish model. I demonstrated that loss of Shoc2 protein expression leads to early embryonic lethality resulting from a significant reduction in the number of circulating erythropoietic and myelopoietic blood cells, underdeveloped neurocranial and pharyngeal cartilages, and a profound delay in calcification of bone structures. Together, this data demonstrates that the Shoc2 scaffolding module transmits ERK1/2 signals in neural crest development and blood cell differentiation.
This dissertation also addresses the mechanistic basis of how allosteric ubiquitination of Shoc2 and RAF-1 is controlled. I have characterized a molecular interaction of Shoc2 with its previously unknown binding partner Valosin-Containing Protein (VCP/p97). These studies demonstrated that hexametric ATPase VCP modulates ubiquitination of Shoc2 and RAF-1 through the remodeling of the scaffolding complex in a spatial-restricted manner. Experiments utilizing fluorescence microscopy and biochemical methods show that VCP/p97 sequesters the E3 ligase HUWE1 from the Shoc2 module, thereby altering the ubiquitination of Shoc2 and RAF-1 as well as the amplitude of ERK1/2 signals. These studies also show that the levels of Shoc2 ubiquitination and ERK1/2 phosphorylation are imbalanced in fibroblasts isolated from Inclusion Body Myopathy with Paget’s disease of bone and Frontotemporal Dementia (IBMPFD) patients harboring VCP germline mutations. This data also suggests that ERK1/2 pathway deregulation is part of IBMPFD pathogenesis.
In summary, these studies make a significant advance in our understanding of the mechanisms by which the Shoc2 scaffold regulates specificity and the dynamics of the ERK1/2 signaling networks. They also make important insights into our understanding of biological activities and targets of Shoc2-mediated ERK1/2 signals at the early stages of embryonic development and disease.
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Szolkiewicz, Michal Jerzy. "Homology-based in silico identification of putative protein-ligand interactions in the malaria parasite." Diss., University of Pretoria, 2014. http://hdl.handle.net/2263/41019.
Full textAbstract:
Malaria is still one of the most proli c communicable diseases in the world with more
than 200 million infections annually, its greatest e ect is felt in the poor nations with-in
sub-saharan Africa and south-east Asia. It is especially fatal for women and children where
out of the 660 000 fatalities in 2010, 86% were below the age of 5.
In the past decade the global fatality rate due to malaria has been signi cantly reduced,
primarily due to proliferation of vector control using treated nets and indoor residual spraying
of DDT. There have, however, been few innovations in anti-malarial therapeutics and with
the threat of the spread of drug resistant strains a need still exists to develop novel drugs to
combat malaria infections. One of the major hinderances to drug development is the huge cost
of the drug development process, where candidate failures late in development are extremely
costly. This is where post-genomic information has the potential of adding great value. By
using all available data pertaining to a disease, one gains higher discerning power to select
good drug candidates and identify risks early in development before serious investments are
made. This need provided the motivation for the development of Discovery; a tool to aid in
the identi cation of protein targets and viable lead compounds for the treatment of malaria.
Discovery was developed at the University of Pretoria to be a platform for a large spectrum
of biological data focused on the malaria causing Plasmodium parasite. It conglomerates
various data types into a web-based interface that allows searching using logical lters or
by using protein or chemical start points. In 2010 it was decided to rebuild Discovery to improve it's functionality and optimize query times. Also, since its inception various new
datasources became available speci cally related to bio-active molecules, these include the ChEMBL database and TCAMS dataset of bio-active molecules and the focus of this project
was the integration of said datasets into Discovery. Large quantities of high quality bioactivity
data have never been available in the public domain and this has opened up the
opportunity to gain even greater insight into the activity of chemical compounds in malaria.
Due to conserved structural/functional similarities of proteins between di erent species it
is possible to derive predictions about a malaria protein or a chemicals activity in malaria
due to experiments carried out on other organisms. These comparisons can be leveraged to
highlight potential new compounds that were previously not considered or prevent wasting
resources persuing potential compounds that pose threats of toxicity to humans. This project
has resulted in a web based system that allows one to search through the chemical space of
the malaria parasite. Allowing them to view sets of predicted protein-ligand interactions for
a given protein based on that proteins similarity to those existing in the bio-active molecule
databases.<br>Dissertation (MSc)--University of Pretoria, 2014.<br>gm2014<br>Biochemistry<br>unrestricted
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Jariwala, Nidhi H. "Characterization of Staphylococcal nuclease and tudor domain containing protein 1 (SND1) as a molecular target in Hepatocellular carcinoma and Non-alcoholic steatohepatitis." VCU Scholars Compass, 2017. https://scholarscompass.vcu.edu/etd/5183.
Full textAbstract:
CHARACTERIZATION OF STAPHYLOCOCCAL NUCLEASE AND TUDOR DOMAIN CONTAINING PROTEIN 1 (SND1) AS A MOLECULAR TARGET IN HEPATOCELLULAR CARCINOMA AND NON-ALCOHOLIC STEATOHEPATITIS
Nidhi Jariwala, PhD
A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Integrative Life Sciences
Virginia Commonwealth University, 2017
Devanand Sarkar, M.B.B.S., PhD.
Associate Professor, Department of Human and Molecular Genetics
Virginia Commonwealth University
Richmond, Virginia
SND1, a subunit of the miRNA regulatory complex RISC, has been implicated as an oncogene in hepatocellular carcinoma (HCC). Oncoprotein SND1 regulates gene expression at a post-transcriptional level in multiple cancers including hepatocellular carcinoma (HCC). In the present study, we characterize oncogenic functions of SND1 in HCC employing a novel transgenic mouse model (Alb/SND1) and present SND1 as a potential molecular target in HCC management. We show that Alb/SND1 mice develop spontaneous HCC with partial penetrance and exhibit more highly aggressive HCC induced by chemical carcinogenesis. Livers from Alb/SND1 mice exhibit a relative increase in inflammatory markers and spheroid-generating tumor initiating cells (TiC). Mechanistic investigations defined roles for Akt and NF-κB signaling pathways in promoting TiC formation in Alb/SND1 mice. Intravenous administration of the selective SND1 inhibitor 3', 5'-deoxythymidine bisphosphate (pdTp) inhibited tumor formation without effects on body weight or liver function. We conclude that SND1 drives pro-oncogenic transcriptomic and proteomic changes in hepatocyte resulting in aggressive HCC. SND1 specific RNA interactome is identified with RNA immunoprecipitation sequencing (RIPSeq) approach. With an adjusted p value of2-fold enrichment over control, 282 mRNAs were identified to significantly associate with SND1 protein. We focused on the tumor suppressor Protein Tyrosine Phosphatase non-receptor type 23 (PTPN23) because its regulation by SND1 and its role in HCC are not known. In current study, we confirm that SND1 post-transcriptionally downregulates PTPN23. Pursuing functional studies with tetracycline inducible overexpression system, we validate that PTPN23 inhibits tyrosine kinase signaling, proliferation, epithelial to mesenchymal transition, migration, invasion and in vivo tumorigenesis.
Alb/SND1 mice also manifest steatosis and fibrosis at one year of age. Coupled with a pro-inflammatory hepatic phenotype, we conclude that Alb/SND1 livers present NASH. High fat diet causes severe NASH and aggressive NASH induced HCC in Alb/SND1 mice. Serum and hepatic lipid profiling shows that hepatocyte specific SND1 overexpression associate with elevated triglyceride and cholesterol LDL levels. Contrarily, hepatocyte specific deletion of SND1 (SND1ΔHEP) in vivo, significantly protects against age dependent steatosis. Association of SND1 in NASH pathology is novel discovery and we present preliminary evidence confirming role of SND1 in promoting NASH.
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Stemm, Mina Catherine. "Computational and combinatorial design of protein-based inhibitors of human tyrosyl-DNA phosphodiesterase /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2005. http://wwwlib.umi.com/cr/ucsd/fullcit?p3166399.
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Matsumiya, Kentaro. "Destabilization of protein-based emulsions caused by bacteriostatic emulsifiers." Master's thesis, Kyoto University, 2014. http://hdl.handle.net/2433/188746.
Full textAbstract:
Kyoto University (京都大学)<br>0048<br>新制・論文博士<br>博士(農学)<br>乙第12820号<br>論農博第2793号<br>新制||農||1025(附属図書館)<br>学位論文||H26||N4815(農学部図書室)<br>31307<br>京都大学農学研究科農学専攻<br>(主査)教授 松村 康生, 教授 裏出 令子, 教授 安達 修二<br>学位規則第4条第2項該当
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Goldflam, Michael. "Combined use of NMR and computational tools for fragment based drug discovery targeting protein-protein interactions VEGF protein surface recognition as a case study." Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/123711.
Full textAbstract:
The capacity of proteins to interact with each other rests at the core of biology. Given the ubiquitous nature of these interactions they have attracted the attention of scientists for the development of inhibitors or biochemical tools.The use of biologics to target protein-protein interfaces is relatively advanced; suffer although from some intrinsic drawbacks as the danger of immunogenicity, the inability to cross biological barriers efficiently and high production costs. Small molecule inhibitors do not necessarily share these drawbacks. Unfortunately the druggability of protein-protein interfaces and strategies to target them with small molecules is under open debate.
In this work we explore the druggability and methods to target the protein-protein interface of VEGF, a model system with therapeutic relevance in the fields of tumor biology and macular degeneration. We focus mainly on a fragment based approach for the following reasons: i.) proved to be successful at least for some particular protein-protein interfaces, ii.) offers a good coverage of the chemical space with small libraries, iii.) may lead to compounds with improved physicochemical properties compared to HTS. NMR, which is an omnipresent method in the field of fragment based drug discovery since the pioneering work of Fesik, was our tool of choice with a strong focus on combination with novel computational approaches.
In the first part of our work we express the required amounts of recombinant VEGF. Then we design and prepared a fragment library after the “SAR by catalog” principle. We developed a new methodology that allowed the preparation of fragment mixtures for NMR based screening with minimized signal overlap. This allowed the direct assessment of nearly all fragment mixtures without the need of mixture deconvolution. Further we developed a program that allowed the automatic evaluation of NMR derived fragment screening data. While being faster than tedious manual interpretation of NMR data it offered a degree of quantitative analysis that would otherwise not be possible in a reasonable amount of time.
Our library consistent of over 500 fragments was screened using STD- and CPMG filtered NMR experiments. The analysis of the NMR data resulted in high hit rates but apparent very weak affinity of identified ligands. We successfully developed a competitive 19F NMR based screening assay to identify ligands that bind to the protein-protein interface of VEGF, however none clear competitors could be identified. A second library of over 350 19F containing molecules was screened which led to low hit rates and identification of ligands with apparent very weak affinity. Finally a computational analysis of VEGF surface predicted a low druggability of the protein-protein interface which was in accordance to our experimental observations.
The characterization of weak binding fragments and their structural evolution was elaboration
was achieved by a combined approach based on NMR and computational experiments. Ligand
binding was assessed by the NMR chemical shift perturbation methodology using as probes both amide backbone N-H groups of the protein and its side chain methionine methyl groups. Binding poses were predicted by induced fit docking with the PELE algorithm under strong guidance by NMR derived restrains. Predicted binding modes were used to select fragment analogs with improved binding parameters. This was performed for three cycles and led finally to the discovery of several scaffold families that bind to or in proximity to the protein-protein interface of VEGF. Finally, we present a preliminary exploration of mRNA display for the selection of novel peptide based VEGF ligands.<br>En el contexto de la presente tesis hemos abordado los siguientes objetivos:
1. El uso de métodos de RMN, basados tanto en la observación del ligando como en la observación de proteína, para estudiar la unión de los compuestos de una quimioteca a la zona de VEGF involucrada en la unión a sus receptores. La interacción VEGF/VEGFR puede ser considerada como un caso de estudio para la evaluación de las interfaces proteína-proteína mediante cribado de fragmentos.
2. Desarrollar herramientas basadas en la combinación de RMN y métodos computacionales para abordar: i) un sistema automático de diseño de mezclas de fragmentos; ii) el análisis automático de datos procedentes de cribados basados en RMN; iii) la evolución de
fragmentos con muy baja afinidad.
3. Explorar el uso de técnicas de “mRNA display” para el descubrimiento de nuevos ligandos peptídicos para VEGF.
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Hunter, Michael. "Molecular investigations of the CMT4D gene N-myc downstream-regulated gene 1 (NDRG1)." University of Western Australia. School of Medicine and Pharmacology, 2006. http://theses.library.uwa.edu.au/adt-WU2007.0034.
Full textAbstract:
[Truncated abstract] Hereditary Motor and Sensory Neuropathy Lom (HMSNL) is a severe autosomal recessive peripheral neuropathy, the most common form of demyelinating Charcot-Marie-Tooth (CMT) disease in the Roma (Gypsy) population. The mutated gene, N-myc downstream-regulated gene 1 (NDRG1) on chromosome 8q24, is widely expressed and has been implicated in a wide range of processes and pathways. In this study we have aimed to assess the overall contribution of this gene to the pathogenesis of peripheral neuropathies, in cases where the most common causes of CMT disease havebeen excluded, as well as to gain clues about its function through the identification of its interactions with other proteins. Sequence analysis of NDRG1 in 104 patients with CMT disease and of diverse ethnicity identified one novel disease-causing mutation, IVS8-1G>A (g.2290787G>A), which affects the splice-acceptor site of IVS8 and results in the skipping of exon 9 . . . The results suggest a defect in Schwann cell lipid trafficking as a major pathogenetic mechanism in CMT4D. At the same time, database searches showed that the chromosomal location of NDRG1 coincides with a reported High-Density Lipoprotein-Cholesterol Quantitive Trait Locus (HDL-CQTL) in humans and in mice. A putative role of NDRG1 in the general mechanisms of HDL-mediated cholesterol transport was supported by biochemical studies of blood lipids, which revealed an association between the Gypsy founder mutation, R148X, and decreased HDL-C levels. These findings suggest that while peripheral neuropathy is the drastic result of NDRG1 deficiency, the primary role of the protein may be related to general mechanisms of lipid transport⁄metabolism.
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Gulati, Sahil Gulati. "Modulating G Protein-Coupled Receptor Signaling Pathways with Selective Chemical- and Protein-Based Effector Molecules." Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1530642105672697.
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Wei, Lixia. "Development of a Novel Protein Based MRI Contrast Agent for Molecular Imaging of Prostate Cancer." Digital Archive @ GSU, 2010. http://digitalarchive.gsu.edu/biology_diss/75.
Full textAbstract:
Molecular Imaging provides new aspects in cancer diagnosis and treatment. With the ap-plication of imaging and biological techniques, molecular imaging can monitor molecular and cellular changes of different diseases. To interpret the mechanism of disease, more and more at-tention is focused on the development of new probes for molecular imaging. Magnetic resonance imaging (MRI) is a powerful, non-invasive clinical diagnostic tool with high spatial resolution without the limitation of the depth of tissues. Applications of MRI contrast agents can amply the MRI signal during imaging. Many studies have been devoted to developing targeted MR contrast agents. Proteins and peptides have been widely used for target-ing cancer cells in cancer diagnosis and treatments. GRP, gastrin-releasing peptide, is one of a well-characterized group of mammalian bombesin-like peptides. GRP acts through its cell surface receptors, GRP receptor (GRPR). It has been reported that there is a high density of GRP receptors in the majority of prostate carci-noma. In contrast, the GRPRs are not highly expressed in normal cells of most tissues. Thus, this tumor specific expression pattern provides an advantage for cancer targeting. A novel class of MRI contrast agent was designed by adding the Gd3+ binding sites into a stable host protein, the domain 1 of rat CD2. Due to the unique features of the designed metal binding properties, the protein contrast agent (ProCA1) exhibits more than 10-fold enhanced MRI relaxivity compared to that of the more commonly used Gd-DTPA. The high relaxivity of the designed protein contrast agent largely overcomes the major barrier of low sensitivity of MRI techniques. A peptide of ten amino acids from the C-terminal of GRP was grafted onto ProCA1. GRP-grafted protein contrast agents (ProCA1.GRPs) showed the targeting capability to the GRPRs which are over-expressed on prostate cancer cells. Cell MRI Imaging demonstrated that ProCA1.GRP(52) grafted between Lys51 and Ser52 had better targeting capability than C-terminal one. Administration of ProCA1.GRP into xenograft tumor model enhances the contrast in the GRPR+ prostate tumor under MRI and optical imaging. Study demonstrated a potential application for disease marker targeted MR imaging by using our developed protein contrast agent.
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Choi, Yoon-Aa. "Molecular engineering of new protein labeling methodology based on rational design and in vitro evolution." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/57981.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2010.<br>Cataloged from PDF version of thesis. Vita.<br>Includes bibliographical references.<br>Site-specific labeling using E coli biotin ligase (BirA) and its 15-amino acid "acceptor peptide" (AP) has been applied to study the function of various cellular proteins. In order to extend the capabilities of biotin ligase-based labeling, we engineered key elements of the labeling platform. First we characterized a novel peptide substrate (called "yeast acceptor peptide" (yAP)) for yeast biotin ligase (yBL) that had been evolved by phage display. Assays performed in vitro and on the yeast surface showed that the yBL/yAP pair was orthogonal to the BirA/AP pair, allowing two-color labeling of different proteins on cells with differently-colored probes. Second, to improve the kinetic efficiency of yAP, we developed a novel selection scheme based on yeast display. Model selections demonstrated up to 1000-fold enrichment, and three rounds of selection on a randomized peptide library were performed. Third, we attempted to improve the kinetic efficiency of BirA through evolution by in vitro compartmentalization (IVC). Because the original IVC protocol based on bead-linked DNA had many technical problems, we developed a novel bead-less IVC protocol. An enrichment factor of 25 was obtained in a model selection. Due to the single-turnover nature of the selection, however, this scheme was not able to enrich highly active catalysts over moderately active ones. In separate work, we turned our attention to the streptavidin-biotin pair. Again using bead-less IVC, we performed a selection for streptavidin mutants that could bind a ketone analog of biotin with high affinity.<br>(cont.) Two rounds of selection were performed but characterization of enriched clones was not completed. Finally, we helped to discover a mutant ligase that could catalyze attachment of a fluorinated aryl azide photocrosslinker to proteins fused to a 17-amino acid peptide tag. The aryl azide probe was tested and shown to be accepted by a W37V mutant of E coli lipoic acid ligase (LplA).<br>by Yoon-Aa Choi.<br>Ph.D.
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Ratnayake, Wishrawana Sarathi Bandara. "Role of Oncogenic Protein Kinase C-iota in Melanoma Progression; A Study Based on Atypical Protein Kinase-C Inhibitors." Scholar Commons, 2019. https://scholarcommons.usf.edu/etd/7895.
Full textAbstract:
Irrespective of plentiful efforts to enhance primary prevention and early detection, the number of melanoma cases in the United States has increased steadily over the past 30 years, thus greatly affecting public health and the economy. We have investigated the effects of five novel aPKC inhibitors; 2-acetyl-1,3-cyclopentanedione (ACPD), 3,4-Diaminonaphthalene-2,7-disulfonic acid (DNDA), [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1T) along with its nucleoside analog 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) and 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) on cell proliferation, apoptosis, migration and invasion of two malignant melanoma cell lines compared to normal melanocyte cell lines. Molecular docking data suggested that both ACPD and DNDA specifically bind to protein kinase C-zeta (PKC-ζ) and PKC-iota (PKC-ι) while both ICA-1 compounds specifically bind to PKC-ι, and ζ-Stat showed a high affinity towards PKC-ζ. Kinase activity assays were carried out to confirm these observations. Results suggest that PKC-ι is involved in melanoma malignancy than PKC-ζ. Both isoforms promote the activation of nuclear factor (NF)-κB and protein kinase B (AKT) thereby supporting survival and progression. In addition, we demonstrated that PKC-ι induced the metastasis of melanoma cells by activating Vimentin, and PKC-ι inhibition downregulated epithilial-mesencymal transition (EMT), while inducing apoptosis. Of note, PKC-ἱ specific inhibitors downregulated the expression of both PKC-ι and phosphorylated PKC-ι, suggesting that PKC-ι plays a role in regulating its own expression in melanoma. We also report the underlaying mechanisms of the transcriptional regulation of PKC-ι (PRKCI gene) expression in melanoma. c-Jun, interferon-stimulated gene factor 3 (ISGF3), paired box gene 3 (PAX3), early growth response protein 1 (EGR1) and forkhead box protein O1 (FOXO1), which bind on or near the promoter sequence of the PRKCI gene, were analyzed for their role in PKC-ι regulation in SK-MEL-2 and MeWo cell lines. We silenced selected transcription factors using siRNA, and the results revealed that the silencing of c-Jun and FOXO1 significantly altered the expression of PRKCI. The levels of both phosphorylated and total PKC-ι increased upon FOXO1 silencing and decreased upon c-Jun silencing, suggesting that c-Jun acts as an upregulator, while FOXO1 acts as a downregulator of PRKCI expression. We also used a multiplex ELISA to analyze multiple pathways other than NF-κB that were affected by treatment with PKC-ι inhibitor. The silencing of NF-κB p65 and PKC-ι by siRNA suggested that the regulation of PKC-ι expression was strongly associated with FOXO1. In addition, we observed a significant decrease in the mRNA levels of both interleukin (IL)-6 and IL-8, with a significant increase in the levels of IL-17E and intercellular adhesion molecule 1 (ICAM-1) upon the knockdown of expression of PKC-ι in both cell lines. This suggested that PKC-ι expression was affected by these cytokines in an autocrine manner. Overall, the findings of this study suggest that PKC-ι inhibition suppresses its own expression, diminishing oncogenic signaling, while upregulating anti-tumor signaling, thus rendering it an effective novel biomarker for use in the design of novel targeted therapeutics for melanoma.
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Pan, Tao. "Genetic and physical interaction of Sgt2 protein with prion-chaperone machinery." Thesis, Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/45765.
Full textAbstract:
The word "Prion" refers to self-perpetuating protein aggregates that cause neurodegenerative diseases in mammals. It is a protein isoform that has undergone a conformational change which converts the normal form of the protein into the infectious form with the same amino acid sequence.
Yeast [PSI+] prion is the prion isoform of Sup35 protein, a translation termination factor eRF3. It has been suggested that prion [PSI+] is controlled by the ensemble of chaperones with Hsp104 playing the major role. The previous work performed in the Chernoffs lab showed that the defective GET pathway caused by get led to the defect in [PSI+] curing by excess Hsp104. The GET pathway is a system responsible for transporting newly synthesized TA-protein to the ER membrane, and the components which have been proven to be involved in this pathway include: Get1, Get2, Get3, Get4, Get5 and Sgt2. In this study we describe the mechanism underlying the effect of the defective GET pathway on [PSI+]. We demonstrate that Sgt2, one of the components of GET pathway, interacts with Sup35 in both [PSI+] and [psi-] strains through its prion domain. Overproduction of Sgt2 and Hsp70-Ssa is triggered by the defective GET pathway and leads to the protection of [PSI+] aggregates from curing by excess Hsp104. We show that the direct interaction between Sgt2 and Hsp70-Ssa is not required for this protective effect.
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Cuzzolin, Alberto. "Novel in silico approaches to depict the protein-ligand recognition events." Doctoral thesis, Università degli studi di Padova, 2016. http://hdl.handle.net/11577/3424818.
Full textAbstract:
The discovery and commercialization of a new drug is a long and expensive process. Such process is divided into different phases during which the phisico-chemical and therapeutic properties of the compounds are determined. In particular, the aim of the first phase is to verify whether the compound recognises and interacts efficiently with the target protein.
In the last decade, several computational tools have been developed and used to support experimentalists. For this purpose, the scientist have to deal with high complex systems that are difficult to study in whole; thus, the methods and algorithms developers have to strongly simplify the system treatment. Moreover, the time required to obtain the results depends on the computational resources (hardware) available. Fortunately, the technological progress have increased the computing power at low cost, resulting in new and more complex techniques development.
During this Ph.D. project we were focused on the development and even the improvement of in silico methods, which allowed to answer certain questions by saving time and money. Furthermore, these methods were implemented in software presenting a Graphical Unit Interface (GUI) with the aim to enhance the user-friendliness.
The computational techniques often require a high understanding of the methodology theoretical aspects and also a good informatics proficiency, like different type files handling and hardware management. For this reason, our developed software were organized as pipelines to automatize the entire process and to make this tools useful also for non-expert users.
Finally, these methodologies were applied in several research projects demonstrating their usefulness by elucidating, for the first time, interesting aspects of the ligand-protein recognition pathway.<br>La scoperta e la commercializzazione di un nuovo farmaco è un processo lungo e dispendioso, che si articola in diverse fasi durante le quali vengono determinate le proprietà fisiche, chimiche e terapeutiche dei composti investigati. In particolare, nella prima fase di questo processo si cerca di verificare che il composto riconosca e interagisca efficacemente con la proteina bersaglio.
A tale scopo, negli ultimi decenni numerosi strumenti computazionali sono stati sviluppati e utilizzati per supportare i ricercatori che si adoperano nella parte sperimentale. I problemi affrontati presentano un alto livello di complessità, che sarebbero difficili da studiare in toto, perciò gli sviluppatori di metodi e algoritmi devono necessariamente adottare notevoli semplificazioni. Inoltre, le risorse di calcolo (hardware) determinano le tempistiche con le quali è possibile ottenere il risultato richiesto. In tal senso, lo sviluppo tecnologico ha portato a un importante aumento della potenza di calcolo a costi accessibili, stimolando l’interesse per lo sviluppo di tecniche sempre più complesse.
Durante questo progetto di dottorato ci si è focalizzati sullo sviluppo e il miglioramento di metodi in silico, che permettono di rispondere ad alcuni interrogativei a costi e tempistiche di molto ridotte.
Inoltre, tali metodi sono stati implementati in software dotati di interfaccia grafica (GUI) al fine di poter aiutare l’utente nel loro utilizzo.
Le tecniche computazionali spesso richiedono un’elevata conoscenza teorica delle metodologie e anche una certa competenza informatica, come la gestione di diversi tipologie di file e delle risorse hardware da impiegare. Per questo motivo i software da noi sviluppati sono stati organizzati in pipelines, in modo da automatizzare l’intero processo e rendere questi strumenti fruibili anhce a persone non esperte.
Infine, l’utilità di queste nuove metodologie è stata comprovata in progetti in cui questi strumenti hanno permesso di delucidare aspetti interessanti e fino ad ora non ancora accessibili nell’ambito del riconoscimento proteina-ligando.
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Düzgün, Ali. "Carbon nanotube based potentiometric aptasensors for protein detection." Doctoral thesis, Universitat Rovira i Virgili, 2013. http://hdl.handle.net/10803/111166.
Full textAbstract:
El diagnóstico rápido de la mayoría de las enfermedades tiene una importancia vital para proporcionar el remedio adecuado y, por lo tanto, el control de problemas de salud. La detección rápida y selectiva de biomoléculas grandes, específicamente proteínas, es uno de los objetivos importantes en este campo. Las técnicas basadas en inmunoensayos son las más comúnmente utilizadas, aunque requieren un marcaje específico. Por lo general, estos métodos también requieren personal altamente capacitado y equipos complejos que se traduce en una metodología relativamente cara y lenta.
En la presente tesis aportamos por primera vez un nuevo tipo de aptasensores potenciométricos de estado sólido basados en nanotubos de carbono que pueden detectar analitos grandes, como por ejemplo proteínas, de manera rápida (casi instantánea), selectiva, y sensible sin necesidad de marcaje químico. Los sensores desarrollados responden correctamente a la proteína analito (α-trombina humana) dentro de los niveles fisiológicos en el suero humano.<br>Rapid diagnosis of most illnesses has a vital importance for providing the appropriate cure and hence controlling public health concerns. Fast and accurate detection of large biomolecules, specifically proteins, is one of the major steps regarding the subject. Over recent years, several detection methodologies have been developed. However, almost all of the developed methods either required very complex techniques to be applied, or a long time to obtain the results. The most commonly used techniques were specific label requiring immunoassays. They generally require highly trained staff and complex equipment which results in an expensive and relatively slow methodology.
With the present thesis we report a new type of label-free potentiometric solid state carbon nanotube based aptasensors that can detect large analytes, as case example proteins, in a rapid (almost instantaneous), selective and sensitive way, for the first time. The developed sensors successfully responded to analyte protein (human α-thrombin) within physiological human serum levels.
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Seeley, Kent W. "Mass Spectrometry-based Methods for the Detection and Characterization of Protein-Tyrosine Nitration." Scholar Commons, 2013. http://scholarcommons.usf.edu/etd/4766.
Full textAbstract:
Protein tyrosine nitration (PTN) is a posttranslational modification resulting from oxidative/nitrosative stress that has been implicated in a wide variety of disease states. Characterization of PTN is challenging due to several factors including its low abundance in a given proteome, preferential site modification, multiple target site proximity within unique peptide sequences, and analytical method and instrument limitations. Current analytical techniques are insufficiently sensitive to identify endogenous nitration sites without incorporation of either nitrotyrosine or target protein enrichment. However, enrichment proficiency can also be inadequate. Chemical derivatization of the nitro- moiety can be incomplete or result in undesirable byproduct formation, while immunoaffinity proficiency is contingent upon antibody specificity. To overcome analytical method and enrichment deficiencies, we aimed to develop a comprehensive nitroproteome-specific workflow using molecular methods combined with mass spectrometry. Our approach was to systematically address all relevant factors contributing to PTN such as primary sequence, protein conformation, solvent accessibility, and nitrating agent concentration. Our ultimate goal was to increase mass spectrometric sensitivity for PTN identification. All putative nitroprotein/nitropeptide identifications were then subjected to rigorous validation by either manual spectrum analyses or peptide synthesis. We further developed MS methods for quantitation of nitropeptides from complex mixtures with minimal sample processing. Successful application of our nitroproteome-specific mass spectrometric workflow is expected to provide powerful tools for comprehensive PTN investigation that will elucidate its role in the onset and progression of a variety of disease states as well as facilitate discovery of therapeutic targets.