Amyloid proteins and fibrils stability

Compared to globular proteins that have a stable native structure, intrinsically disordered peptides (IDP) sample an ensemble of structures without folding into a native conformation.One example of IDP is the amyloid-beta(Abeta) protein which is the main constituent of senile plaques in the brain of Alzheimer\u27s patients.Understanding the process by which IDPs undergo structural changes to form oligomers that eventually aggregate into senile plaques/amyloid fibrils may significantly advance the development of novel therapeutic methods to treat neurodegenerative diseases, for which there is no cure to date. This dissertation has two main objectives. The first one is to investigate and identify structural conformations of Abeta monomer which are precursor to aggregation. The second objective is to understand the underlying mechanisms of amyloid fibril stability using atomistic molecular dynamics simulations in explicit water. The aggregation of Abeta peptides into amyloid fibrils in Alzheimer\u27s patients depends on the spectrum of conformations adopted by monomers of this peptides. These conformations are strongly affected by properties of the aqueous environment. In the first part of this dissertation, conformations of Abeta in environments that promote and inhibit fibril formation are studied. Micro-second Replica Exchange Molecular Dynamics (REMD) simulations are performed for that purpose. A comparative study of the set of conformations in each environment is performed using contact maps, cluster analysis and by studying the network of the backbone hydrogen bonds of Abeta. A specific in-register strand-loop-strand conformation is found in the environment that promotes fibril formation, which is not observed in environments that inhibit fibril formation. It is proposed here that this conformation may act as intermediate structure in fibril formation. Inhibiting the formation of this conformation might be helpful in developing drugs for Alzheimer\u27s disease. In the second part of this dissertation, the molecular mechanisms of amyloid fibril stability are investigated using a thermodynamic framework. Understanding the atomic interactions responsible for fibril stability may be useful in designing novel therapeutic methods to disrupt fibrils and plaques in neurodegenerative diseases. However, despite numerous studies on amyloid fibrils, a thorough understanding of fibril stability is still missing. A combination of enhanced sampling methods is used to simulate all-atom models in explicit solvent in order to investigate the stability of non-polar (Abeta16-21) and polar (IAPP28-33) amyloid fibrils. Umbrella sampling is performed jointly with replica exchange molecular dynamics to determine the free energy of peptide addition to a pre-formed amyloid fibril at different temperatures.Results from these simulations show that the non-polar fibril becomes more stable with increasing temperature and its stability is dominated by entropy. In contrast, the polar fibril becomes less stable as temperature increases while it is stabilized by enthalpy. These findings suggest that the stability of fibrils can be customized by the choice of amino acid sequence in the dry core of the amyloid fibrils, e.g., proteins can be modified to transition between fibril and monomer state at a designated temperature. Such fine-tuned amyloid fibrils can be used as scaffolds for drug delivery and other biomaterials

Advanced Image Acquisition, Processing Techniques and Applications

"Advanced Image Acquisition, Processing Techniques and Applications" is the first book of a series that provides image processing principles and practical software implementation on a broad range of applications. The book integrates material from leading researchers on Applied Digital Image Acquisition and Processing. An important feature of the book is its emphasis on software tools and scientific computing in order to enhance results and arrive at problem solution

Optimization methods for side-chain positioning and macromolecular docking

This dissertation proposes new optimization algorithms targeting protein-protein docking which is an important class of problems in computational structural biology. The ultimate goal of docking methods is to predict the 3-dimensional structure of a stable protein-protein complex. We study two specific problems encountered in predictive docking of proteins. The first problem is Side-Chain Positioning (SCP), a central component of homology modeling and computational protein docking methods. We formulate SCP as a Maximum Weighted Independent Set (MWIS) problem on an appropriately constructed graph. Our formulation also considers the significant special structure of proteins that SCP exhibits for docking. We develop an approximate algorithm that solves a relaxation of MWIS and employ randomized estimation heuristics to obtain high-quality feasible solutions to the problem. The algorithm is fully distributed and can be implemented on multi-processor architectures. Our computational results on a benchmark set of protein complexes show that the accuracy of our approximate MWIS-based algorithm predictions is comparable with the results achieved by a state-of-the-art method that finds an exact solution to SCP. The second problem we target in this work is protein docking refinement. We propose two different methods to solve the refinement problem. The first approach is based on a Monte Carlo-Minimization (MCM) search to optimize rigid-body and side-chain conformations for binding. In particular, we study the impact of optimally positioning the side-chains in the interface region between two proteins in the process of binding. We report computational results showing that incorporating side-chain flexibility in docking provides substantial improvement in the quality of docked predictions compared to the rigid-body approaches. Further, we demonstrate that the inclusion of unbound side-chain conformers in the side-chain search introduces significant improvement in the performance of the docking refinement protocols. In the second approach, we propose a novel stochastic optimization algorithm based on Subspace Semi-Definite programming-based Underestimation (SSDU), which aims to solve protein docking and protein structure prediction. SSDU is based on underestimating the binding energy function in a permissive subspace of the space of rigid-body motions. We apply Principal Component Analysis (PCA) to determine the permissive subspace and reduce the dimensionality of the conformational search space. We consider the general class of convex polynomial underestimators, and formulate the problem of finding such underestimators as a Semi-Definite Programming (SDP) problem. Using these underestimators, we perform a biased sampling in the vicinity of the conformational regions where the energy function is at its global minimum. Moreover, we develop an exploration procedure based on density-based clustering to detect the near-native regions even when there are many local minima residing far from each other. We also incorporate a Model Selection procedure into SSDU to pick a predictive conformation. Testing our algorithm over a benchmark of protein complexes indicates that SSDU substantially improves the quality of docking refinement compared with existing methods

Signal and image processing methods for imaging mass spectrometry data

Imaging mass spectrometry (IMS) has evolved as an analytical tool for many biomedical applications. This thesis focuses on algorithms for the analysis of IMS data produced by matrix assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer. IMS provides mass spectra acquired at a grid of spatial points that can be represented as hyperspectral data or a so-called datacube. Analysis of this large and complex data requires efficient computational methods for matrix factorization and for spatial segmentation. In this thesis, state of the art processing methods are reviewed, compared and improved versions are proposed. Mathematical models for peak shapes are reviewed and evaluated. A simulation model for MALDI-TOF is studied, expanded and developed into a simulator for 2D or 3D MALDI-TOF-IMS data. The simulation approach paves way to statistical evaluation of algorithms for analysis of IMS data by providing a gold standard dataset. [...

Targeted and untargeted metabolomic profiling of human serum, urine and brain by liquid chromatography mass spectrometry (LC-MS)

Four projects were undertaken in the current study to apply, and in some cases develop, metabolomic profiling methods based on LC-HRMS for application to different human biofluid and tissue samples.;In the first study concerns the potential of metabolic changes to give an indication of physical fitness. Urine samples were collected from ten physically active subjects who underwent two cycling exercise trials. Firstly, their aerobic capacity (VO2max) was determined and the second was a 45 min submaximal exercise test. One hundred and twenty urine samples were collected in total over two days at regular time intervals: on day 1 where there was no exercise intervention; on day 2 where there was an exercise session and on day 3 where there was no exercise intervention.;The metabolites affected by the exercise included a range of purine metabolites and several acyl carnitines. Some metabolites including bile acids and several amino acids were subject to diurnal variation during the rest day (day 1). Using OPLS modelling it proved possible to identify a single abundant urinary metabolite provisionally identified as oxo-aminohexanoic acid (OHA) as being strongly correlated with VO2max.;Secondly, metabolomic profiling was carried out on 53 post-mortem brain samples from subjects diagnosed with schizophrenia (S), depression (D), bipolar (B), diabetes (Di) and controls (C) in order determine whether or not there were clear metabolites distinguishing these conditions. The most important metabolites producing discrimination were the lipophilic amino acids leucine/isoleucine, proline, methionine, phenylalanine, and tyrosine; the neurotransmitters gamma-aminobutyric acid (GABA) and N-acetyl aspartyl glutamate (NAAG) and sugar metabolites sorbitol, gluconic acid, xylitol, ribitol, arabinotol, and erythritol.;For the determination of the sugar polyols it was necessary to develop and new GC-MS method. There appears some commonality between metabolic perturbations resulting from diabetes and from SDB. This study concluded that the changes in sugar alcohol levels which were quantified in physiological fluids confirm the metabolic similarity between mental illness and diabetes. This leads to the question regarding whether or not antidiabetic drugs might have a role in treating mental illness?;In a third project a reductive amination method was used in combination with hydrophilic interaction liquid chromatography and high resolution mass spectrometry to analyse the sugars in post-mortem human brain. The samples were collected from six control brain samples and eighteen samples from individuals who had suffered from schizophrenia, depression and bipolar (SDB) disorder. This method was applied to confirm the important role of sugar metabolism through evaluation of relation between the changes in sugar levels and psychiatric.;The best separation and quantification of the common hexoses (glucose, fructose, mannose and galactose) by this method was achieved when 2H5-aniline was used as a tagging agent. Additionally, it succeeded in tagging a range of other sugars including pentoses (ribose and xylose) and sugar derivatives (N-acetylneuraminic acid and glucuronic acid). The results highlighted a relationship between the increases in the levels of sugars and psychiatric disorders (SDB). This relation which matched with other studies once again supported our results project 2 suggesting that in that sugar metabolism has an important role in distinguishing the control and SDB brains.;Oxidised fatty acids have such as oxylipins and prostaglandins have profound effects on human physiology and are present a low levels in biological fluids. In project 4 a novel tagging method for the analysis of oxidised fatty acids was developed. Fatty acids give a relatively weak response in negative ion ESI-MS. In untargeted metabolomics studies, is important to obtain a good response from molecules of interest. A derivatisation method using choline as a tag to put a positive charge on the analytes succeeded for the quantification of oxidised fatty acids, especially oxylipins, in plasma samples collected from seven participants before and after taking of beetroot juice (strong anti-oxidant and a source of nitrate).;The current results concluded that this derivatisation method improved the sensitivity and selectivity of LC-MS analysis by increasing the response factors for the positively tagged oxidised fatty acids by more than 100 times in comparison to those for the untagged compounds in negative ion. There was no clear effect of beetroot juice on the levels of oxidised fatty acids in plasma.Four projects were undertaken in the current study to apply, and in some cases develop, metabolomic profiling methods based on LC-HRMS for application to different human biofluid and tissue samples.;In the first study concerns the potential of metabolic changes to give an indication of physical fitness. Urine samples were collected from ten physically active subjects who underwent two cycling exercise trials. Firstly, their aerobic capacity (VO2max) was determined and the second was a 45 min submaximal exercise test. One hundred and twenty urine samples were collected in total over two days at regular time intervals: on day 1 where there was no exercise intervention; on day 2 where there was an exercise session and on day 3 where there was no exercise intervention.;The metabolites affected by the exercise included a range of purine metabolites and several acyl carnitines. Some metabolites including bile acids and several amino acids were subject to diurnal variation during the rest day (day 1). Using OPLS modelling it proved possible to identify a single abundant urinary metabolite provisionally identified as oxo-aminohexanoic acid (OHA) as being strongly correlated with VO2max.;Secondly, metabolomic profiling was carried out on 53 post-mortem brain samples from subjects diagnosed with schizophrenia (S), depression (D), bipolar (B), diabetes (Di) and controls (C) in order determine whether or not there were clear metabolites distinguishing these conditions. The most important metabolites producing discrimination were the lipophilic amino acids leucine/isoleucine, proline, methionine, phenylalanine, and tyrosine; the neurotransmitters gamma-aminobutyric acid (GABA) and N-acetyl aspartyl glutamate (NAAG) and sugar metabolites sorbitol, gluconic acid, xylitol, ribitol, arabinotol, and erythritol.;For the determination of the sugar polyols it was necessary to develop and new GC-MS method. There appears some commonality between metabolic perturbations resulting from diabetes and from SDB. This study concluded that the changes in sugar alcohol levels which were quantified in physiological fluids confirm the metabolic similarity between mental illness and diabetes. This leads to the question regarding whether or not antidiabetic drugs might have a role in treating mental illness?;In a third project a reductive amination method was used in combination with hydrophilic interaction liquid chromatography and high resolution mass spectrometry to analyse the sugars in post-mortem human brain. The samples were collected from six control brain samples and eighteen samples from individuals who had suffered from schizophrenia, depression and bipolar (SDB) disorder. This method was applied to confirm the important role of sugar metabolism through evaluation of relation between the changes in sugar levels and psychiatric.;The best separation and quantification of the common hexoses (glucose, fructose, mannose and galactose) by this method was achieved when 2H5-aniline was used as a tagging agent. Additionally, it succeeded in tagging a range of other sugars including pentoses (ribose and xylose) and sugar derivatives (N-acetylneuraminic acid and glucuronic acid). The results highlighted a relationship between the increases in the levels of sugars and psychiatric disorders (SDB). This relation which matched with other studies once again supported our results project 2 suggesting that in that sugar metabolism has an important role in distinguishing the control and SDB brains.;Oxidised fatty acids have such as oxylipins and prostaglandins have profound effects on human physiology and are present a low levels in biological fluids. In project 4 a novel tagging method for the analysis of oxidised fatty acids was developed. Fatty acids give a relatively weak response in negative ion ESI-MS. In untargeted metabolomics studies, is important to obtain a good response from molecules of interest. A derivatisation method using choline as a tag to put a positive charge on the analytes succeeded for the quantification of oxidised fatty acids, especially oxylipins, in plasma samples collected from seven participants before and after taking of beetroot juice (strong anti-oxidant and a source of nitrate).;The current results concluded that this derivatisation method improved the sensitivity and selectivity of LC-MS analysis by increasing the response factors for the positively tagged oxidised fatty acids by more than 100 times in comparison to those for the untagged compounds in negative ion. There was no clear effect of beetroot juice on the levels of oxidised fatty acids in plasma

Improving the resolution of interaction maps: A middleground between high-resolution complexes and genome-wide interactomes

Protein-protein interactions are ubiquitous in Biology and therefore central to understand living organisms. In recent years, large-scale studies have been undertaken to describe, at least partially, protein-protein interaction maps or interactomes for a number of relevant organisms including human. Although the analysis of interaction networks is proving useful, current interactomes provide a blurry and granular picture of the molecular machinery, i.e. unless the structure of the protein complex is known the molecular details of the interaction are missing and sometime is even not possible to know if the interaction between the proteins is direct, i.e. physical interaction or part of functional, not necessary, direct association. Unfortunately, the determination of the structure of protein complexes cannot keep pace with the discovery of new protein-protein interactions resulting in a large, and increasing, gap between the number of complexes that are thought to exist and the number for which 3D structures are available. The aim of the thesis was to tackle this problem by implementing computational approaches to derive structural models of protein complexes and thus reduce this existing gap. Over the course of the thesis, a novel modelling algorithm to predict the structure of protein complexes, V-D2OCK, was implemented. This new algorithm combines structure-based prediction of protein binding sites by means of a novel algorithm developed over the course of the thesis: VORFFIP and M-VORFFIP, data-driven docking and energy minimization. This algorithm was used to improve the coverage and structural content of the human interactome compiled from different sources of interactomic data to ensure the most comprehensive interactome. Finally, the human interactome and structural models were compiled in a database, V-D2OCK DB, that offers an easy and user-friendly access to the human interactome including a bespoken graphical molecular viewer to facilitate the analysis of the structural models of protein complexes. Furthermore, new organisms, in addition to human, were included providing a useful resource for the study of all known interactomes

Functional characterization of bacterial sRNAs involved in stress responses and quorum sensing of bacterial pathogens

Pathogenic bacteria such as Vibrio cholerae, the causative agent of cholera disease, thrive in host and non-host environments. This lifestyle requires constant monitoring of environmental signals to mediate adaptation through changes in gene expression. Furthermore, V. cholerae employs quorum sensing systems to synchronize group behaviors, such as biofilm formation and virulence gene expression. Both processes frequently involve regulation of gene expression by small regulatory RNAs (sRNAs). Bacterial sRNAs base-pair to trans encoded target mRNAs to alter their stability or translation. Recent studies in V. cholerae identified 107 putative sRNAs with yet undescribed functions. The present work aimed to characterize candidate sRNAs with respect to their involvement in stress responses and quorum sensing. Specifically, a bioinformatical approach aiming to identify binding sites of the envelope stress related, alternative sigma factor σE, identified the MicV sRNA as a member of the σE regulon. The σE stress response of V. cholerae involves regulation by another sRNA, VrrA. Expression of both sRNAs is activated under high cell density conditions or by treatment with membrane damaging agents. Global transcriptome analyses revealed that both sRNAs act together to control outer membrane protein expression, and to maintain envelope homeostasis under membrane damaging conditions. We pinpoint collective functions of both sRNAs to the presence of a conserved base-pairing domain in both sRNAs. Finally, laboratory selection experiments employing a library of synthetic sRNAs revealed that regulation of a single porin is sufficient to mediate stress relief. The third sRNA studied in this thesis, VadR, was identified using a genetic screen to assess sRNAs affecting cell curvature of V. cholerae. Analyses of the vadR promoter revealed that vadR expression is activated in response to cell wall damage by the VxrAB two component system. Transcriptome analyses revealed that VadR regulates a large gene cluster responsible for synthesis of the biofilm matrix, and biofilm formation was inhibited in cells overexpressing vadR. Additionally, we found that VadR regulates cell curvature by inhibiting translation of crvA, encoding the major curvature determinant of V. cholerae. Finally, we pinpoint regulation of crvA by VadR to be critical to mediate resistance to cell wall damage. Expression of the fourth sRNA analyzed in this study, VqmR, is controlled by quorum sensing system, involving the autoinducer DPO. Analyses aiming to identify additional mRNA targets for the VqmR sRNA found five additional targets, including the low cell density master regulator aphA. VqmR base-pairs to aphA using an unusual site located in the rho-independent terminator of the sRNA. Regulation of aphA by VqmR resulted in reduced virulence gene expression. Finally, the present work found that the interplay of all three quorum sensing systems is required to achieve a full quorum sensing response.Bakterielle Pathogene wie Vibrio cholerae, der Erreger der Cholera, finden sich in der Umwelt und in Wirtsorganismen. Dieser Lebenszyklus setzt ein konstantes Überwachen von Umweltsignalen voraus, um sich durch entsprechende Genexpression anzupassen. Zudem verwendet V. cholerae bakterielle Kommunikationssysteme um Gruppenverhalten, wie das Bilden von Biofilmen und die Expression von Virulenzgenen, in der Population zu synchronisieren. Diese beiden Prozesse beinhalten häufig die Regulation der Genexpression durch kleine regulatorische RNAs (sRNAs). Bakterielle sRNAs interagieren über Basen-paarungen mit trans-kodierten mRNAs, um deren Stabilität oder Translation zu regulieren. Jüngste Studien haben 107 potenzielle sRNAs, mit bisher ungeklärten Funktionen, in V. cholerae identifiziert. Ziel der vorliegenden Arbeit war es diese sRNAs, unter Berücksichtigung ihrer Beteiligung an Stressantworten und bakterieller Kommunikation, zu charakterisieren. Im Einzelnen wurde ein bioinformatischer Ansatz verwendet, um die Bindestellen des Membranstress-Sigmafaktors σE zu finden und dabei wurde die sRNA MicV als Teil des σE-Regulons identifiziert. Die σE Stressantwort in V. cholerae beinhaltet Regulation durch eine weitere sRNA, VrrA. Die Expression beider sRNAs wird bei hohen Zelldichten, oder durch Behandlung mit membranschädigenden Substanzen, aktiviert. Transkriptomanalysen zeigten das beide sRNAs gemeinsam die Expression von Proteinen der äußeren Membran regulieren, um die Membranhomeostase unter Membranstressbedingungen zu gewährleisten. Die überlappenden Funktionen beider sRNAs begründen sich durch das Vorhandensein einer konservierten Domäne zur Basenpaarung. Abschließend zeigten wir durch Selektionsexperimente mit einer Sammlung von synthetischen sRNAs, dass die Regulation eines einzigen Porins ausreicht, um Membranstress zu lindern. Die dritte sRNA in dieser Studie, VadR, wurde durch einen genetischen Screen für sRNAs die die Zellkrümmung von V. cholerae beeinflussen gefunden. Analysen des vadR Promotors zeigten das die Expression von vadR bei Zellwandstress durch das VxrAB Zwei-komponentensystem aktiviert wird. Transkriptomanalysen zeigten das VadR ein großes Gencluster, verantwortlich für die Synthese der Biofilm Matrix, reguliert. Demzufolge war die Bildung von Biofilmen in Zellen die vadR überexprimieren inhibiert. Zusätzlich regulierte VadR durch Inhibieren der Translation der crvA mRNA, welche für eine wichtige Zellkrümmungs-determinante kodiert, die Zellkrümmung. Letztendlich zeigten wir das die Regulation von crvA durch VadR entscheidend für die Resistenz gegen Zellwandstress ist. Die Expression der vierten sRNA in dieser Studie, VqmR, wird durch ein bakterielles Kommunikationssystem und den Autoinducer DPO reguliert. Durch Analysen, die darauf abzielten neue Ziel-mRNAs der VqmR sRNA zu identifizieren, konnten fünf weitere Ziel-mRNAs identifiziert werden, welche unter anderem, für den Hauptregulator bei niedriger Zelldichte, AphA, kodieren. Die Basenpaarung zwischen VqmR und aphA ist ungewöhnlich, und benötigte eine Region im Rho-unabhängigen Terminator der sRNA. Die Regulation von aphA durch VqmR resultierte in einer Reduktion der Virulenzgenexpression. Schlussendlich konnte die vorliegende Arbeit zudem zeigen das drei bakterielle Kommunikationssysteme zusammenwirken, um ihre volle Wirkung zu entfalten