Implementation and development of X-ray and electron diffraction methods for the structural characterisation of nanocrystalline materials

The present PhD thesis focuses on the development and application of innovative methodologies for structural characterization of materials with emphasis on proteins of pharmaceutical interest. Specifically, the polymorphism of human insulin molecule has been extensively studied and the structure of specific insulin polymorphs has been solved using X-ray crystallography. For the purpose of this thesis, several crystallization experiments of insulin have been performed under various conditions (pH, organic ligands). Additionally the effect of oxidative stress on insulin’s structure has been examined as well (Chapters 2 & 7), after constructing successfully an innovative system for artificially producing hydroxyl radicals. The later study has been performed in collaboration with the Biochemistry research group, of the section of Genetics, Cell Biology & Development, Department of Biology, at the University of Patras.The crystals obtained from the above mentioned crystallisations were employed in several X-ray diffraction experiments, which were initially performed using a laboratory source and later on at the European Synchrotron Radiation Facility, ESRF, in France. The main technique employed for the crystallographic studies was the X- Ray powder diffraction (XRPD) method, which has been proven to be an important tool for determining the properties and structure of polycrystalline protein precipitants during the last decade. In parallel, some single crystal X-ray diffraction experiments have been performed as well in order to validate our results and extract further information by combining both the two techniques. Our results regarding insulin’s co-crystallisation with ligands include the complete characterization of all insulin polymorphs obtained, whereas the distinct insulin conformations observed and their potential pharmaceutical importance have been stated. Among the various polymorphs obtained, two novel insulin crystalline polymorphs have been identified with unit cell parameters which have not been published before and thus their three dimensional structures have been deposited in Protein Data Bank.Regarding the impact that oxidative stress and more specifically of hydroxyl (˙OH) radicals, on insulin’s structure, the design and the construction of an innovative system for artificially producing hydroxyl radicals has been successfully built and can be furthermore applied to investigate several protein systems. The need for constructing such system has arisen from the fact that the only available artificial in vitro ˙OH production system used nowadays, is the Fenton system or its variants. However, all these systems are not providing specifically only the ˙OH radical, as other forms of ROS (Reactive Oxygen Species) exist and more importantly reduced/oxidized metal ions, antioxidants and ˙OH scavengers coexist in the same system. Thus, with such systems it wouldn’t be possible to produce exclusively selected radicals and isolate their effect on the proteins studied.Our results, after exposing human insulin to the novel suggested system, demonstrate some amino acids which have undergone changes due to hydroxyl radicals’ presence whereas the oxidation level versus exposure time has been defined, as obtained through spectrophotometry and fluorescence spectroscopy. In addition the effects caused in the three dimensional structure of human insulin have been examined through X- ray crystallography. By identifying some sensitive-to-radicals amino acids we can suggest the development of insulin analogues having amino acids least prone to radicals so that insulin can remain functional even under conditions of oxidative stress. Lastly, in terms of characterizing materials structurally, several diffraction experiments have been performed using transmission electron microscopy and the Manual Diffraction Tomography (MDT) method, for characterizing materials of high scientific importance due to their superconducting properties, li lithium-rich minerals of great geochemical importance, as well as samples of high archaeological importance. The Precession Electron Diffraction (PED) method and the diffraction tomography were combined in order to drastically reduce the dynamical scattering effect (particularly intense during electron diffraction), allowing to correctly record the reflections in three dimensional reciprocal space and extract accurate structural information.Η παρούσα Διδακτορική Διατριβή αφορά στην ανάπτυξη και εφαρμογή καινοτόμων μεθοδολογιών για τον δομικό χαρακτηρισμό υλικών εστιάζοντας σε πρωτεΐνες φαρμακευτικού ενδιαφέροντος. Συγκεκριμένα, πραγματοποιήθηκε μελέτη του πολυμορφισμού της ινσουλίνης και επίλυση της δομής πολυμόρφων της μέσω κρυσταλλογραφίας ακτίνων-Χ. Για το σκοπό αυτό πραγματοποιήθηκαν πολυάριθμα πειράματα κρυστάλλωσης της ινσουλίνης υπό διαφορετικές συνθήκες (pH, οργανικοί προσδέτες). Επιπλέον πραγματοποιήθηκε μελέτη της επίδρασης του οξειδωτικού στρες στη δομή της ανθρώπινης ινσουλίνης (Κεφάλαια 2 και 7), για τη μελέτη της οποίας καταστευάσθηκε εκ νέου σύστημα ελεγχόμενης παραγωγής ελεύθερων ριζών. Η τελευταία μελέτη πραγματοποιήθηκε σε συνεργασία με την ομάδα Βιοχημείας του Τομέα Γενετικής, Βιολογίας Κυττάρου & Ανάπτυξης, του Τμήματος Βιολογίας του Πανεπιστημίου Πατρών. Οι κρύσταλλοι ινσουλίνης που προέκυψαν είτε μεταβάλλοντας τις συνθήκες κρυστάλλωσης είτε λόγω του οξειδωτικού στρες, μελετήθηκαν μέσω της περίθλασης ακτίνων-Χ αρχικά με τη χρήση εργαστηριακού περιθλασίμετρου και στη συνέχεια στο Ευρωπαϊκό σύγχροτρον, ESRF, της Γαλλίας. Η βασική τεχνική που ακολουθήθηκε για τα πειράματα κρυσταλλογραφίας ήταν η περίθλαση ακτίνων-Χ από πολυκρυσταλλικά δείγματα (X-Ray Powder Diffraction-XRPD), η οποία τα τελευταία χρόνια έχει αναδειχθεί σε ένα σημαντικό εργαλείο χαρακτηρισμού των πρωτεϊνικών πολυκρυσταλλικών ιζημάτων. Παράλληλα διεξήχθησαν και κάποια πειράματα περίθλασης ακτίνων-Χ από μονοκρυσταλλικά δείγματα (Single crystal diffraction) ώστε να εξαχθούν περαιτέρω πληροφορίες χρησιμοποιώντας συνδυαστικά τις δυο τεχνικές.Από τη μελέτη συγκρυστάλλωσης της ινσουλίνης με τους οργανικούς προσδέτες, προσδιορίστηκαν εκτενώς τα κρυσταλλογραφικά χαρακτηριστικά των πολυμόρφων που προέκυψαν, ενώ εξήχθησαν σημαντικά συμπεράσματα για τις διαμορφώσεις που λαμβάνει η ινσουλίνη παρουσία αυτών, καθώς και η φαρμακευτική σημασία τους. Ανάμεσα στα πολύμορφα που εντοπίσθηκαν, ταυτοποιήθηκαν δυο νέα πολύμορφα ινσουλίνης που χαρακτηρίζονται κρυσταλλογραφικά από πλεγματικές σταθερές που δεν έχουν καταχωρηθεί στην παγκόσμια τράπεζα πρωτεϊνικών δομών (Protein Data Bank) όπου και καταθέσαμε τις ακριβείς συντεταγμένες των πρωτεϊνικών δομών όπως προέκυψαν πειραματικά.Για την εκπόνηση της μελέτης της επίδρασης του οξειδωτικού στρες στην δομή της ινσουλίνης, πραγματοποιήθηκε μελέτη και κατασκευή ενός καινοτόμου συστήματος επιτυχούς παραγωγής ελεύθερων ριζών με ελεγχόμενο ρυθμό, το οποίο μπορεί να χρησιμοποιηθεί για τη μελέτη της επίδρασης συγκεκριμένων ριζών σε ποικίλα πρωτεϊνικά συστήματα. Η ανάγκη αυτή προέκυψε καθώς το μοναδικό τεχνητό διαθέσιμο σύστημα παραγωγής ριζών (σύστημα Fenton) περιέχει παρεμβάλλοντα οξειδωτικά ή αντιοξειδωτικά συστατικά με αποτέλεσμα να μην επιτρέπει εξειδικευμένα την παραγωγή ελευθέρων ριζών και τη μελέτη της επίδρασής τους. Συγκεκριμένα από τη μελέτη στην ινσουλίνη, εντοπίσθηκαν κάποια από τα αμινοξέα που επηρεάζονται από τη ρίζα υδροξυλίου και ο βαθμός οξείδωσής τους συναρτήσει του χρόνου έκθεσης μέσω τεχνικών φωτομετρίας, ενώ επιπλέον η ινσουλίνη εξετάσθηκε μέσω της κρυσταλλογραφίας ακτίνων-Χ για τον εντοπισμό ενδεχόμενων αλλοιώσεων που προκαλούνται στη στερεοδιαμόρφωσή της. Ο εντοπισμός των αμινοξέων που επηρεάζονται μέγιστα από τη ρίζα υδροξυλίου αποτελεί σημαντικό εύρημα καθώς αντικατάσταση (μέσω μετάλλαξης ή χημικής σύνθεσης) των ευπρόσβλητων αμινοξέων, θα μπορούσε να προστατεύσει την ινσουλίνη, καθιστώντας τη λειτουργική ακόμα και υπό συνθήκες οξειδωτικού στρες. Τέλος, στα πλαίσια του δομικού χαρακτηρισμού υλικών, πραγματοποιήθηκαν πολυάριθμα πειράματα περίθλασης ηλεκτρονίων με τη χρήση ηλεκτρονικού μικροσκοπίου διελεύσεως και την τεχνική της ηλεκτρονικής τομογραφίας (Manual Diffraction Tomography, MDT) σε υλικά υψηλού ερευνητικού ενδιαφέροντος λόγω των υπεραγώγιμων ιδιοτήτων τους, σε ορυκτά λιθίου τα οποία παρουσιάζουν ιδιαίτερο γεωχημικό ενδιαφέρον καθώς και σε δείγματα αρχαιολογικής σημασίας. Η τεχνική της μετάπτωσης ηλεκτρονικής δέσμης (Precession Electron Diffraction, PED) η οποία χρησιμοποιήθηκε συνδυαστικά με την ηλεκτρονική τομογραφία, μείωσε δραστικά το φαινόμενο της δυναμικής σκέδασης (ιδιαίτερα έντονο κατά την περίθλαση ηλεκτρονίων) επιτρέποντας τη σαφή καταγραφή ανακλάσεων στον τρισδιάστατο αντίστροφο χώρο και την εξαγωγή δομικής πληροφορίας με μεγάλη ακρίβεια

Ένας Αλγόριθμος για το Σχεδιασμό Προσανατολισμένων Γράφων

The use of images and diagrams is most of the times the most effective method for the transmission of abstract as well as concrete information, due to the inherent human ability to perceive more easily information displayed graphically. Graphs are a form of graphical representation of information. This thesis deals with the design and implementation of a graph layout algorithm for hierarchically drawn directed graphs. The algorithm is given as input the list of the edges of the graph and produces in the output the coordinates where each node should be put in order to produce an aesthetically pleasing drawing of the graph. A well known graph layout algorithm for directed graphs is the algorithm which was developed by Sygiyama, Tagawa and Toda (STT algorithm), and was a major concern for scientists during the past decade. We initially implemented this algorithm, which is time inefficient when drawing large graphs and it often creates unwanted edge bends. The new algorithm initially selects a spanning tree of the graph. Then, it traverses the spanning tree and rearranges its nodes in order to reduce the number of crossings between edges of the graph. In the end, it uses a tree layout algorithm which computes the coordinates of the nodes of the spanning tree. We present the algorithm we designed and evaluate it by comparing it with the STT algorithm. Our algorithm achieves layout times under 1 second for small and medium sized graphs and it can be as much as ten times faster that the STT algorithm for large graphs. Furthermore it minimizes the number of edge bends

ArrayTracer: A Parallel Performance Analysis Tool

ArrayTracer is a high-level, low-overhead performance analysis tool for parallel applications. It provides the selective tracing facilities at a user-defined grain. The tracing technique used is program instrumentation. Instrumentation code is inserted at source code level during a source-to-source translation. The tool allows tracing of application's high-level concepts (e.g. program variables, subroutine calls, interprocess communication events, etc.) rather than tracing of low-level events (e.g. memory location accesses, disk accesses, network accesses, etc.). ArrayTracer is heavily based on compiletime information and attempts to off-load the execution time tracing overhead by extracting as much information as possible during the static analysis of application's source code. The tool's operations are divided into five stages: user interaction to determine trace parameters, instrumentation of source code during the compilation phase, collection of traces, processing of traces and an..

In Quest for Improved Drugs against Diabetes: The Added Value of X-ray Powder Diffraction Methods

Human insulin (HI) is a well-characterized natural hormone which regulates glycose levels into the blood-stream and is widely used for diabetes treatment. Numerous studies have manifested that despite significant efforts devoted to structural characterization of this molecule and its complexes with organic compounds (ligands), there is still a rich diagram of phase transitions and novel crystalline forms to be discovered. Towards the improvement of drug delivery, identification of new insulin polymorphs from polycrystalline samples, simulating the commercially available drugs, is feasible today via macromolecular X-ray powder diffraction (XRPD). This approach has been developed, and is considered as a respectable method, which can be employed in biosciences for various purposes, such as observing phase transitions and characterizing bulk pharmaceuticals. An overview of the structural studies on human insulin complexes performed over the past decade employing both synchrotron and laboratory sources for XRPD measurements, is reported herein. This review aims to assemble all of the recent advances in the diabetes treatment field in terms of drug formulation, verifying in parallel the efficiency and applicability of protein XRPD for quick and accurate preliminary structural characterization in the large scale

Applications of X-ray Powder Diffraction in Protein Crystallography and Drug Screening

Providing fundamental information on intra/intermolecular interactions and physicochemical properties, the three-dimensional structural characterization of biological macromolecules is of extreme importance towards understanding their mechanism of action. Among other methods, X-ray powder diffraction (XRPD) has proved its applicability and efficiency in numerous studies of different materials. Owing to recent methodological advances, this method is now considered a respectable tool for identifying macromolecular phase transitions, quantitative analysis, and determining structural modifications of samples ranging from small organics to full-length proteins. An overview of the XRPD applications and recent improvements related to the study of challenging macromolecules and peptides toward structure-based drug design is discussed. This review congregates recent studies in the field of drug formulation and delivery processes, as well as in polymorph identification and the effect of ligands and environmental conditions upon crystal characteristics. These studies further manifest the efficiency of protein XRPD for quick and accurate preliminary structural characterization

Solid solutions along the synthetic LiAlSi2O6 (spodumene)-LiFeSi2O6 (ferri-spodumene) join: a general picture of solid solutions, bond lengths, lattice strains, steric effects, symmetries and chemical compositions of Li clinopyroexenes

Seven clinopyroxene compositions along the joinM2LiM1AlTSi2O6(spodumene) toM2LiM1Fe3+TSi2O6(ferri-spodumene) were synthesized at 2 GPa, 800 °C under highly oxidizing conditions (using H2O2fluid) in an end-loaded piston cylinder. In addition, the LiFe3+TSi2O6composition was also synthesized under the intrinsically reducing conditions in a piston cylinder, to check the effect of fO2 on iron speciation. The run products were characterized by field emission scanning electron microscope (FE-SEM), Rietveld refinements on XRPD synchrotron data, and space groups were assigned using SAED-TEM patterns. Run products are composed mainly of lithium clinopyroxene (Li-Cpx), plus minor amounts of hematite (magnetite under reducing condition) and corundum, as independently detected by image analysis (area%) and Rietveld refinements (wt%); moreover, Rietveld results were used to derive cell parameters, M1-site occupancy (Al vs. Fe3+), atomic positions, and average bond lengths of all these Li-Cpx indexed in the C2/c space groups according to SAED-TEM. Li-Cpx with Al and Fe3+amounts close to 50:50 are actually slightly richer in Al apfu than nominal; the LiFe3+Si2O6grown under very oxidized and reducing conditions have very similar cell parameters, indicating that fO2 is unable to induce a significant incorporation of Fe2+ in these Li-Cpx. The replacement of Al with Fe3+induces a linear (%) increase of the cell edges following b ≥ a > c, whereas β is roughly constant and the cell volume increases linearly. Furthermore, the substitution of Al with Fe3+ only weakly affects the T-O average length ( b > a > c, whereas ? is roughly constant except for Ti-end-member and P21/c compositions. Lattice strains induced by X, T, and P for Li-Cpx in the C2/c stability field show that when the M1 site is progressively filled with a large cation, ϵ1 axis (ϵ1ϵ2ϵ3) increases along b, whereas ϵ2and ϵ3are nearly parallel to a and at about 30° from c. Conversely, T will provoke a similar enlargement of ϵ1and ϵ2along b and a edges, respectively, whereas ϵ3is again oriented at about 30° from c; the increasing of P will instead shorten all strain tensor components (ϵ1, ϵ2, and ϵ3) with a similar percentage amount; notably, high-P is the only stress that induces a strain component to be almost parallel to c edge. Moreover, finite lattice strains and orientation in C2/c LiMe3+Si2O6Li-Cpx induced by Me3+: Al-Fe3+, Fe3+-Sc, Sc-In are slightly different, with &1 invariably lying along b; conversely, Li-Na cation substitution is completely different with the highest and lowest deformations on the ac plane and ϵ2along b; ϵ3vector is negative and oriented at about 30° from T-chains. The ideal replacement of Al with larger cations up to In in Li-Cpx induces the M1-O, M2-O, and T-O average bond lengths to increase by 10.6, 4.3, and <0.5%. Steric effects in LiM1Me3+Si2O6and NaM1Me3+Si2O6 Cpx are significant and very similar, whereas several other Me1+and Me2+substitutions in Cpx at both the M1 and M2 site, keeping fixed the other site, display less or even the absence of steric effects. Our new data also better elucidate relationships between Li-Cpx composition, symmetry at room and non-Ambient conditions and Tc. The aggregate cation radii at the M1 site does not exclusively control the stability of C2/c and P21/c polymorphs; instead valence electrons can profoundly favor the stabilization of a polymorph

Unit-cell response of tetragonal hen egg white lysozyme upon controlled relative humidity variation

Variation of relative humidity (rH) greatly affects the internal order of solvent-based protein crystals, and the rearrangement of molecules can be efficiently recorded in distinct diffraction patterns. This study focuses on this topic, reporting the effect of rH variation experiments on hen egg white lysozyme (HEWL) polycrystalline precipitates of tetragonal symmetry using X-ray powder diffraction (XRPD). In situ XRPD data were collected on HEWL specimens during dehydration and rehydration processes using laboratory instrumentation. A known polymorph [space group P43212, a = 79.07181 (1), c = 38.0776 (1) Å] was identified during gradual dehydration from 95 to 63% rH and vice versa. Pawley analysis of collected data sets and accurate extraction of unit-cell parameters indicated a characteristic evolution of the tetragonal axes with rH. In addition, there is a low humidity level below which samples do not retain their crystallinity. This work illustrates the accuracy of laboratory XRPD as a probe for time-resolved studies of proteins and in situ investigations of gradual structural modifications upon rH variation. These experiments provide essential information for improving production and post-production practices of microcrystalline protein-based pharmaceuticals. © International Union of Crystallography, 201