Aceleración de algoritmos de procesamiento de imágenes para el análisis de partículas individuales con microscopia electrónica

Tesis Doctoral inédita cotutelada por la Masaryk University (República Checa) y la Universidad Autónoma de Madrid, Escuela Politécnica Superior, Departamento de Ingeniería Informática. Fecha de Lectura: 24-10-2022Cryogenic Electron Microscopy (Cryo-EM) is a vital field in current structural biology. Unlike X-ray crystallography and Nuclear Magnetic Resonance, it can be used to analyze membrane proteins and other samples with overlapping spectral peaks. However, one of the significant limitations of Cryo-EM is the computational complexity. Modern electron microscopes can produce terabytes of data per single session, from which hundreds of thousands of particles must be extracted and processed to obtain a near-atomic resolution of the original sample. Many existing software solutions use high-Performance Computing (HPC) techniques to bring these computations to the realm of practical usability. The common approach to acceleration is parallelization of the processing, but in praxis, we face many complications, such as problem decomposition, data distribution, load scheduling, balancing, and synchronization. Utilization of various accelerators further complicates the situation, as heterogeneous hardware brings additional caveats, for example, limited portability, under-utilization due to synchronization, and sub-optimal code performance due to missing specialization. This dissertation, structured as a compendium of articles, aims to improve the algorithms used in Cryo-EM, esp. the SPA (Single Particle Analysis). We focus on the single-node performance optimizations, using the techniques either available or developed in the HPC field, such as heterogeneous computing or autotuning, which potentially needs the formulation of novel algorithms. The secondary goal of the dissertation is to identify the limitations of state-of-the-art HPC techniques. Since the Cryo-EM pipeline consists of multiple distinct steps targetting different types of data, there is no single bottleneck to be solved. As such, the presented articles show a holistic approach to performance optimization. First, we give details on the GPU acceleration of the specific programs. The achieved speedup is due to the higher performance of the GPU, adjustments of the original algorithm to it, and application of the novel algorithms. More specifically, we provide implementation details of programs for movie alignment, 2D classification, and 3D reconstruction that have been sped up by order of magnitude compared to their original multi-CPU implementation or sufficiently the be used on-the-fly. In addition to these three programs, multiple other programs from an actively used, open-source software package XMIPP have been accelerated and improved. Second, we discuss our contribution to HPC in the form of autotuning. Autotuning is the ability of software to adapt to a changing environment, i.e., input or executing hardware. Towards that goal, we present cuFFTAdvisor, a tool that proposes and, through autotuning, finds the best configuration of the cuFFT library for given constraints of input size and plan settings. We also introduce a benchmark set of ten autotunable kernels for important computational problems implemented in OpenCL or CUDA, together with the introduction of complex dynamic autotuning to the KTT tool. Third, we propose an image processing framework Umpalumpa, which combines a task-based runtime system, data-centric architecture, and dynamic autotuning. The proposed framework allows for writing complex workflows which automatically use available HW resources and adjust to different HW and data but at the same time are easy to maintainThe project that gave rise to these results received the support of a fellowship from the “la Caixa” Foundation (ID 100010434). The fellowship code is LCF/BQ/DI18/11660021. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 71367

MC 2019 Berlin Microscopy Conference - Abstracts

Das Dokument enthält die Kurzfassungen der Beiträge aller Teilnehmer an der Mikroskopiekonferenz "MC 2019", die vom 01. bis 05.09.2019, in Berlin stattfand

Self-assembly of rod-like colloids at the air-water interface

Two-dimensional (2D) colloidal materials and their assembly are of scientific significance and industrial importance. The development of 2D colloidal structures is a key stepping stone towards three-dimensional (3D) structures in relation to controlled chemical composition, morphology, assembly and so on. Nowadays, uniform colloidal structures with complexity in both shape and interactions have become a popular topic in fundamental colloid science and applications. Being motivated by this, in this thesis, micro-scale colloidal rods and self-assembled dipeptides have been studied experimentally at the air-water interface. Monolayers containing these colloidal materials were created in a Langmuir trough. Surface pressure measurements, microscopic observations and many other techniques were combined for the investigation. The aim of this work is to understand the phase behaviours in complex monolayers, including the phase transitions during compression, the flipping dynamics of micro-rods, the contribution of dipole-dipole interactions between magnetic rods, and the interfacial self-assembly process of dipeptide molecules. Iron oxide micro-rods (β-FeOOH @silica) with different aspect ratios have been synthesized to create the monolayers at an air-water interface. Microscopic observations reveal a sequence of phase transitions by compressing the monolayers. It has been proved that the aspect ratio of the rods plays an important role in the phase transitions, —short rods flip into a perpendicular position relative to the interface to relieve the compressional stress, while longer rods form multilayers under compression. Magnetic rods (Fe3O4) were converted from the synthesized FeOOH rods. They can be aligned in an external field, which further induces the reorganization at the interface. To study these magnetic rods, differential dynamic microscopy (DDM) was carried out to measure the magnetic moment. Their interfacial properties were investigated in an external field applied perpendicular and parallel to the interface, respectively. A magnetic field-induced flipping process has been observed, which proves the theoretical prediction. Besides rod-like particles, naphthalene dipeptides have been successfully trapped at the interface of a low pH subphase, self-assembling into a hydrogel film. The mechanism of interfacial self-assembly has been studied. Both FTIR spectra and AFM images are used to investigate the fibrous structures of the film. The film has elastic properties and buckles under compression. Moreover, dipeptide hydrogel induced by metal ions has been used to create a wet foam system, which owns the advantages of long-term stability (more than two weeks), low cost, and easy preparation

Functionalization of Nanoparticles with Tyrosine Hydroxylase : Biotechnological and Therapeutic Implications

Tyrosine hydroxylase (TH) er et viktig enzym for nervesystemet, fordi det katalyserer det første steget i syntesen av dopamin, noradrenalin og adrenalin. Nivået på TH og dopamin synker hos Parkinsons pasienter pga. den gradvise celledøden i den hjernedelen som heter substantia nigra. Vanlig behandling går ut på å ta levodopa som er forløperen til dopamin, men effekten avtar etter hvert, og pasientene får alvorlige bivirkninger ved høye doser. Det trengs altså et bedre behandlingstilbud. En mulighet kan være å tilføre mer TH vha. en enzymerstatningsterapi som også vil gjenopprette dopaminnivået. Hovedformålet med denne avhandlingen er å finne ut hvordan TH kan bli brukt som biologisk medisin. Vi har derfor utviklet forskjellige nanopartikkel-baserte formuleringer som kan stabilisere og levere TH, og evaluert det terapeutiske potensiale til TH-lastede nanopartikler. Vi begynte med å produsere TH sammen med forskjellige fusjonspartnere og fikk et stabilt enzym som vi målte strukturen av. Så valgte vi porøst silisium og maltodekstrin nanopartikler som mulige bærere av TH. De første forsøkene med porøst silisium viste at det er en sammenheng mellom fotoluminescensen og frigjøringen av et modellprotein som kan være nyttig i sporing av legemiddelleveringen i kroppen. Videre fant vi at TH kunne bindes i nanopartikler av porøst silisium, men at dette førte til aggregering av TH. Maltodekstrin-nanopartikler kunne derimot absorbere store mengder TH samtidig som de forhindret eller forsinket TH aggregeringen. Vi observerte at disse nanopartiklene kunne levere TH til nerveceller og hjernevev og dermed økte den intracellulære TH aktiviteten. Alt i alt har denne avhandlingen gitt et godt innblikk i de strukturelle mekanismene og de funksjonelle forutsetningene som trengs for å kunne lage vellykkede nanopartikkel-baserte formuleringer av TH. TH-lastede nanopartikler har muligheten til å bli videreutviklet til enzymerstatningsterapi for sykdommer hvor det er for lite aktivt TH, som f.eks. ved Parkinsons sykdom.Tyrosine hydroxylase (TH) is important for neuronal function as it is the rate-limiting enzyme in the synthesis of dopamine, noradrenaline, and adrenaline. In Parkinson’s disease, the levels of TH and dopamine decrease, due to progressive loss of the dopaminergic neurons in a part of the midbrain called substantia nigra. Treatment is typically with the dopamine precursor, levodopa, but its pharmacological effect wears off, and the patients develop serious side effects, so there is a need for better treatment options. One alternative could be to replace the lacking TH with an enzyme replacement therapy and thereby restore the dopamine levels. The main goal of this thesis has been to investigate how TH can be pharmacologically developed into a potential biological drug. We have therefore studied different nanoparticle (NP)-based formulations to stabilize and deliver TH and evaluated the therapeutic potential of TH-loaded NPs. We started out by using fusion tags in the preparation of TH to obtain a stable enzyme of which we determined the full-length solution structure. Then we selected porous silicon and maltodextrin NPs as potential carriers of TH. Initial characterization revealed that the photoluminescent properties of porous silicon can be tuned to correlate with the release of a model protein, which can be useful in tracking of drug delivery. Furthermore, we found that TH loading in porous silicon NPs occurred through electrostatic interactions, but that it also induced TH aggregation. On the other hand, maltodextrin NPs absorbed large amounts of TH while preventing or delaying its aggregation. We observed functional delivery of TH by these NPs to neuronal cells and tissue, which significantly increased the intracellular TH activity. All in all, this thesis has given insights into the structural mechanisms and functional prerequisites necessary for successful formulations of TH with NPs, which shows the therapeutic potential of enzyme replacement therapy with TH-loaded NPs.Doktorgradsavhandlin

A Modular and Open-Source Framework for Virtual Reality Visualisation and Interaction in Bioimaging

Life science today involves computational analysis of a large amount and variety of data, such as volumetric data acquired by state-of-the-art microscopes, or mesh data from analysis of such data or simulations. The advent of new imaging technologies, such as lightsheet microscopy, has resulted in the users being confronted with an ever-growing amount of data, with even terabytes of imaging data created within a day. With the possibility of gentler and more high-performance imaging, the spatiotemporal complexity of the model systems or processes of interest is increasing as well. Visualisation is often the first step in making sense of this data, and a crucial part of building and debugging analysis pipelines. It is therefore important that visualisations can be quickly prototyped, as well as developed or embedded into full applications. In order to better judge spatiotemporal relationships, immersive hardware, such as Virtual or Augmented Reality (VR/AR) headsets and associated controllers are becoming invaluable tools. In this work we present scenery, a modular and extensible visualisation framework for the Java VM that can handle mesh and large volumetric data, containing multiple views, timepoints, and color channels. scenery is free and open-source software, works on all major platforms, and uses the Vulkan or OpenGL rendering APIs. We introduce scenery's main features, and discuss its use with VR/AR hardware and in distributed rendering. In addition to the visualisation framework, we present a series of case studies, where scenery can provide tangible benefit in developmental and systems biology: With Bionic Tracking, we demonstrate a new technique for tracking cells in 4D volumetric datasets via tracking eye gaze in a virtual reality headset, with the potential to speed up manual tracking tasks by an order of magnitude. We further introduce ideas to move towards virtual reality-based laser ablation and perform a user study in order to gain insight into performance, acceptance and issues when performing ablation tasks with virtual reality hardware in fast developing specimen. To tame the amount of data originating from state-of-the-art volumetric microscopes, we present ideas how to render the highly-efficient Adaptive Particle Representation, and finally, we present sciview, an ImageJ2/Fiji plugin making the features of scenery available to a wider audience.:Abstract Foreword and Acknowledgements Overview and Contributions Part 1 - Introduction 1 Fluorescence Microscopy 2 Introduction to Visual Processing 3 A Short Introduction to Cross Reality 4 Eye Tracking and Gaze-based Interaction Part 2 - VR and AR for System Biology 5 scenery — VR/AR for Systems Biology 6 Rendering 7 Input Handling and Integration of External Hardware 8 Distributed Rendering 9 Miscellaneous Subsystems 10 Future Development Directions Part III - Case Studies C A S E S T U D I E S 11 Bionic Tracking: Using Eye Tracking for Cell Tracking 12 Towards Interactive Virtual Reality Laser Ablation 13 Rendering the Adaptive Particle Representation 14 sciview — Integrating scenery into ImageJ2 & Fiji Part IV - Conclusion 15 Conclusions and Outlook Backmatter & Appendices A Questionnaire for VR Ablation User Study B Full Correlations in VR Ablation Questionnaire C Questionnaire for Bionic Tracking User Study List of Tables List of Figures Bibliography Selbstständigkeitserklärun

Structural And Intrinsic Disorder In The Regulation Of Protein-Protein Interactions

University of Minnesota Ph.D. dissertation. June 2019. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: David Thomas. 1 computer file (PDF); xx, 174 pages.This thesis applied spectroscopy and molecular dynamics simulation to study the structural biology of actin-binding domains (ABDs) from the spectrin superfamily of proteins as well as an intrinsically disordered region (IDR) of an integral membrane protein called synaptotagmin 1. In the former case, the structural hypothesis being tested was that actin-binding domains exist in distinct conformational states that are either permissive to or inhibitory towards binding of actin filaments. This question was probed using pulsed-EPR, which measured distances between the calponin homology (CH) domains that make up the ABD as proxy for conformation in the presence or absence of actin or with and without disease-causing mutation. The initial hypothesis of a closed compact state being unable to bind actin and an open extended state being binding-competent was largely supported by the data. However, the hypothesis was ultimately refined to conclude that an “open” state is likely to still be a fairly collapsed structure that is dynamically disordered. With this model, future efforts will be able use the model to look for small molecules that perturb the conformational equilibrium of ABDs harboring disease-causing mutations in potentially therapeutically efficacious ways. Moreover, the model can be tested in other ABDs of the protein superfamily to assess similarities and differences in mechanism. In the case of the intrinsically disordered region of synaptotagmin 1, it was hypothesized that a post-translational modification, specifically phosphorylation of a threonine residue, caused a structural change in the IDR that then results in a change in neurotransmitter release. This hypothesis was also tested with spectroscopic methods, mainly FRET and circular dichroism, but also with molecular dynamics. It was found that mimicking the low dielectric environment of the membrane with co-solvents in solution and artificially in silico caused the synaptotagmin 1 IDR to fold into helical structure. The post-translational modification, however, was found to interfere with the formation of helical structure, providing a still incomplete but novel molecular explanation for the effect it has on potentiation of neurotransmitter release observed in vivo. At the very least, the structural model provides a working hypothesis that can be further explored in further work

Towards a review of the EC Recommendation for a definition of the term "nanomaterial"; Part 1: Compilation of information concerning the experience with the definition

In October 2011 the European Commission (EC) published a Recommendation on the definition of nanomaterial (2011/696/EU). The purpose of this definition is to enable determination when a material should be considered a nanomaterial for regulatory purposes in the European Union. In view of the upcoming review of the current EC Definition of the term 'nanomaterial' and noting the need expressed by the EC Environment Directorate General and other Commission services for a set of scientifically sound reports as the basis for this review, the EC Joint Research Centre (JRC) prepares three consecutive reports, of which this is the first. This Report 1 compiles information concerning the experience with the definition regarding scientific-technical issues that should be considered when reviewing the current EC definition of nanomaterial. Based on this report and the feedback received, JRC will write a second, follow-up report. In this Report 2 the JRC will provide a detailed assessment of the scientific-technical issues compiled in Report 1, in relation to the objective of reviewing the current EC nanomaterial definition.JRC.I.4-Nanobioscience

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin