Imaging amyloid fibers at the nanoscale: Method development and applications for hybrid materials and biomedicine

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Física de la Materia Condensada. Fecha de lectura: 13-12-2019Esta tesis tiene embargado el acceso al texto completo hasta el 13-06-2021In the last decades, advanced imaging techniques have improved our ability to analyze biological systems at the nanoscale, enabling the observation of structural and molecular components. Different imaging tools are specialized in the characterization of a specific aspect of the sample and, when they are combined, complementary information is obtained providing a more comprehensive understanding of the system. This thesis focuses on the application of (super-resolution) fluorescence microscopy in combination with atomic force microscopy (AFM) for revealing specific chemical information in a high-resolution topography map. Particularly, correlative microscopy is applied to the characterization of amyloid fibers, which are misfolded protein aggregates with interest in nanomaterials research and biomedicine. This manuscript is organized in seven chapters. Chapter 1 introduces the imaging techniques used in the thesis. It also gives a general overview on amyloid fibers, their application as hybrid materials, their importance in biomedicine for being involved in different diseases, and the phototherapeutic approaches available to treat them. In Chapter 2, the general materials and methods used during the thesis are explained. Chapter 3 provides a detailed discussion about technical aspects of correlative super-resolution fluorescence microscopy and AFM such as sample preparation, data analysis and image alignment. Furthermore, the advantage of using AFM as a “ground truth” to evaluate different aspects of super-resolution techniques, such as labeling or image reconstruction, is highlighted. In Chapter 4, the methodology developed in Chapter 3 is applied to evaluate the functionalization of amyloid fibers with quantum dots or organic fluorophores. Thus, correlative microscopy is presented as a useful technique for characterizing luminescent hybrid materials at the nanoscale. In the context of biomedicine, amyloid aggregates are important for being involved in different diseases (e.g. Alzheimer or Parkinson). Photochemical strategies to degrade amyloid structures are becoming an interesting alternative. In this thesis, a thioflavin T (ThT) derivative (ROS-ThT), which is able to target pathogenic aggregates in the presence of functional proteins, is used to study photodamage effects on amyloid fibers. In addition to fluorescence, this photocatalyst or photosensitizer produces singlet oxygen Abstract upon blue light exposure, affecting amyloid structures through oxidation. The purpose of Chapter 5 is to select a useful amyloid model to evaluate photodamage at the nanoscale, and therefore different fibers were produced, fibrillated and characterized. In Chapter 6, the selected amyloid model is used to study photodamage induced by ROS-ThT at the single-fiber level through imaging techniques, and complemented by classical biochemical assays. These experiments highlight that the combination of fluorescence microscopy and AFM is useful to probe the heterogeneity of amyloid material and to disentangle the complex dependence between photocatalyst binding/activity and fiber morphology and/or composition. The aim of Chapter 7 is to provide coherence and perspective to the main results of the thesis, as well as an outlook on how advanced microscopy methods may impact the study of amyloids in different fields of researchQuiero agradecer también al Ministerio de Economía y Competitividad por financiar mi trabajo con la beca FPI BES-2016-076293 dentro del proyecto MAT2015-66605-P y al Ministerio de Ciencia, Innovación y Universidades por financiar el proyecto PCI2018‐093064

Uso de superficies free-form para simular los cambios locales de indice de refracción en lentes de plástico a partir de su medida polariscópica

In this paper we propose a method to convert the local variations of refractive index into local changes of one surface of the lens, which then can then be described as a free-form surface, easy to introduce in conventional simulation tools. The proposed method tries to provide an equivalent model of the lens with refractive index variations where the index changes have been replaced for local thickness variations on the lens' surface. The local thickness variations can be fitted at a freeform description that holds all the local index variations through the OPD (Optical patch difference) using a B-Spline description and a simplex algorithm to find the best fitted surface.Peer ReviewedPostprint (published version

The reduction of the metabolyc syndrome in Navarra-Spain (RESMENA-S) study; a multidisciplinary strategy based on chrononutrition and nutritional education, together with dietetic and psychological control

Introduction: The high prevalence of metabolic syndrome (MS) in Spain requires additional efforts for prevention and treatment. Objective: The study RESMENA-S aims to improve clinical criteria and biomarkers associated with MS though an integral therapy approach. Methods: The study is a randomized prospective parallel design in which is expected to participate a total of 100 individuals. The RESMENA-S group (n = 50) is a personalized weight loss (30% energy restriction) diet, with a macronutrient distribution (carbohydrate / fat / protein) of 40/30/30, high meal frequency (7 / day), low glycemic index/load and high antioxidant capacity as well as a high adherence to the Mediterranean diet. The control group (n = 50) is assigned to a diet with the same energy restriction and based on the American Heart Association pattern. Both experimental groups are under dietary and psychological control during 8 weeks. Likewise, for an additional period of 16 weeks of self-control, is expected that volunteers will follow the same pattern but with no dietary advice. Results: Anthropometrical data and body composition determinations as well as blood and urine samples are being collected at the beginning and end of each phase. This project is registered at www.clinicaltrials.gov with the number NCT01087086 and count with the Research Ethics Committee of the University of Navarra approval (065/2009). Conclusions: Intervention trials to promote the adoption of dietary patterns and healthy lifestyle are of great importance to identify the outcomes and nutritional mechanisms that might explain the link between obesity, metabolic syndrome and associated complications

Imaging amyloid fibers at the nanoscale: method development and applications for hybrid materials and biomedicine

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid. Fecha de lectura: 13-12-2019

Uso de superficies free-form para simular los cambios locales de indice de refracción en lentes de plástico a partir de su medida polariscópica

In this paper we propose a method to convert the local variations of refractive index into local changes of one surface of the lens, which then can then be described as a free-form surface, easy to introduce in conventional simulation tools. The proposed method tries to provide an equivalent model of the lens with refractive index variations where the index changes have been replaced for local thickness variations on the lens' surface. The local thickness variations can be fitted at a freeform description that holds all the local index variations through the OPD (Optical patch difference) using a B-Spline description and a simplex algorithm to find the best fitted surface.Peer Reviewe

Long-term STED imaging of amyloid fibers with exchangeable Thioflavin T

We report the use of the amyloid probe Thioflavin T (ThT) as a specific and exchangeable fluorophore for stimulated emission depletion (STED) super-resolution imaging of amyloid fibers. This method achieves a spatial resolution in the range of 60–70 nm, low image background and increased photostability that enables long-term STED imaging. These results expand the widespread uses of ThT and can be potentially extended to other common amyloid fluorescent probes, providing new tools for the study of amyloid diseases.This research was supported by the Ministerio de Ciencia, Innovación y Universidades through projects PCI2018-093064 and PGC2018-094802-B-I00 (C. F.), SAF2017-87305-R (B. S.) and SEV-2016-0686 (IMDEA Nanociencia), and the Comunidad Autónoma de Madrid project S2017/BMD-3867 (C.F.)

Mechanically Induced Bacterial Death Imaged in Real Time: A Simultaneous Nanoindentation and Fluorescence Microscopy Study

Mechano-bactericidal nanomaterials rely on their mechanical or physical interactions with bacteria and are promising antimicrobial strategies that overcome bacterial resistance. However, the real effect of mechanical versus chemical action on their activity is under debate. In this paper, we quantify the forces necessary to produce critical damage to the bacterial cell wall by performing simultaneous nanoindentation and fluorescence imaging of single bacterial cells. Our experimental setup allows puncturing the cell wall of an immobilized bacterium with the tip of an atomic force microscope (AFM) and following in real time the increase in the fluorescence signal from a cell membrane integrity marker. We correlate the forces exerted by the AFM tip with the fluorescence dynamics for tens of cells, and we find that forces above 20 nN are necessary to exert critical damage. Moreover, a similar experiment is performed in which bacterial viability is assessed through physiological activity, in order to gain a more complete view of the effect of mechanical forces on bacteria. Our results contribute to the quantitative understanding of the interaction between bacteria and nanomaterials.This work was supported by the Spanish Ministerio de Ciencia, Innovacion y Universidades (MAT2015-66605-P, PGC2018-094802-B-I00, and SEV-2016-0686), and the Comunidad Autonoma de Madrid (PEJD-2016/IND-2774, S2017/BMD- ́3867). We thank Prof. Piet A. J. de Boer (Case Western Reserve University) for the gift of plasmid pDR122 (GFPMinD) and Prof. Pedro J. de Pablo (Universidad Autonoma de Madrid) for insightful discussions, and Dr. Pawel Hermanowicz for assistance with AtomicJ and the implementation of version 2.1.2

Analys av gynnsamma affärsammansättningar för bildäck

Abstract The average price of commodities has over the past years increased significantly and already erased the real price fall seen during the 20th century. Humanity is today consuming resources corresponding to 1,5 earths which puts pressure on manufacturers to develop more sustainable and resource conservative business models. The current market for car tires isstanding before a potential change in the tires conceptual design, which opens up for manufacturers to take advantage of the situation to develop a more sustainable proposition to customers. Research up until today has mainly focused on the supply chain without including the necessary business model. Lately the holistic concept Resource Conservative Manufacturing (ResCoM) has been developed to take the full grasp of the problem. This thesis therefore focuses on finding a range of favourable business model compositions for cartires from the perspective ResCoM. Initially, this thesis investigates the ResCoM model and its requirements on a business model. Furthermore the market for car tires and the product itself is investigated thoroughly to find elements that affect the business model composition. Afterwards a tool for evaluation of thejoint effect of the products as well as the markets individual effects on the business model composition is developed in the form of a key figure. The key figure is used as an indicatorfor favourable business model compositions to meet the products, the markets and theResCoM models requirements. Finally the key figure is analysed and a top and bottom border for the range of favourable business model compositions is defined. The result illustrates that the bottom border for favourable business model composition for car tires is located at rental, whereas the top boarder stretches to, but does not include, functional sales. The developed tool has someconstraints when used and therefore the conclusion is that the purpose of the thesis has beenfulfilled to a limited extent.Sammanfattning Det genomsnittliga priset på råvaror har under det senaste århundradet ökat signifikant och raderat ut den reella prissänkning som skedde under 1900-talet. Idag använder mänskligheten resurser som motsvarar 1,5 jordklot vilket sätter krav på tillverkare att utveckla mer hållbara och resurssnåla affärsmodeller. Marknaden för bildäck står idag inför en potentiell ändring av bildäckets konceptuella design vilket öppnar upp för tillverkare att dra nytta av denna situation och utveckla ett mer hållbart erbjudande till kunder. Forskning har fram till idag framförallt fokuserat på försörjningskedjor utan att inkludera lämpliga affärsmodeller. Det holistiska konceptet Resource Conservative Manufacturing (ResCoM) har den senaste tiden utvecklats genom att fullständigt angripa problemet och dess omfattning. Denna uppsats fokuserar således på att hitta gynnsamma affärssammansättningar för bildäck utifrån perspektivet ResCoM. Inledningsvis fokuserar uppsatsen på modellen ResCoM och dess krav på en affärsmodell. Vidare är marknaden för bildäck samt produkten i sig grundligt undersökt för att hitta element som påverkar affärs sammansättningen. Därefter utvecklas ett verktyg för evaluering av de sammanlagda effekterna av de produkt- samt marknadsrelaterade faktorerna på affärssammansättningen i form av ett nyckeltal. Nyckeltalet används som en indikator för gynnsamma affärssammansättningar i syfte att möta kraven från produkten, marknaden och även ResCoM. Slutligen analyseras nyckeltalet där en övre och undre gräns definierar ett spann av gynnsamma affärssammansättningar. Resultatet illustrerar att spannet för gynnsamma affärssammansättningar börjar vid uthyrning och sträcker sig till, men inkluderar inte, funktionsbaserad försäljning. Det utvecklade verktyget kan även konstateras ha vissa begränsningar varpå det slutligen kan fastslås att syftet med arbetet i viss utsträckning uppnåtts

Short-term role of the dietary total antioxidant capacity in two hypocaloric regimes on obese with metabolic syndrome symptoms: the RESMENA randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Dietary strategies seem to be the most prescribed therapy in order to counteract obesity regarding not only calorie restriction, but also bioactive ingredients and the composition of the consumed foods. Dietary total antioxidant capacity (TAC) is gaining importance in order to assess the quality of the diet.</p> <p>Methods</p> <p>Ninety-six obese adults presenting metabolic syndrome (MetS) symptoms completed an 8-week intervention trial to evaluate the effects of a novel dietary program with changes in the nutrient distribution and meal frequency and to compare it with a dietary pattern based on the American Heart Association (AHA) guidelines.</p> <p>Anthropometric and biochemical parameters were assessed at baseline and at the endpoint of the study, in addition to 48-hours food dietary records.</p> <p>Results</p> <p>Both diets equally (<it>p</it> > 0.05) improved MetS manifestations. Dietary TAC was the component which showed the major influence on body weight (<it>p</it> = 0.034), body mass index (<it>p</it> = 0.026), waist circumference (<it>p</it> = 0.083) and fat mass (<it>p</it> = 0.015) reductions. Transaminases (ALT and AST) levels (<it>p</it> = 0.062 and <it>p</it> = 0.004, respectively) were associated with lower TAC values.</p> <p>Conclusion</p> <p>RESMENA diet was as effective as AHA pattern for reducing MetS features. Dietary TAC was the most contributing factor involved in body weight and obesity related markers reduction.</p> <p>Trial registration</p> <p><url>http://www.clinicaltrials.gov; NCT01087086</url></p