Techniques for the realization of ultra- reliable spaceborne computer Final report

Bibliography and new techniques for use of error correction and redundancy to improve reliability of spaceborne computer

Caracterización de la respuesta inmunitaria inducida por nuevas vacunas contra la tuberculosis basadas en nanopartículas

Lung diseases are a common and diverse group of illnesses that represent a health problem affecting millions of people worldwide. The microenvironment and the immune system in the lung are very important but they are also the most challenging immunological dilemma for the host. On one hand, lung location and function are constantly exposed to pathogens; but on the other hand should be sterile and preserve lung homeostasis through the balance between tolerance and inflammation. For this reason is extremely important to characterize and understand the mechanisms and populations involved in a protective immune response, and find better targets to design vaccines. The aim of this project is to define and understand the immunological profiles that correlate with greater protection generated by preventive and therapeutic vaccines in the lung. Preventive vaccines are generally administered to healthy individuals to prevent or ameliorate infectious diseases, but therapeutic cancer vaccines are administrated once the individual has already developed the disease to strength patient's own immune responses. We will focus on two different lung diseases: an infection caused by a bacteria and lung cancer. To study a bacterial infection, we will use as a model Mycobacterium tuberculosis (Mtb), responsible for Tuberculosis (TB). TB is a serious global health problem and the only licensed vaccine against TB is "Mycobacterium bovis Bacille Calmette-Guérin" (BCG). But BCG has a poor efficacy. To achieve a better understanding of the type of immune responses that confer protection, we will use a mouse model to assess the changes in the immune system produced in the lung by vaccines and in response to Mtb infection. Lung cancer is a devastating disease and a major therapeutic burden with poor survival rates. Cancer cells have been in the focus of the research, but the importance of the immune system is becoming more important. Syngenic murine models entailing the injection of immunological compatible cancer cells in immunocompetent mice will allow us to study of the modulation of the immune system performed by cancer cells. Moreover, we will use therapeutic vaccines against tumor cells to activate anti-tumor T cells to recognize and destroy the tumor, increasing the effectiveness when used alone or in combination with conventional therapies. In both approaches, the immune cell populations present in the bronchoalverolar lavage (BAL) and in the lung parenchyma, and the ones induced by the different experimental conditions, will be analyzed and characterized by flow cytometry and ELISA. Also, cells will be obtained to perform proteomic and gene expression profiles to better define the pathways with impact in controlling infection or cancer cells. The outputs generated by our research would lead in the future to a more rational design of protective prophylactic and therapeutic vaccines against lung diseases.Las enfermedades pulmonares son un grupo diverso de enfermedades que afectan a millones de personas en todo el mundo. El microambiente y el sistema inmunitario en el pulmón son muy importantes a la vez que suponen un dilema para el huésped. Por un lado, por su ubicación y función, el pulmón está constantemente expuesto a patógenos; por otro lado debe ser estéril y preservar la homeostasis a través del equilibrio entre tolerancia e inflamación. Por ello es extremadamente importante caracterizar y comprender los mecanismos y las poblaciones involucradas en una respuesta inmunitaria protectora, y encontrar mejores dianas para diseñar vacunas. El objetivo de este proyecto es definir y comprender los perfiles inmunitarios que correlacionan con una mayor protección generada por vacunas preventivas y terapéuticas en pulmón. Las vacunas preventivas se administran a individuos sanos para prevenir o mejorar enfermedades infecciosas, mientras que las vacunas terapéuticas contra el cáncer se administran, para fortalecer la propia respuesta inmunitaria del paciente. Nos centraremos en dos enfermedades pulmonares: una infección causada por una bacteria y cáncer de pulmón. Para estudiar la infección bacteriana, utilizaremos como modelo Mycobacterium tuberculosis (Mtb), responsable de la tuberculosis. La tuberculosis es un grave problema de salud mundial. La única vacuna autorizada contra ella es "Mycobacterium bovis Bacille Calmette-Guérin" (BCG), que tiene una baja eficacia. Para comprender mejor el tipo de respuestas inmunológicas que confieren protección, utilizaremos un modelo de ratón para evaluar los cambios en el sistema inmunitario producidos por diferentes vacunas y la infección de Mtb en el pulmón. El cáncer de pulmón es una enfermedad devastadora, con una carga terapéutica importante y baja tasa de supervivencia. La investigación se ha centrado en las células tumorales, pero el sistema inmunitario se está volviendo cada vez más importante. Los modelos murinos sinogénicos permiten la inyección de células cancerosas inmunológicamente compatibles en ratones inmunocompetentes para estudiar la modulación del sistema inmunitario por las células tumorales. Además, utilizaremos vacunas terapéuticas para activar las células T a fin de que reconozcan y destruyan el tumor, aumentando su eficacia solas o en combinación con terapias convencionales. En ambos casos, las poblaciones celulares inmunitarias presentes en el lavado broncoalverolar y el parénquima pulmonar en las diferentes condiciones experimentales serán analizadas y caracterizadas por citometría de flujo y ELISA. Se obtendrán además células para estudios proteómicos y de expresión génica, para definir mejor las vías implicadas en el control de la infección o de las células cancerosas. Los resultados obtenidos podrían conducir en el futuro a un diseño más racional de vacunas profilácticas y terapéuticas contra enfermedades pulmonares.As enfermidades pulmonares son un grupo diverso de enfermidades que afectan a millóns de persoas en todo o mundo. O microambiente e o sistema inmunitario nos pulmóns son moi importantes ao mesmo tempo que representan un dilema para o hospedeiro. Por unha banda, debido á súa localización e función, o pulmón está constantemente exposto a patóxenos; por outra banda, debe ser estéril e preservar a homeostase a través do equilibrio entre tolerancia e inflamación. Por iso, é moi importante caracterizar e comprender os mecanismos e poboacións implicados nunha resposta inmune protectora e atopar mellores obxectivos para o deseño de vacinas. O obxectivo deste proxecto é definir e comprender os perfís inmunolóxicos que se correlacionan con maior protección xerada por vacinas preventivas e terapéuticas no pulmón. As vacinas preventivas son xeralmente administradas a individuos sans para previr ou mellorar doenzas infecciosas, mentres que as vacinas terapéuticas contra o cancro son administradas unha vez o individuo desenvolve a enfermidade para fortalecer a súa resposta inmunitaria. Centrarémonos en dúas enfermidades pulmonares: unha infección bacteriana e cancro de pulmón. Para estudar unha infección bacteriana, usaremos como modelo Mycobacterium tuberculosis (MTB), responsable da tuberculose (TB), un grave problema de saúde global. A única vacina con licenza actualmente é "Mycobacterium bovis Bacille Calmette-Guérin" (BCG), que ten unha baixa eficacia. Para entender mellor o tipo de respostas inmunolóxicas que confiran protección, usaremos un modelo de rato para avaliar cambios no sistema inmunitario causadas por vacinas e infección por MTB no pulmón. O cancro de pulmón é unha enfermidade devastadora, cunha importante carga terapéutica e baixa taxa de supervivencia. As células tumorais foron o foco da investigación, pero o sistema inmunolóxico é cada vez máis importante. Os modelos murinos sinxénicos, que inclúen a inxección de células de cancro imunolóxicamente compatibles en ratos inmunocompetentes, permítennos estudar a modulación do sistema inmunitario por células tumorais. Ademáis, utilizaremos vacinas terapéuticas para activar as células T que recoñezan e destrúan o tumor, aumentando a súa eficacia cando se usa só ou en combinación con terapias convencionais. En ambos casos, as poboacións de células inmunitarias presentes no lavado broncoalverolar e o parénquima pulmonar nas diferentes condicións experimentais analizaranse e caracterízanse por citometría de fluxo e ELISA. As células tamén se obterán para estudos proteómicos e de expresión xénica, para definir mellor as vías implicadas no control da infección ou as células cancerosas. Os resultados obtidos poderían levar no futuro a un deseño máis racional de vacinas profilácticas e terapéuticas contra as enfermidades pulmonares.Xunta de Galicia | Ref. ED431C 2016/041Xunta de Galicia and the European Regional Development Fund (ERDF) | Ref. ED431G2019/06Fundação para a Ciência e a Tecnologia | Ref. PTDC/SAU-INF/28463/2017Fundação para a Ciência e a Tecnologia | Ref. UIDB/50026/2020Fundação para a Ciência e a Tecnologia | Ref. UIDP/50026/2020FEDER | Ref. NORTE-01-0145-FEDER-000013FEDER | Ref. NORTE-01-0145- FEDER-00002

Winchell Papers

Abstracts of the Winchell Papers

Photophysical and Photocatalytic Properties of Covalent Organic Frameworks

This dissertation is most interested in how a class of materials known as covalent organic frameworks (COFs) can be designed to capture photon energy to initiate chemical reactions. Different COF designs change how long the energy is held, how it migrates, and how it is dispersed – and these differences can be used to change their performance as artificial photosynthesis platforms. Thus, it is helpful to have an informative discussion about the processes behind natural photosynthesis, that is, nature’s light harvesting strategies and photocatalytic schemes (Section 1.2) and will lead into an introduction of COFs and why they possess unique potential as artificial photosynthesis platforms (Section 1.3). Their beneficial physical qualities are complemented by understanding their electronic structures from theoretically predicted properties with specific focus on topological symmetry (Section 1.4). Synthesizing and characterizing COF systems then becomes an important consideration (Section 1.5) along with how their excited state behaviors are probed and interpreted at reaction timescales by ultrafast spectroscopic techniques (Section 1.6). Finally, a look is taken at how COF structure versatility adds unique potential in catalyst engineering (Section 1.7). The main body of this dissertation will present five main research projects that seek to test theoretical predictions, assess the impact of COF planarity, or fine tune electronic structures. To test theoretical predictions, “Tuning Photoexcited Charge Transfer in Imine-Linked Two-Dimensional Covalent Organic Frameworks, which involves exploring nodal symmetry in topologically similar COFs by varying monomers, is reported. This work has implications on charge separation characteristics of COFs which is important to retain activated catalytic sites for chemical reactions. The second project, “Impact of πConjugation Length on the Excited-State Dynamics of Star-Shaped Carbazole-π-Triazine Organic Chromophores,” doesn’t directly probe COF systems, but looks at the role of dihedral angles on intersystem crossing (ISC) rates in organic chromophores with similar star-shaped motifs like those often found in COFs. Another study on planarity is “Conjugation- and Aggregation-Directed Design of Covalent Organic Frameworks as White-Light-Emitting Diodes” that explores planar and non-planar COFs and the how this affects the deactivation of their photoexcited states. “Wavelength Dependent Excitonic Properties of Imine-Linked Covalent Organic Frameworks,” explores how subtle changes in donor-acceptor arrangements can lead to differences in excited state populations. Finally, the seminal work in this dissertation, “Imine Reversal Mediates Charge Separation and CO2 Photoreduction in Covalent Organic Frameworks,” explores the effect of the imine bond on photophysical and photocatalytic properties

Engineering derivatives from biological systems for advanced aerospace applications

The present study consisted of a literature survey, a survey of researchers, and a workshop on bionics. These tasks produced an extensive annotated bibliography of bionics research (282 citations), a directory of bionics researchers, and a workshop report on specific bionics research topics applicable to space technology. These deliverables are included as Appendix A, Appendix B, and Section 5.0, respectively. To provide organization to this highly interdisciplinary field and to serve as a guide for interested researchers, we have also prepared a taxonomy or classification of the various subelements of natural engineering systems. Finally, we have synthesized the results of the various components of this study into a discussion of the most promising opportunities for accelerated research, seeking solutions which apply engineering principles from natural systems to advanced aerospace problems. A discussion of opportunities within the areas of materials, structures, sensors, information processing, robotics, autonomous systems, life support systems, and aeronautics is given. Following the conclusions are six discipline summaries that highlight the potential benefits of research in these areas for NASA's space technology programs

Transparent polycrystalline ceramics at visible and infrared wavelenghts

Tato práce se zabývá přípravou průhledných keramických plátů vyrobených ze sub-µm prášku oxidu hlinitého. Zhutnělá keramická tělesa jsou připraveny ultrafialovým (UV) vytvrzováním UV vytvrzovací pryskyřice obsahující keramický prášek s následným vypálením organické složky za zvýšené teploty. Vysoká relativní hustota takto vypálených těles je nezbytná ke snížení smršťování během následujícího slinovacího procesu. Proto jsou použity vysoce plněné disperse obsahující > 57 obj.% keramických částic. K dosažení transparentního chování, porozita uvnitř plátů musí být úplně odstraněna. Proto jako závěrečné operace je použito isostatického stlačování za zvýšené teploty. Nakonec jsou prezentovány měření světelné propustnosti a tvrdosti. Možnosti výroby mikrostruktur s vysokým rozlišením za použití bezmaskové litografie a návrhy na použití UV vytvrzovací metody pro produkci tvarově náročných 3D struktur jsou krátce zmíněny.his thesis deals with preparation of transparent ceramic sheets made out of sub-µm alumina powder. Green bodies are prepared by ultraviolet (UV) curing of UV curable resin containing ceramic powder followed by debinding of organic parts at elevated temperature. High relative density of green bodies is essential for reduction of shrinkage during subsequent sintering process. Therefore high solids loading dispersions containing > 57 vol% ceramic particles are used. To reach transparent behaviour, porosity within the sheets must be reduced completely. Therefore hot isostatic pressing (HIP) is used as a final operation. Finally, light transmission and hardness measurements are presented. Possibilities of making high resolution microstructures using maskless lithography and some suggestions for use of the UV curing technique for production of complex-shaped 3D structures are briefly mentioned.

Metal-organic frameworks for water adsorption applications in the automotive filtration industry

In dieser Arbeit werden verschiedene MOF Materialien die sich für die Wasseradsorption eignen hinsichtlich Ihrer Wasseradsorptionseigenschaften untersucht. Das vielversprechendste Material wird ausgewählt und an einem Prüfstand für Lufttrocknerkartuschen untersucht. Für diese Messungen ist eine geeignete Formgebung des Pulvers von Nöten, welche eine wichtige Rolle in dieser Arbeit spielt. Das Material Cu3(BTC)2 wurde hier zu monolithischen Formkörpern verarbeitet. Eine weitere Art der Formgebung war das Pressen von Papieren sowie das direkte Kristallwachstum auf Zellulose Fasern. Desweiteren wurden die Materialien hinsichtlich der Trocknung von n-Heptan untersucht, was hier als Referenz für Dieselkraftstoffe herangezogen wurde. Die Analytik wurde mittels Karl-Fischer Titration duchgeführt. MOF Materialien wurden in beiden Fällen mit kommerziell verwendeten Zeolithen und Silikagel verglichen.Metal-organic frameworks (MOFs) were investigated for their possible use in drying of compressed air in air braking systems for commercial vehicles. Another possible application was the drying of diesel fuel. In this context, n-heptane was chosen as a reference for diesel fuel. Selected metal-organic frameworks were characterized regarding the water adsorption properties by recording water adsorption isotherms. The most promising material was further investigated on a air-drying cartridge test rig. In order to perform these tests, the powder had to be processed to monolithic structures, beads or paper sheets,i.e. a shape that is suitable for the given application. The MOF Cu3(BTC)2 was sucessfully extruded to monolithic structures, which were used in the test rig experiments. Another possibility for immobilization of Cu3(BTC)2 was the crystal growth on pulp fibers. N-heptane drying isotherms were measured on selected samples making use of Karl-Fischer coulometric titration. In both applications, MOF materials were compared with silica based desiccants

Spatial and temporal features of neutrophils in homeostasis from the perspective of computational biology

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 22-07-2022Neutrophils are myeloid cells that originate in the Bone Marrow and enter circulation to patrol for infectious agents. An important part of the “nonspecific” immune system consists on Neutrophils infiltrating challenged tissues, and the established belief was that they stay away from steady-state organs to avoid the risk of exposing them to their cytotoxic content. In the papers presented in this thesis, we show that neutrophils can in fact be found in almost all tissues under homeostasis. We further present proof that they undergo shifts in DNA accessibility, RNA expression and protein content in the infiltrated tissues. Using functional annotation we predict distinct roles depending on the tissue. While in hematopoietic organs the transcriptomic signatures of neutrophils align with canonical functions like immune response and migration, in other tissues such as the skin we find non-canonical functions i.e, epithelial and connective tissue growth or pro-angiogenic roles in the gut and the lung. This predicted pro-angiogenic role was indeed confirmed for the lung. We finally describe that infiltration in tissues follows circadian dynamics, and that once it has occurred, neutrophils experience changes in transcription depending on the time of the day. The analyses of circadian rhythms on mammalian models are often hindered by the inherent difficulty of performing exhaustive sampling (i.e.: every hour for at least 48h). Hence, I implemented CircaN as an R package, which outperforms existing tools in most scenarios. To provide the most complete analysis possible, we provide a full mode analysis option, in which we run CircaN and the two most used algorithms and provide integrated results. We present proof-of-concept results showing that combining various tools yields the best true positive to false positive ratio for most purposesEsta Tesis ha sido financiada por el Ministerio de Ciencia, Innovación y Universidades (MICINN

Guiding clinical malaria vaccine development using immune cell monitoring and controlled human malaria infection

Despite being treatable and preventable, malaria kills about half a million people each year, mostly in Africa. Notwithstanding many attempts to develop a malaria vaccine, only the vaccine RTS,S/AS01 (Mosquirix), has been fully developed and was recommended by the World Health Organization for use in children in malaria endemic areas on October 6th in 2021. RTS,S is a subunit vaccine and provides only partial protection. Manufacturing of pharmaceutical grade Plasmodium falciparum (Pf) sporozoites (SPZ) has been achieved in the past decade, making the development of plasmodial whole cell vaccines possible. This may be a novel and better platform to design malaria vaccines. In addition, the availability of cryopreserved infectious sporozoites has boosted possibilities to assess vaccine efficacy by using controlled human malaria infection (CHMI) trials. The most effective vaccination strategy to date is to inoculate PfSPZ while using chemoprophylactic antimalarials such as chloroquine – the PfSPZ Chemoprophylaxis Vaccine (PfSPZ-CVac). Although CHMI can test vaccine efficacy (VE), the immune mechanisms underneath are not fully understood. Particularly, the cellular immune response is poorly characterised compared to the humoral response (i.e. antibody titers). Thus, the systematic monitoring of cellular immune responses during immunization in combination with CHMI might be a valuable step to guide the clinical development of malaria vaccine candidates and identify immune mechanisms causally related to protection. In Chapter 1, I investigated the influence of two accelerated regimens of PfSPZ-CVac on the generation of pro-inflammatory Pf-specific CD4+ T helper cells and their suitability as a surrogate of protection. The trial was conducted in healthy, malaria-naïve adults in Tübingen. To detect antigen-specific cells in peripheral blood with increased sensitivity, Plasmodium specific T cells were enriched by magnetic-activated cell sorting followed by staining and multiparameter flow cytometry (MFC). Measurements were done using the flow cytometer FACS Canto II and stimulations were done with infected red blood cells (iRBCs). Uninfected red blood cells and Staphylococcal enterotoxin B (SEB) were also as negative and positive controls, respectively. Unexpectedly, I found that a condensed vaccination schedule, where the three vaccinations were given within 10 days induced higher frequencies of Plasmodium specific CD40L+CD4+ TNF-α+/IFN-γ+ cells than a 28-day regimen. The response was also qualitatively different. The shorter regimen led to a polarized Th1 response with more CD45RO+CCR7- effector memory T cells. This may lead to higher numbers of memory cells in the liver. A tendency towards higher frequencies of specific CD40L+CD4+ TNF-α+/IFN-γ+ effector memory T cells was present in protected volunteers but no reliable correlate of protection could be identified. Future research will be needed to identify effector and regulatory responses that predict vaccine efficacy. It will be particularly important to include the characterization of Plasmodium-specific cytotoxic T cells. More than 94% of the estimated malaria cases globally occur in Africa. Therefore, every malaria vaccine must be tested in malaria exposed volunteers to be sure that it has a significant public health impact. Unfortunately, for many vaccines, VE is lower in Africa. This effect is particularly strong in malaria vaccines. The GMZ2 vaccine was developed to prevent malaria and its complications by mimicking naturally acquired immunity. The vaccine antigen consists of a fusion protein between fragments of the merozoite surface protein-3 and the glutamate rich protein. In Chapter 2, I investigated the immunogenicity of GMZ2 adjuvanted with two different immune modulators: Alhydrogel or CAF01. The study was performed in healthy, adult, lifelong malaria-exposed volunteers from Lambaréné, Gabon. MFC was used to systematically measure the T and B cell response, and to compare immune response patterns before and after immunization. Peripheral blood mononuclear cells were cryopreserved and measured upon completion of the trial using a Sony SP6800 Spectral Analyzer. Monitoring of GMZ2-stimulated CD4+ T, and CD20+ B cells showed that GMZ2 did not induce significant immune responses beyond the baseline. The low response to vaccination was unexpected as was the similar performance of the two adjuvants. VE in CHMI was similar between the groups, hence these findings may be expected. Notwithstanding the negative result, this study will help guiding the development of the next generation of blood stage malaria vaccines in malaria-exposed volunteers, where baseline responses play a major role in generating successful immune responses following vaccination. In summary, the work presented as part of the thesis shows that systematic monitoring of the cellular immune responses by MFC, in combination with CHMI studies is a valid, and stringent approach to measure VE and identify correlates of protection, surrogate markers, and the effect of schedule and pre-existing immunity on vaccine responses. More work will be required to replace CHMI with immunological surrogate endpoints and understand vaccine-induced antimalarial immunity

Predictors of outcome and immunological markers in patients with Systemic lupus erythematosus

Background Systemic lupus erythematosus (SLE) is the prototypic multisystem autoimmune disease, with a broad spectrum of clinical presentations encompassing almost all organs and a chronic course, which can vary from mild to life-threatening. There is a major unmet need for outcome prediction enabling tailoring management and therapeutic interventions for SLE patients. Objectives To improve outcome prediction in SLE, the specific aims of this thesis were: (1) to evaluate the performance of the ACR and the SLICC classification criteria sets for SLE; (2) to evaluate the effect of the classification criteria fulfilled at time of SLE diagnosis and other predictors on long-term outcomes of damage and mortality; (3) to identify clinical predictors for SLE flares of disease activity; (4) to increase knowledge about the relationships between immunological markers and SLE disease activity. Methods We conducted a cross-sectional observational study of 2055 patients with a clinical diagnosis of SLE followed at 17 centers and registered in the Portuguese and Spanish national registries; the sensitivity of the ACR and SLICC classification criteria was compared using the McNemar’s test; the sensitivity of the two classification sets was further examined in five subgroups defined according to disease duration. We conducted a prospective inception cohort study of 192 SLE patients from time of diagnosis and followed up to 10 years at the CHUC Lupus Clinic; we assessed with multivariate Cox models the 10-year outcomes of damage and mortality, according to SLE classification status (fulfilling the ACR or only the SLICC criteria) at inception, and adjusting for potential baseline confounders. We conducted a prospective cohort study of 202 SLE patients followed up to 24 months at the CHUC Lupus Clinic over 1083 visits; we evaluated potential clinical predictors for disease activity flares with multivariate Cox regression models adjusting for potential confounders factors and estimating hazard ratios. We conducted cross-sectional studies of two groups of SLE patients, one with clinically active and another with inactive disease recruited at the CHUC Lupus Clinic and a group of healthy subjects enrolled at the same site; one peripheral blood sample was collected from each participant and analyzed with flow cytometry multiparametric immunophenotyping protocols to define relationships between immunological markers in B cells, Th17 cells and NK cells and SLE classification and disease activity status. Results The cross-sectional study on performance of classification criteria showed that the sensitivity for SLE clinical diagnosis was higher with the SLICC than with the ACR classification criteria (93.2% versus 85.6%, p<0.0001). From patients not fulfilling the ACR criteria, 62.8% could be classified with the SLICC. Patients within 5 years since disease onset presented the largest difference in sensitivity between the SLICC and the ACR criteria (respectively 89.3% and 76.0%, p<0.0001). In the 10-year prospective inception cohort study, patients meeting the ACR criteria compared to those with only the SLICC criteria at inception presented during follow-up with more cases of lupus nephritis (35.1% versus 13.8%, p<0.01), but less thrombotic antiphospholipid syndrome (4.5% versus 17.2%, p<0.01). The Cox models showed no significant differences in risk for damage or death between groups. In the 24-month prospective cohort study, the multivariate Cox models demonstrated that the risk of flare was more than two-fold, four-fold and three-fold higher for patients with SLE diagnosis up to 25 years, previous lupus nephritis or baseline immunosuppressant treatment, respectively. In the cross-sectional immunophenotyping studies, analysis of B cell subsets showed that differential expression of BAFFR, CD81 and CD38 in the transitional B cells allowed identifying two major clusters: the cluster 1 integrated all healthy subjects and 79% of SLE patients with clinically inactive disease, while in the cluster 2 there was only patients with SLE and 82% of those with clinically active disease. The analysis of Th17 cells showed no significant differences in the frequency of Th17 among healthy subjects and SLE patients, as well as among patients with clinically inactive and active disease. The analysis of NK cells showed a lower number and frequency of NK cells in SLE patients as compared to healthy subjects, regardless of disease activity status, and a lower frequency of CD56dim NK cells expressing CXCR3 was a marker of clinically active SLE (12.5% versus 24.1% in the active and inactive SLE group, respectively, p<0.01). Conclusions The SLICC classification criteria are more sensitive and may allow a SLE classification earlier in the disease course than the previous ACR criteria. Patients fulfilling at inception either of the classification criteria present no differences in the major long-term outcomes of organ damage and mortality. Patients with a SLE diagnosis before age 25, lupus nephritis or immunosuppressant treatment/severe SLE present higher risk for flares of disease activity; patients fulfilling at inception only the SLICC classification criteria may present higher risk of thrombotic antiphospholipid syndrome: these clinical predictors thus provide a basis for tailoring management strategies of SLE patients. Immunophenotyping studies suggested that SLE patients present: an upregulation of B cells, with subset clusters able to differentiate SLE patients from healthy subjects and clinically active from inactive SLE; a downregulation of NK cells, and less clear changes of Th17 cells. We provide proof-of-concept that a panel of immunological markers may provide a basis for biologic validation of clinical definitions for SLE disease activity states.Introdução O Lúpus Eritematoso sistémico (LES) representa o paradigma das doenças autoimunes multissistémicas, apresentando um largo espectro de manifestações clínicas que potencialmente envolvem quase todos os órgãos e sistemas, com um curso clínico crónico e gravidade clínica variada, entre ligeira a severa e com risco de vida. Existe uma necessidade fundamental por cumprir de identificar preditores de prognóstico que permitam a individualização da monitorização e intervenções terapêuticas para os doentes com LES. Objetivos Identificar preditores de prognóstico em doentes com LES, através dos objetivos específicos desta tese: (1) avaliar o desempenho dos critérios de classificação ACR e SLICC para a identificação de doentes com LES; (2) avaliar o efeito dos critérios de classificação preenchidos à data de diagnóstico de LES e outros preditores no prognóstico a longo prazo em termos de dano irreversível e mortalidade; (3) identificar preditores clínicos para agudizações da atividade clínica do LES; (4) contribuir para o conhecimento das relações entre marcadores imunológicos e a atividade clínica do LES. Métodos Efetuámos um estudo observacional transversal de 2055 doentes com diagnóstico clínico de LES, seguidos em 17 centros e integrados nos registos nacionais de Portugal e Espanha; a sensibilidade dos critérios de classificação ACR e SLICC foi comparada através do teste de McNemar; a sensibilidade dos dois sistemas de classificação foi ainda analisada em 5 subgrupos definidos de acordo com a duração da doença. Realizámos um estudo prospetivo de coorte, incluindo 192 doentes com LES avaliados desde a data de diagnóstico e seguidos até 10 anos na CHUC Lupus Clinic; analisámos através de regressão multivariada de Cox o prognóstico a 10 anos, em termos de dano irreversível e mortalidade, em grupos definidos de acordo com os critérios de classificação cumpridos à data de diagnóstico (critérios ACR ou apenas os critérios SLICC) e ajustando para potenciais confundidores definidos à data de diagnóstico. Conduzimos um estudo prospetivo de coorte incluindo 202 doentes com LES seguidos até 24 meses na CHUC Lupus Clinic ao longo de 1083 consultas; analisámos potenciais preditores clínicos de agudizações da atividade do LES, aplicando regressão multivariada de Cox ajustada a potenciais confundidores e com estimativa dos hazard ratios dos preditores. Efetuámos estudos transversais incluindo dois grupos de doentes com LES, um com doença clinicamente ativa e outro com doença inativa, recrutados na CHUC Lupus Clinic e um grupo de indivíduos saudáveis recrutados no mesmo local; colhemos uma amostra de sangue periférico de cada participante, que foi processada através de protocolos de imunofenotipagem e analisada com citometria de fluxo multiparamétrica, para identificar relações entre marcadores imunológicos em células B, Th17 e NK e a classificação de LES e seus estados de atividade clínica. Resultados O estudo transversal do desempenho dos critérios de classificação demonstrou que a sensibilidade para o diagnóstico clínico de LES é mais elevado com o sistema de classificação SLICC do que com o ACR (93,2% e 85,6%, respetivamente, p<0,0001). Entre os doentes que não cumpriam os critérios ACR, 62,8% preencheram os critérios SLICC. Os pacientes com duração de doença até 5 anos apresentaram a maior diferença em sensibilidade entre o sistema SLICC e ACR de classificação (respetivamente 89,3% e 76,0%, p<0001). O estudo prospetivo de coorte de LES inicial e seguimento até 10 anos, mostrou que os doentes que preenchiam à data de diagnóstico os critérios ACR de classificação apresentaram ao longo do seguimento mais casos de nefrite lúpica do que aqueles cumprindo apenas critérios SLICC (35,1% e 13,8%, respetivamente, p<0,01), mas menos casos de síndrome antifosfolípido trombótico (4,5% e 17,2%, respetivamente, p<0,01). Os modelos multivariados de Cox não mostraram diferenças entre grupos no risco de dano irreversível nem de mortalidade. No estudo prospetivo de coorte com seguimento a 24 meses, os modelos multivariados de Cox demonstraram que o risco de agudizações clínicas do LES é mais de 2 vezes, 4 vezes e três vezes mais elevado para os doentes com diagnóstico de LES até aos 25 anos, com nefrite lúpica prévia ou sob terapêutica com imunossupressores à data de inclusão, respetivamente. Nos estudos transversais de imunofenotipagem, a análise da linhagem de células B demonstrou que a expressão diferencial de BAFFR, CD81 e CD38 nas células B de transição permite a identificação de dois principais clusters: o cluster 1, que integrou todos os indivíduos saudáveis e 79% dos doentes com LES clinicamente inativo, enquanto o cluster 2 incluiu apenas doentes com LES e 82% daqueles com doença clinicamente ativa. A análise das células Th17 mão mostrou diferenças significativas na frequência de Th17 entre o grupo de controlos e o de doentes com LES, nem entre pacientes com doença clinicamente inativa ou ativa. A análise das células NK revelou menor número e frequência de células NK em doentes com LES comparativamente aos controlos, independentemente da atividade clínica da doença; uma frequência mais baixa de células NK CD56dim expressando CXCR3 revelou ser um marcador de LES clinicamente ativo (12,5% e 24,1% no grupo com doença ativa e inativa, respetivamente, p<0,01). Conclusões Os critérios de classificação SLICC apresentam maior sensibilidade e podem permitir estabelecer a classificação como LES mais precocemente no curso da doença do que o prévio sistema de classificação ACR. Os doentes preenchendo à data de diagnóstico qualquer dos dois sistemas de classificação não apresentam diferenças no prognóstico a longo prazo em termos de dano irreversível nem de mortalidade. Os doentes com diagnóstico de LES até aos 25 anos, com nefrite lúpica ou necessitando terapêutica imunossupressora, apresentam risco mais elevado de sofrer agudizações clínicas do LES; pacientes preenchendo à data de diagnóstico apenas os critérios de classificação SLICC podem apresentar maior risco de síndrome anti-fosfolípido trombótico: estes preditores clínicos fornecem uma base para individualizar estratégias de monitorização e tratamento de doentes com LES. Os estudos de imunofenotipagem sugerem que os doentes com LES apresentam: hiperatividade de células B, com clusters de marcadores imunológicos em subtipos de células B que permitem diferenciar doentes com LES de indivíduos saudáveis e LES clinicamente ativo de inativo; hipoatividade das células NK e anomalias menos consistentes das células Th17. Fizemos prova do conceito que um painel de marcadores imunológicos pode providenciar uma base de validação biológica para definições clínicas de estados de atividade do LES