Sammanfattning Intäktsredovisningen har länge varit ett komplicerat område. Flera olika standarder har funnits och detta har lett till stora redovisningsskandaler. IASB och FASB började tillsammans att utveckla de två intäktsstandarderna IFRS 15 och ASC 606. Detta till följd av de tidigare skandalerna, målet var att ersätta tidigare standarder med IFRS 15 och ASC 606. Harmoniseringsarbetet syftade till att redovisningen skulle bli likadan världen över med hjälp av dessa standarder.    Denna studies syfte var att jämföra de båda standarderna, för att se hur långt harmoniseringsarbetet har kommit. För att lyckas med detta går studien igenom likheter mellan IFRS 15 och ASC 606 samt fortsätta skillnader. Sedan konstruerades sju typfall inspirerade från byggbranschen. Detta på grund av de ofta komplexa och långa arbetena i branschen. Målet var att se hur valet av standard påverkar ett företags resultaträkning, således hur harmoniseringen ser ut standarderna emellan.   Resultaten av typfallen visar på att det i stor utsträckning är harmoniserat standarderna emellan. Det finns vissa skillnader, det beror ofta på den friare tolkningen i IFRS 15. I några av typfallen innebär val av standard en differens i resultaträkningen eller andra delar av ett företags redovisning. Slutsatsen är att det är harmoniserat mellan standarderna trots att vissa skillnader består. Vi anser att det finns fördelar med bägge standarderna. För de som arbetar med redovisning kan IFRS 15 vara mer fördelaktigt på grund av den friare tolkningen. Externa parter såsom investerare, potentiella samarbetspartners och så vidare föredrar troligtvis ASC 606 hårdare riktlinjer då redovisningen blir mer likvärdigt företag emellan. / Abstract Revenue accounting has long been a complicated area. There have been several different standards, this has led to big scandals in revenue accounting. IASB and FASB started to develop the two revenue standards IFRS 15 and ASC 606 together. This as a result of the previous scandals, the goal was to replace previous standards with IFRS 15 and ASC 606. The harmonization work aimed at making accounting the same all over the world with the help of this standards. This studies purpose was to compare the both standards, this to see how far the harmonization work has come. To succeed with this the studie goes through similarities between IFRS 15 and ASC 606 and continued differences. After these seven hypothetical cases were constructed inspired by the construction industry. This because of the complex and long-time work spans in the industry. The goal was to see how the choice of standard affects a company’s income statement, thus how the harmonization looks between the standards. The results of the hypothetical cases shows that the standards are harmonized to a large extent. There are still some differences. This depends on the often-freer interpretation in IFRS 15. In some of the hypothetical cases the choice of standard makes differences in the income statement or other parts of the company`s accounting. The conclusion is that the standards are harmonized even though some differences persist. We believe that there are advantages with both standards. For the people that works with accounting IFRS 15 may be more advantageous because of the freer interpretation. External parts such as investors, potential partners and so on probably prefers ASC 606 tougher guidelines as accounting becomes more equal between companies.

A hiperalgesia inflamatória é o processo pelo qual ocorre a sensibilização dos neurônios nociceptores aferentes primários por mediadores químicos inflamatórios, gerando assim uma diminuição do limiar nociceptivo e como consequência episódios de dor. Entre os principais mediadores envolvidos com a sensibilizacão das fibras nociceptivas periféricas está a prostaglandina E2 (PGE2), que é liberada como um produto final de uma cascata de mediadores inflamatórios. Dentro desta cascata de liberação hierárquica podemos destacar a interleucina -1? (IL)-1?, uma citocina importante na gênese da dor inflamatória, devido à sua capacidade de induzir a produção da enzima cicloxigenase-2 (COX-2), e consequentemente PGE2. O mecanismo de controle da produção da IL-1 ? envolvem dois passos intracelulares: a indução da expressão de uma forma protêica inativa (a pró-IL-1 ?) e a geração da forma biologicamente ativa (IL-1?) a partir da pró-IL-1 ?. Este último passo envolve a ação de uma cisteína-protease ativada em decorrência de um processo inflamatório, conhecida como Caspase-1, a qual cliva a pró-IL-1? em IL1?. Recentemente, nosso grupo demonstrou que a caspase-1 tem um papel importante na gênese da dor inflamatória, sendo crucial para a geração de IL-1? e consequentemente COX2/PGE2. Porém, não são conhecidos os mecanismos de ativação da caspase-1 na hiperalgesia inflamatória. Sabe-se que a ativação da Caspase-1 e clivagem da pro-IL-1? são dependentes de uma plataforma molecular intracelular denominada inflamassoma. Os principais inflamassomas ativadores de caspase-1 são formados pelas proteínas NLRP3, IPAF (NLRC4) e por sua molécula adaptadora ASC. O objetivo desse trabalho então foi avaliar a participação do inflamassoma na gênese da dor inflamatória. Nós identificamos que as moléculas IPAF e ASC, mas não o NLRP3, participa no desenvolvimento da hiperalgesia inflamatória mecânica e térmica induzida pela carragenina. Observou-se que estas moléculas são cruciais para a ativação da Caspase-1 e, consequentemente, para a produção da IL-1? ativa. Estes resultados evidenciam pela primeira vez um papel importante do inflamassoma no desenvolvimento da hiperalgesia inflamatória. / The inflammatory hyperalgesic is the process by which occurs the sensitization of nociceptors primary afferent neurons by inflammatory chemical mediators, that generating a decreased nociceptive threshold and result in episodes of pain. Among the main of nociceptive mediators involved with sensitization of peripheral nociceptive fibers are prostaglandin E2 (PGE2), which is released as a final product of a cascade of inflammatory mediators. Within this hierarchical cascade of release can highlight interleukin-1? (IL)-1?, a cytokine important in the genesis of inflammatory pain due to their ability to induce the production of the enzyme cyclooxygenase-2 (COX-2) and consequently PGE2. The control mechanism production of intracellular IL-1 ? involved two steps: induction of expression of a protein inactive form (pro-IL-1 ?) and the generation of the biologically active form from pro-IL-1 ? (IL-1?). This last step involves the action of a cysteine protease-activated due to an inflammatory process, known as Caspase-1, which cleaves pro-IL-1? to IL-1?. Recently our group has demonstrated that caspase-1 plays an important role in the genesis of inflammatory pain, crucial for the generation of IL-1? and consequently COX2/PGE2. However, there aren\'t known mechanisms of activation of caspase-1 in inflammatory hyperalgesic. It is known that the activation of Caspase-1 cleavage and pro-IL-1? are dependent on an intracellular molecular platform called inflammassome. The main inflammassome activators of caspase-1 proteins are formed by NLRP3, IPAF (NLRC4) and its adapter molecule ASC. The aim of this study was to evaluate the inflammassome participation in the genesis of inflammatory pain. We have identified molecules IPAF and ASC, but not NLRP3, is participate in the development of mechanical and thermal inflammatory hyperalgesic induced by carrageenan. It was observed that these molecules are crucial for the activation of Caspase-1 and thus for the production of active IL-1?. These results demonstrate for the first time an important role of the inflammassome in the development of inflammatory hyperalgesic.

A hiperalgesia inflamatória é o processo pelo qual ocorre a sensibilização dos neurônios nociceptores aferentes primários por mediadores químicos inflamatórios, gerando assim uma diminuição do limiar nociceptivo e como consequência episódios de dor. Entre os principais mediadores envolvidos com a sensibilizacão das fibras nociceptivas periféricas está a prostaglandina E2 (PGE2), que é liberada como um produto final de uma cascata de mediadores inflamatórios. Dentro desta cascata de liberação hierárquica podemos destacar a interleucina -1? (IL)-1?, uma citocina importante na gênese da dor inflamatória, devido à sua capacidade de induzir a produção da enzima cicloxigenase-2 (COX-2), e consequentemente PGE2. O mecanismo de controle da produção da IL-1 ? envolvem dois passos intracelulares: a indução da expressão de uma forma protêica inativa (a pró-IL-1 ?) e a geração da forma biologicamente ativa (IL-1?) a partir da pró-IL-1 ?. Este último passo envolve a ação de uma cisteína-protease ativada em decorrência de um processo inflamatório, conhecida como Caspase-1, a qual cliva a pró-IL-1? em IL1?. Recentemente, nosso grupo demonstrou que a caspase-1 tem um papel importante na gênese da dor inflamatória, sendo crucial para a geração de IL-1? e consequentemente COX2/PGE2. Porém, não são conhecidos os mecanismos de ativação da caspase-1 na hiperalgesia inflamatória. Sabe-se que a ativação da Caspase-1 e clivagem da pro-IL-1? são dependentes de uma plataforma molecular intracelular denominada inflamassoma. Os principais inflamassomas ativadores de caspase-1 são formados pelas proteínas NLRP3, IPAF (NLRC4) e por sua molécula adaptadora ASC. O objetivo desse trabalho então foi avaliar a participação do inflamassoma na gênese da dor inflamatória. Nós identificamos que as moléculas IPAF e ASC, mas não o NLRP3, participa no desenvolvimento da hiperalgesia inflamatória mecânica e térmica induzida pela carragenina. Observou-se que estas moléculas são cruciais para a ativação da Caspase-1 e, consequentemente, para a produção da IL-1? ativa. Estes resultados evidenciam pela primeira vez um papel importante do inflamassoma no desenvolvimento da hiperalgesia inflamatória. / The inflammatory hyperalgesic is the process by which occurs the sensitization of nociceptors primary afferent neurons by inflammatory chemical mediators, that generating a decreased nociceptive threshold and result in episodes of pain. Among the main of nociceptive mediators involved with sensitization of peripheral nociceptive fibers are prostaglandin E2 (PGE2), which is released as a final product of a cascade of inflammatory mediators. Within this hierarchical cascade of release can highlight interleukin-1? (IL)-1?, a cytokine important in the genesis of inflammatory pain due to their ability to induce the production of the enzyme cyclooxygenase-2 (COX-2) and consequently PGE2. The control mechanism production of intracellular IL-1 ? involved two steps: induction of expression of a protein inactive form (pro-IL-1 ?) and the generation of the biologically active form from pro-IL-1 ? (IL-1?). This last step involves the action of a cysteine protease-activated due to an inflammatory process, known as Caspase-1, which cleaves pro-IL-1? to IL-1?. Recently our group has demonstrated that caspase-1 plays an important role in the genesis of inflammatory pain, crucial for the generation of IL-1? and consequently COX2/PGE2. However, there aren\'t known mechanisms of activation of caspase-1 in inflammatory hyperalgesic. It is known that the activation of Caspase-1 cleavage and pro-IL-1? are dependent on an intracellular molecular platform called inflammassome. The main inflammassome activators of caspase-1 proteins are formed by NLRP3, IPAF (NLRC4) and its adapter molecule ASC. The aim of this study was to evaluate the inflammassome participation in the genesis of inflammatory pain. We have identified molecules IPAF and ASC, but not NLRP3, is participate in the development of mechanical and thermal inflammatory hyperalgesic induced by carrageenan. It was observed that these molecules are crucial for the activation of Caspase-1 and thus for the production of active IL-1?. These results demonstrate for the first time an important role of the inflammassome in the development of inflammatory hyperalgesic.

Macrophages are innate immune cells responsible for engulfing microbes and cell debris through phagocytosis and orchestrating immune responses to maintain homeostasis. While conducting immune surveillance over all types of organs and tissues, macrophages face inherently heterogeneous microenvironments with unique biophysical features. For instance, microglia residing in the brain, Kupffer cells living in the skin and bone osteoclasts are exposed to very distinct tissue stiffnesses. Despite the research done in the last decade clearly indicates that macrophages are sensitive to physical factors, how mechanical cues modulate their inflammatory response remains poorly understood. The present study aims at investigating how microenvironment stiffness influences the pro-inflammatory behaviour of macrophages. Besides characterising the regulatory effect on pro-inflammatory gene expression and cytokine production, this work examines the impact of stiffness on the inflammasome, one of the main macrophage signalling platforms. For this, an in vitro system based in 2D polyacrylamide hydrogels whose stiffness can be independently tuned was established. Using substrates with an elastic moduli between 0.2 and 33.1 kPa, bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff polyacrylamide. Upon priming with lipopolysaccharide, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels, yet there were no significant differences in the expression of other major pro-inflammatory genes. Additionally stimulating macrophages with the ionophore nigericin revealed higher secreted protein levels of IL-1β and IL-6 on compliant substrates. Interestingly, macrophages challenged on compliant polyacrylamide also displayed an enhanced formation of the NLRP3 inflammasome as well as increased levels of pyroptotic cell death. The upregulation of inflammasome assembly on compliant hydrogels was not primarily attributed to the reduced cell spreading, since spatially confining cells on micropatterns led to a decrease of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, these results show that substrate stiffness affects the pro-inflammatory response of macrophages and for the first time describe that the NLRP3 inflammasome is one of the signalling components affected by stiffness mechanosensing. The work presented here expands our understanding of how microenvironment stiffness affects macrophage behaviour and which elements of their machinery might contribute to integrate mechanical cues into the regulation of their inflammatory functions. The onset of pathological processes or the implant of foreign bodies represent immune challenges in which macrophages can face a mechanically changing environment. Therefore, a better insight on how macrophages detect and process biophysical signals could potentially provide a basis for new strategies to modulate inflammatory responses.:INTRODUCTION 1.1 Macrophage cell biology 1.1.1 The origin of macrophages 1.1.2 The macrophage: a swiss army knife 1.1.3 The macrophage pro-inflammatory response 1.2 Immunobiophysics: the force of the immune system 1.2.1 Exertion of immune cell forces 1.2.2 Immune cell mechanosensing 1.3 Cellular mechanosensing and mechanotransduction 1.3.1 Cell adhesions to the extracellular matrix 1.3.2 Nuclear mechanotransduction 1.3.3 Membrane mechanosensing elements 1.4 Macrophage mechanosensing AIMS AND SCOPE OF THE THESIS RESULTS 3.1 Morphol. characterisation of macrophages cultured on substrates of varying stiffness 3.1.1 BMDMs adhere and can be cultured on polyacrylamide hydrogels 3.1.2 Macrophage morphology is influenced by substrate stiffness 3.1.3 PEG-Hep hydrogels induce similar morphological differences as PAA substrates but do not constitute a suitable macrophage culture platform 3.1.4 Substrate stiffness affects membrane architecture 3.2 Impact of substrate stiffness on the pro-inflammatory response of macrophages 3.2.1 The morphol. differences induced by different stiffness persist after macrophage priming 3.2.2 Tuning substrate stiffness does not cause major changes in the expression of pro-inflammatory genes 3.2.3 Lower substrate stiffness upregulates the secretion of the cytokines IL-6 and IL-1β 3.2.4 Stiffer substrates diminish macrophage pyroptotic cell death 3.2.5 Compliant substrates enhance NLRP3 inflammasome formation 3.3 Investigation of macrophage mechanotransducing elements 3.3.1 Limiting cell spreading alone does not recapitulate the effects induced by stiffness on inflammasome formation 3.3.2 Actomyosin contractility may play a role in transducing the mechanical cues given by substrate stiffness DISCUSSION AND CONCLUSIONS 4.1 Compliant substrates enhance the macrophage pro-inflammatory response 4.2 Substrate stiffness influences the formation of the NLRP3 inflammasome 4.3 Exclusively altering cell spreading does not explain the differences induced by substrate stiffness 4.4 Actomyosin contractility as a potential macrophage mechanotransducer element 4.5 Potential impact of the study in the context of cancer 4.6 Potential impact of the study in the context of implant design 4.7 Conclusions of the study MATERIALS AND METHODS 5.1 Production of polyacrylamide (PAA) hydrogels 5.2 Production of polyethylenglycol-heparin (PEG-Hep) hydrogels 5.3 Mechanical characterisation of hydrogels and macrophages 5.4 Isolation and culture of bone marrow-derived macrophages (BMDMs) 5.5 Fluorescence confocal microscopy 5.6 Scanning electron microscopy (SEM) 5.7 Gene expression analysis using quantitative real-time PCR (qRT-PCR) 5.8 Cytokine quantification assays 5.9 Cell viability assay 5.10 Culture of BMDMs on micropatterns 5.11 Optical diffraction tomography (ODT) 5.12 Statistical analysis and data visualisation APPENDIX LIST OF ACRONYMS AND ABBREVIATIONS LIST OF FIGURES BIBLIOGRAPHY ACKNOWLEDGEMENTS / Als Teil des angeborenen Immunsystems sind Makrophagen dafür verantwortlich Pathogene und Zellrückstände durch Phagozytose zu beseitigen. Sie orchestrieren Immunantworten um homöostatische Bedingungen von Organen und Geweben aufrechtzuerhalten. Dabei sind sie extrem heterogenen Mikroumgebungen ausgesetzt, welche sich jeweils durch eine einzigartige Kombination von (bio)chemischen und mechanischen Eigenschaften, vor allem Gewebesteifigkeiten, auszeichnen. Dies veranschaulichen beispielsweise im Gehirn residierende Mikroglia, Kupffer-Zellen in der Haut und Osteoklasten in Knochen. Obwohl diverse Studien aus dem letzten Jahrzehnt eindeutig zeigen, dass Makrophagen auf mechanische Signale reagieren, ist der zugrunde liegende Mechanismus, wie diese Signale eine Entzündungsreaktion modulieren, noch immer unzureichend verstanden. Die vorliegende Studie beinhaltet die systematische Untersuchung, wie die Steifigkeit der Mikroumgebung das proinflammatorische Verhalten von Makrophagen beeinflusst. Neben der Charakterisierung der regulatorischen Wirkung auf die proinflammatorische Genexpression und Zytokinproduktion untersucht diese Arbeit auch den Einfluss der Steifigkeit auf das Inflammasom; eine der wichtigsten Signalplattformen für Makrophagen. Zu diesem Zweck wurde zunächst ein Zellkultursystem mit 2D-Polyacrylamid-Hydrogelen als Zellsubstrat entwickelt, bei dem das Elastizitätsmodul der Gelsubstrate gezielt eingestellt werden kann. Unter Verwendung von Substraten mit einem Elastizitätsmodul zwischen 0,2 kPa und 33,1 kPa zeigt die mikroskopische Analyse, dass aus Knochenmark stammende Makrophagen im Vergleich zu steifem Polyacrylamid eine weniger ausgebreitete und rundere Morphologie annehmen. Nach dem Primen mit Lipopolysaccharid waren die Expressionsniveaus des Gens, das für TNF-α kodiert, auf deformierbareren Hydrogelen höher, jedoch gab es keine signifikanten Unterschiede in der Expression anderer wichtiger pro-inflammatorischer Gene. Eine zusätzliche Stimulierung von Makrophagen mit dem Ionophor Nigericin bewirkte höhere sekretierte Proteinspiegel von IL-1β und IL-6 auf deformierbaren Substraten. Makrophagen, die deformierbarem Polyacrylamid ausgesetzt waren, zeigten auch eine verstärkte Bildung des NLRP3-Inflammasoms sowie ein erhöhtes Ausmaß an pyroptotischem Zelltod. Die Hochregulierung der Inflammasom-Assemblierung auf deformierbaren Hydrogelen wurde nicht primär auf die reduzierte Zellausbreitung zurückgeführt, da räumlich begrenzte Zellen auf Mikromustern zu einer Abnahme von Inflammasom-positiven Zellen im Vergleich zu stark ausgebreiteten Zellen führten. Schließlich verringerte eine Störung der Aktomyosin-Kontraktilität die Unterschiede in der Inflammasombildung zwischen deformierbaren und steifen Substraten. Zusammenfassend zeigen diese Ergebnisse, dass die Substratsteifigkeit die proinflammatorische Reaktion von Makrophagen beeinflusst und beschreiben erstmalig, dass das NLRP3-Inflammasom eine der Signalkomponenten ist, die von der zellulären Steifheitswahrnehmung beeinflusst werden. Die hier vorgestellte Arbeit erweitert unser Verständnis davon, wie die Steifigkeit der Mikroumgebung das Verhalten von Makrophagen beeinflusst und welche Elemente ihrer Maschinerie dazu beitragen könnten mechanische Signale in die Regulierung ihrer Entzündungsfunktionen zu integrieren. Das Einsetzen pathologischer Prozesse oder die Implantation von Fremdkörpern stellen Immunherausforderungen dar, bei denen Makrophagen einer sich mechanisch verändernden Umgebung ausgesetzt sein können. Daher könnte ein besserer Einblick in die Art und Weise, wie Makrophagen biophysikalische Signale erkennen und verarbeiten, möglicherweise eine Grundlage für neue Strategien zur Modulation von Entzündungsreaktionen bieten.:INTRODUCTION 1.1 Macrophage cell biology 1.1.1 The origin of macrophages 1.1.2 The macrophage: a swiss army knife 1.1.3 The macrophage pro-inflammatory response 1.2 Immunobiophysics: the force of the immune system 1.2.1 Exertion of immune cell forces 1.2.2 Immune cell mechanosensing 1.3 Cellular mechanosensing and mechanotransduction 1.3.1 Cell adhesions to the extracellular matrix 1.3.2 Nuclear mechanotransduction 1.3.3 Membrane mechanosensing elements 1.4 Macrophage mechanosensing AIMS AND SCOPE OF THE THESIS RESULTS 3.1 Morphol. characterisation of macrophages cultured on substrates of varying stiffness 3.1.1 BMDMs adhere and can be cultured on polyacrylamide hydrogels 3.1.2 Macrophage morphology is influenced by substrate stiffness 3.1.3 PEG-Hep hydrogels induce similar morphological differences as PAA substrates but do not constitute a suitable macrophage culture platform 3.1.4 Substrate stiffness affects membrane architecture 3.2 Impact of substrate stiffness on the pro-inflammatory response of macrophages 3.2.1 The morphol. differences induced by different stiffness persist after macrophage priming 3.2.2 Tuning substrate stiffness does not cause major changes in the expression of pro-inflammatory genes 3.2.3 Lower substrate stiffness upregulates the secretion of the cytokines IL-6 and IL-1β 3.2.4 Stiffer substrates diminish macrophage pyroptotic cell death 3.2.5 Compliant substrates enhance NLRP3 inflammasome formation 3.3 Investigation of macrophage mechanotransducing elements 3.3.1 Limiting cell spreading alone does not recapitulate the effects induced by stiffness on inflammasome formation 3.3.2 Actomyosin contractility may play a role in transducing the mechanical cues given by substrate stiffness DISCUSSION AND CONCLUSIONS 4.1 Compliant substrates enhance the macrophage pro-inflammatory response 4.2 Substrate stiffness influences the formation of the NLRP3 inflammasome 4.3 Exclusively altering cell spreading does not explain the differences induced by substrate stiffness 4.4 Actomyosin contractility as a potential macrophage mechanotransducer element 4.5 Potential impact of the study in the context of cancer 4.6 Potential impact of the study in the context of implant design 4.7 Conclusions of the study MATERIALS AND METHODS 5.1 Production of polyacrylamide (PAA) hydrogels 5.2 Production of polyethylenglycol-heparin (PEG-Hep) hydrogels 5.3 Mechanical characterisation of hydrogels and macrophages 5.4 Isolation and culture of bone marrow-derived macrophages (BMDMs) 5.5 Fluorescence confocal microscopy 5.6 Scanning electron microscopy (SEM) 5.7 Gene expression analysis using quantitative real-time PCR (qRT-PCR) 5.8 Cytokine quantification assays 5.9 Cell viability assay 5.10 Culture of BMDMs on micropatterns 5.11 Optical diffraction tomography (ODT) 5.12 Statistical analysis and data visualisation APPENDIX LIST OF ACRONYMS AND ABBREVIATIONS LIST OF FIGURES BIBLIOGRAPHY ACKNOWLEDGEMENTS

[ES] Esta tesis doctoral titulada "El microambiente tumoral inflamatorio como objetivo en el diseño de nanoconjugados para el tratamiento del cáncer de mama avanzado" se centra en la evaluación de un nuevo inhibidor del inflamasoma (MM01) como herramienta química para estudiar el papel del inflamasoma en modelos de inflamación y cáncer. El capítulo I incluye una descripción general del sistema inmune, los inflamasomas dependientes de ASC y el papel que juegan en el desarrollo de enfermedades. También se profundiza en el papel de los inflamasomas y de la proteína ASC en la progresión del cáncer de mama. Además, se incluyen conceptos básicos de nanotecnología, nanomedicina y polímeros terapéuticos. Finalmente, se abordan las ventajas de utilizar nanomedicinas como terapia, las interacciones de las nanomedicinas con los sistemas biológicos, los nanofármacos descritos en la literatura, así como sus posibilidades de traslación a la práctica clínica. En los capítulos de resultados, delineamos un novedoso mecanismo de acción para MM01, un modulador de la actividad del inflamasoma recientemente identificado: la inhibición de la oligomerización del ASC y el subsiguiente procesamiento reducido de la pro-caspasa-1 y la inhibición de la actividad de la caspasa-1. Demostramos que MM01 interrumpe el proceso de oligomerización de ASC asociado a la actividad de varios inflamasomas e inhibe la liberación de IL-1ß y la piroptosis en varios modelos celulares de inflamación. MM01 también reduce la infiltración de neutrófilos y la acumulación de citoquinas pro-inflamatorias en un modelo in vivo de peritonitis. Dada la implicación de la función de ASC en múltiples complejos del inflamasoma, el tratamiento con MM01 puede representar un enfoque terapéutico eficaz para tratar aquellas enfermedades en las que está implicada la activación de múltiples inflamasomas. Teniendo en cuenta los resultados obtenidos, empleamos nuestro inhibidor del inflamasoma, MM01, para estudiar el papel del inflamasoma en la progresión tumoral en diferentes modelos de cáncer de mama tanto in vitro como in vivo. Demostramos que diferentes líneas celulares de cáncer de mama responden de forma diferente al tratamiento con MM01. Desarrollamos un ensayo funcional que comprende la evaluación de la migración de las células de cáncer de mama en respuesta al secretoma pro-inflamatorio de los macrófagos M1 (estímulo inflamatorio) en presencia de MM01. Ciertas líneas celulares (como la línea celular EO771) mostraron un aumento de la migración en respuesta al estímulo inflamatorio y una disminución de la migración en respuesta al tratamiento con MM01; sin embargo, también identificamos líneas celulares que responden negativamente al tratamiento con MM01 (como la línea celular 4T1). Por último, demostramos la eficacia de este experimento funcional in vivo demostrando que el tratamiento con MM01 redujo el tamaño del tumor en el modelo ortotópico EO771 pero aumentó el tamaño del tumor y la metástasis pulmonar en el modelo ortotópico 4T1. Estos dos modelos, que recapitulan respuestas contradictorias al tratamiento con nuestro inhibidor del inflamasoma, podrán utilizarse en el futuro para determinar biomarcadores que predigan la respuesta. Por último, desarrollamos una estrategia sintética para obtener un nuevo nanomedicamento que mejora la solubilidad y la orientación tumoral del MM01 en un modelo de cáncer de mama. Implementamos un enfoque híbrido de conjugación-complejación que comprende la conjugación de ß-ciclodextrina con un ácido lineal poli-L-glutámico (PGA) (L-PGA-ßCD) para proporcionar la capacidad de atrapar MM01 dentro de los anillos de ciclodextrina. El nanosistema obtenido mostró una mejor solubilidad en soluciones acuosas en comparación con la forma libre de MM01. Nuestro nanosistema demostró una mejor eficacia en un modelo ortotópico de cáncer de mama al producir una mayor reducción del tamaño del tumor en aquellos ratones tratados con la nanomedicina L-PGA-CD-MM01. / [CA] Aquesta tesi doctoral titulada "El microambient tumoral inflamatori com a objectiu en el disseny de nanoconjugats per al tractament del càncer de mama avançat" se centra en l'avaluació d'un nou inhibidor del inflamasoma (MM01) com a eina química per a estudiar el paper del inflamasoma en models d'inflamació i càncer. El capítol I inclou una descripció general del sistema immune, els inflamasomas dependents de ASC i el paper que juguen en el desenvolupament de malalties. També s'aprofundeix en el paper dels inflamasomas i de la proteïna ASC en la progressió del càncer de mama. A més, s'inclouen conceptes bàsics de nanotecnologia, nanomedicina i polímers terapèutics. Finalment, s'aborden els avantatges d'utilitzar *nanomedicinas com a teràpia, les interaccions de les nanomedicines amb els sistemes biològics, els nanofármacs descrits en la literatura, així com les seues possibilitats de translació a la pràctica clínica. En els capítols de resultats, delineem un nou mecanisme d'acció per a MM01, un modulador de l'activitat del s*inflamasoma recentment identificat: la inhibició de la oligomerización del ASC i el subsegüent processament reduït de la pro-caspasa-1 i la inhibició de l'activitat de la caspasa-1. Vam demostrar que MM01 interromp el procés de oligomerización de ASC associat a l'activitat de diversos inflamasomas i inhibeix l'alliberament de IL-*1ß i la piroptosis en diversos models cel·lulars d'inflamació. MM01 també redueix la infiltració de neutròfils i l'acumulació de citocines pro-inflamatòries en un model in vivo de peritonitis. Donada la implicació de la funció de ASC en múltiples complexos del *inflamasoma, el tractament amb MM01 pot representar un enfocament terapèutic eficaç per a tractar aquelles malalties en les quals està implicada l'activació de múltiples *inflamasomas. Tenint en compte els resultats obtinguts, emprem el nostre inhibidor del *inflamasoma, MM01, per a estudiar el paper del *inflamasoma en la progressió tumoral en diferents models de càncer de mama tant in vitro com in vivo. Vam demostrar que diferents línies cel·lulars de càncer de mama responen de forma diferent del tractament amb MM01. Desenvolupem un assaig funcional que comprén l'avaluació de la migració de les cèl·lules de càncer de mama en resposta al secretoma pro-inflamatori dels macròfags M1 (estímul inflamatori) en presència de MM01. Unes certes línies cel·lulars (com la línia cel·lular EO771) van mostrar un augment de la migració en resposta a l'estímul inflamatori i una disminució de la migració en resposta al tractament amb MM01; no obstant això, també identifiquem línies cel·lulars que responen negativament al tractament amb MM01 (com la línia cel·lular 4T1). Finalment, vam demostrar l'eficàcia d'aquest experiment funcional in vivo demostrant que el tractament amb MM01 va reduir la grandària del tumor en el model ortotòpic EO771 però va augmentar la grandària del tumor i la metàstasi pulmonar en el model ortotòpic 4T1. Aquests dos models, que recapitulen respostes contradictòries al tractament amb el nostre inhibidor del *inflamasoma, podran utilitzar-se en el futur per a determinar biomarcadors que prediguen la resposta. Finalment, desenvolupem una estratègia sintètica per a obtindre un nou nanomedicament que millora la solubilitat i l'orientació tumoral del MM01 en un model de càncer de mama. Implementem un enfocament híbrid de conjugació-complexació que comprén la conjugació de ß-ciclodextrina amb un àcid lineal *poli-L-glutàmic (PGA) (L-PGA-ßCD) per a proporcionar la capacitat d'atrapar MM01 dins dels anells de ciclodextrina. El nanosistema obtingut va mostrar una millor solubilitat en solucions aquoses en comparació amb la forma lliure de MM01. El nostre nanosistema va demostrar una millor eficàcia en un model ortotòpic de càncer de mama en produir una major reducció de la grandària del tumor en aquells ratolins tractats amb la nanomedicina L-PGA-CD-MM01. / [EN] This PhD thesis entitled "The inflammatory tumor microenvironment as a target in the design of nanoconjugates for the treatment of advanced breast cancer" focuses on the evaluation of a novel inflammasome inhibitor (MM01) as a chemical tool to study the role of the inflammasome in models of inflammation and cancer. Chapter I includes an overview of the immune system, ASC-dependent inflammasomes and the role they play in disease development. It also delves into the role of inflammasomes and ASC protein in breast cancer progression. In addition, basic concepts of nanotechnology, nanomedicine and therapeutic polymers are included. Finally, the advantages of using nanomedicines as therapeutics, the interactions of nanomedicines with biological systems, the nanodrugs described in the literature, as well as their translation possibilities to clinical practice are discussed. In the results chapters, we delineate a novel mechanism of action for MM01, a recently identified modulator of inflammasome activity: inhibition of ASC oligomerization and subsequent reduced pro-caspase-1 processing and inhibition of caspase-1 activity. We demonstrate that MM01 disrupts the ASC oligomerization process associated with the activity of several inflammasomes and inhibits IL-1ß release and pyroptosis in several cellular models of inflammation. MM01 also reduces neutrophil infiltration and pro-inflammatory cytokine accumulation in an in vivo model of peritonitis. Given the involvement of ASC function in multiple inflammasome complexes, treatment with MM01 may represent an effective therapeutic approach to treat those diseases in which multiple inflammasome activation is involved. Considering the results obtained, we employed our inflammasome inhibitor, MM01, to study the role of the inflammasome in tumor progression in different breast cancer models both in vitro and in vivo. We demonstrate that different breast cancer cell lines respond differently to MM01 treatment. We developed a functional assay involving the assessment of breast cancer cell migration in response to the pro-inflammatory M1 macrophage secretome (inflammasome stimulus) in the presence of MM01. Certain cell lines (such as the EO771 cell line) showed increased migration in response to the inflammatory stimulus and decreased migration in response to MM01 treatment; however, we also identified cell lines that respond negatively to MM01 treatment (such as the 4T1 cell line). Finally, we demonstrated the efficacy of this functional experiment in vivo by showing that MM01 treatment reduced tumor size in the orthotopic EO771 model but increased tumor size and lung metastasis in the orthotopic 4T1 model. These two models, which recapitulate conflicting responses to treatment with our inflammasome inhibitor, may be used in the future to determine biomarkers predictive of response. Finally, we developed a synthetic strategy to obtain a novel nanomedicine that enhances the solubility and tumor targeting of MM01 in a breast cancer model. We implemented a hybrid conjugation-complexation approach comprising the conjugation of ß-cyclodextrin to a linear poly-L-glutamic acid (PGA) (L-PGA-ßCD) to provide the ability to trap MM01 within the cyclodextrin rings. The obtained nanosystem showed improved solubility in aqueous solutions compared to the free form of MM01. Our nanosystem demonstrated improved efficacy in an orthotopic breast cancer model by producing a greater reduction in tumor size in those mice treated with the L-PGA-CD-MM01 nanomedicine. / Soriano Teruel, P. (2023). Inflammatory Tumor Microenvironment as target in the design of nanoconjugates for the treatment of advanced breast cancer [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/192498

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-04-25T11:14:43Z No. of bitstreams: 1 2012 - Liane Caroline S Nascimento.pdf: 1534381 bytes, checksum: a4191d92ac386756692de94030e95ef7 (MD5) / Made available in DSpace on 2017-04-25T11:14:43Z (GMT). No. of bitstreams: 1 2012 - Liane Caroline S Nascimento.pdf: 1534381 bytes, checksum: a4191d92ac386756692de94030e95ef7 (MD5) Previous issue date: 2012-06-25 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / This paper describes the investigation of the centesimal composition, chemical and antioxidant activity of Morinda citrifolia L. (Rubiaceae), native of Southeast Asia, popularly known as noni, which has emerged in Brazil as a plant with great healing potential. The empirical knowledge of the population has meant that the fruit is constantly present in the diet of Maranh?o. Early in the second half of 2010 seedlings of the species were planted in two experimental areas of the Federal Institute of Maranh?o (IFMA), Campus of Z? Doca-MA, broken down by planting time. After three months of the planting in the first area the fruiting process was initiated, and it was possible to perform bromatologic analyzes. For the centesimal composition was found in pulp, seed and mixtures of both, respectively: the ash content (0,82%?0,01; 0,60%?0,01 e 0,79%?0,03), moisture (90%?0,01; 28,34%?0,01; 80,64%?0,05), proteins (4,2%?0,01; 7,47%?0,06; 4,70%?0,02), total lipids (0,34%?0,04; 0,79%?0,03; 0,63%?0,07), carbohydrates (2,68%?0,01; 25,83%?0,02; 13,24%?0,01) and calorific value (30,58kcal/100g?0,02; 140,31 kcal/100g?0,03; 77,43 kcal/100g?0,03). It was found in the pulp and seed values soluble dietary fiber (0,51?0,04; 0,93%?0,03) and insoluble (1,44?0,05; 36,04%?0,38). For the chemical characterization it was found in the pulp, seed and mixtures of both: ? Brix (8,17?0,05;-), the total acidity (0,54?0,02; 0,28?0,01; 0,62?0,02%) and pH (3,95?0,07; 4,5?0,04; 4,1?0,05), the relationship TA / SS pulp was 14.97?0,07. The content of ascorbic acid was calculated by the method Tilmans, pulp and seed fractions (117,33?0,01; 24,33?0,03), expressed in mg/100 g sample. The antioxidant capacity analyzes were performed in three different parts: the pulp, peel and seed using alcoholic extracts (11,63?0,07; 10,03?0,04; 11,19?0,01) and aqueous extract (7,20?0,07; 6,98?0,07; 7,60?0,01) through the sequestration of free radical DPPH, expressed in mM of Trolox / g sample. It was further evaluated the antioxidant capacity of different extracts of the three parts of the fruit and alcoholic extract (98.78% in pulp, 84.76% in peel and 94.96% in the seed) showed much higher antioxidant capacity than the aqueous extract ( 8.08% in the pulp, 5.22% in peel and 22.66% in the seed) and the prominence given to the pulp, followed by seed and peel, respectively. It was demonstrated that the noni fruit grown in the municipality of Z? Doca presented a rich source of nutrition important fact that justifies its inclusion in the diet. It should be noted that the pulp showed a high content of vitamin C and antioxidant activity, and seed a significant amount of insoluble fiber, suggesting that this product can be inserted in the consumer market for being a nutritious food with great herbal medicine potential / Este trabalho descreve a investiga??o da composi??o centesimal, qu?mica e atividade antioxidante de Morinda citrifolia L. (Rubiaceae), origin?rio do sudoeste da ?sia, popularmente conhecida como noni, que vem se configurando no territ?rio brasileiro como um vegetal com grande potencial fitoter?pico. O conhecimento popular tem feito com que o fruto esteja presente constantemente na dieta alimentar maranhense. No in?cio do segundo semestre de 2010 foram plantadas mudas da esp?cie em duas ?reas experimentais do Instituto Federal do Maranh?o (IFMA), Campus Z? Doca-MA, divididas por ?poca de plantio. Ap?s tr?s meses do plantio na primeira ?rea o processo de frutifica??o foi iniciado, sendo poss?vel realizar as an?lises bromatol?gicas. Para composi??o centesimal verificou-se na polpa, semente e mistura de ambos, respectivamente: o teor de cinzas (0,82%?0,01; 0,60%?0,01 e 0,79%?0,03); a umidade (90%?0,01; 28,34%?0,01; 80,64%?0,05); prote?nas (4,2%?0,01; 7,47%?0,06; 4,70%?0,02); lip?deos totais (0,34%?0,04; 0,79%?0,03; 0,63%?0,07); carboidratos (2,68%?0,01; 25,83%?0,02; 13,24%?0,01) e valor cal?rico (30,58 kcal/100g?0,02; 140,31 kcal/100g?0,03; 77,43 kcal/100g?0,03). Verificou-se na polpa e semente os valores de fibras alimentares sol?veis (0,51?0,04; 0,93%?0,03) e insol?veis (1,44?0,05; 36,04%?0,38). Para caracteriza??o qu?mica verificou-se na polpa, semente e mistura de ambos: o ?Brix (8,17?0,05;-), a acidez total (0,54?0,02; 0,28?0,01; 0,62?0,02%) e o pH (3,95?0,07; 4,5?0,04; 4,1?0,05), a rela??o ATT/SS na polpa foi de 14,97?0,07. O Teor de ?cido asc?rbico foi calculado pelo m?todo Tilmans, nas fra??es polpa e semente (117,33?0,01; 24,33?0,03), expressos em mg/100g amostra. As an?lises da capacidade antioxidante foram realizadas em tr?s partes diferentes: polpa, casca e semente utilizando extrato alco?lico (11,63?0,07; 10,03?0,04; 11,19?0,01) e extrato aquoso (7,20?0,07; 6,98?0,07; 7,60?0,01), atrav?s do sequestro do radical livre DPPH, expressos em ?M de Trolox/g amostra. Foi ainda avaliada a capacidade antioxidante dos diferentes extratos, das tr?s partes do fruto e o extrato alco?lico (98,78% na polpa; 84,76% na casca e 94,96% na semente) apresentou capacidade antioxidante bem maior que o extrato aquoso (8,08% na polpa, 5,22% na casca e 22,66% na semente) sendo o destaque dado ? polpa, seguido da semente e da casca, respectivamente. Demonstrou-se que os frutos do noni cultivados no munic?pio de Z? Doca apresentaram uma rica fonte nutricional importante fato que justifica sua inser??o na dieta alimentar. Cabe destacar que a polpa apresentou um elevado teor de vitamina C, e atividade antioxidante, e a semente um relevante teor de fibras insol?veis, sugerindo que esse produto pode ser inserido no mercado consumidor por ser um alimento nutritivo e com grande potencial fitoter?pico.

La niche des cellules souches hématopoïétiques représente un compartiment complexe et radiosensible. Sa protection est nécessaire pour la restauration de l'hématopoïèse faisant suite à la myélosuppression due à l'exposition aux rayonnements ionisants. Nous avons dans un premier temps étudié l'effet des RI sur les progéniteurs endothéliaux et mésenchymateux de la niche par une étude de radiosensiblilité et une étude d'évaluation de la mort cellulaire. Nous avons proposé par la suite une stratégie innovante de thérapie génique basée sur la sécrétion locale et à court terme du morphogène Sonic hedgehog visant à favoriser la réparation de niche vasculaire et de stimuler les cellules souches hématopoïétiques et les cellules progénitrices résiduelles. Nous avons étudié la réponse hématopoïétique des singes irradiés à 8-Gy gamma après une seule injection intra-osseuse de cellules souches mésenchymateuses xénogéniques, multipotentes et d'origine adipocytaire transfectées avec un plasmide pIRES2-eGFP codant la protéine Shh. La durée de thrombocytopénie et celle de neutropénie ont été significativement réduites chez les animaux greffés et les clonogènes sont normalisés à partir du 42e jour. Les aires sous la courbe des numération des plaquettes et des neutrophiles entre 0 et 30 jours ont été significativement plus élevée chez les animaux traités que chez les témoins. La greffe d'explants de MatrigelTM colonisés ou non avec des ASC chez des souris immunodéprimées a démontré une activité pro-angiogénique notable des ASC transfectées avec le plasmide Shh . Le suivi à long terme (180 à 300 jours) a confirmé une reconstitution durable dans les quatre singes greffés. Globalement cette étude suggère que la greffe de cellules souches multipotentes Shh-peut représenter une nouvelle stratégie pour la prise en charge des dommages radio-induits de la niche.

<p>Articular cartilage is the thin, load-bearing connective tissue that lines the ends of long bones in diarthroidal joints, providing predominantly a mechanical function. Because cartilage is avascular and aneural, it has little capacity for self-repair if damaged. One repair strategy is through a functional tissue engineering approach using adipose-derived stem cells (ASCs). ASCs are an abundant progenitor cell source easily obtained through a minimally invasive liposuction procedure. When appropriately stimulated, ASCs have demonstrated significant potential for chondrogenic differentiation. Though studies have demonstrated the ability of ASCs to synthesize cartilage-specific macromolecules, a more thorough understanding of factors that modulate ASC chondrogenesis is required. Accordingly, the central aim of this dissertation was to study the chondrogenic response of ASCs to biochemical, biomechanical, and biomaterial factors.</p><p>We hypothesized that factors, other than TGF-beta and dexamethasone, would improve ASC chondrogenesis. BMP-6 emerged as a potent regulator of ASC chondrogenesis, particularly in early culture, as noted by significant upregulation of cartilage-specific extracellular matrix (ECM) genes and downregulation of cartilage hypertrophy markers.</p><p>Hypothesizing that biomechanical factors would accelerate the formation of cartilage-specific macromolecules, we designed and manufactured an instrument to apply dynamic deformational loading to ASC seeded constructs. Dynamic loading significantly inhibited ASC metabolism and downregulated cartilage-specific ECM genes. However, 21 days of dynamic loading induced the production of type II collagen, a principal component of articular cartilage.</p><p>We hypothesized that a biomaterial derived from cartilage would serve as a bioactive scaffold and induce chondrogenic differentiation. The novel, ECM-derived scaffold promoted the most robust differentiation of ASCs relative to both biochemical and biomechanical factors, particularly noted by a type II collagen-rich matrix after 28 days of culture. After 42 days of culture, biphasic mechanical testing revealed an aggregate modulus of 150 kPa, approaching that of native cartilage. These data suggest that the ECM-derived scaffold may retain important signaling molecules to drive differentiation or that ASC differentiation is dependent on proper cell anchorage.</p><p>In summary, we have shown that biochemical, biomechanical, and biomaterial factors have strong influences on the chondrogenic potential of ASCs. Optimization of these factors will ultimately be required to successfully engineer a functional tissue.</p> / Dissertation

Chlamydia trachomatis (C. trachomatis) is the most prevalent sexually transmitted bacteria with devastating reproductive consequences that lead to tubal factor infertility (TFI). Recent studies have implicated apoptosis – associated speck – like protein containing a caspase recruitment domain (ASC) as an adaptor of inflammasomes that stimulate IL – 1β and IL – 18 secretion, pro – inflammatory cytokines with critical functions in host defense against a variety of pathogens. Therefore, for the first time, we are reporting the use of ASC-/- mice in a mouse model of Chlamydia infection that might provide some information on the role of inflammasomes in the pathogenesis of Chlamydia infection. In this study, wild type (WT) and ASC-/- mice were infected with Chlamydia. Infectivity, pathology of the upper genital tract (UGT), and, fertility were evaluated. In addition, expression of ASC – dependent inflammasomes and the activation of immune cells within the genital tract (GT) were studied. Results showed that Chlamydia infectivity in ASC-/- mice was significantly higher (p-/- mice which, when compared to infected WT mice, was exhibited by decrease in average number of pups and percent pregnancy. There was also severe UGT damage in ASC-/- mice compared to WT mice, correlating with the higher number of hydrosalpinx observed on the UGT of Chlamydia infected ASC-/- mice. Furthermore, IL – 1β and IL – 18 production as well as immune cell activation were down regulated in the GT of Chlamydia infected ASC-/- mice. This finding indicates that in absence of ASC, host innate and adaptive immunity is impaired. Results imply that ASC plays a protective role in the mucosal immunity against GT Chlamydia infection.