9,211 research outputs found

    Identifizierung prädiktiver und prognostischer Biomarker in unterschiedlichen Tumorkompartimenten des ösophagealen Adenokarzinoms

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    Das ösophageale Adenokarzinom zeigt eine global steigende Inzidenz und hat mit einer 5-Jahres-Überlebensrate von weniger als 25% eine schlechte Prognose. Personalisierte Therapieansätze sind selten und prognostische/prädiktive Biomarker des Tumormikromilieus sind unzureichend charakterisiert. Die kumulative Promotion nähert sich dieser Problematik in drei unterschiedlichen Schwerpunkten. 1. Zur Identifizierung Kompartiment-spezifischer Biomarker wurde eine Methode entwickelt, welche als kostengünstige Alternative zum sc-Seq Expressionsprofile individueller Zelltypen generiert. Dabei erfolgt die Extraktion der RNA nicht aus Einzelzellen, sondern aus flowzytometrisch-getrennten Zellkompartimenten. Die Separation der Proben in Epithelzellen, Immunzellen und Fibroblasten wurde durch verschiedene Verfahren validiert und eine suffiziente Ausbeute an RNA auch für kleine Gewebemengen gezeigt. 2. Biomarker des Immunzellkompartiments als therapeutische Angriffspunkte wurden in einem Patientenkollektiv von bis zu 551 Patienten auf ihre Bedeutung beim EAC überprüft. Es zeigte sich eine Expression der Immuncheckpoints LAG3, VISTA und IDO auf TILs durch IHC und RNA-Sonden basierte Verfahren in einem relevanten Anteil (LAG3: 11,4%, VISTA: 29%, IDO: 52,6%). Es konnte eine prognostisch günstige Bedeutung der VISTA, LAG3 und IDO Expression gezeigt werden. Durch den Vergleich von Genexpressionsprofilen aus therapienaiven und vorbehandelten Tumoren konnte zudem ein immunsuppressiver Effekt von neoadjuvanten Therapiekonzepten auf das Tumormikromilieu des EACs gezeigt werden. Dabei kam es zur verminderten Expression von Checkpoints und Anzahl TILs nach (Radio-) Chemotherapie. 3. Im Tumorzellkompartiment wurde die Rolle von Amplifikationen in ErbB-Rezeptor abhängigen Signalwegen durch FISH-Technik und Immunhistochemie evaluiert. Es fanden sich KRAS Amplifikationen in 17,1%, PIK3CA Amplifikationen in 5% sowie eine HER2/neu-Überexpression in 14,9% der untersuchten Tumore

    Integrative multi-omics analysis for the effect of genetic alterations in cancer xenograft and organoid models

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    Department of Biomedical EngineeringDNA damage is a well-recognized factor in the development and progression of cancer. Numerous studies on genetic changes associated with cancer or the DNA repair pathway have been conducted, however, there is still a need for additional research on their function. The establishment of patient-derived xenografts or organoids for the purpose of testing functional genomic approaches is the subject of ongoing research. According to model-specific characteristics, it is not fully understood how these attempts to simulate patient cancer differ from original cancer. To comprehend the distinction between genuine patient cancer and these patient-derived disease models in more depth, multi-omics analysis is required to comprehend the overall genotypes, phenotypes, and environmental variables. Depending on the characteristics of each disease model, distinct omics analysis approaches and factors must be considered. In addition, care must be taken to avoid technical errors when integrating omics data generated by different sequencing equipment. There is currently no golden rule for data integration, but several approaches are being developed. It is crucial to determine the function of genes linked with the DNA repair pathway because these genes contribute to the induction or prevention of cancer. In chapter 1, I identified the interaction between MRE11 and TRIP13 through proximity labeling combined with the SILAC method which is quantitative proteomics using metabolic labeling. TRIP13 depletion doesn???t affect the nuclease activity and conformation of the MRN complex but directly inhibits the interaction of MDC1 with MRN complex and MDC1 recruitment on the DNA damage site. TRIP13 degradation with mirin treatment shows additive effects on ATM signaling activation. In conclusion, TRIP13 regulates immediate-early DNA damage sensing through MRE11 and ATM signaling independently of mirin. When assessing the functional genomic approach using patient-derived disease models, it is essential to determine which aspects of the models' correlation to actual cancer should be properly considered. In chapter 2, I found there are a few overlapped deleterious somatic mutations of the PDX model and their original tumor. I suspected novel mutagen exposure during PDX establishment or sample contamination. However, germline mutations of PDX models are well conserved from original tumors, and their mutational signatures of PDX also mimic that of their tumor. Though the number of overlapped mutations between the PDX model and their tumor was few, brain tumor-specific mutations are found in PDX samples. Especially, histone methylation- and cilia-related gene mutations are enriched in PDX samples. While it suggested these mutated genes are needed for maintaining the stemness of brain tumor PDX model or PDX model would be more appropriate for the samples with high heterogeneity, I have presented precautions and considerations in PDX model genome analysis. Multi-omics analysis that takes into consideration genetic, expressive, and clinical aspects can provide important information for the study of diseases with complicated etiologies, such as cancer, and can contribute to the development of diagnosis and treatment. To utilize colorectal cancer organoids for Companion Diagnostics (CDx), in chapter 3, I characterized patient-derived colorectal cancer (CRC) organoids through well-known genomic markers such as Tumor mutation burden (TMB), Microsatellite instability (MSI) and propose a novel grouping method using sharing same mutation site. The classification of CRC patients was more detailed combined with consensus molecular subtype (CMS) classifications. Additionally, I extract the expression features of the patients who experience recurrence or metastasis after first-line chemotherapy treatment with reference to clinical data. Drug response of CRC organoids by patient group and knockdown of the extracted features in the selected organoids would be validated in further study. In summary, with this dissertation, I conducted functional research on the DNA repair pathway of cancer-related genes, as well as the genetic analysis between patient-derived xenograft and original tumors, and introduced a novel perspective on the diagnosis and treatment of colorectal cancer patients using patient-derived organoids through multi-omics analysis.ope

    Anuário científico da Escola Superior de Tecnologia da Saúde de Lisboa - 2021

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    É com grande prazer que apresentamos a mais recente edição (a 11.ª) do Anuário Científico da Escola Superior de Tecnologia da Saúde de Lisboa. Como instituição de ensino superior, temos o compromisso de promover e incentivar a pesquisa científica em todas as áreas do conhecimento que contemplam a nossa missão. Esta publicação tem como objetivo divulgar toda a produção científica desenvolvida pelos Professores, Investigadores, Estudantes e Pessoal não Docente da ESTeSL durante 2021. Este Anuário é, assim, o reflexo do trabalho árduo e dedicado da nossa comunidade, que se empenhou na produção de conteúdo científico de elevada qualidade e partilhada com a Sociedade na forma de livros, capítulos de livros, artigos publicados em revistas nacionais e internacionais, resumos de comunicações orais e pósteres, bem como resultado dos trabalhos de 1º e 2º ciclo. Com isto, o conteúdo desta publicação abrange uma ampla variedade de tópicos, desde temas mais fundamentais até estudos de aplicação prática em contextos específicos de Saúde, refletindo desta forma a pluralidade e diversidade de áreas que definem, e tornam única, a ESTeSL. Acreditamos que a investigação e pesquisa científica é um eixo fundamental para o desenvolvimento da sociedade e é por isso que incentivamos os nossos estudantes a envolverem-se em atividades de pesquisa e prática baseada na evidência desde o início dos seus estudos na ESTeSL. Esta publicação é um exemplo do sucesso desses esforços, sendo a maior de sempre, o que faz com que estejamos muito orgulhosos em partilhar os resultados e descobertas dos nossos investigadores com a comunidade científica e o público em geral. Esperamos que este Anuário inspire e motive outros estudantes, profissionais de saúde, professores e outros colaboradores a continuarem a explorar novas ideias e contribuir para o avanço da ciência e da tecnologia no corpo de conhecimento próprio das áreas que compõe a ESTeSL. Agradecemos a todos os envolvidos na produção deste anuário e desejamos uma leitura inspiradora e agradável.info:eu-repo/semantics/publishedVersio

    Neuroanatomical and gene expression features of the rabbit accessory olfactory system. Implications of pheromone communication in reproductive behaviour and animal physiology

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    Mainly driven by the vomeronasal system (VNS), pheromone communication is involved in many species-specific fundamental innate socio-sexual behaviors such as mating and fighting, which are essential for animal reproduction and survival. Rabbits are a unique model for studying chemocommunication due to the discovery of the rabbit mammary pheromone, but paradoxically there has been a lack of knowledge regarding its VNS pathway. In this work, we aim at filling this gap by approaching the system from an integrative point of view, providing extensive anatomical and genomic data of the rabbit VNS, as well as pheromone-mediated reproductive and behavioural studies. Our results build strong foundation for further translational studies which aim at implementing the use of pheromones to improve animal production and welfare

    The place where curses are manufactured : four poets of the Vietnam War

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    The Vietnam War was unique among American wars. To pinpoint its uniqueness, it was necessary to look for a non-American voice that would enable me to articulate its distinctiveness and explore the American character as observed by an Asian. Takeshi Kaiko proved to be most helpful. From his novel, Into a Black Sun, I was able to establish a working pair of 'bookends' from which to approach the poetry of Walter McDonald, Bruce Weigl, Basil T. Paquet and Steve Mason. Chapter One is devoted to those seemingly mismatched 'bookends,' Walt Whitman and General William C. Westmoreland, and their respective anthropocentric and technocentric visions of progress and the peculiarly American concept of the "open road" as they manifest themselves in Vietnam. In Chapter, Two, I analyze the war poems of Walter McDonald. As a pilot, writing primarily about flying, his poetry manifests General Westmoreland's technocentric vision of the 'road' as determined by and manifest through technology. Chapter Three focuses on the poems of Bruce Weigl. The poems analyzed portray the literal and metaphorical descent from the technocentric, 'numbed' distance of aerial warfare to the world of ground warfare, and the initiation of a 'fucking new guy,' who discovers the contours of the self's interior through a set of experiences that lead from from aerial insertion into the jungle to the degradation of burning human feces. Chapter Four, devoted to the thirteen poems of Basil T. Paquet, focuses on the continuation of the descent begun in Chapter Two. In his capacity as a medic, Paquet's entire body of poems details his quotidian tasks which entail tending the maimed, the mortally wounded and the dead. The final chapter deals with Steve Mason's JohnnY's Song, and his depiction of the plight of Vietnam veterans back in "The World" who are still trapped inside the interior landscape of their individual "ghettoes" of the soul created by their war-time experiences

    Bioactive Compounds from Marine Heterobranchs

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    The natural products of heterobranch molluscs display a huge variability both in structure and in their bioactivity. Despite the considerable lack of information, it can be observed from the recent literature that this group of animals possesses an astonishing arsenal of molecules from different origins that provide the molluscs with potent chemicals that are ecologically and pharmacologically relevant. In this review, we analyze the bioactivity of more than 450 compounds from ca. 400 species of heterobranch molluscs that are useful for the snails to protect themselves in different ways and/or that may be useful to us because of their pharmacological activities. Their ecological activities include predator avoidance, toxicity, antimicrobials, antifouling, trail-following and alarm pheromones, sunscreens and UV protection, tissue regeneration, and others. The most studied ecological activity is predation avoidance, followed by toxicity. Their pharmacological activities consist of cytotoxicity and antitumoral activity; antibiotic, antiparasitic, antiviral, and anti-inflammatory activity; and activity against neurodegenerative diseases and others. The most studied pharmacological activities are cytotoxicity and anticancer activities, followed by antibiotic activity. Overall, it can be observed that heterobranch molluscs are extremely interesting in regard to the study of marine natural products in terms of both chemical ecology and biotechnology studies, providing many leads for further detailed research in these fields in the near future

    Estudo da remodelagem reversa miocárdica através da análise proteómica do miocárdio e do líquido pericárdico

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    Valve replacement remains as the standard therapeutic option for aortic stenosis patients, aiming at abolishing pressure overload and triggering myocardial reverse remodeling. However, despite the instant hemodynamic benefit, not all patients show complete regression of myocardial hypertrophy, being at higher risk for adverse outcomes, such as heart failure. The current comprehension of the biological mechanisms underlying an incomplete reverse remodeling is far from complete. Furthermore, definitive prognostic tools and ancillary therapies to improve the outcome of the patients undergoing valve replacement are missing. To help abridge these gaps, a combined myocardial (phospho)proteomics and pericardial fluid proteomics approach was followed, taking advantage of human biopsies and pericardial fluid collected during surgery and whose origin anticipated a wealth of molecular information contained therein. From over 1800 and 750 proteins identified, respectively, in the myocardium and in the pericardial fluid of aortic stenosis patients, a total of 90 dysregulated proteins were detected. Gene annotation and pathway enrichment analyses, together with discriminant analysis, are compatible with a scenario of increased pro-hypertrophic gene expression and protein synthesis, defective ubiquitinproteasome system activity, proclivity to cell death (potentially fed by complement activity and other extrinsic factors, such as death receptor activators), acute-phase response, immune system activation and fibrosis. Specific validation of some targets through immunoblot techniques and correlation with clinical data pointed to complement C3 β chain, Muscle Ring Finger protein 1 (MuRF1) and the dual-specificity Tyr-phosphorylation regulated kinase 1A (DYRK1A) as potential markers of an incomplete response. In addition, kinase prediction from phosphoproteome data suggests that the modulation of casein kinase 2, the family of IκB kinases, glycogen synthase kinase 3 and DYRK1A may help improve the outcome of patients undergoing valve replacement. Particularly, functional studies with DYRK1A+/- cardiomyocytes show that this kinase may be an important target to treat cardiac dysfunction, provided that mutant cells presented a different response to stretch and reduced ability to develop force (active tension). This study opens many avenues in post-aortic valve replacement reverse remodeling research. In the future, gain-of-function and/or loss-of-function studies with isolated cardiomyocytes or with animal models of aortic bandingdebanding will help disclose the efficacy of targeting the surrogate therapeutic targets. Besides, clinical studies in larger cohorts will bring definitive proof of complement C3, MuRF1 and DYRK1A prognostic value.A substituição da válvula aórtica continua a ser a opção terapêutica de referência para doentes com estenose aórtica e visa a eliminação da sobrecarga de pressão, desencadeando a remodelagem reversa miocárdica. Contudo, apesar do benefício hemodinâmico imediato, nem todos os pacientes apresentam regressão completa da hipertrofia do miocárdio, ficando com maior risco de eventos adversos, como a insuficiência cardíaca. Atualmente, os mecanismos biológicos subjacentes a uma remodelagem reversa incompleta ainda não são claros. Além disso, não dispomos de ferramentas de prognóstico definitivos nem de terapias auxiliares para melhorar a condição dos pacientes indicados para substituição da válvula. Para ajudar a resolver estas lacunas, uma abordagem combinada de (fosfo)proteómica e proteómica para a caracterização, respetivamente, do miocárdio e do líquido pericárdico foi seguida, tomando partido de biópsias e líquidos pericárdicos recolhidos em ambiente cirúrgico. Das mais de 1800 e 750 proteínas identificadas, respetivamente, no miocárdio e no líquido pericárdico dos pacientes com estenose aórtica, um total de 90 proteínas desreguladas foram detetadas. As análises de anotação de genes, de enriquecimento de vias celulares e discriminativa corroboram um cenário de aumento da expressão de genes pro-hipertróficos e de síntese proteica, um sistema ubiquitina-proteassoma ineficiente, uma tendência para morte celular (potencialmente acelerada pela atividade do complemento e por outros fatores extrínsecos que ativam death receptors), com ativação da resposta de fase aguda e do sistema imune, assim como da fibrose. A validação de alguns alvos específicos através de immunoblot e correlação com dados clínicos apontou para a cadeia β do complemento C3, a Muscle Ring Finger protein 1 (MuRF1) e a dual-specificity Tyr-phosphoylation regulated kinase 1A (DYRK1A) como potenciais marcadores de uma resposta incompleta. Por outro lado, a predição de cinases a partir do fosfoproteoma, sugere que a modulação da caseína cinase 2, a família de cinases do IκB, a glicogénio sintase cinase 3 e da DYRK1A pode ajudar a melhorar a condição dos pacientes indicados para intervenção. Em particular, a avaliação funcional de cardiomiócitos DYRK1A+/- mostraram que esta cinase pode ser um alvo importante para tratar a disfunção cardíaca, uma vez que os miócitos mutantes responderam de forma diferente ao estiramento e mostraram uma menor capacidade para desenvolver força (tensão ativa). Este estudo levanta várias hipóteses na investigação da remodelagem reversa. No futuro, estudos de ganho e/ou perda de função realizados em cardiomiócitos isolados ou em modelos animais de banding-debanding da aorta ajudarão a testar a eficácia de modular os potenciais alvos terapêuticos encontrados. Além disso, estudos clínicos em coortes de maior dimensão trarão conclusões definitivas quanto ao valor de prognóstico do complemento C3, MuRF1 e DYRK1A.Programa Doutoral em Biomedicin

    Towards personalized immunotherapy : development of in vitro models for imaging natural killer cell behavior in the tumor microenvironment

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    Tremendous advances in the tumor immunology field have transformed immunotherapy from a promising approach to a standard clinical practice. However, a subset of cancer patients is non-responsive to immunotherapy. More research is therefore needed to understand the mechanisms underlying tumor resistance to immunotherapeutic treatments. The aim of this doctoral work was to develop new tools to study the mechanisms of cancer immunosurveillance and to test immunotherapeutic treatments in vitro. In this thesis, I describe the methods developed, and I discuss the main biological findings obtained by using these methods. The thesis is organized as follows. A short historical background of immunotherapy is provided in Chapter 1. Chapter 2 describes the principles of NK cell-mediated cancer immunosurveillance, and provides an overview on rare cancers, mainly focusing on sarcoma. The research aims are listed in Chapter 3. In Chapter 4, I describe the cell culture methods and cell analysis techniques relevant for my doctoral work. In Chapter 5, I describe the methods we developed to culture tumor spheroids in vitro using ultrasonic standing waves in microwell chips, focusing on the theory, design, and applications. Chapter 6 and Chapter 7 focus on the biological findings obtained using our platform in combination with traditional immunological methods, followed by future implementations discussed in Chapter 8. The constituent papers are provided at the end of the thesis. In Paper I, we combined the use of the microwell chip, ultrasonic standing waves and a protein-repellent polymer coating to enable the production of spheroids from multiple cell types. In absence of cell adhesion to the chip, spheroids could be collected and further analyzed by off-the-chip techniques. In Paper II, we designed a novel multichambered microwell chip to perform multiplexed fluorescence screening of two- or three-dimensional cell cultures. The platform allows the direct assessment of drug or immune cell cytotoxic efficacy, making it a promising tool for individualized cytotoxicity tests for personalized medicine. In Paper III, we investigate the function of PVR receptors in NK cells interacting with renal carcinoma spheroids, and the impact of PVR in NK cell-based cellular immunotherapy. We demonstrated that variations in PVR expression are primarily recognized by the inhibitory receptor TIGIT, while DNAM-1 strongly contributes to NK cell activation mainly through PVR-independent mechanisms. We performed NK cell-based cytotoxicity assays against renal carcinoma spheroids in the microwell chip. Anti-TIGIT treatment was effective only for TIGIThigh NK cells both when used as monotherapy or in combination with other drugs, suggesting that only a fraction of patients might respond to anti-TIGIT therapy. In Paper IV, a similar approach was used with primary sarcomas. We cultured patient-derived sarcoma spheroids and tested NK cell-based immunotherapy in the microwell chip, either alone or in combination with antibody therapy, and we identified promising treatment combinations. In Paper V, we applied the use of expansion microscopy to visualize NK cells infiltrating renal carcinoma spheroids. In conclusion, our multi-disciplinary work shows the development of new imaging-based platform and its use to study the mechanisms of NK cell-mediated tumor surveillance and for personalized therapy
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