13,909 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

    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

    Bridging technology and educational psychology: an exploration of individual differences in technology-assisted language learning within an Algerian EFL setting

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    The implementation of technology in language learning and teaching has a great influence onthe teaching and learning process as a whole and its impact on the learners’ psychological state seems of paramount significance, since it could be either an aid or a barrier to students’ academic performance. This thesis therefore explores individual learner differences in technology-assisted language learning (TALL) and when using educational technologies in higher education within an Algerian English as a Foreign Language (EFL) setting. Although I initially intended to investigate the relationship between TALL and certain affective variables mainly motivation, anxiety, self-confidence, and learning styles inside the classroom, the collection and analysis of data shifted my focus to a holistic view of individual learner differences in TALL environments and when using educational technologies within and beyond the classroom. In an attempt to bridge technology and educational psychology, this ethnographic case study considers the nature of the impact of technology integration in language teaching and learning on the psychology of individual language learners inside and outside the classroom. The study considers the reality constructed by participants and reveals multiple and distinctive views about the relationship between the use of educational technologies in higher education and individual learner differences. It took place in a university in the north-west of Algeria and involved 27 main and secondary student and teacher participants. It consisted of focus-group discussions, follow-up discussions, teachers’ interviews, learners’ diaries, observation, and field notes. It was initially conducted within the classroom but gradually expanded to other settings outside the classroom depending on the availability of participants, their actions, and activities. The study indicates that the impact of technology integration in EFL learning on individual learner differences is both complex and dynamic. It is complex in the sense that it is shown in multiple aspects and reflected on the students and their differences. In addition to various positive and different negative influences of different technology uses and the different psychological reactions among students to the same technology scenario, the study reveals the unrecognised different manifestations of similar psychological traits in the same ELT technology scenario. It is also dynamic since it is characterised by constant change according to contextual approaches to and practical realities of technology integration in language teaching and learning in the setting, including discrepancies between students’ attitudes and teacher’ actions, mismatches between technological experiences inside and outside the classroom, local concerns and generalised beliefs about TALL in the context, and the rapid and unplanned shift to online educational delivery during the Covid-19 pandemic situation. The study may therefore be of interest, not only to Algerian teachers and students, but also to academics and institutions in other contexts through considering the complex and dynamic impact of TALL and technology integration at higher education on individual differences, and to academics in similar low-resource contexts by undertaking a context approach to technology integration

    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

    In vitro investigation of the effect of disulfiram on hypoxia induced NFκB, epithelial to mesenchymal transition and cancer stem cells in glioblastoma cell lines

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    A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy.Glioblastoma multiforme (GBM) is one of the most aggressive and lethal cancers with a poor prognosis. Advances in the treatment of GBM are limited due to several resistance mechanisms and limited drug delivery into the central nervous system (CNS) compartment by the blood-brain barrier (BBB) and by actions of the normal brain to counteract tumour-targeting medications. Hypoxia is common in malignant brain tumours such as GBM and plays a significant role in tumour pathobiology. It is widely accepted that hypoxia is a major driver of GBM malignancy. Although it has been confirmed that hypoxia induces GBM stem-like-cells (GSCs), which are highly invasive and resistant to all chemotherapeutic agents, the detailed molecular pathways linking hypoxia, GSC traits and chemoresistance remain obscure. Evidence shows that hypoxia induces cancer stem cell phenotypes via epithelial-to-mesenchymal transition (EMT), promoting therapeutic resistance in most cancers, including GBM. This study demonstrated that spheroid cultured GBM cells consist of a large population of hypoxic cells with CSC and EMT characteristics. GSCs are chemo-resistant and displayed increased levels of HIFs and NFκB activity. Similarly, the hypoxia cultured GBM cells manifested GSC traits, chemoresistance and invasiveness. These results suggest that hypoxia is responsible for GBM stemness, chemoresistance and invasiveness. GBM cells transfected with nuclear factor kappa B-p65 (NFκB-p65) subunit exhibited CSC and EMT markers indicating the essential role of NFκB in maintaining GSC phenotypes. The study also highlighted the significance of NFκB in driving chemoresistance, invasiveness, and the potential role of NFκB as the central regulator of hypoxia-induced stemness in GBM cells. GSC population has the ability of self-renewal, cancer initiation and development of secondary heterogeneous cancer. The very poor prognosis of GBM could largely be attributed to the existence of GSCs, which promote tumour propagation, maintenance, radio- and chemoresistance and local infiltration. In this study, we used Disulfiram (DS), a drug used for more than 65 years in alcoholism clinics, in combination with copper (Cu) to target the NFκB pathway, reverse chemoresistance and block invasion in GSCs. The obtained results showed that DS/Cu is highly cytotoxic to GBM cells and completely eradicated the resistant CSC population at low dose levels in vitro. DS/Cu inhibited the migration and invasion of hypoxia-induced CSC and EMT like GBM cells at low nanomolar concentrations. DS is an FDA approved drug with low toxicity to normal tissues and can pass through the BBB. Further research may lead to the quick translation of DS into cancer clinics and provide new therapeutic options to improve treatment outcomes in GBM patients

    The developing maternal-infant relationship: a qualitative longitudinal study

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    Aim The study aimed to explore maternal perceptions and the use of knowledge relating to their infant’s mental health over time using qualitative longitudinal research. Background There has been a growing interest in infant mental health over recent years. Much of this interest is directed through the lens of infant determinism, through knowledge regarding neurological development resulting in biological determinism. Research and policy in this field are directed toward individual parenting behaviours, usually focused on the mother. Despite this, there is little attention given to maternal perspectives of infant mental health, indicating that a more innovative approach to methodology is required. Methods This study took a qualitative longitudinal approach, and interviews were undertaken with seven mothers from the third trimester of pregnancy and then throughout the first year of the infant’s life. Interviews were conducted at 34 weeks of pregnancy, and then when the infant was 6 and 12 weeks, 6, 9, and 12 months, alongside the collection of researcher field notes—a total of 41 interviews. Data were analysed by creating case profiles, memos, and summaries, and then cross-comparison of the emerging narratives. A psycho-socially informed approach was taken to the analysis of data. Findings Three interrelated themes emerged from the data: evolving maternal identity, growing a person, and creating a safe space. The theme of evolving maternal identity dominated the other themes of growing a person and creating a safe space in a way that met perceived socio-cultural requirements for mothering and childcare practices. Participants’ personal stories give voice to their perceptions of the developing maternal-infant relationship in the context of their socio-cultural setting, relationships with others, and experiences over time. Conclusions This study adds new knowledge by giving mothers a voice to express how the maternal-infant relationship develops over time. The findings demonstrate how the developing maternal-infant relationship grows in response to their mutual needs as the mother works to create and sustain identities for herself and the infant that will fit within their socio-cultural context and individual situations. Additionally, the findings illustrate the importance of temporal considerations, social networks, and intergenerational relationships to this evolving process. Recommendations for practice, policy, and education are made that reflect the unique relationship between mother and infant and the need to conceptualise this using an ecological approach

    Desenvolvimento de testes genéticos por PCR em tempo-real para diagnóstico rápido de LHON e surdez

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    Mitochondrial cytopathies are a set of diseases caused by a disturbance in the cell energy production. Mitochondrial dysfunction impairs efficiency of the mitochondrial respiratory chain (MRC) and ATP production, affecting the organism’s energetic equilibrium. Pathogenic sequence variants in mitochondrial DNA (mtDNA) that lead to these pathologies are more frequent in tissues that need higher energy levels to function. The presented work looks into two such diseases: Leber’s Hereditary Optic Neuropathy and mitochondrial non-syndromic Hearing Loss (MNSHL). LHON is characterized by presence of genetic alterations in mtDNA, with three main primary pathogenic sequence variants existing, which represent 90-95% of LHON cases with an identified genetic cause: m.3460G>A, in ND1 subunit gene; m.11778G>A, in ND4 subunit gene; and m.14484T>C, in ND6 subunit gene. All of these are subunits of the MRC’s complex I. These mtDNA variations lead to mitochondrial dysfunction in complex I, creating ATP depletion, reactive oxygen species (ROS) increase and oxidative stress. LHON is commonly characterized by a sequential vision loss and, within 1 year of symptoms starting, 97% of patients with vision loss in one eye develop loss in the second. Therapy administration yields good outcomes, if done in a short-time span after first vision loss. It is essential to quickly and reliably scan for pathogenic sequence variants, in order to act timely and rescue function. Mitochondrial non-syndromic hearing loss and deafness (MNSHL) is characterized by sensorineural hearing loss (SNHL). This type of hearing loss, particularly when induced by aminoglycosides, has also three primary pathogenic sequence variants associated with ototoxicity: m.1494C>T and m.1555A>G, both in the MTRNR1 gene, and m.7445A>G, in the MTCO1 and MTTS1 genes. These are responsible for ATP depletion, an increase of ROS and oxidative stress, due to alterations in the mitochondrial ribosome or tRNA. In MNSHL, the cochlea is the affected tissue. With this disorder the principal modifier factor is the administration of aminoglycosides, a type of antibiotics, which trigger a cascade, that leads the individual permanently deaf. The best course of action is prevention, and to ensure clinical action is not dramatically slowed down, results that show whether administration is safe or not need to be quick. The aim of this work, for both diseases, is the development of a screening method characterized by fast and reliable approach for genetic assessment, to be used for clinical guidance, particularly in therapeutics. For LHON, the screening method is based on real-time PCR with High-Resolution Melting (HRM) analysis, for detection of the TOP-3 pathogenic sequence variants, by assessing the amplicon’s Tm. In this case, 94 samples were analyzed, including LHON suspected patients, relatives, other mitochondrial disease patients and healthy controls. All samples were previously classified by another method, having then been blinded before the performance of this work. For analysis, Real-Time PCR was run in triplicates, to allow for a more robust HRM analysis. The software had the ability to classify samples as different variants, wild-type or mutant; information which was then crossed with the previous classification of the sample to assess the success of the software classification. Samples were correctly assigned. This approach provides results in a quick fashion that guides clinical action in a timely fashion. The presence of other polymorphisms in the amplicons might be a hindrance to the robustness of the results provided by this technique and their effect on variant classification needs to be considered. For this, a predictive in-silico analysis was performed, regarding all described variants’ presence in the sequences in analysis. Accordingly, an additional complementary method may be necessary for assurance of result’s specificity. For MNSHL, the screening method was also real-time PCR based, but this one was performed with Amplification-Refractory Mutation System (ARMS) primers, designed for the pathogenic sequence variants previously associated in literature for the MNSHL. Discrimination of results was done based on amplification in positive cases and lack of it in negative cases. This approach analyzed 32 samples, including MNSHL suspected patients, their relatives, other mitochondrial disease patients and healthy controls, but only results concerning the m.1555A>G were obtained timely. All samples were previously classified by another method, having then been blinded before performance of this work. For optimization, Real-Time PCR was run in duplicates, to increase robustness of analysis. The Real-Time software showed if samples amplified as wild-type or mutant, with classification following. This data was crossed with previous known classification of the samples to assess the success of the approach. All analyzed samples were correctly identified with this approach. However, two of the three pathogenic sequence variants did not achieve implementation within the timeframe necessary for their inclusion, namely m.1494C>T and m.7445A>G. The optimization of their screening was not possible and further work is necessary to optimize and implement the approach concerning the analysis for these variants. In conclusion, it was possible to implement an analysis method for LHON’s TOP-3 pathogenic sequence variants within 24h, which represents a big step in precision medicine for diagnosis of this disease. On the other hand, although the implementation was not concluded, a similar approach was started for MNSHL – that, when concluded, will have an enormous impact in preventing aminoglycoside induced HL. This work represents a high impact scientific contribution in reverse translational research.As citopatias mitocondriais são um conjunto de doenças causadas por um distúrbio na produção de energia celular. A disfunção mitocondrial prejudica a eficiência da cadeia respiratória mitocondrial (CRM) e a produção de ATP, afetando o equilíbrio energético do organismo. As variações de sequência patogénicas no DNA mitocondrial (mtDNA) que levam a estas patologias são mais frequentes em tecidos que necessitam de maiores níveis de energia para funcionar. O presente trabalho explora duas dessas doenças: Neuropatia ótica hereditária de Leber (LHON) e Surdez mitocondrial induzida por aminoglicosídeos. A LHON é caracterizada pela presença de alterações genéticas do mtDNA, existindo três variações de sequência patogénicas primárias principais, que representam 90-95% de casos de LHON com identificação da causa genética: m.3460G>A, no gene que codifica a subunidade ND1; m.11778G>A, no gene que codifica a subunidade ND4; e m.14484T>C, no gene que codifica a subunidade ND6. Todas estas subunidades pertencem ao complexo I da CRM. Estas alterações no mtDNA levam a disfunção mitocondrial no complexo I, criando depleção de ATP, aumento de espécies reativas de oxigénio (ROS) e stresse oxidativo. A LHON é comummente caracterizada pela perda sequencial de visão e, 1 ano após o início dos sintomas, 97% dos casos com perda de visão num olho desenvolvem perda de visão no segundo. A administração de terapia produz bons resultados, quando realizada num curto período de tempo após a primeira perda de visão. Assim, é essencial pesquisar variações de sequência patogénicas genéticas de forma rápida e fiável, para atuar rapidamente e recuperar a função visual. A Surdez mitocondrial não-sindrómica (MNSHL), em particular a induzida por aminoglicosídeos, tem também três mutações principais associadas à perda de audição: m.1494C>T e m.1555A>G, ambas no gene MTRNR1, e m.7445A>G, nos genes MTCO1 e MTTS1. Estas são responsáveis pela depleção de ATP, aumento de ROS e stresse oxidativo, devido a alterações no ribossoma ou no tRNA mitocondrial. Aqui, o tecido afetado é a cóclea. Nesta doença, o fator modificador em destaque é a administração de antibióticos de tipo aminoglicosídeos, que despoletam uma cascata de acontecimentos, levando à surdez permanente. A melhor estratégia passa pela prevenção, enquanto ao mesmo tempo se garante que a ação clínica não sofre atrasos. Desta forma, são necessários resultados rápidos, que demonstrem se a administração será segura ou não. O objetivo deste trabalho, para ambas as doenças, é o desenvolvimento de um método de screening, caracterizado por uma abordagem rápida e fiável, usado para guiar a decisão clínica, particularmente na terapêutica. Para a LHON, o método de screening é baseado em PCR em tempo-real com análise de High-Resolution Melting (HRM), para deteção das variantes patogénicas TOP-3, avaliando as Tm dos amplicons. Neste caso, foram analisadas 94 amostras, incluindo doentes com suspeita de LHON, familiares, outros doentes com suspeita de outra doença mitocondrial e controlos saudáveis. Todas as amostras foram previamente classificadas por outro método, tendo sido sujeitas a anonimização antes da realização do trabalho. Para a análise, a PCR em tempo-real foi realizada em triplicados, para permitir uma análise de HRM mais robusta. O software teve a capacidade de classificar amostras como diferentes variantes, ou seja, normal ou mutante. Esta informação foi cruzada com as classificações previamente existentes para avaliar o sucesso da classificação pelo software. As amostras foram corretamente classificadas. Esta abordagem fornece resultados de forma rápida, podendo guiar a ação clínica em tempo útil. A presença de outros polimorfismos nos amplicons poderão obstruir a robustez dos resultados fornecidos por esta técnica e o seu efeito na classificação de variantes precisa de ser considerado. Por esta razão, foi realizada uma análise de previsão in-silico, considerando a presença de todas as variantes descritas. Nesse sentido, pode ser necessário um método complementar de análise para assegurar a especificidade dos resultados. Para a Surdez mitocondrial não-sindrómica, o método de screening baseou-se também na PCR em tempo-real, mas foi realizada com primers de Amplification-Refractory mutation system (ARMS), desenhados para as variantes de sequência patogénicas associadas à MNSHL induzida por aminoglicosídeos, previamente descritas na literatura para esta doença. A discriminação de resultados foi feita com base na presença/ausência de amplificação para cada variante. Foram analisadas 32 amostras com esta abordagem, incluindo doentes com suspeita de MNSHL, seus familiares, doentes com suspeita de outra doença mitocondrial e controlos saudáveis, mas apenas foram obtidos resultados em tempo útil para a m.1555A>G. Todas as amostras tinham sido previamente classificadas por outro método, tendo sido anonimizadas antes da realização do trabalho. Para a otimização, a PCR em tempo-real foi realizada em duplicados, aumentando a robustez da análise. O software de tempo-real mostrou quais as amostras que amplificaram como normais ou mutantes, permitindo a classificação das mesmas. Os dados foram comparados com as classificações previamente conhecidas, para avaliar o sucesso da abordagem em estudo. Todas as amostras em análise foram corretamente identificadas. No entanto, duas das três variantes patogénicas não foram implementadas em tempo útil para inclusão neste trabalho. Para a m.1494C>T e a m.7445A>G, a otimização não foi possível, e será necessário trabalho adicional no futuro, para a implementação da análise destas variantes. Em conclusão, foi possível implementar um método da análise das variantes genéticas TOP-3 da LHON em 24h, o que representa um grande passo na medicina de precisão para diagnóstico desta doença. Por outro lado, apesar de não ter sido concluída a implementação, iniciou-se uma abordagem semelhante para a MNSHL – que, quando for concluída, terá um enorme impacto para evitar a perda auditiva por exposição a aminoglicosídeos. Este trabalho representa uma contribuição científica de alto impacto na investigação translacional reversa.O Laboratório de Biomedicina Mitocondrial e Teranóstica recebeu apoio financeiro da Santhera Pharmaceuticals que permitiu implementação do projeto nacional “Investigação Translacional Epidemiológica, Bigenómica e Funcional nas Atrofias Ópticas” (IP Professora Doutora Manuela Grazina). Apoio financeiro do CNC.IBILI no âmbito do Plano Estratégico UID/NEU/04539/2019.Mestrado em Biologia Aplicad

    Development of in-vitro in-silico technologies for modelling and analysis of haematological malignancies

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    Worldwide, haematological malignancies are responsible for roughly 6% of all the cancer-related deaths. Leukaemias are one of the most severe types of cancer, as only about 40% of the patients have an overall survival of 10 years or more. Myelodysplastic Syndrome (MDS), a pre-leukaemic condition, is a blood disorder characterized by the presence of dysplastic, irregular, immature cells, or blasts, in the peripheral blood (PB) and in the bone marrow (BM), as well as multi-lineage cytopenias. We have created a detailed, lineage-specific, high-fidelity in-silico erythroid model that incorporates known biological stimuli (cytokines and hormones) and a competing diseased haematopoietic population, correctly capturing crucial biological checkpoints (EPO-dependent CFU-E differentiation) and replicating the in-vivo erythroid differentiation dynamics. In parallel, we have also proposed a long-term, cytokine-free 3D cell culture system for primary MDS cells, which was firstly optimized using easily-accessible healthy controls. This system enabled long-term (24-day) maintenance in culture with high (>75%) cell viability, promoting spontaneous expansion of erythroid phenotypes (CD71+/CD235a+) without the addition of any exogenous cytokines. Lastly, we have proposed a novel in-vitro in-silico framework using GC-MS metabolomics for the metabolic profiling of BM and PB plasma, aiming not only to discretize between haematological conditions but also to sub-classify MDS patients, potentially based on candidate biomarkers. Unsupervised multivariate statistical analysis showed clear intra- and inter-disease separation of samples of 5 distinct haematological malignancies, demonstrating the potential of this approach for disease characterization. The work herein presented paves the way for the development of in-vitro in-silico technologies to better, characterize, diagnose, model and target haematological malignancies such as MDS and AML.Open Acces
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