13 research outputs found

    LITERATURE MINING SUSTAINS AND ENHANCES KNOWLEDGE DISCOVERY FROM OMIC STUDIES

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    Genomic, proteomic and other experimentally generated data from studies of biological systems aiming to discover disease biomarkers are currently analyzed without sufficient supporting evidence from the literature due to complexities associated with automated processing. Extracting prior knowledge about markers associated with biological sample types and disease states from the literature is tedious, and little research has been performed to understand how to use this knowledge to inform the generation of classification models from ‘omic’ data. Using pathway analysis methods to better understand the underlying biology of complex diseases such as breast and lung cancers is state-of-the-art. However, the problem of how to combine literature-mining evidence with pathway analysis evidence is an open problem in biomedical informatics research. This dissertation presents a novel semi-automated framework, named Knowledge Enhanced Data Analysis (KEDA), which incorporates the following components: 1) literature mining of text; 2) classification modeling; and 3) pathway analysis. This framework aids researchers in assigning literature-mining-based prior knowledge values to genes and proteins associated with disease biology. It incorporates prior knowledge into the modeling of experimental datasets, enriching the development process with current findings from the scientific community. New knowledge is presented in the form of lists of known disease-specific biomarkers and their accompanying scores obtained through literature mining of millions of lung and breast cancer abstracts. These scores can subsequently be used as prior knowledge values in Bayesian modeling and pathway analysis. Ranked, newly discovered biomarker-disease-biofluid relationships which identify biomarker specificity across biofluids are presented. A novel method of identifying biomarker relationships is discussed that examines the attributes from the best-performing models. Pathway analysis results from the addition of prior information, ultimately lead to more robust evidence for pathway involvement in diseases of interest based on statistically significant standard measures of impact factor and p-values. The outcome of implementing the KEDA framework is enhanced modeling and pathway analysis findings. Enhanced knowledge discovery analysis leads to new disease-specific entities and relationships that otherwise would not have been identified. Increased disease understanding, as well as identification of biomarkers for disease diagnosis, treatment, or therapy targets should ultimately lead to validation and clinical implementation

    Liver Tumors

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    This book is oriented towards clinicians and scientists in the field of the management of patients with liver tumors. As many unresolved problems regarding primary and metastatic liver cancer still await investigation, I hope this book can serve as a tiny step on a long way that we need to run on the battlefield of liver tumors

    Frameshift mutations at the C-terminus of HIST1H1E result in a specific DNA hypomethylation signature

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    BACKGROUND: We previously associated HIST1H1E mutations causing Rahman syndrome with a specific genome-wide methylation pattern. RESULTS: Methylome analysis from peripheral blood samples of six affected subjects led us to identify a specific hypomethylated profile. This "episignature" was enriched for genes involved in neuronal system development and function. A computational classifier yielded full sensitivity and specificity in detecting subjects with Rahman syndrome. Applying this model to a cohort of undiagnosed probands allowed us to reach diagnosis in one subject. CONCLUSIONS: We demonstrate an epigenetic signature in subjects with Rahman syndrome that can be used to reach molecular diagnosis

    From Birds to Drug-Resistant Cancer, a novel In situ Methodology to Explore Divergent Genome Evolution

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    Fluorescent hybridisatio nmethodologies have not changed in principles over the past 30 years, with the increase of computational sequencing technologies causing the replacement of in situ hybridisations. Fluorescence in situ hybridisation (FISH) is in need of a refresh to be a worthwhile tool in a modern day cytogenetic laboratory to overcome short comings of these new methods. The creation of the novel multilayer FISH protocol has effectively eliminated many negative aspects of classic FISH based experiments, such as a large reduction in cost and is no longer as limited by fluorophore availability. Here presented within this thesis is the creation of this methodology and application to a wide variety of cytogenetic hypothesises. Key species from the Galliform order were investigated in order to detect previously missed intrachromosomal rearrangements within their macrochromosomes, a premise formerly overlooked. Rearrangements were found within chromosomes of the galliforme species used such as E.chinensis which displays a intrachromosomal inversion on the p-arm of chromosome 2. Furthermore, the creation of an interphase state folding prediction tool has been used to assess the arrangement of macrochromosomes during cellular growth stages within G.gallus. Here it is noted that there are particular arrangements identified which are similar across chromosomes studied. The chicken lymphoma cell line DT40 is of great importance in B-cell receptor studies along with gene disruption experiments. Presented here is an updated karyotype for the cell line. Here shows contrasting and more in-depth evidence of aberrations to further develop our understanding of the genomic arrangement of this useful cell line. The level of tumour heterogeneity in a cancer is a diagnostic tool allowing clinicians to comment on therapeutic choices and prognosis of the disease. Found to be dominant in recurrent cancers, cytotoxic resistant tumour cell populations may indeed exist within initial primary tumours at low frequency to be positively selected during chemotherapy. Within a neuroblastoma cell line,and cyto-toxic resistant derivatives lines,there has been identified a level of genomic heterogeneity which may give clues towards the generation of drug resistance mechanisms

    Meiosis

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    Meiosis, the process of forming gametes in preparation for sexual reproduction, has long been a focus of intense study. Meiosis has been studied at the cytological, genetic, molecular and cellular levels. Studies in model systems have revealed common underlying mechanisms while in parallel, studies in diverse organisms have revealed the incredible variation in meiotic mechanisms. This book brings together many of the diverse strands of investigation into this fascinating and challenging field of biology

    Mechanisms of osteoblast reprogramming and differentiation during zebrafish caudal fin regeneration

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    Regeneration is an impressive biological process that allows the replacement of lost body parts due to damage or injury, restoring both tissue architecture and function. It is well documented that while mammals have a limited capacity to regenerate lost tissues, other vertebrates, such as amphibians and teleost fish, exhibit a remarkable capacity to regenerate organs, like the heart and the retina, and large sections of the body, such as the limb and the fin. Zebrafish has become an important model system to study vertebrate regeneration and the adult caudal fin is one of the most used tissues to comprehend how tissues are restored. This structure is easily accessible to surgery, amenable to live imaging, its amputation does not compromise survival and regeneration is particularly fast, occurring over the course of two weeks. Fin regeneration is an epimorphic process since it relies on a specialized structure called blastema, which is composed of a proliferative heterogeneous population of dedifferentiated cells with restrictive lineage potential. After caudal fin amputation, a regenerative program is activated and occurs in three sequential phases: wound healing, blastema formation, and regenerative outgrowth. These events comprise a tight coordination between proliferation, patterning and differentiation to reconstitute the architecture and the size of the original tissue. The adult caudal fin is composed of multiple tissues, including blood vessels, nerves, mesenchyme and the structural support, the bony-rays (skeletal elements). Each bony-ray is surrounded and maintained by an outer and inner monolayer of bone secreting cells, the osteoblasts. Many studies have focused on bone regeneration since the zebrafish caudal fin provides a unique model to understand bone formation and osteoblast dynamics upon tissue damage and regeneration. After caudal fin amputation, formation of the new bone elements depends greatly on tissue plasticity (changes in cellular identity). This is achieved through the activation of two complementary processes that enable the assembly of an osteoblast progenitor pool during blastema formation: dedifferentiation of resident mature osteoblasts and commitment of joint-associated osteoblast progenitors. Complex regulatory mechanisms subsequently maintain and expand the osteoblast progenitor pool and promote their redifferentiation into mature osteoblasts to restore the skeletal tissue. Therefore, both osteoblast progenitor assembly and redifferentiation are critical aspects of caudal fin bony-ray regeneration. Interestingly, ablation of mature osteoblasts prior to caudal fin amputation does not affect normal bone regeneration, suggesting that de novo bone formation can rely solely on the commitment of joint-associated osteoblast precursors or on new osteoblast progenitors arising from alternative sources.In this PhD thesis, I aimed to unravel key aspects of caudal fin bone regeneration, focusing on new regulators of osteoblast dedifferentiation and redifferentiation and alternative sources for de novo osteoblast formation in osteoblast-depleted fins. To investigate novel regulators of osteoblast dedifferentiation, we performed a genome-wide gene expression analysis of osteoblasts undergoing dedifferentiation. With this analysis, we concluded that this process occurs much earlier in the regenerative process than what was previously thought. Furthermore, we characterized the molecular basis of osteoblast dedifferentiation regarding epigenetic modulation, signal transduction, cell adhesion reorganization, epithelial to mesenchymal transition and acquisition of migratory behaviour and proliferation. Particularly, we observed that osteoblasts change their metabolic signature upon injury. This was predicted based on the upregulation of several glycolytic and lactate producing enzymes, which is followed by an increase in the expression of oxidative phosphorylation electron transport chain components. We hypothesize that osteoblast dedifferentiation relies on a bivalent metabolism that uses both glycolysis and oxidative phosphorylation, which may reflect an adaption to the energetic demands of regeneration. Since the link between metabolic adaptation and regeneration remains poorly understood, we decided to address it by inhibiting the glycolytic influx. We observed major defects in the regenerative process, including impaired assembly of the wound epidermis, a major signalling centre during regeneration, and fewer cells re-entering the cell cycle. In addition, we showed that several osteoblast markers were downregulated and that osteoblast populations became disorganized. This suggests that metabolic adaptation plays an important role in regeneration, in particular during osteoblast dedifferentiation. In addition to the transcriptional analysis, we followed a targeted approach. We examined the role of the Hippo signalling pathway as a potential regulator of osteoblast dedifferentiation, by inhibiting Yap (Hippo pathway effector). This prevented mature osteoblasts to migrate, re-enter the cell cycle and to assemble the osteoblast progenitor pool. In parallel, we evaluated the role of this pathway in mediating osteoblast redifferentiation during regenerative outgrowth. We noticed that Yap inhibition leads to a decrease in the number of differentiating osteoblasts and to the misregulation of key signalling pathways, such as Bmp and Wnt signalling. We provide evidence that Yap not only promotes osteoblast differentiation through activation of Bmp signalling via bmp2a expression but also restricts the osteoblast progenitor pool by inhibiting Wnt signalling to the differentiation front by regulating dkk1a. Altogether, these results lead us to propose that the Hippo/Yap signalling pathway regulates osteoblast dedifferentiation as well as redifferentiation. This reveals a previously unknown duality if the Hippo/Yap pathway in controlling two different aspects of osteoblast biology during caudal fin regeneration. Lastly, we provide evidence into the cellular and molecular mechanisms that regulate de novo osteoblast formation in osteoblast-depleted caudal fins. We identified an additional osteoblast progenitor population that arises at the outer and inner bone surfaces adjacent to the epidermal and mesenchymal compartments, respectively. These cells are not part of a uniform population but seem to form two distinct osteoblast progenitor populations with different origins or expression profiles. Lineage tracing experiments revealed that mesenchymal cells within the intraray compartment, but not epidermal cells, contribute to generate new osteoblasts in osteoblast-depleted caudal fins. This provides new evidence of an additional source of osteoblasts for regeneration. Moreover, we showed that both Retinoic Acid and Bmp signalling pathways are activated in this osteoblast progenitor population and are important to induce their commitment and recruitment during caudal fin regeneration. Thus, we elucidate potentially dormant regenerative mechanisms that emerge to ensure correct bone formation in caudal fins lacking mature osteoblasts. Taken together, this PhD thesis provides novel insights into new regulators of bone formation and alternative cells that can contribute to correct bone regeneration upon injury. We expect that defining the mechanisms regulating tissue plasticity, reprogramming and fate specification during bone reconstitution have major implications not only to understand the basic mechanisms that regulate tissue regeneration but also to the field of regenerative medicine and bone cancer biology.A regeneração é um processo biológico notável que permite a restituição de tecidos após lesão ou amputação, que incluí a recuperação da função, forma e tamanho do tecido original. Enquanto que os mamíferos possuem uma capacidade limitada de regenerar tecidos, outros vertebrados, como anfíbios e alguns peixes teleósteos, são dotados de uma capacidade extraordinária de regenerar órgãos, como o coração e a retina, e grandes superfícies corporais, como membros e barbatanas. O peixe-zebra (Danio rerio) é utilizado como modelo para o estudo de processos regenerativos em vertebrados. A barbatana caudal do peixe-zebra surgiu como uma das estruturas mais utilizadas para estudar regeneração. Esta estrutura é de fácil acesso à cirurgia de amputação, passível ao uso de técnicas de microscopia, não comprometendo a sobrevivência do animal, sendo que o seu processo regenerativo é consideravelmente rápido. A regeneração da cauda é um processo epimórfico, uma vez que depende da formação de uma estrutura especializada designada blastema. Esta estrutura é composta por uma população heterogénea de células com capacidade proliferativa. Após amputação da cauda, o programa regenerativo é caracterizado por três fases sequenciais: fecho da ferida, formação do blastema e, por fim, crescimento e diferenciação. Durante o processo regenerativo, a coordenação entre proliferação e diferenciação é de grande importância para assegurar e alcançar a estrutura e tamanho iniciais. A barbatana caudal é constituída por vários tecidos, incluindo vasos sanguíneos, nervos, tecido mesenquimal e tecido ósseo, este último sendo constituído por raios ósseos que providenciam estabilidade à cauda. Cada um destes raios ósseos é revestido externa e internamente por uma monocamada de células produtoras de osso, os osteoblastos. Muitos estudos têm-se focado na regeneração destes elementos ósseos presentes na barbatana caudal, uma vez que este sistema possibilita a compreensão do processo regenerativo do osso e a dinâmica dos osteoblastos neste contexto. Após amputação da cauda, a formação do novo tecido ósseo depende consideravelmente da plasticidade celular (alterações na identidade celular). Isto é alcançado durante a formação do blastema, através da ativação de dois processos complementares que permitem a formação de um conjunto de progenitores de osteoblastos: desdiferenciação de osteoblastos maduros presentes no tecido não danificado e diferenciação de progenitores presentes na zona da articulação. Posteriormente, mecanismos regulatórios mantêm e expandem o grupo de progenitores e promovem a sua diferenciação em osteoblastos completamente diferenciados, capazes de produzir matriz óssea e de reconstituir o tecido ósseo. Desta forma, a formação dos progenitores de osteoblastos assim como a sua correta diferenciação são essenciais para promover a regeneração dos raios ósseos da barbatana caudal. Curiosamente, a ablação de osteoblastos maduros antes do início do processo regenerativo não afeta a regeneração dos elementos ósseos, o que sugere que, neste contexto, que a sua formação depende unicamente de progenitores associados à articulação ou de fontes celulares alternativas ainda por descobrir. Durante esta tese de doutoramento, procurei elucidar aspetos chave da regeneração do osso da barbatana caudal do peixe-zebra, nomeadamente os processos regulatórios que controlam o programa de desdiferenciação dos osteoblastos maduros bem como a sua posterior diferenciação, e as fontes alternativas de progenitores de osteoblastos em caudas desprovidas de osteoblastos maduros. Com o intuito de investigar novos mecanismos regulatórios do processo de desdiferenciação, recorremos a uma análise global do transcriptoma dos osteoblastos nesta fase da regeneração. Esta análise revelou que o processo de desdiferenciação ocorre muito cedo durante o processo regenerativo. Para além disso, caracterizámos a base molecular do programa de desdiferenciação, no que diz respeito a mecanismos epigenéticos, metabolismo, vias de transdução de sinal, adesão celular, transição epitélio-mesênquima, migração e proliferação. Em particular, observámos que várias enzimas glicolíticas e produtoras de lactato exibem a sua expressão aumentada no início da desdiferenciação, seguidas de um aumento na expressão de componentes da cadeia transportadora de eletrões. Estes resultados demonstram que os osteoblastos alteram significativamente o seu metabolismo em resposta à amputação. Desta forma, propomos a hipótese de que a desdiferenciação dos osteoblastos depende da aquisição de um metabolismo bivalente no qual as vias glicolíticas e de fosforilação oxidativa são usadas para melhor adaptar os osteoblastos aos novos requisitos do processo regenerativo. Uma vez que a ligação entre adaptação metabólica e regeneração ainda está pouco explorada, decidimos investigar como é que o processo regenerativo é influenciado pela glicólise. Ao inibirmos o fluxo glicolítico, verificámos que o crescimento do tecido regenerativo é significativamente reduzido. Neste contexto, vários fenótipos foram observados: inibição da proliferação; desorganização da população de osteoblastos dentro do blastema; alterações na expressão de vários marcadores de osteoblastos; e deformação da epiderme especializada que se forma durante a regeneração, cuja função secretora de moléculas sinalizadoras é essencial para a desdiferenciação. Estes resultados sugerem que esta adaptação metabólica tem um papel importante durante a regeneração, em particular no processo de desdiferenciação. Para além da análise de transcriptoma, estudámos também uma via de sinalização em particular, a via Hippo, através da manipulação genética do seu efetor Yap. Descobrimos que a inibição de Yap durante a fase de desdiferenciação impede a migração e proliferação de osteoblastos maduros e a formação de novos progenitores. Para além disso, a inibição desta via durante a fase de rediferenciação leva a uma diminuição substancial dos osteoblastos em diferenciação e a uma alteração na expressão de componentes de vias de sinalização cruciais, bmp2 (via BMP) e dkk1 (via Wnt). Estes dados evidenciam que Yap promove a diferenciação dos osteoblastos através da ativação da via BMP e restringe o grupo de progenitores através da inibição da via Wnt. Estes resultados permitem-nos propor que a via de sinalização Hippo/Yap regula quer a desdiferenciação dos osteoblastos quer a sua subsequente rediferenciação, o que revela a dualidade do mecanismo de ação desta via em fases diferentes do processo regenerativo da barbatana caudal. Por último, revelamos a existência de mecanismos celulares e moleculares que regulam a formação de novos progenitores de osteoblastos em caudas desprovidas de osteoblastos maduros. Identificámos assim uma fonte adicional de progenitores que emerge na interface da matriz óssea com os tecidos adjacentes, nomeadamente a epiderme e o mesênquima. Estes progenitores não parecem formar uma população homogénea, mas sim duas populações distintas com diferentes origens ou com diferentes padrões de expressão. Recorrendo a técnicas de seguimento de linhagem, conseguimos identificar o mesênquima, mas não a epiderme, como uma fonte de novos osteoblastos em caudas desprovidas de osteoblastos maduros. Estes resultados põem em evidência uma origem adicional de osteoblastos que contribui para o processo regenerativo. Para além disso, demonstramos que as vias de sinalização do Ácido retinóico e Bmp estão ativas nesta população de progenitores e têm um papel crucial na formação e recrutamento desta fonte adicional de osteoblastos durante o processo regenerativo. Assim, este trabalho permite revelar que, em barbatanas caudais desprovidas de osteoblastos maduros, mecanismos regenerativos que se encontram normalmente inativos, são estimulados e asseguram a formação correta dos elementos ósseos neste contexto. De uma forma geral, esta tese de doutoramento identifica novos mecanismos de regulação da formação do tecido ósseo e fontes celulares alternativas que contribuem e garantem a correta regeneração do osso após lesão. Antevemos que o conhecimento dos mecanismos que regulam a plasticidade celular, reprogramação e especificação de linhagem durante a regeneração do osso possam ter implicações fulcrais não só para o conhecimento dos processos básicos que promovem regeneração, mas também no campo da medicina regenerativa e neoplasias
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