Las células tumorales presentan alteraciones metabólicas. Una de las diferencias con las células no transformadas es que los tumores usan más glucosa incluso en condiciones de normoxia, lo que se utiliza actualmente para visualizar tumores mediante la técnica de PET. Ultimamente se están desarrollando terapias basadas en estas particularidades metabólicas de las células tumorales, que las hacen más sensibles a inhibición del metabolismo glicolítico. Aquí estudiamos la muerte producida por la falta de glucosa en sarcomas y otros tipos tumorales. La privación de glucosa induce apoptosis o necrosis dependiendo de la línea celular. Mostramos cómo la privación de glucosa induce actividad caspasa en células deficientes de Bax/Bak, mientras que en las líneas de rabdomiosarcoma induce necrosis. Además la caspasa-8 está implicada en la muerte por ausencia de glucosa en estas células deficientes en Bax/Bak y también en células humanas HeLa. Investigamos el uso de 2-deoxiglucosa como inductor de muerte celular frente a líneas de rabdomiosarcoma alveolar y embrional. La 2-deoxiglucosa promueve muerte celular en líneas de rabdomiosarcoma alveolar. También induce diferenciación acompañada por una bajada de PAX3/FOXO1a, una proteína surgida de la translocación cromosómica en las células de rabdomiosarcoma alveolar y crítica en el desarrollo oncogénico de las mismas. Caracterizamos la muerte celular apoptótica inducida por 2-deoxiglucosa en líneas de sarcoma, in vitro. Estudiamos las moléculas de las rutas de apoptosis que determinan la sensibilidad de estas células a la 2-deoxiglucosa, con especial interés en las proteínas de la familia del oncogén Bcl-2. La muerte celular inducida por el tratamiento está asociada a la activación de Bax y Bak. Observamos que la sobre-expresión de proteínas anti-apoptóticas de la familia Bcl-2 como Bcl-xL y de Mcl-1 previene la apoptosis, indicando que la muerte sucede a través de la ruta mitocondrial. Enseñamos que la bajada de Mcl-1 y la subida de Noxa son críticos en la muerte por 2-DG. Además, la 2-DG promueve estrés reticular acompañado de la inducción de ATF4 y chaperonas. Al bajar los niveles de ATF4, protegemos a las células de la muerte inducida por 2-DG y prevenimos la pérdida de Mcl-1. Incubamos células en presencia de manosa, que revierte el estrés inducido por la 2-DG y previnimos la muerte sin subir los niveles de ATP. Así, el estrés energético causado por la 2-DG no es la principal causa de muerte celular. También la 2-DG promueve la fosforilación de eIF2α, un inductor de ATF4, y la inactivación de mTOR. Nuestros resultados sugieren que el uso de inhibidores glicolíticos como la 2-DG pueden ser efectivos en el tratamiento de los rabdomiosarcoma alveolares y que Noxa podría ser un marcador pronóstico de la eficiencia de estas drogas. Por otro lado, caracterizamos las respuestas a la falta de glucosa y 2-DG, en particular la respuesta autofágica que puede determinar que las células mueran o no en respuesta a la falta de nutrientes. Observamos que las células están sensibilizadas al tratamiento con 2-DG tras el uso de cloroquina. Sin embargo, la presencia de cloroquina no afecta a la privación de glucosa. Intentamos clarificar si la falta de glucosa está induciendo macroautofagia. El flujo de LC3 por western blot nos indica que no hay más macroautofagia ni en células DKO ni en las Rh4 en ausencia de glucosa. Por microscopía confocal y analizando células HeLa y Rh4, tampoco vemos mayores acúmulos de GFP-LC3 tras la retirada de glucosa comparando con tratamientos clásicos de inducción de autofagía como la retirada de aminoácidos o con rapamicina. Estos datos sugieren que la combinación de 2-DG con inhibidores de la autofagia podría ser útil en el tratamiento contra rabdomiosarcomas y que la falta de glucosa no induce autofagia. / Characterization of cell death induced by inhibition of glucose metabolism Rhabdomyosarcoma (RMS) is the most common soft-tissue tumor of childhood characterized by high malignancy, local invasiveness and a marked predisposition to metastasize. Recently, a number of therapies targeting tumor cell metabolism are being developed, since oncogenic changes make tumors more sensitive to inhibition of glycolytic metabolism. We observed that the glycolytic inhibitor 2-deoxyglucose (2-DG) can efficiently promote cell death in p53-deficient alveolar rhabdomyosarcoma (the rhabdomyosarcoma subtype with poorer prognosis), but not in embryonal rhabdomyosarcoma. Differential expression of HIF-1 could not explain the different sensitivity of tumor subtypes. 2-deoxyglucose induced nuclear chromatin condensation, cleavage of caspase substrates and DNA degradation which was inhibited by caspase inhibitors. Cell death triggered by 2-DG was associated with its ability to activate Bax and Bak and was prevented by overexpression of the anti-apoptotic Bcl-2 homologs Bcl-xL and Mcl-1. Conversely, siRNA against Bcl-xL or Mcl-1 accelerated death. 2-DG promoted downregulation of the anti-apoptotic protein Mcl-1 and accumulation of two BH3-only proteins, Noxa and Bim. siRNA against these proteins indicated that Noxa mediates 2-DG-induced cell death. Addition of different carbon/energy sources indicated that apoptosis is not associated with loss of ATP but rather with endoplasmic reticulum stress and the eIF2-alpha-ATF4 pathway. 2-DG promoted Mcl-1 loss, probably due to general inhibition of translation, since both eIF2-alpha phosphorylation and inactivation of the mTOR pathway were observed. All these events (ER stress, energetic stress and inhibition of the mTOR pathway) are known to induce autophagy. Indeed, 2-DG induces autophagy, and inhibition of autophagy at different levels sensitizes cells to 2-DG. Together, our findings suggest that glycolysis inhibitors such as 2-DG may be effective in treating alveolar rhabdomyosarcoma.

The von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome that is transmitted in an autosomal dominant manner. The disease is characterized by the formation of highly angiogenic tumors in many organs but the main causes of mortality are renal cell carcinomas and hemangioblastomas. Mutations in the VHL protein are responsible for the pathogenesis of the disease. VHL associates with elongin Band C to form the VBC complex. The cullin 2 protein (CUL2) and ring box protein 1 (RBX1) also associate with the VBC complex to form an E3 ubiquitin ligase involved in the ubiquitination and subsequent degradation of the hypoxia inducible transcription factor (HIF2alpha). Mutations in VHL that abrogate its E3 ligase activity lead to increased levels ofHIF2alpha and the subsequent accumulation of pro-proliferative and pro-angiogenic HIF2alpha target genes. VHL also has an important function in the regulation of extracellular matrix (ECM) assembly which is independent of its HIF2alpha regulation pathway. VHL's regulation of ECM assembly was shown to have important consequences for tumor angiogenesis and cell invasion. It was shown to be necessary for the proper assembly of a fibronectin matrix and was most recently found to interact with collagen IV alpha 2 (COL4A2). The aim of this thesis is to further characterize the VHL-COL4A2 interaction. VHL was shown to interact directly and specifically to COL4A2 and is necessary for proper COL4A2 matrix assembly. The association of VHL with COL4A2 appears to be independent of its functions as an E3 ubiquitn ligase and CUL2 was identified as part of the VBC complex that associates with collagen IV (COL4). Furthermore, a strategy to identify the binding site of VHL on COL4A2 has been employed and is in progress. These experiments represent the beginning of investigations into the novel interaction between VHL and COL4A2.

A estimulação vibrotátil tem sido utilizada para promover a comunicação alternativa em pessoas com deficiência visual e/ou auditiva. Por meio dela, ativam-se os receptores da pele que promovem a transdução do estímulo mecânico em uma resposta eletrofisiológica enviada ao SNC. O objetivo do presente trabalho é o de investigar a resposta à estimulação tátil por jato de CO2 como método de comunicação alternativa. Materiais e Métodos: desenvolveu-se um sistema de estimulação tátil por jato de CO2, no qual pressão e fluxo do gás são controlados e o gás é aplicado à pele por meio de uma agulha, cuja velocidade de deslocamento pode ser ajustada entre 20 e 100 mm/s. O instrumento atua como um plotter tátil. Foram realizados testes com 25 voluntários sem deficiências físicas e/ou cognitivas, nos quais avaliaram-se as respostas psicofísica e cognitiva e a taxa de reconhecimento correto de dezenove letras aplicadas na região abdominal, com intensidade estimulatória equivalente a três vezes o limiar de percepção. Resultados: o limiar de percepção da força encontrado foi de 60 mgf e a estimulação fluxotátil foi realizada utilizando-se a força de 180 mgf. Em testes cegos, a melhor taxa de reconhecimento correto das letras atingiu a média de 68 (±17) %, e ocorreu na velocidade de 40 mm/s e frequência de 1,33 Hz. As letras D, O, M e N apresentaram as menores taxas médias de reconhecimento, com 29%, 37%, 47% e 51%, respectivamente, devido à morfologia das mesmas gerar dúvida na diferenciação entre elas, enquanto as melhores taxas foram observadas sobre as letras I, U, J e B com 87%, 71%, 68% e 65%, respectivamente. Conclusões: o sistema desenvolvido possibilitou a execução dos testes e a coleta das informações, necessárias à realização da pesquisa. Os resultados mostraram que a resposta psicofísica à estimulação fluxotátil está relacionada à faixa de frequência de resposta dos discos de Merkel, com resposta máxima em 1,33 Hz, correspondente a velocidade de deslocamento da agulha de estimulação em 40 mm/s. A morfologia das letras e a velocidade de deslocamento da agulha de estimulação influenciaram nas taxas de reconhecimento das letras. / Vibrotactile stimulation has been used to promote alternative communication for people with visual and hearing impairments. The stimuli activate the receptors of the skin that promote the transduction of mechanical stimuli into an electrophysiological response sent to the CNS. Objective: to investigate the response to tactile stimulation by a CO2 jet as a method of alternative. Materials and Methods: a cutaneous stimulation system was developed which yields jets of CO2 on the subject’s skin at stimulating speeds varying from 20 up to 100 mm/s, by means of an injection needle. The instrument acts as a tactile plotter. Tests were conducted in twenty five volunteers without physical and/or cognitive disabilities in whose the psychophysical and cognitive responses were evaluated as well as the rate of correct recognition of nineteen characters applied to the abdominal region, with the stimulus intensity equivalent to three times the threshold of perception. Results: a force perception threshold of 60 mgf was found and the fluxtactile stimulation was performed using a force of 180 mgf. In blind tests, the best rate of correct character recognition had an average of 68 (± 17)%, and occurred at the speed of 40 mm/s and frequency of 1.33 Hz. The characters D, O, M and N have shown the lower average rate of recognition with 29%, 37%, 47% and 51%, respectively, due to the uncertainty generated by their similar morphology, whereas the best rates were observed for letters I, U, J, and B with 87%, 71%, 68% and 65%, respectively. Conclusions: the developed system enabled the experimental research and the acquisition of information necessary for carrying out the research. The results have shown that the psychophysical responses to fluxtactile stimuli are related to the frequency response of the Merkel discs, with maximal performance at 1.33 Hz, corresponding to the speed of the stimulation needle of 40 mm/s. The morphology of the graphical characters and the stimulation needle speed influenced the rates of letters recognition.

As leucemias são desordens proliferativa nas células tronco hematopoiéticas pluripotentes caracterizada por mutações que comprometem seus precursores mielóides ou linfóides levando ao desenvolvimento de leucemias similares quanto ao fenótipo (Mielóide ou Linfóide) e forma (Crônica ou Aguda). Descrito em 1960, o cromossomo philadelphia (Ph) foi a primeira anomalia cromossômica associada a uma neoplasia, a Leucemia Mielóide Crônica (LMC). Em 1973, foi demonstrado que o cromossomo Ph resulta da translocação recíproca entre os braços longos do cromossomo 9 e 22 [t(9;22)(q34;q11)], produzindo um gene quimérico BCR-ABL, encontrado em 95% dos casos de LMC. A proteína quimérica codificada, BCR-ABL, com atividade tirosina quinase descontrolada, é o fator causal da LMC. Durante os anos 1990, uma série de moléculas sintéticas foram desenvolvidas e testadas para inibir especificamente a atividade tirosina quinase da proteína BCR-ABL, o Imatinib (STI-571 ou Gleevec) foi a primeira dessas drogas aprovadas e usadas como agente terapêutico contra a LMC, demonstrando redução do número de células leucêmicas em quase todos pacientes. No entanto muitos pacientes com resistências primárias ou secundárias foram frequentemente relatados. Os mecanismos de resistência são provavelmente heterogêneos, e no mínimo dois mecanismos envolvendo o gene BCR-ABL foram descritos em estudos realizados in vivo: amplificação do gene e ocorrência de mutações que resultam em substituições de aminoácidos associadas com o fenótipo de resistência. Diferentes níveis de resistência são frequentemente associadas a mutações pontuais recorrentes encontradas no domínio quinase da proteína c-ABL em regiões como: sítio catalítico, alça de ativação (A-loop) e alça de fosforilação (P-loop). Os primeiros estudos computacionais foram realizados com o mutante Thr334Ile e mutações na região do P-loop, e ajudaram a entender num nível molecular, os mecanismos de resistência ao Imatinib. Neste trabalho, realizamos um estudo estrutural e termodinâmico das interações entre os ligantes Imatinib e Nilotinib no sítio ativo da proteína c-ABL selvagem e em 12 proteínas com mutações específicas, além de determinar a importância do solvente (com particular interesse na molécula de água denominada Cw - que medeia a interação fármaco-proteína) no processo de resistência aos fármacos. No presente trabalho, utilizamos o método semi-empírico de energia de interação linear (LIE) de maneira alternativa: analisando as interações entre diferentes mutantes da proteína c-ABL e um inibidor (Imatinib ou Nilotinib). Os coeficientes de LIE obtidos foram os seguintes: com Cw fixa (restrição de Cw na posição inicial) = 0,2169; = 0,0654; = 1,4159; e Cw livre (sem restrição de Cw na posição inicial) = 0,0394; = 0,0077; = -2,0503. O melhor ajuste dos coeficientes de LIE foram obtidos nas simulações sem restrição da posição inicial de uma molécula de água conservada (Cw), essa escolha foi baseada no valor de energia livre absoluta da proteína c-ABL selvagem ( ) ser sempre um número mais negativo quando comparado com o valor da energia livre absoluta de cada proteína c-ABL mutada. O uso do método LIE (Linear Interaction Energy) com essa estratégia foi eficiente para analisar o efeito da interação entre diferentes proteínas c-ABL mutantes e um ligante (Imatinib ou Nilotinib). Até o momento não existem simulações de dinâmica molecular na presença de outros domínios da proteína BCR-ABL: SH2 e SH3.

Muitos autores associam as doenças alcoólicas hepáticas às deficiências nutricionais. Por outro lado, trabalhos experimentais estabelecem que a hepatotoxidade alcoólica relaciona-se especialmente à geração de espécies reativas através do sistema microsomal que oxida etanol via citocromo 450, principalmente o CYP2E1. O CYP2E1 hepático tem a capacidade de ativar algumas drogas comumente utilizadas, como o acetaminofeno, em seus metabólitos mais tóxicos e promover carcinogênese. Além. disso, o metabolismo pelo CYP2E1 resulta num aumento na produção de espécies reativas, com diminuição nos sistemas de defesa antioxidantes, estabelecendo o estresse oxidativo. Como a expressão do CYP2E1 é muito influenciada por fatores nutricionais e hormonais, este trabalho descreve os efeitos do tratamento com etanol nos níveis de CYP2E1 e sua relação com alguns parâmetros pró- e antioxidantes, considerando três modelos experimentais diferentes. Ratos machos Sprague Dawley com cerca de três meses de idade receberam ad lib. ração Purina (Purina Ind., Brasil) e, separadamente, uma solução 25 % etanol-20%sacarose durante 1, 2, 3 ou 4 semanas. Os grupos controles foram isocaloricamente pareados aos animais que consumiram etanol, ou receberam quantidades de sacarose equivalentes às calorias recebidas com o consumo de etanol. 18 h antes do sacrifício os animais foram mantidos em abstinência alcoólica, recebendo água e ração ou apenas água ad lib. Os resultados indicam que o consumo de etanol pode ser associado à estabilização do CYP2E1. No entanto, nas nossas condições experimentais, a presença da isoforma não está associada ao estresse oxidativo. Esses resultados indicam que as deficiências nutricionais, especialmente o baixo consumo de carboidratos, são fundamentais na potenciação do estresse oxidativo induzido pelo etanol. / Many authors have attributed alcoholic liver disease to dietary deficiencies. On the other hand, experimental studies have established that alcohol hepatotoxicity is especially related to the generation of oxidant species through its microsomal metabolism via cytochrome P-450, mainly CYP2E1. Liver CYP2E1 has a high capacity to activate some commonly used drugs, such as acetaminophen, to their toxic metabolites, and to promote carcinogenesis. Moreover, metabolism by CYP2E1 results in a significant reactive oxygen species (ROS) release, accompanied by the defense systems decrease against oxidative stress. Since the expression of CYP2E1 is very much influenced by hormonal and nutritional factors, this study describes the effects of ethanol treatment on CYP2E 1 levels and their relationship with some pro and antioxidant parameters considering three experimental models. Male Sprague-Dawley rats were fed ad. lib. for 1, 2, 3 or 4 weeks a commercial diet (Purina Ind., Brazil) plus a 25% ethanol-20% sucrose solution. Control groups were isocalorically pair-feed to the leading ethanol-consuming animals, or received isocaloric amounts of sucrose for pairing only ethanol calories. Eighteen hours before sacrifice ethanol was withdrawal and animals had only free access to tap water or they were offered food and water ad. lib. Results have shown that ethanol administration was associated with CYP2E1 stabilization although under our experimental condition it was not associated with any oxidative stress. These findings indicate that dietary deficiencies, especially low carbohydrate intake are crucial in the potentiation of the ethanol-induced oxidative stress.

Das Glioblastom ist der häufigste maligne Tumor des zentralen Nervensystems. Trotz leitliniengerechter Therapie, bestehend aus mikrochirurgischer Resektion, Strahlentherapie und ergänzender Chemotherapie mit Temozolomid, beträgt die 2-Jahres-Überlebensrate nur ca. 17%. Daher sind dringend neue Therapieansätze erforderlich. Dem natürlich vorkommenden Dipeptid Carnosin, welches vor über 100 Jahren erstmals isoliert wurde, konnten viele physiologische Funktionen zugeschrieben werden. Zu Beginn unserer Arbeiten war bekannt, dass das Dipeptid das Wachstum von Krebszellen inhibiert, wobei die genauen Mechanismen der antineoplastischen Wirkungsweise weitgehend unbekannt waren. Die Untersuchungen im Rahmen der vorliegenden Habilitationsarbeit setzten sich mit möglichen Wirkmechanismen des Dipeptides auseinander, wobei ebenfalls Fragestellungen zur klinischen Anwendung von Carnosin bearbeitet wurden. Im ersten Abschnitt werden die Transportmechanismen von Carnosin in Glioblastom-Zellen beschrieben. Weiterhin wird die Frage beantwortet, ob das Dipeptid die biologisch aktive Verbindung ist oder ob L-Histidin von Carnosin abgespalten werden muss, um die antineoplastische Wirkung zu entfalten. Der zweite Abschnitt beschäftigt sich mit den Einflüssen von Carnosin auf die Signaltransduktion und Genexpression. Im dritten Abschnitt wird unter anderem mit einem Metabolomics-Ansatz der Stoffwechsel von Glioblastom-Zellen charakterisiert und der Einfluss von Carnosin auf diesen bestimmt. Im vierten Abschnitt wird ein neuartiges Ko-Kultur Modell zur Untersuchung von Carnosins Einfluss auf Glioblastom-Zell-Migration und Koloniebildung vorgestellt. Weiterhin untersuchten wir die möglichen Interaktionen des Dipeptides mit der Standardtherapie von Glioblastomen. Zusammenfassend zeigten wir, dass Carnosin durch drei verschiedene Transporter aufgenommen werden kann. Das Dipeptid hemmt sowohl Proliferation und Migration von Glioblastom-Zellen. Die Spaltung des Dipeptides ist für seine antineoplastische Wirkung nicht notwendig. In die Zelle aufgenommen, wirkt Carnosin inhibitorisch auf den Pentosephosphatweg. Eine mögliche Erklärung dafür lieferte die beobachte nicht-enzymatische Reaktion von Glycerinaldehyd-3-phosphat mit dem Dipeptid. Weiterhin zeigten unsere Experimente zum ersten Mal eine Carnosin-bedingte Veränderung der Histonacetylierung und eine damit einhergehende Beeinflussung der Genexpression. Da das Dipeptid den Effekt der Radio-/Chemotherapie verstärkt, sollte die Wirkung von Carnosin in einer klinischen Studie an Glioblastom-Patienten untersucht werden. / Glioblastoma is the most common malignant tumour of the central nervous system. Only ~17% of patients undergoing standard therapy, including microsurgical resection, radiotherapy and adjuvant chemotherapy using temozolomide survive two years after diagnosis. Hence, new therapeutic approaches are urgently needed. The naturally occurring dipeptide carnosine was discovered more than 100 years ago. Since then, many physiological functions and beneficial effects have been ascribed to it. Previous studies demonstrated that carnosine inhibits growth of cancer cells. However, at the beginning of our investigations were the mechanisms behind carnosine’s anti-neoplastic effect mostly unknown. The present work addresses possible modes of action of carnosine and issues regarding the clinical application of the dipeptide. In the first paragraph we describe the transport mechanisms of carnosine in glioblastoma cells. Furthermore, we deal with the problem whether carnosine is the biological active compound or release of L-histidine from the dipeptide is required to deploy its anti-neoplastic effect. The second paragraph addresses the influence of carnosine on glioblastoma cell signal transduction and gene expression. In the third paragraph we characterise the metabolism of glioblastoma cells and how it is influenced by carnosine by using a metabolomics approach. The fourth paragraph introduces a novel co-culture model which allows the analysis of carnosine’s impact on glioblastoma cell migration and colony formation. Furthermore, the possible interaction of the dipeptide with the glioblastoma standard therapy is investigated. In conclusion, we demonstrated that three different transporters are capable for the uptake of carnosine in glioblastoma cells. The dipeptide inhibited in addition to proliferation also migration of glioblastoma cells. Moreover, cleavage of carnosine was not required for its anti-neoplastic effect. After taken up by the cell, carnosine inhibits the pentose phosphate pathway. The observed non-enzymatic reaction of glyceraldehyde-3-phosphate with the dipeptide could possibly explain this effect. Furthermore, our experiments showed for the first time that carnosine influences gene expression by an effect on histone acetylation. As the administration of carnosine arguments the effects of radio-/chemotherapy, we encourage the clinical evaluation of the dipeptide for glioblastoma patients.

<p>Specialized metabolites are chemical tools produced by organisms to aid in their interaction with the surrounding environment. These diverse compounds can often function as metabolic weapons (<em>e.g.</em> antibiotics), structural components (<em>e.g.</em> lignins), or even attractants (<em>e.g.</em> flavonoids). Because of their frequent utilization in niche environments, specialized metabolite production is often lineage- or even species-specific. Therefore, knowledge between specialized metabolic systems is often nontransferable, which poses a major obstacle in the characterization of these bioactive and commercially relevant compounds. Beyond resolving the chemical composition of a specialized metabolite, the identification of responsible pathway genes and the evolutionary processes responsible for their formation is an arduous task. These gaps in knowledge are further widened by the lack of genomic resources available for specialized metabolite producing species. In this work, we present the genome assemblies of two organisms, each with unique specialized metabolic pathways: the Chinese medicinal plant <em>Lithospermum erythrorhizon </em>and the toxic golden alga <em>Prymnesium parvum. </em>Leveraging the predicted proteome of <em>L. erythrorhizon</em>, we investigated the evolutionary history of specialized metabolic genes responsible for the production of shikonin, a 1,4-naphthoquinone specialized metabolite. We identified a retrotransposition-mediated duplication event responsible for the creation of the core shikonin biosynthesis gene, <em>PGT</em>. In addition, we performed a global coexpression network analysis to identify regulatory and enzymatic gene candidates involved in the shikonin biosynthesis pathway. We also built phylogenetic trees of known and candidate shikonin genes to reveal patterns of lineage-specific gene duplication and retroduplication. Like plants, unicellular algae are known for their production of diverse, often toxic, specialized metabolites. However, these species are often enigmatic. For example, previous studies have documented large phenotypic variation in both toxin chemotypes and levels among different strains of <em>P. parvum</em>. To investigate the genetic basis of this variation, we generated near chromosome level assemblies of two <em>P. parvum </em>strains and performed a broad genome survey of thirteen additional strains. As a result, we identified a commonly studied reference strain, UTEX 2797, as a hybrid with two distinct subgenomes. We also provide evidence of significant variation in haploid genome size across the species. Collectively, these studies supply genetic resources for the future study of these organisms, as well as provide insight into the evolution of their specialized metabolic pathways.</p>

No description available.

<p>Liposarcoma (LPS) arises from adipocytes and is a rare malignancy among all cancer types, but represents the most common form of soft tissue sarcoma, with approximately 2,000 new cases reported annually. Clinically, liposarcomas are classified into four subtypes based on histological analysis: well-differentiated liposarcoma (WDLPS), dedifferentiated liposarcoma (DDLPS), myxoid/round cell liposarcoma, and pleomorphic liposarcoma. Although histological analysis provides useful information for identifying various liposarcoma subtypes, treatment options rely on a fundamental understanding of driver mutations and molecular mechanisms underlying tumorigenesis. This thesis focuses on elucidating important driver mutations and therapeutic targets to eradicate DDLPS. Notch signaling is an evolutionarily conserved signaling pathway essential for organ development and stem cell function. Aberrant Notch signaling underlies the tumorigenesis of many cancers including LPS. However, the specific role of Notch signaling in development of LPS remains elusive. In Chapter 2, I provide evidence demonstrating that Notch signaling plays a key role in cancer stem cells (CSCs), also referred to as tumor-initiating cells (TICs), that drive aggressive DDLPS. I used serial transplantation to enrich and generate a murine DDLPS cell line with constitutively activated Notch signaling (NICDOE). My analyses revealed that NICDOE DDLPS cells are heterogeneous and contain TICs that express cancer stem cell markers. Chapter 3 elucidates how Notch signaling regulates CSCs of LPS. I analyzed human LPS samples to establish a strong correlation between Notch signaling activation and tumor marker expression and prognosis. I further performed gene expression and metabolic analyses of NICDOE DDLPS cells. These assays revealed that NICDOE reduced mitochondrial respiration in DDLPS cells, which was associated with diminished expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), a master regulator of mitochondrial biogenesis. CRISPR/CAS9-mediated deletion of the NICDOE cassette rescued the expression of PGC-1α and mitochondrial respiration in DDLPS cells. Similarly, overexpression of PGC-1α was sufficient to rescue mitochondrial biogenesis in DDLPS cells. Together, these data demonstrate that Notch signaling regulates CSCs, at least partially by controlling PGC-1α mediated mitochondria biogenesis.</p>

Metabolic alterations in hepatocellular carcinoma (HCC) are fundamental for the development of diagnostic screening and therapeutic intervention since energy metabolism plays a central role in differentiated hepatocytes. In HCC research, hepatoma cell lines (HCLs) like HepG2 and Huh7 cells are still the gold standard. In this study, we characterized the metabolic profiles of primary human hepatoma cells (PHCs), HCLs and primary human hepatocytes (PHHs) to determine their differentiation states. PHCs and PHHs (HCC-PHHs) were isolated from surgical specimens of HCC patients and their energy metabolism was compared to PHHs from non-HCC patients and the HepG2 and Huh7 cells at different levels (transcript, protein, function). Our analyses showed successful isolation of PHCs with a purity of 50–73% (CK18+). The transcript data revealed that changes in mRNA expression levels had already occurred in HCC-PHHs. While many genes were overexpressed in PHCs and HCC-PHHs, the changes were mostly not translated to the protein level. Downregulated metabolic key players of PHCs revealed a correlation with malign transformation and were predominantly pronounced in multilocular HCC. Therefore, HCLs failed to reflect these expression patterns of PHCs at the transcript and protein levels. The metabolic characteristics of PHCs are closer to those of HCC-PHHs than to HCLs. This should be taken into account for future optimized tumor metabolism research.