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Oral squamous cell carcinoma (OSCC) is a malignant tumour of Head and Neck Cancer (HNC). The recent therapeutic approaches used to treat cancer have adverse side effects. The natural agents exhibiting anticancer activities are generally considered to have a robust therapeutic potential. Curcuminoids, one of the major active compounds of the turmeric herb, are used as a therapeutic agent for several diseases including cancer. In this study, the cytotoxicity of curcuminoids was investigated against OSCC cell line HNO97. Our data showed that curcuminoids significantly inhibits the proliferation of HNO97 in a time and dose-dependent manner (IC50=35 μM). Cell cycle analysis demonstrated that curcuminoids increased the percentage of G2/M phase cell populations in the treated groups. Treating HNO97 cells with curcuminoids led to cell shrinking and increased detached cells, which are the typical appearance of apoptotic cells. Moreover, flow cytometry analysis revealed that curcuminoids significantly induced apoptosis in a time-dependent manner. Furthermore, as a response to curcuminoids treatment, comet tails were formed in cell nuclei due to the induction of DNA damage. Curcuminoids treatment reduced the colony formation capacity of HNO97 cells and induced morphological changes. Overall, these findings demonstrate that curcuminoids can in vitro inhibit HNC proliferation and metastasis and induce apoptosis. Resumo O carcinoma de células escamosas oral (OSCC) é um tumor maligno do câncer de cabeça e pescoço (HNC). As recentes abordagens terapêuticas usadas para tratar o câncer têm efeitos colaterais adversos. Os agentes naturais que exibem atividades anticâncer são geralmente considerados como tendo um potencial terapêutico robusto. Curcuminoides, um dos principais compostos ativos da erva cúrcuma, são usados como agente terapêutico para várias doenças, incluindo câncer. Neste estudo, a citotoxicidade dos curcuminoides foi investigada contra a linha de células OSCC HNO97. Nossos dados mostraram que os curcuminoides inibem significativamente a proliferação de HNO97 de forma dependente do tempo e da dose (IC50 = 35 μM). A análise do ciclo celular demonstrou que os curcuminoides aumentaram a porcentagem de populações de células da fase G2 / M nos grupos tratados. O tratamento das células HNO97 com curcuminoides levou ao encolhimento celular e ao aumento das células destacadas, que são a aparência típica das células apoptóticas. Além disso, a análise de citometria de fluxo revelou que os curcuminoides induziram significativamente a apoptose de uma maneira dependente do tempo. Além disso, em resposta ao tratamento com curcuminoides, caudas de cometa foram formadas nos núcleos das células devido à indução de danos ao DNA. O tratamento com curcuminoides reduziu a capacidade de formação de colônias das células HNO97 e induziu alterações morfológicas. No geral, esses achados demonstram que os curcuminoides podem inibir in vitro a proliferação e metástase de HNC e induzir apoptose.

Las roturas de doble cadena del DNA (DSBs) son las lesiones que más afectan a la supervivencia celular y a la integridad del genoma. Las células presentan diferentes mecanismos de reparación, entre los que encontramos un mecanismo menos estudiado denominado unión de extremos no homólogos alternativa (A-NHEJ). En este destaca la participación de la DNA polimerasa theta (POLθ), la cual se encuentra sobreexpresada en muchos cánceres, siendo necesaria para la supervivencia de las células tumorales deficientes en la recombinación homóloga (HR). Además, diferentes estudios sugieren la importancia de la polimerasa incluso en células con HR funcional, por lo que es una diana terapéutica de gran potencial. Tras comprobar previamente que las células tumorales prostáticas estudiadas no eran deficientes en la HR, el objetivo de este trabajo consistió en determinar el posible papel de POLθ frente al estrés replicativo en dichas células, las cuales dependen de POLθ para su supervivencia según resultados previos del grupo. Para ello, se realizó el silenciamiento de POLQ y el tratamiento posterior con agentes inductores de estrés replicativo (camptotecina, etopósido e hidroxiurea) analizando después el daño producido en las células mediante la inmunodetección por western-blot de la histona γH2AX, marcador de DSBs. También se llevó a cabo el análisis del efecto producido en el ciclo celular. Estos resultados mostraron diferencias notables entre las células silenciadas y las células control, de tal forma que la inhibición de POLθ promovió una acumulación de células en las fases G1 y S, indicando una dificultad aumentada a la hora de replicar el DNA y continuar el ciclo. Adicionalmente, en este trabajo se llevó a cabo el clonaje de un plásmido inducible por doxiciclina denominado LT3-POLQ capaz de silenciar POLQ.

Quiescence, the ability to temporarily halt proliferation, is a conserved process that initially allowed survival of unicellular organisms during inhospitable times and later contributed to the rise of multicellular organisms, becoming key for cell differentiation, size control and tissue homeostasis. In this Review, we explore the concept of cancer as a disease that involves abnormal regulation of cellular quiescence at every step, from malignant transformation to metastatic outgrowth. Indeed, disrupted quiescence regulation can be linked to each of the so-called ‘hallmarks of cancer’. As we argue here, quiescence induction contributes to immune evasion and resistance against cell death. In contrast, loss of quiescence underlies sustained proliferative signalling, evasion of growth suppressors, pro-tumorigenic inflammation, angiogenesis and genomic instability. Finally, both acquisition and loss of quiescence are involved in replicative immortality, metastasis and deregulated cellular energetics. We believe that a viewpoint that considers quiescence abnormalities that occur during oncogenesis might change the way we ask fundamental questions and the experimental approaches we take, potentially contributing to novel discoveries that might help to alter the course of cancer therapy.

Little is known about Nima‐related kinase (NEKs), a widely conserved family of kinases that have key roles in cell‐cycle progression. Nevertheless, it is now clear that multiple NEK family members act in networks, not only to regulate specific events of mitosis, but also to regulate metabolic events independently of the cell cycle. NEK5 was shown to act in centrosome disjunction, caspase‐3 regulation, myogenesis, and mitochondrial respiration. Here, we demonstrate that NEK5 interacts with LonP1, an AAA+ mitochondrial protease implicated in protein quality control and mtDNA remodeling, within the mitochondria and it might be involved in the LonP1‐TFAM signaling module. Moreover, we demonstrate that NEK5 kinase activity is required for maintaining mitochondrial mass and functionality and mtDNA integrity after oxidative damage. Taken together, these results show a new role of NEK5 in the regulation of mitochondrial homeostasis and mtDNA maintenance, possibly due to its interaction with key mitochondrial proteins, such as LonP1., mtDNA mutations have been increasingly observed in human cancers. Cumulative mtDNA damage declines cell and tissue function and underpins the aging process. Protein kinases selectively modify proteins and a plethora of signaling pathways, leading to a specific physiological responses. Here, we demonstrate how NEK5 kinase can contribute to the maintenance and repair of mtDNA.

The host hormone melatonin is known to modulate the asexual cell-cycle of the human malaria parasite Plasmodium falciparum and the kinase PfPK7 is fundamental in the downstream signaling pathways. The nuclear protein PfMORC displays a histidine kinase domain and is involved in parasite cell cycle control. By using a real-time assay, we show a 24 h (h) rhythmic expression of PfMORC at the parasite asexual cycle and the expression is dramatically changed when parasites were treated with 100 nM melatonin for 17 h. Moreover, PfMORC expression was severely affected in PfPK7 knockout (PfPK7−) parasites following melatonin treatment. Parasites expressing 3D7morc-GFP shows nuclear localization of the protein during the asexual stage of parasite development. Although the PfMORC knockdown had no significant impact on the parasite proliferation in vitro it significantly changed the ratio of the different asexual intraerythrocytic stages of the parasites upon the addition of melatonin. Our data reveal that in addition to the upstream melatonin signaling pathways such as IP3 generation, calcium, and cAMP rise, a nuclear protein, PfMORC is essential for the hormone response in parasite synchronization.

Esta tesis consiste en el estudio del ciclo celular de células madre pluripotentes (CMP) en diferenciación y los mecanismos moleculares que pueden almacenar memoria de un estímulo externo. Pretende mostrar cómo existen factores hereditarios que determinan el ciclo, y estudiar los mecanismos de herencia y memoria molecular dentro de las redes de regulación génica. Se divide en 3 capítulos. El primero aborda el estudio del ciclo celular y sus fases durante el proceso de diferenciación temprana en las CMP de embrión de ratón. Estas células fueron genéticamente modificadas para fluorescer en dos colores marcando las fases del ciclo, y ser filmadas por microscopía de time-lapse. Los análisis incluyen medidas del ciclo celular a nivel de célula única, construcción de dendrogramas de linajes celulares y el diseño y aplicación de algoritmos para probar si las relaciones familiares determinan la duración del ciclo celular. El resultado principal es que, durante la diferenciación, el ciclo se acorta y las células proliferan a mayor velocidad. Además, la duración del ciclo de una célula está determinada por las células progenitoras, probando que existen factores parentales que controlan la tasa de proliferación. Sin embargo, una de las dificultades a la hora de encontrar cuáles son estos factores, encontrar candidatos y compararlos es, por un lado, la falta de cuantificadores de memoria almacenada en las redes de regulación génica y, por otro, la falta de un catálogo de mecanismos moleculares capaces de almacenar memoria. El segundo capítulo, por su parte, consiste en el desarrollo de un cuantificador matemático de la memoria molecular en redes complejas, aplicado principalmente a vías de señalización celular y redes de regulación génica, que guardan información de estímulos transitorios. Una vez definido el cuantificador, se utilizó para buscar mecanismos o motifs de 1, 2 y 3 nodos que fueran capaces de almacenar información de estos estímulos. Los resultados principales consisten en que los ciclos de retroalimentación positiva son esenciales para dar multiestabilidad y memoria a las redes. Además, las redes capaces de oscilar de manera autónoma también pueden guardar información en la fase de la oscilación. El tercer y último capítulo consiste en una serie de experimentos en las mismas células que se utilizaron en el primer capítulo. El objetivo es detectar la existencia de mecanismos capaces de almacenar memoria. Para esto, tratamos a las células con un estímulo de diferenciación transitorio y luego aplicamos el cuantificador desarrollado en el segundo capítulo. El resultado es que, luego de solo un día expuestas al estímulo de diferenciación, las células lo recuerdan y continúan con el proceso de diferenciación. Asimismo, es probable que esta memoria se deba a la existencia de alguna retroalimentación positiva entre los factores de transcripción marcadores de la pluripotencia. This thesis is focused on the study of cell cycle in pluripotent stem cells (PSC) during differentiation and on the molecular mechanisms which can store the memory of an external stimulus. It aims to show if there are hereditary factors that determine it, and to study the mechanisms of inheritance and molecular memory within gene regulatory networks. It is organized into three chapters. The first chapter deals with the study of the cell cycle and its phases during the early differentiation process in mouse embryo PSCs. These cells were genetically modified to fluoresce in two colors marking the phases of the cycle, and to be filmed by time-lapse microscopy. Analyzes include cell cycle measurements at the single cell level, construction of cell lineage dendrograms, and the design and application of algorithms to test whether familial bonds determine cell cycle length. The main result is that, during differentiation, the cycle is shortened, and cells proliferate at a faster rate. Furthermore, the length of a cell cycle is determined by the progenitor cells, proving that there are hereditary factors that control the rate of proliferation. However, one of the needs in finding out what these factors are, obtain candidates and compare them is, on one hand, the lack of memory quantifiers stored in gene regulation networks and, on the other hand, some catalog of the molecular mechanisms capable of storing memory. This issue is addressed in the next chapter. The second chapter deals with the development of mathematical quantifier to account for molecular memory in complex networks, applied mainly to signaling and gene regulatory networks which store information of transient stimuli. Once defined, we use the quantifier to look for 1,2 & 3 nodes mechanisms or motifs capable of stimuli information storage. The main results consist of positive feedbacks being essential to provide multistability and memory to networks. Furthermore, oscillating networks can also store information in their phase. The third and last chapter is a description of experiments performed on the same cells used in the first chapter. The aim of these experiments is to detect mechanisms capable of storing memory. To accomplish this, we give a differentiation stimulus and apply the memory quantifier developed in the second chapter. The main result is that, after only one day exposed to a differentiation stimulus, cells remember it and continue its progression through the differentiation process. In addition, this memory is probably due to the existence of a positive feedback between pluripotency transcription factors. Fil: Sevlever, Federico. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Cells maintain their size within limits over successive generations to maximize fitness and survival. Sizer, timer, and adder behaviors have been proposed as possible alternatives to coordinate growth and cell cycle progression. Regarding budding yeast cells, a sizer mechanism is thought to rule cell cycle entry at Start. However, while many proteins controlling the size of these cells have been identified, the mechanistic framework in which they participate to achieve cell size homeostasis is not understood. We show here that intertwined APC and SCF degradation machineries with specific adaptor proteins drive cyclic accumulation of the G1 Cdk in the nucleus, reaching maximal levels at Start. The mechanism incorporates Mad3, a centromeric-signaling protein that subordinates G1 progression to the previous mitosis as a memory factor. This alternating-degradation device displays the properties of a timer and, together with the sizer device, would constitute a key determinant of cell cycle entry., This work was supported by Agencia Estatal de Investigación Spain PID2019-109638GB-I00 (to M.A.), Agencia Estatal de Investigación Spain PGC2018-096597-B-I00 (to J.C.), Agència de Gestió d'Ajuts Universitaris i de Recerca Catalonia 2019FI_B 00452 (to A.P.P.), and Agència de Gestió d'Ajuts Universitaris i de Recerca Catalonia 2014FI_B 00074 (to D.F.M.).

Among skin cancers, melanoma has the highest mortality rate. The heterogeneous genetic melanoma background leads to a tumor-propagating capacity particularly important in maintaining therapeutic resistance, and tumor recurrence. The identification of efficient molecules able to control melanoma progress represents an important opportunity for new therapeutic strategies, particularly in combination with the current standard-of-care treatments. In this context, several studies have reported the antitumor effects of melatonin against different types of cancer, including melanoma. Here, we describe the underlying mechanisms associated with melatonin’s activity in human melanoma cell lines, focusing on cell cycle and cytoskeleton remodeling. Interestingly, while melatonin induced melanocyte DNA replication, melanoma cells exhibited cell cycle arrest in the G1-phase. This phenomenon was associated with cyclin-D1 downregulation or p21 overexpression. The efficacy of melatonin on melanoma cells survival and proliferation was detected using the clonogenic assay, with a decrease in both the number and size of colonies. Additionally, melatonin induced a dramatic cytoskeleton remodeling in all melanoma cell lines, leading to a star-like morphology or cell swelling. The role of melatonin on melanoma cytoskeleton was associated with the actin disruption, with thinning and/or broken actin fibers, and weak and/or loss of paxillin along stress fibers. These data support the observed findings that melatonin impairs melanoma invasion in skin reconstructed models. Together, our results suggest that melatonin could be used to control melanoma growth and support basic and clinical studies on melatonin as a promising immunometabolic adjuvant for melanoma therapy.

Schizosaccharomyces pombe initiates sexual development under nitrogen starvation, changing the transcription patterns of many genes to allow sexual differentiation. MBF transcription factor recognize specific sequences present in genes required for mitotic and meiotic S phase and activates their transcription through binding to their promoters. Genes involved in recombination (rec genes) are also activated by MBF, but in a different and independent wave of transcription. Res2 plays an important role in the transcription of this new wave, as higher levels of this protein are required to initiate recombination. Another form of gene expression regulation takes place through splicing. Spliceosome is in charge of these transesterification reactions and requires a correct recognition of intron-exon boundaries to achieve proper splicing efficiency. Auxiliary factors are essential for a correct splicing of introns with degenerated sequences, as Prp2, being the last nucleotide of the upstream exon what determines the dependency of an intron on Prp2. Schizosaccharomyces pombe inicia el proceso de meiosis bajo la restricción de nutrientes, cambiando los patrones de transcripción de muchos genes. El factor de transcripción MBF reconoce secuencias específicas presentes en genes necesarios para la fase de replicación del DNA mitótica y meiótica, y activa su transcripción a través de la unión a sus promotores. Los genes encargados de la recombinación meiótica también son activados por MBF, pero en una ola de transcripción diferente e independiente a la de la fase de replicación. Res2 juega un papel importante en la transcripción de esta nueva ola, puesto que se requieren niveles altos de esta proteína para iniciar la recombinación. Otra forma de regulación de la expresión génica tiene lugar a través del splicing. Una adecuada definición de los bordes intrón-exón es necesaria para que el espliceosoma se forme correctamente. Los factores de splicing tienen un papel fundamental en el reconocimiento de los sitios de splicing con secuencias no-consenso, como Prp2, siendo el último nucleótido del exón upstream lo que determina la dependencia de Prp2 de un determinado intrón.

[eng] SLIMP (Seryl-tRNA Synthetase-Like Insect Mitochondrial Protein) was identified during the generation of a mitochondrial human disease fly model. SLIMP was described as a previously uncharacterized paralog of the Drosophila mitochondrial seryl-tRNA synthetase (SerRS2), which became an essential protein, universally distributed in insects, echinoderms, and molluscs. Notably, SLIMP constitutes an aaRS-like protein that can bind to tRNAs, but it has lost the aminoacylation activity (Guitart, Bernardo, Sagalés, et al., 2010). Interestingly, we recently reported that SLIMP plays an essential role in the mitochondria by simultaneously regulating the mitochondrial protein synthesis and mtDNA copy number through interacting with SerRS2 and LON, respectively (Picchioni, Antolin-Fontes, et al., 2019). On the other hand, two reported studies pointed to SLIMP as an essential cell cycle regulator (Ambrus et al., 2009; Liang et al., 2014). Previous experiments in our group showed a G2 accumulation phenotype in SLIMP-KD cells, which can be rescued by overexpressing SLIMP without the mitochondria-targeting signal. Additionally, transcriptional upregulation of a core set of E2F1-target genes was detected upon SLIMP depletion. All these data point to a cell cycle-related role of SLIMP, potentially carried out from outside the organelle. Interestingly, here we first experimentally characterized the SLIMP MSP and proved that it is essential for driving the protein into the organelle. Moreover, we discovered an extra-mitochondrial population of SLIMP potentially shuttling between the cytosol and the nucleus, presumably in charge of the cell cycle role of SLIMP. Furthermore, our results suggest a role of SLIMP repressing the transition from the G1 to S phase during the cell cycle progression in an E2F1-independent pathway. Finally, interestingly, we demonstrate that the CDK1-P levels are increased upon SLIMP depletion, suggesting an activation of the G2/M checkpoint. In balance, the data presented in this thesis reinforces the idea of SLIMP playing a non-canonical function outside the mitochondria regulating the cell cycle progression near the R point. Thus, SLIMP would represent a single molecule at the crossroad between the mitochondrial homeostasis and cell cycle regulation., [cat] SLIMP (Seryl-tRNA Synthetase-Like Insect Mitochondrial Protein) es va identificar com un paraleg prèviament desconegut de la seril-tRNA sintetasa mitocondrial de Drosòfila (SerRS2), durant la generació d’un model de malaltia mitocondrial humana. Segurament SLIMP és fruit d’una duplicació gènica a la base dels animals i s’ha convertit en una proteïna essencial i universalment distribuïda en artròpodes, mol·luscs i equinoderms. A més, SLIMP ha perdut l’activitat aminoaciladora, tot i que encara conserva la capacitat d’unir-se a ARNs de transferència (Guitart, Bernardo, Sagalés, et al., 2010). El nostre laboratori ha demostrat recentment que SLIMP desenvolupa una funció clau a la mitocòndria, regulant simultàniament la traducció gènica a l’orgànul i el número de copies de ADN mitocondrial, a traves de la interacció amb SerRS2 i LON respectivament (Picchioni, Antolin-Fontes, et al., 2019). D’altre banda, dos estudis publicats en els últims anys vinculen SLIMP amb la regulació del cicle cel·lular (Ambrus et al., 2009; Liang et al., 2014). En aquest sentit, experiments prèviament realitzats al grup demostren que quan els nivells d’SLIMP baixen, les cèl·lules s’acumulen en la fase G2 del cicle. Curiosament, aquest fenotip es pot revertir sobre-expressant un forma truncada d’SLIIMP que no pot entrar a la mitocòndria. A més, en aquestes condicions, també s’observa una pujada a nivell transcripcional d’alguns gens regulats per E2F1. Conjuntament aquestes dades apunten a una possible funció d’SLIMP relacionada amb la regulació del cicle cel·lular potencialment portada a terme des de fora de la mitocòndria. En aquesta tesi, nosaltres, primer determinem la seqüència del pèptid senyal mitocondrial d’SLIMP experimentalment, i demostrem que es essencial i suficient per dirigir la proteïna cap a l’orgànul. A més, detectem una petita població d’SLIMP fora de la mitocòndria, present tant en el nucli com en el citoplasma. És important remarcar, que varis experiments presentats en aquesta tesi suggereixen que SLIMP estaria reprimint la progressió del cicle cel·lular a nivell de la transició G1-S, mitjançant un mecanisme alternatiu a E2F1. Finalment, demostrem que els nivells de CDK1-P son mes elevats en les cèl·lules que els hi manca SLIMP, cosa que indica una activació del punt de control de G2/M i que podria explicar la acumulació en G2 observada en aquestes cèl·lules. En conjunt les dades presentades en aquesta tesi, reforcen la idea que SLIMP desenvolupa una funció no canònica des de fora de la mitocòndria, reprimint l’entrada en la fase S del cicle cel·lular. Per tant, SLIMP representaria una molècula que vincula directament la homeòstasi mitocondrial i la regulació del cicle cel·lular.

Prostate cancer (PCa) is the second most common cancer in men, only behind lung cancer. The development of precision medicine has become one of the main objectives for treating it and it seems increasingly crucial to find biomarkers that allow both to stratify patients and to propose a personalized treatment. Previous studies have described the transcriptional coactivator PPAR gamma co-activator 1 alpha (PGC1⍺) as a possible biomarker for the stratification of patients with PCa. However, despite PGC1⍺’s role in the stratification, it remains necessary to study its molecular mechanisms so that it can be targeted in personalized treatment. Taking into account that one of the characteristics of cancer is its uncontrolled cell proliferation due to cell cycle deregulation, the aim of this work is to analyze if PGC1⍺ may regulate the progression of the cycle and the G1-to-S phase transition targeting E2F transcription factor. For that, first we analyze the transcriptional program associated with PGC1⍺ and related to cell cycle; second, the activity of CDK4 and associated proteins; and lastly, the correlations between PGC1⍺ and its transcriptional program in patients.

Dissertação de mestrado em Genética Molecular, A resistência do nosso genoma é testada diariamente não só por agentes exógenos, de natureza física, química e biológica, mas também por fatores endógenos, que podem causar genotoxicidade e citotoxicidade. Para contra-atacar estes efeitos nocivos, as células possuem vários mecanismos capazes de reparar o ADN ferido, no entanto, em algumas situações os danos estão para além da reparação e a patogénese ocorre. Os investigadores têm a oportunidade de criar novas estratégias terapêuticas com plantas que têm sido usadas na medicina tradicional desde há séculos. Esta possibilidade pode ser muito benéfica para o bem-estar dos seres humanos, por exemplo, para travar ou retardar o desenvolvimento de várias doenças associadas ao envelhecimento. Uma dessas plantas de interesse é a Dittrichia viscosa, típica dos países que circundam o mar Mediterrâneo e consequentemente empregada pela sua população na medicina tradicional. O objetivo deste estudo é verificar se o extrato aquoso de Dittrichia viscosa subsp. revoluta tem atividades biológicas, concretamente, atividade antigenotóxica. E se assim for, compreender o seu mecanismo de ação. Os resultados dos ensaios in vitro, da atividade antioxidante pela captura do radical livre DPPH e da quantificação do conteúdo fenólico total, sugerem que o extrato estudado tem atividade antioxidante devido ao seu conteúdo fenólico, em parte. Além disso, foram realizadas experiências in vivo com o organismo modelo, Schizosaccharomyces pombe, onde foram avaliados as viabilidades, a morfologia nuclear e os comprimentos das leveduras tratadas com o extrato da planta, hidroxiureia e a combinação de ambas. Os resultados obtidos desses testes indicam que o extrato da planta afetou a atividade de HU, um inibidor da replicação do ADN, após um longo período de tempo. Em conclusão, a o extrato aquoso de Dittrichia viscosa subsp. revoluta exibiu atividade antigenotóxica parcialmente explicada pela sua atividade antioxidante., The resistance of our genome is tested on daily basis by not only exogenous agents of physical, chemical and biological nature, but also, by endogenous factors, that can cause genotoxicity and cytotoxicity. To counterattack these harmful effects, cells possess several mechanisms capable of repair injured DNA, nevertheless, in some situations the damage is beyond reparation and pathogenesis takes place. Investigators have the opportunity to create new therapeutic strategies with plants that have been used in traditional medicine from centuries ago. This possibility could be very beneficial for the wellbeing of humans, for example, to halt or retard the development of several diseases associated with ageing. One of those plants of interest is Dittrichia viscosa, typical from countries surrounding the Mediterranean Sea and consequently employed by their population in traditional medicine. The objective of this study is to verify if Dittrichia viscosa subsp. revoluta aqueous extract has biological activities, concretely, antigenotoxic activity. And if so, comprehend its mechanism of action. The results from in vitro assays, DPPH radical scavenging activity and the quantification of total phenolic content, suggest that the studied extract has antioxidant activity due to their phenolic content, in part. Moreover, in vivo experiments were performed with the model organism, Schizosaccharomyces pombe, where the viability, nuclear morphology and lengths of yeasts cells treated with the plant extract, hydroxyurea and the combination of both, were assessed. The obtained results from those tests point out that the plant extract affected the HU activity, an inhibitor of DNA replication, after a long period of time. In conclusion the Dittrichia viscosa subsp. revoluta aqueous extract had antiogentotoxic activity partially explained by its antioxidant activity.

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 28-09-2021, Esta tesis tiene embargado el acceso al texto completo hasta el 28-03-2023, Durante la edad adulta, en el cerebro de mamíferos se generan nuevas neuronas a partir de poblaciones de células madre neurales adultas (NSCs). Este proceso denominado neurogénesis, en condiciones normales, se encuentra restringido a dos regiones especializada del cerebro adulto, la zona subgranular del giro dentado del hipocampo (SGZ) y la zona ventricular-subventricular o subependimaria (SEZ). En condiciones normales, existe un equilibrio entre el estado de quiescencia y activación de las NSCs, de manera que el control estricto de su modo de división y su potencial de diferenciación, mantienen las reservas de NSCs y preservan la capacidad neurogénica del tejido. Los nichos neurogénicos presentan una cierta capacidad para activarse tras una lesión (por ejemplo, isquémica), aunque la capacidad de regenerar el tejido dañado es mínima en condiciones normales. Sin embargo, la ablación experimental de la neurogénesis provoca el empeoramiento de la lesión, por lo que se ha postulado la relevancia en este contexto del mantenimiento del proceso neurogénico en adultos. Actualmente se están buscando estrategias que potencien la neurogénesis endógena, y promover así la formación y supervivencia de las nuevas neuronas. El equilibrio entre el mantenimiento de la quiescencia, la autorrenovación y la diferenciación de los distintos tipos celulares implicados en el proceso de la neurogénesis adulta están regulados por un conjunto de factores extrínsecos e intrínsecos que a su vez interaccionan entre ellos de forma coordinada y compleja. Entre estos factores, los reguladores del ciclo celular son claves en la regulación de la neurogénesis, ya que controlan la proliferación de los reservorios de células madre y orquestan la salida de ciclo con la diferenciación. Entre éstos, los menos estudiados son los mitóticos. Plk1 (Polo like kinase 1), una serina/treonina quinasa altamente conservada con un papel esencial en la regulación del ciclo celular se ha visto implicada en el control de la autorrenovación de las células madre neurales y la neurogénesis en Drosophila m. pero su papel en la neurogénesis en mamíferos, y más concretamente en la neurogénesis adulta está aún por esclarecer. En esta Tesis Doctoral hemos investigado el papel Plk1 sobre la neurogénesis adulta in vitro e in vivo, estudiando los dos nichos neurogénicos adultos, que se hipotetiza se encuentran conservados en humanos. Utilizando modelos genéticos de pérdida y ganancia de función, así como inhibidores farmacológicos. Nuestros resultados indican que Plk1 es un nuevo regulador intrínseco de la activación y del modo división de las NSCs, restringiendo su división simétrica y la autorrenovación para acotar la capacidad neurogénica de las NSCs. Además, proporcionamos evidencias de que Plk1 puede realizar estas funciones mediante el control de los niveles y la actividad del factor de transcripción Ascl1/Mash1 (Achaete-Scute Family BHLH Transcription Factor 1), crucial para el mantenimiento del estado activado de las NSCs y el control de la neurogénesis, que de confirmarse sería una diana novedosa de esta quinasa. Consideramos que estos resultados tienen gran relevancia ya que Plk1 es una molécula modulable farmacológicamente con pequeñas moléculas, algunas de ellas probadas en ensayos clínicos contra distintos tipos de cáncer. Nuestros resultados indican que la modulación de la actividad quinasa de Plk1 puede ser una estrategia para incrementar la generación de nuevas neuronas en situaciones de demanda, como puede ocurrir tras la depleción de la progenie neural tras daño

Submitted by Stefanie Oliveira de Sousa (stefanie.sousa@unesp.br) on 2021-09-21T01:15:13Z No. of bitstreams: 1 sousa_sod_me_sjrp.pdf.pdf: 2711828 bytes, checksum: 23e3a59d9d86f49efe34ca96920263e3 (MD5) Approved for entry into archive by Elza Mitiko Sato null (elzasato@ibilce.unesp.br) on 2021-09-22T00:42:40Z (GMT) No. of bitstreams: 1 sousa_so_me_sjrp.pdf: 2711828 bytes, checksum: 23e3a59d9d86f49efe34ca96920263e3 (MD5) Made available in DSpace on 2021-09-22T00:42:40Z (GMT). No. of bitstreams: 1 sousa_so_me_sjrp.pdf: 2711828 bytes, checksum: 23e3a59d9d86f49efe34ca96920263e3 (MD5) Previous issue date: 2021-09-03 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) O carcinoma espinocelular de cabeça e pescoço com acometimento em regiões anatômicas do trato aerodigestivo superior representa o sexto tipo mais comum de câncer no mundo. O seu tratamento inclui cirurgia, terapia por irradiação e quimioterapia, entretanto, estes métodos são altamente invasivos, com um significativo comprometimento funcional. Em função disso, tratamentos fitoterápicos contra o câncer têm despertado interesse científico, e a Piper nigrum, uma planta utilizada no tratamento de diversas doenças, tem sido estudada por possuir moléculas bioativas com propriedades farmacológicas anticâncer. Desse modo, o presente trabalho teve como objetivo geral avaliar o potencial efeito do extrato total da folha nas células sobre a morfologia, proliferação e migração celular, citotoxicidade, genotoxicidade, apoptose e necrose, ciclo celular, expressão gênica (PTGS2, PTGER3, PTGER4, MMP2, MMP9, TIMP1 e TIMP2) e proteica (PTGS2, PTGER4, MMP2), observando como o composto age e como essas alterações participam do processo tumoral. Para isso foram utilizadas duas linhagens de células tumorais (HEp-2 e SCC-25) e células normais orais (fibroblastos), após o tratamento com a Piper nigrum em três concentrações (10 µg/mL, 50 µg/mL e 100 µg/mL), por 4, 24, 48 e 72 horas. Os resultados evidenciaram que o extrato total da folha de Piper nigrum não altera a morfologia celular, mas diminui a proliferação e migração das células, não altera a viabilidade das células normais, e possui efeitos genotóxico, provavelmente por indução de apoptose celular, e ainda modula a expressão de genes e proteínas envolvidas com o processo inflamatório. Dessa maneira, o extrato total da folha de Piper nigrum parece apresentar uma sinergia entre seus metabólitos atuando diretamente nas células tumorigênicas por mecanismos relacionados às vias antiproliferativas, apoptóticas e/ou inflamatórias e abrem novas possibilidades de estudos futuros para terapias associadas no câncer de cabeça e pescoço. Head and neck squamous cell carcinoma with involvement in anatomical regions of the upper aerodigestive tract represents the sixth most common type of cancer in the world. The treatment includes surgery, irradiation therapy and chemotherapy, however, these methods are highly invasive, with significant functional impairment. In function of this, herbal treatments against cancer have aroused scientific interest, and Piper nigrum, a plant used in the treatment of various diseases, has been studied for having bioactive molecules with anti-cancer pharmacological properties. Therefore, the present work had as objective to evaluate the potential effect of total leaf extract on cells on morphology, proliferation and migration, cytotoxicity, genotoxicity, apoptosis and necrosis, cell cycle, gene and protein expression, observing how the compound acts and how these changes participate in the tumoral process. For this, two tumorigenic cell lines (HEp-2 and SCC-25) and normal oral cells (Fibroblasts) were used, after treatment with Piper nigrum in three concentrations (10 µg / mL, 50 µg / mL and 100 µg / mL), for 4, 24, 48 and 72 hours. The results showed that the total extract of the leaf of Piper nigrum does not alter the cell morphology, but decreases the proliferation and migration of the cells, does not alter the viability of normal cells, and has genotoxic effects, probably by inducing cell apoptosis, and it also modulates the expression of genes and proteins involved with the inflammatory process. Thus, the total extract of the leaf of Piper nigrum seems to present a synergy between its metabolites acting directly on tumorigenic cells by mechanisms related to antiproliferative, apoptotic and/ or inflammatory pathways and open up new possibilities for future studies for associated therapies in cancer of head and neck. CAPES: 88882.434449/2019-01 FAPESP: 2017/02100-3

The gastric mucosa is disturbed when breastfeeding is interrupted, and such early weaning (EW) condition permanently affects the differentiation of zymogenic cells. The aim of the study was to evaluate the immediate and long-term effects of EW on gastric cell proliferation, considering the molecular markers for cell cycle, inflammation, and metaplasia. Overall, we investigated the lifelong adaptation of gastric growth. Wistar rats were divided into suckling-control (S) and EW groups, and gastric samples were collected at 18, 30, and 60 days for morphology, RNA, and protein isolation. Inflammation and metaplasia were not identified, but we observed that EW promptly increased Ki-67-proliferative index (PI) and mucosa thickness (18 days). From 18 to 30 days, PI increased in S rats, whereas it was stable in EW animals, and such developmental change in S made its PI higher than in EW. At 60 days, the PI decreased in S, making the indices similar between groups. Spatially, during development, proliferative cells spread along the gland, whereas, in adults, they concentrate at the isthmus-neck area. EW pushed dividing cells to this compartment (18 days), increased PI at the gland base (60 days), but it did not interfere in expression of cell cycle molecules. At 18 days, EW reduced Tgfβ2, Tgfβ3, and Tgfbr2 and TβRII and p27 levels, which might regulate the proliferative increase at this age. We demonstrated that gastric cell proliferation is immediately upregulated by EW, corroborating previous results, but for the first time, we showed that such increased PI is stable during growth and aging. We suggest that suckling and early weaning might use TGFβs and p27 to trigger different proliferative profiles during life course.

Orientadores: Lair Zambon, Fernando Ferreira Costa Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas Resumo: Considerada uma doença rara no início do século XX, o carcinoma de pulmão hoje é a neoplasia visceral mais comum e a principal causa de morte por câncer. O estudo dos eventos moleculares envolvidos no câncer de pulmão é importante para o conhecimento do processo de carcinogênese, e para a determinação do espectro mutacional dos genes relacionados ao carcinoma brônquico. No futuro poderemos empregar as técnicas de biologia molecular no diagnóstico precoce e na correlação entre variáveis clínicas, tumorais e genéticas para definir os fatores de prognóstico e a abordagem terapêutica. Realizamos a pesquisa de mutações nos genes p53 e K-ras em pacientes com neoplasia de pulmão. Em relação ao gene p53, pesquisamos mutações nos exons 5 a 10 em 38 pacientes com carcinoma de pulmão não pequenas células, e nos exons 5 a 9 em nove pacientes com carcinoma de pequenas células. A freqüência de mutações foi, respectivamente, de 21 e 33%. A pesquisa de mutações no gene K-ras foi realizada em 8 pacientes com adenocarcinoma e em quatro com carcinoma de grandes células. Não encontramos mutações, provavelmente, devido ao tamanho da amostra. Nos pacientes com carcinoma não pequenas células a presença de mutação no gene p53 não interferiu na sobrevida (p=0,53), no estadiamento (p=0,67) e no tempo de sintomatologia (p=0,15). Comparamos as mutações encontradas no gene p53 com o banco de dados mantido pela Organização Mundial de Saúde (International Agency for Research on Cancer). Considerando que o banco de dados da IARC foi atualizado em março/2002 (16285 entradas), provavelmente somos o primeiro grupo a descrever a mutação no codon 237 (ATGàAAG) em carcinoma brônquico, independente do tipo histológico; da mutação no codon 337 (CGCàCAC) em carcinoma de grandes células e da mutação silenciosa no codon 295 (CCT>CCC) (IARC, 2002) Abstract: Considered a rare illness in the beginning of 20th century, the lung cancer today is the more common visceral neoplasia and the main cause of death by cancer. The study of the molecular events involved in lung cancer is important for the knowledge of the process of carcinogenesis and for the determination of the mutational spectrum of the genes related to lung cancer. In the future we will be able to use the techniques of molecular biology in the early diagnosis, and the correlation between clinical, tumoral and genetic variables will be useful for us to define the factors of prognostic and therapeutic approach. We carried out the study of mutations in the genes p53 and K-ras in patients with lung cancer. In relation to the p53 gene, we have looked for mutations in exons 5 to 10 in 38 patients with non-small cell lung cancer, and in exons 5 to 9 in nine patients with small cell lung cancer. The frequency of mutations was, respectively, 21 and 33%. The analysis of mutations in the K-ras gene was carried out in 8 patients with adenocarcinoma and four with large cell carcinoma. We didn¿t find mutations, probably, due to the size of the sample. In patients with non-small cell lung cancer, the presence of mutation in the p53 gene did not correlate with survival (p=0,53), staging (p=0,67) and time of symptoms (p=0,15). We compared the mutations found in the p53 gene with the database kept by the World Health Organization (International Agency for Research on Cancer). Taking into account that the IARC data bank was updated in March/2002 (16,285 entries), we are probably the first group to describe the transversion on codon 237 (ATGàAAG) in bronchial cancer, independent of the histological type and the mutation on codon 337 in large cell cancer (IARC, 2002) Mestrado Clínica Médica Mestre em Clínica Médica