This thesis describes the characterization of ceramide (Cer) biosynthesis by mammalian cells. The possibility that Cer undergo developmental changes was explored using mouse embryonic stem cells versus embryoid bodies by analysis of the Cer subspecies by liquid chromatography, electrospray ionization-tandem mass spectrometry (LC ESI-MS/MS) and of the transcript levels for enzymes involved in Cer biosynthesis by qRT-PCR. Cer of embroid bodies had higher proportions of very-long-chain fatty acids, which correlated with the relative expression of mRNA for the respective Cer synthases (CerS) and fatty acyl-CoA elongases, as well as changes in the fatty acyl-CoA's of the cells. Therefore, it is clear that Cer subspecies change during embryogenesis, possibly for functionally important reasons. One CerS isoform, CerS2, was studied further because it has the broadest tissue distribution and a remarkable fatty acyl-CoA specificity, utilizing longer acyl-chain CoAs (C20-C26) in vitro. The fatty acid chain selectivity was refined by analysis of the Cer from livers from CerS2 null mice, which displayed very little Cer with fatty acyl chains with 24 + 2 carbons. Another interesting structural variation was discovered in studies of cells treated with fumonisin B1 (FB1), which inhibits CerS. Under these conditions, cells in culture and animals accumulate substantial amounts of a novel sphingoid base that was identified as 1-deoxysphinganine. This compound arises from utilization of L-alanine instead of L-serine by serine palmitoyltransferase (SPT) based on the inability of LYB cells, which lack SPT, to make 1-deoxysphinganine. In the absence of FB1, 1-deoxysphinganine is primarily acylated to 1-deoxydihydroceramides. These are an underappreciated category of bioactive sphingoid bases and "ceramides" that might play important roles in cell regulation and disease. In summary, cells contain a wide variety of Cer subspecies that are determined by changes in expression of CerS, enzymes that produce co-substrates (such as fatty acyl-CoAs), and the types of amino acids utilized by SPT, the initial enzyme of de novo sphingolipid biosynthesis. One can envision how these changes might impact membranes structure as well as signaling by this family of highly bioactive compounds.

Univ., Diss.--Greifswald, 2005. / Literaturverz. S. 383 - 401.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2010. / Härtill 4 uppsatser.

The zygote and blastomeres of cleavage stage mouse embryos have the capacity to differentiate to the embryonic and both extra-embryonic lineages and are considered functionally totipotent. Until now, it has not been possible to establish stable cell lines that resemble these totipotent-like cells. In this work, I hypothesised that by modulating signalling pathways known to be important in early embryonic development it may be possible to capture in vitro a self-renewing state that possessed features of pre-implantation blastomeres. I succeeded in formulating a novel hypothesis driven cell culture medium which allowed the establishment of a stem cell state that possessed expanded differentiation potential to the embryonic and both extra-embryonic lineages in vitro and in vivo. These cells were isolated directly from in vitro culture of mouse pre-implantation embryos or single cell blastomeres, reprogrammed from somatic cells or converted from mouse ES cells. With these cells, I generated single cell chimeras which demonstrated extensive contribution to all lineages in the developing organism providing additional evidence that this chemical medium maintained a homogenous stem cell population. I demonstrated that the transcriptome of these cells was enriched with an early pre-implantation blastomere signature, distinct from other rare published totipotent-like cells. Finally, I demonstrated that the same chemical formulation permitted the establishment in vitro of a human stem cell state that possessed expanded differentiation potential to the embryonic and extra-embryonic lineage in vitro. My work has shown for the first time that through chemical modulation of pathways implicated to be involved in pre-implantation development, a novel homogenous stem cell state that possesses a pre-implantation transcriptional signature and expanded differentiation potential to both the embryonic and extra-embryonic lineage can be established and maintained in vitro in both mouse and human, suggesting a possible interspecies conservation of the signalling networks involved in early embryonic development.

There is a huge unmet clinical need to treat damaged articular cartilage such as that caused by osteoarthritis (OA) with an estimated 8.75 million people in the UK having sought treatment for OA (ARUK 2013). Embryonic stem cells (ESCs) offer a promising alternative therapeutic approach, potentially providing an unlimited source of chondrocytes capable of regenerating the damaged cartilage however this is limited by the efficiency of the chondrogenic differentiation protocol. An improved understanding of the posttranscriptional regulation of chondrogenesis by microRNAs (miRNAs) may enable us to improve hESC chondrogenesis. Also the recent discovery that miRNAs are selectively packaged into exosomes which can then be transferred to and be functionally active within neighbouring cells suggests they may have a role in cell-cell communication. This project investigated the regulation of miRNA expression in relation to the transcriptome during hESCs-directed chondrogenesis and the possible role for exosomes during differentiation and in stem cell maintenance of hESCs. Small RNA-seq and whole transcriptome sequencing was performed on distinct stages of hESC-directed chondrogenesis using the Directed Differentiation Protocol (DDP) developed in our lab. Also small RNA-seq was performed on exosomes isolated from hESCs and chondroprogenitors along with the donor cells that the exosomes originated from. This revealed significant changes in the expression of several miRNAs during hESC-directed chondrogenesis including: upregulation of miRNAs transcribed from the four Hox complexes, known cartilage associated miRNAs and the downregulation of pluripotency associated miRNAs. Overall miRome and transcriptome analysis revealed the two hESC lines exhibited slightly different miRome and transcriptome profiles during chondrogenesis, with Man7 displaying larger changes in miRNA and mRNA expression as it progressed through the DDP suggesting it may be more predisposed to undergo chondrogenesis. Integration of miRomes and transcriptomes generated during hESC-directed chondrogenesis identified four key functionally related clusters of co-expressed miRNAs and protein coding genes: pluripotency associated cluster, primitive streak cluster, limb development cluster and an extracellular matrix cluster. Further investigation of these gene/miRNA clusters allowed the identification of several potential novel regulators of hESC-directed chondrogenesis. In accordance with the reported literature the exosomal miRNAs from hESCs and hESC-chondroprogenitors were enriched with a guanine rich motif. Notably, several of these were enriched with targets associated with embryonic skeletal system development suggesting they may play a role in regulating differentiation. Preliminary functional experiments examining pluripotency-associated exosomes suggests they may have a role in regulating hESC stem cell maintenance. However the molecular mechanism by which this is achieved has not been investigated. This research identified main miRome and transcriptome changes during hESC-directed chondrogenesis leading to the identification of several potential novel regulators of chondrogenesis and pluripotency which can be further investigated. This project has also highlighted the potential of exosomal miRNAs to regulate hESC stem cell maintenance and differentiation.

No description available.

Telomeres are fundamental structures found at the end of all eukaryotic chromosomes that function to protect the end of chromosomes from end-to-end fusion, erosion and subsequent telomere dysfunction. Telomerase and alternative lengthening of telomere (ALT) mechanisms maintain the telomeres by compensating natural telomeric loss. ALT is found to be present in 15% of human tumours lines and it may be expressed at low levels in the normal mouse tissues. However, the exact mechanism behind ALT depression and/or activation in the mammalian cells is not fully understood. Previous studies have highlighted the role of BRCA1 in telomere dysfunction. Also, it has recently been shown that BRCA1 co-localises at telomeres in the ALT + human cells through BLM and Rad50. However, it is still unclear whether BRCA1 plays a direct role on telomere length maintenance and integrity. The aim of this project was to examine the role of BRCA1 in telomere maintenance associate with ALT in BRCA1 defective mammalian cells. Therefore to achieve this, we have set up series of experiments to look at, (a) hallmarks of ALT activity at the cytological level, (b) measuring of ALT activity using biochemical and immunocytochemistry techniques and (c) understanding the role of BRCA1 in DNA damage response mechanism and telomere dysfunction. Firstly, we found elevated levels of recombination at telomeres in the two human BRCA1 carrier cell lines and mouse embryonic stem cell with deficiency in Brca1-/-. Secondly, our data showed that human and mouse BRCA1 defective cells are significantly more sensitive to ionizing radiation in line with the DNA repair function of BRCA1. Moreover, we found persistent DNA damage at telomeres in the BRCA1 defective environment when after exposure of cells to ionizing radiation. Thirdly, we found evidence of ALT activity in some mouse cell lines, and elevated ALT in mouse cells defective in Brca1. Finally, we examined some other ALT markers using immunofluorescence. Our data indicate differences between human and mouse cells in regulating ALT. Taken together data presented in this thesis revealed that (i) BRCA1 plays a major role in telomere maintenance and defective BRCA1 mammalian cells show evidence of telomere dysfunction and telomere length shortening in line with previous publish data, (ii) BRCA1 defective mouse cells have elevated levels of ALT, (iii) the mouse lymphoblastoid LY-S cells have complete absence of ALT.

abstract: Researchers in the twentieth and twenty-first centuries identify the study of the intrinsic and external factors that influence human aging as senescence. A commonly held belief in the year 2015 is that at least some kinds of cells can replicate over long periods or even indefinitely, thereby meaning the cell does not undergo senescence (also known as replicative senescence) and is considered immortal. This study aims to provide information to answer the following question: While some scientists claim they can indefinitely culture a stem cell line in vitro, what are the consequences of those culturing practices? An analysis of a cluster of articles from the Embryo Project Encyclopedia provides information to suggest possible solutions to some potential problems in cell culturing, recognition of benefits for existing or historical culturing practices, and identification of gaps in scientific knowledge that warrant further research. Recent research suggests that hESCs, and immortalized cell lines in general, do not escape the effects of senescence. While there exists a constant change in the practices of cell culturing, a large portion of scientists still rely on practices established before modern senescence research: research that seems to suggest that cultured hESCs, among other immortal cell lines, are not truly immortal. / Dissertation/Thesis / Masters Thesis Biology 2015

Made available in DSpace on 2014-06-11T19:29:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-19Bitstream added on 2014-06-13T20:59:34Z : No. of bitstreams: 1 oliveira_cs_me_jabo.pdf: 1799127 bytes, checksum: 5a858a3a6eed3c27cf1b8f8968db5a50 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O estudo dos processos de diferenciação em células tronco embrionárias (CTE) representa uma importante ferramenta para o entendimento das vias moleculares que os regem, apresentando grande aplicação tanto na ciência básica quanto na engenharia de tecidos e medicina regenerativa. Pouco é conhecido sobre as marcas epigenéticas existentes na cromatina destas células, e de que forma a regulação da expressão gênica ocorre no momento da diferenciação. O presente trabalho teve como objetivo o estudo dos efeitos da hiperacetilação das histonas causada pela droga tricostatina A (TSA), uma inibidora das enzimas histona desacetilases, sobre a diferenciação destas células em estádios iniciais e avançados. Para tanto, a hiperacetilação induzida pela droga foi estimada por reações de imunocitoquímica para AcLys9H3. Os efeitos anti-proliferativos da TSA foram mensurados pelo teste de TUNEL e contagem de células. Ainda, foram conduzidos experimentos de diferenciação in vitro de CTE e análise da expressão de proteínas características de linhagens celulares diferenciadas por reações de imunocitoquímica (Oct3/4, nestina, âIII tubulina, desmina e troponina I), em cultivos tratados com TSA em diferentes concentrações e em diferentes momentos. Desta forma, foi estimada a população de tipos celulares oriundos dos folhetos embrionários ectodérmico e mesodérmico, como neurônios, e células musculares, quando foi promovida a hiperacetilação das histonas nas CTE, em diferentes momentos da diferenciação celular in vitro. A TSA induziu apoptose em níveis superiores aos do grupo controle, e retardou/inibiu a divisão celular. Promoveu hiperacetilação dose-dependente nos períodos estudados, e estimulou a diferenciação de precursores mesodérmicos (50nM d5) e ectodérmicos (15nMd0-5 e 50nMd5), cardiomiócitos (50nMd5 e 100nMd13) e neurônios (15nMd0-5, 50nMd5, 100nMd5, 100nMd13). / Studies on embryonic stem cells (ESC) differentiation represents an important tool leading to understanding of its molecular pathways, with many applications both on basic research and tissue engineering / regenerative medicine. Little is known about epigenetic marks on ESC chromatin, and how gene expression occurs at differentiation time. The aim of this work was to study effects of histone hiperacetylation, induced by cell treatment with trichostatin A (TSA), an histone deacetylase inhibitor, on both initial and late differentiation. For that, drug-induced hyperacetylation was studied by AcLys9H3 immunocitochemistry. TSA anti-proliferative effects were analysed by TUNEL test and cell counts. Experiments on ESC in vitro differentiation and immunocitochemistry for specific cell types proteins (Oct3/4, nestin, âIII tubulin, desmin and troponin I) were performed, in treated and control groups, at different moments. This analysis showed specific cell types populations derived from embryonic ectodermal and mesodermal, such as neurons and cardiomyocytes, when histone hyperacetylation were induced, on both initial and late diferentiation. Our results showed that TSA induces apoptosis and inhibits cellular proliferation. Also, TSA promoted dose-dependent histone hyperacetylation at studied moments, and stimulated mesodermal (50nM d5) and ectodermal (15nMd0-5 e 50nMd5) precursors, cardiomyocytes (50nMd5 e 100nMd13) and neurons (15nMd0-5, 50nMd5, 100nMd5, 100nMd13) differentiation.

Le contrôle de l'expression des programmes géniques orchestrant le développement précoce et l'homéostasie des cellules souches fait l'objet de recherches intenses. En effet, les cellules souches embryonnaires (CSE) sont caractérisées par des propriétés comme leur clonogénicité (la capacité à proliférer dans le même état indifférencié) et leur pluripotence (la capacité à se différencier et à former les tissus embryonnaires et adultes). Au niveau moléculaire, l'identité des CSE est orchestrée par le contrôle de l'expression génique aux niveaux épigénétique, transcriptionnel, post- transcriptionnel et traductionnel en réponse à l'activation de voies de signalisation spécifiques. Dans ce contexte, des données récentes suggèrent un rôle de la machinerie traductionnelle les ribosomes et de la régulation de leur biogenèse, dans le maintien de l'homéostasie de cellules souches de différentes espèces. À partir de l'analyse de données transcriptomiques à haut débit (RNAseq), mon équipe d'accueil a ainsi identifié un ensemble de protéines associées aux ribosomes (PaR) significativement enrichies dans les cellules souches embryonnaires murines (CSEm) en comparaison à des lignées cellulaires murines différenciées et à des tissus. Parmi ces candidats, mes travaux de thèse ont consisté à la caractérisation d'une PaR particulièrement enrichie : Rsl24d1. Rsl24d1 est une protéine de biogenèse des ribosomes décrites exclusivement chez la levure. Son profil d'expression dans différentes lignées de CSEm suggère une fonction spécifique: enrichissement au niveau transcriptionnel et protéique dans les CSE à l'état de pluripotence naïf et diminution importante au cours de la différenciation. En effet, des approches de perte d'expression de Rsl24d1 m'ont permis d'établir l'importance de cette PaR dans l'homéostasie des CSEm. Rsl24d1 contribue au maintien de la prolifération cellulaire des CSE, de leur clonogénicité et plus modérément à leur pluripotence. Rsl24d1 semble être une protéine majoritairement nucléaire mais également associée aux sous- unités 60S libres des ribosomes cytoplasmiques. D'autre part, la perte d'expression de Rsl24d1 affecte spécifiquement la biogenèse des particules ribosomiques 60S. Ainsi, comme chez la levure, dans les CSEm, Rsl24d1 est un facteur navette orchestrant la maturation des particules ribosomiques pré-60S. Par ailleurs, Rsl24d1 semble permettre le maintien d'un taux de synthèse protéique élevé permettant notamment le renouvellement des protéines ayant une demi-vie courte parmi lesquels on recense des facteurs de transcription de la pluripotence comme Oct4 (Oct3/4), Nanog et Esrrb. Mes travaux de thèse ont donc permis d'identifier et de caractériser un facteur de biogenèse de la sous-unité 60S, Rsl24d1, impliqué dans l'homéostasie des CSEm / Embryonic stem cells (ESC) possess clonogenic and pluripotency abilities i.e. they are able to self-renew indefinitely in the same developpemental state and to differentiate in all the cell types composing embryonic and adult tissues. ESC homeostasis is coordinated by complex networks which are regulated at different levels of gene expression regulation, including epigenetic, transcriptional and post-transcriptional levels. Furthermore, emerging evidences point out that the translational machinery, ribosomes, are directly implicated in the control of adult and embryonic stem cell homeostasis in different model organisms. Along this line, we have identified Rsl24d1, a ribosomal associated protein (RaP), which is strongly expressed in naïve murine ESCs compared to their differentiated progenies. We demonstrated that Rsl24d1 actively contributes to ESC homeostasis and its expression is essential for ESC proliferation and clonogenic capacities. Finally, we have also demonstrated that Rsl24d1, like Rlp24 its yeast ortholog, is associated to pre-ribosomes in ESCs from the nucleus to the cytoplasm and is required for the biogenesis of the large ribosomal subunit