Embryonic thymic epithelium differentiation in chicken: study of molecular signals involved in lymphoid progenitor cells colonization

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2012O timo é o órgão linfóide primário responsável pela diferenciação de progenitores hematopoiéticos linfóides (PHL) em linfócitos T maduros. Após o nascimento, os PHL entram no timo através dos vasos sanguíneos e iniciam a timopoiese, processo complexo de diferenciação em linfócitos T. Os PHL começam por especificar-se na linhagem T e depois iniciam o seu longo processo de diferenciação adquirindo coreceptores de membrana característicos das células T maduras, como os marcadores CD3, CD4 e CD8. A timopoiese é essencial para a constituição de um sistema imunitário saudável. De facto, os linfócitos T são o componente principal do sistema imunitário adaptativo, capazes de responder a agentes infecciosos, e aumentando a capacidade de defesa do organismo com cada exposição a antigénios específicos. O timo é um órgão constituído por células epiteliais tímicas (CET), células mesênquimais derivadas da crista neural (CN), células dos vasos sanguíneos e células hematopoiéticas. O seu desenvolvimento está intimamente ligado ao das glândulas paratiróides, uma vez que os seus epitélios partilham a mesma origem embrionária: a endoderme das terceira e quarta bolsas faríngicas (3/4 BF)1,2. A origem endodérmica das células epiteliais foi pela primeira vez demonstrada por Le Douarin e Jotereau utilizando o modelo de quimeras galinhacodorniz3. Neste trabalho também mostraram que distintos mesênquimas ectópicos, são capazes de suportar (mesênquima permissivo, da somatopleura) ou não (mesênquima não permissivo, do sómito ou do botão do membro) o desenvolvimento da endoderme na formação dum timo funcional, revelando a importância das interações celulares entre a endoderme e o mesênquima adjacente, nas fases iniciais do desenvolvimento tímico3 Durante esta primeira fase do desenvolvimento tímico, ocorre a especificação das CET2. Os territórios presuntivos do timo e das glândulas paratiróides são definidos pela expressão de factores de transcrição distintos nas bolsas faríngicas, o gene Foxn1 (forkhead box N1) e o gene Gcm2 (glial cells missing-2), respectivamente4. Assim, em galinha, o rudimento do timo foi identificado ao dia 4,5 de desenvolvimento embrionário (E4,5) (E4 em codorniz) na região dorsal das 3/4 BF enquanto que o domínio das paratiróides ocupa um território mais anterior e ventral das mesmas5. A segunda fase do desenvolvimento do timo depende da sua colonização por PHL e de interações entre estes e as células epiteliais tímicas. Estas interações são essenciais para a diferenciação das CET em duas linhagens celulares, cortical e medular, originando consequentemente a formação de dois compartimentos tímicos: o córtex e a medula6–9. Durante a embriogénese, Le Douarin descreveu três ondas de colonização do epitélio tímico (ET) por PHL, no modelo de galinha10. A primeira onda ocorre ao E6,5 (E6 em codorniz), antes da vascularização do rudimento tímico, entrando os progenitores hematopoiéticos no rudimento tímico através da sua migração pelo mesênquima adjacente3,11,12. As restantes ondas realizam-se através dos vasos sanguíneos, ao E12 e E1810. As interações entre os PHL e as TEC são mediadas por vários factores solúveis, como citocinas (estimulam a proliferação dos timócitos) e quimiocinas (importantes na migração dos PHL para o rudimento tímico), e por vias de sinalização como a sinalização Notch. A importância do microambiente Notch para a correcta especificação de progenitores hematopoiéticos nas diferentes linhagens linfóides foi demonstrada pela primeira vez em 2001 pelo grupo de L. Parreira13. Além disso, os genes envolvidos na sinalização Notch (receptores, ligandos e genes-alvo) são expressos de forma distinta nos diferentes territórios do timo adulto, reforçando a importância desta via de sinalização na função do mesmo14. Recentemente, o grupo de H. Neves observou em embriões de galinha que os genes envolvidos na sinalização Notch estão diferencialmente expressos na endoderme das bolsas faríngicas, em estádios prévios à formação do rudimento tímico. Em estudos recentes, este mesmo grupo, utilizando o modelo de quimeras galinha-codorniz, observou que células da endoderme das 3/4 BF de codorniz (dador; E3) quando enxertadas num ambiente permissivo (mesênquima da somatopleura) dum embrião de galinha (receptor; E2,5), especificam em ET (Foxn1+). Também constataram que, 5 dias após o enxerto, o epitélio tímico se encontra colonizado por PHL com origem no embrião receptor (galinha)5. Contudo, quando a endoderme é enxertada num ambiente não permissivo (mesênquima do botão embrionário do membro posterior), nas mesmas janelas temporais, esta expressa Foxn1 mas não é colonizada por células hematopoiéticas5. Estes resultados sugerem que, quando enxertada num ambiente não permissivo, a endoderme não possui e/ou não recebe os sinais do mesmo (mesênquima adjacente) necessários à sua colonização por PHL. De facto, em codorniz, a primeira vaga de colonização do rudimento tímico por PHL (E6) ocorre durante a janela temporal em análise. Assim, este sistema representa um novo modelo de estudo para identificar os factores envolvidos na migração dos PHL e na colonização do rudimento tímico pelos mesmos, etapas fundamentais ao desenvolvimento do timo. Este trabalho teve como primeiro objectivo identificar os sinais moleculares da via de sinalização Notch envolvidos na fase de colonização do rudimento tímico (CET) pelos PHL. Para este efeito utilizou-se o modelo de quimeras galinha-codorniz acima descrito. Embriões quiméricos, com 3 e 5 dias de desenvolvimento após o enxerto da endoderme, foram sacrificados e estudados por hibridação in situ com genes envolvidos na sinalização Notch, especificamente os ligandos-Notch, Delta1 e Delta4. Os resultados obtidos mostram que a endoderme, quando enxertada no ambiente permissivo, expressa ambos os ligandos, nos dias 3 e 5 após o enxerto. Em contraste, quando a endoderme é enxertada num ambiente não permissivo, embora expresse Delta4, apresenta uma expressão diminuída ou inexistente de Delta1 (3 e 5 dias após o enxerto). Assim, os resultados sugerem que Delta1 poderá ser o ligando Notch específico envolvido na mediação do sinal Notch na interação entre o epitélio (ligando Delta1) e os PHL (receptor Notch) nas fases iniciais de colonização do epitélio tímico. Em conformidade, no timo adulto de ratinho foi descrita a expressão de Delta1 na junção cortico-medular, local de entrada dos PHL após o nascimento14. Atualmente, decorrem estudos para aprofundar o papel deste ligando e caracterizar a sua sinalização durante o processo de colonização do epitélio tímico. De futuro, também serão estudadas outras moléculas possivelmente envolvidas neste processo, tais como quimiocinas, e componentes de outras vias de sinalização. Em paralelo, neste trabalho caracterizou-se o desenvolvimento do timo e das glândulas paratiróides em galinha (entre os dias 5 e 18 de desenvolvimento), ao nível histológico e ao nível da diferenciação celular dos linfócitos T no rudimento tímico (por análise de citometria de fluxo). A análise histológica foi realizada através da expressão in situ dos genes Foxn1 e Gcm2. Em cortes seriados de parafina, ao longo dos estádios estudados (E5 a E13), observou-se a expressão de Foxn1 e Gcm2 nos rudimentos do timo e das glândulas paratiróides, respectivamente. Concluiu-se que a expressão destes factores de transcrição é mantida até ao dia E13. Mostrou-se também que as glândulas paratiróides apresentam uma morfologia adulta a partir de E9, e que o timo ao dia 13 de desenvolvimento se encontra ao longo do pescoço e diferenciado em dois compartimentos (córtex e medula). Analisou-se a dinâmica de diferenciação das populações de timócitos presentes no rudimento tímico, entre E11 e E18, por citometria de fluxo, utilizando os marcadores de membrana CD3, CD4 e CD8. Os resultados desta análise mostram que até E13 existem duas populações imaturas de timócitos, CD8+CD4-CD3- e CD3+CD8-CD4-, pouco proliferativas, e derivadas da primeira onda de colonização, sugerindo que podem ter um papel na especificação do epitélio tímico. De facto, no momento da segunda onda de colonização (E12-13) a medula começa a ser definida, evidência morfológica do processo prévio de especificação das células epiteliais em linhagens cortical e medular. Paralelamente, após a segunda onda de colonização (>E15) observa-se uma forte expansão de células duplas positivas, CD4+CD8+, sugerindo que estes timócitos têm uma maior capacidade de se diferenciarem (e expandirem) e possivelmente de contribuir para a maturação dos dois compartimentos tímicos.The thymus is the primary lymphoid organ where maturation of lymphoid progenitors cells (LPCs) into T-cell occurs. This maturation depends on interactions between the LPCs and thymic epithelium (TE). TE derives from endoderm of the 3rd and 4th Pharyngeal Pouches (3/4 PPs) (in chicken) and depends on epithelial-mesenchymal interactions and on LPCs colonization to become functional. Using the quail-chick model, our group showed that distinct mesenchymal tissues are permissive (somatopleure) or non-permissive (limb bud) to 3/4 PPs endoderm specification and early development. In addition, only in the permissive environment the endoderm is colonized by LPCs. In this work, we aimed to identify the molecular cues, namely Notch signaling ligands, involved in TE colonization by LPCs. For that, we used the quail-chick chimeric model. Using immunohistochemistry combined with in situ hybridization techniques we showed that Delta1, as opposed to Delta4, is down-regulated in the endoderm developed in the non-permissive mesenchyme (with no LPCs colonization). Together, these results suggest that mesenchymal-epithelial interactions are important to establish a proper environment to thymus formation and that Delta1 is involved in TE colonization by LPCs. In parallel, we described thymus and parathyroid glands development through the in situ expression analysis of specific transcription factors for each organ (Foxn1 and Gcm2, respectively). We observed that the expression of both transcription factors is maintained from E5 to E13 in the respective developing organs. We then characterized thymocyte populations during thymic organogenesis by flow cytometry analysis. We identified two immature populations, single positive for CD3 or CD8, at early thymic development (<E13), suggesting that these thymocytes may be important in TECs specification into different lineages (cortical and medullary), and subsequent maturation of thymic compartments. With this work, we hope to clarify important events in thymus organogenesis: colonization by LPCs and TECs specification

Linking Oxidative Stress and DNA Damage to Changes in the Expression of Extracellular Matrix Components

Cells are subjected to endogenous [e.g., reactive oxygen species (ROS), replication stress] and exogenous insults (e.g., UV light, ionizing radiation, and certain chemicals), which can affect the synthesis and/or stability of different macromolecules required for cell and tissue function. Oxidative stress, caused by excess ROS, and DNA damage, triggered in response to different sources, are countered and resolved by specific mechanisms, allowing the normal physiological equilibrium of cells and tissues to be restored. One process that is affected by oxidative stress and DNA damage is extracellular matrix (ECM) remodeling, which is a continuous and highly controlled mechanism that allows tissues to readjust in reaction to different challenges. The crosstalk between oxidative stress/DNA damage and ECM remodeling is not unidirectional. Quite on the contrary, mutations in ECM genes have a strong impact on tissue homeostasis and are characterized by increased oxidative stress and potentially also accumulation of DNA damage. In this review, we will discuss how oxidative stress and DNA damage affect the expression and deposition of ECM molecules and conversely how mutations in genes encoding ECM components trigger accumulation of oxidative stress and DNA damage. Both situations hamper the reestablishment of cell and tissue homeostasis, with negative impacts on tissue and organ function, which can be a driver for severe pathological conditions.info:eu-repo/semantics/publishedVersio

Plasmid Interactions Can Improve Plasmid Persistence in Bacterial Populations

It is difficult to understand plasmid maintenance in the absence of selection and theoretical models predict the conditions for plasmid persistence to be limited. Plasmid-associated fitness costs decrease bacterial competitivity, while imperfect partition allows the emergence of plasmid-free cells during cell division. Although plasmid conjugative transfer allows mobility into plasmid-free cells, the rate of such events is generally not high enough to ensure plasmid persistence. Experimental data suggest several factors that may expand the conditions favorable for plasmid maintenance, such as compensatory mutations and accessory genes that allow positive selection. Most of the previous studies focus on bacteria that carry a single plasmid. However, there is increasing evidence that multiple plasmids inhabit the same bacterial population and that interactions between them affect their transmission and persistence. Here, we adapt previous mathematical models to include multiple plasmids and perform computer simulations to study how interactions among them affect plasmid maintenance. We tested the contribution of different plasmid interaction parameters that impact three biological features: host fitness, conjugative transfer and plasmid loss - which affect plasmid persistence. The interaction affecting conjugation was studied in the contexts of intracellular and intercellular interactions, i.e., the plasmids interact when present in the same cell or when in different cells, respectively. First, we tested the effect of each type of interaction alone and concluded that only interactions affecting fitness (epistasis) prevented plasmid extinction. Although not allowing plasmid maintenance, intracellular interactions increasing conjugative efficiencies had a more determinant impact in delaying extinction than the remaining parameters. Then, we allowed multiple interactions between plasmids and concluded that, in a few cases, a combined effect of (intracellular) interactions increasing conjugation and fitness lead to plasmid maintenance. Our results show a hierarchy among these interaction parameters. Those affecting fitness favor plasmid persistence more than those affecting conjugative transfer and lastly plasmid loss. These results suggest that interactions between different plasmids can favor their persistence in bacterial communities.info:eu-repo/semantics/publishedVersio

NFIXing Cancer: The Role of NFIX in Oxidative Stress Response and Cell Fate

NFIX, a member of the nuclear factor I (NFI) family of transcription factors, is known to be involved in muscle and central nervous system embryonic development. However, its expression in adults is limited. Similar to other developmental transcription factors, NFIX has been found to be altered in tumors, often promoting pro-tumorigenic functions, such as leading to proliferation, differentiation, and migration. However, some studies suggest that NFIX can also have a tumor suppressor role, indicating a complex and cancer-type dependent role of NFIX. This complexity may be linked to the multiple processes at play in regulating NFIX, which include transcriptional, post-transcriptional, and post-translational processes. Moreover, other features of NFIX, including its ability to interact with different NFI members to form homodimers or heterodimers, therefore allowing the transcription of different target genes, and its ability to sense oxidative stress, can also modulate its function. In this review, we examine different aspects of NFIX regulation, first in development and then in cancer, highlighting the important role of NFIX in oxidative stress and cell fate regulation in tumors. Moreover, we propose different mechanisms through which oxidative stress regulates NFIX transcription and function, underlining NFIX as a key factor for tumorigenesis.info:eu-repo/semantics/publishedVersio

Dominance Between Plasmids Determines the Extent of Biofilm Formation

Bacterial biofilms have an impact in medical and industrial environments because they often confer protection to bacteria against harmful agents, and constitute a source from which microorganisms can disperse. Conjugative plasmids can enhance bacterial ability to form biofilms because conjugative pili act as adhesion factors. However, plasmids may interact with each other, either facilitating or inhibiting plasmid transfer. Accordingly, we asked whether effects on plasmid transfer also impacts biofilm formation. We measured biofilm formation of Escherichia coli cells harboring two plasmid types, or when the two plasmids were present in the same population but carried in different cells. Using eleven natural isolated conjugative plasmids, we confirmed that some indeed promote biofilm formation and, importantly, that this ability is correlated with conjugative efficiency. Further we studied the effect of plasmid pairs on biofilm formation. We observed increased biofilm formation in approximately half of the combinations when both plasmids inhabited the same cell or when the plasmids were carried in different cells. Moreover, in approximately half of the combinations, independent of the co-inhabitation conditions, one of the plasmids alone determined the extent of biofilm formation - thus having a dominant effect over the other plasmid. The molecular mechanisms responsible for these interactions were not evaluated here and future research is required to elucidate them.info:eu-repo/semantics/publishedVersio

Characterization of Coelomic Fluid Cell Types in the Starfish Marthasterias glacialis Using a Flow Cytometry/Imaging Combined Approach

Funding: To the “Maristem COST Action” (CA16203), supported by COST (European Cooperation in Science and Technology), for funding PM STSM visits (February 2019 and November 2020) to the AVC Laboratory.Coelomocytes is the generic name for a collection of cellular morphotypes, present in many coelomate animals, and highly variable among echinoderm classes. The roles attributed to the major types of these free circulating cells present in the coelomic fluid of echinoderms include immune response, phagocytic digestion and clotting. Our main aim in this study was to characterize coelomocytes found in the coelomic fluid of Marthasterias glacialis (class Asteroidea) by using a combination of flow cytometry (FC), imaging flow cytometry (IFC) and fluorescence plus transmission electron microscopy (TEM). Two coelomocyte populations (P1 and P2) identified through flow cytometry were subsequently studied in terms of abundance, morphology, ultrastructure, cell viability and cell cycle profiles. Ultrastructurally, P2 diploid cells were present as two main morphotypes, similar to phagocytes and vertebrate thrombocytes, whereas the smaller P1 cellular population was characterized by low mitotic activity, a relatively undifferentiated cytotype and a high nucleus/cytoplasm ratio. In the present study we could not rule out possible similarities between haploid P1 cells and stem-cell types in other animals. Additionally, we report the presence of two other morphotypes in P2 that could only be detected by fluorescence microscopy, as well as a morphotype revealed via combined microscopy/FC. This integrative experimental workflow combined cells physical separation with different microscopic image capture technologies, enabling us to better tackle the characterization of the heterogeneous composition of coelomocytes populations.publishersversionpublishe

Archaeology of the Pleistocene-Holocene transition in Portugal: synthesis and prospects

The Tardiglacial of Portugal has been associated with the Magdalenian culture and lithic industries characterized by tool miniaturization, a diversity of microlith types, and the absence of a intentional blade production. The technological characterization, the chronology and the phasing of the Portuguese Magdalenian have been defined based on data recovered from open-air sites of the Estremadura region (Central Portugal). This paper presents an overview of the research undertaken over the last twenty-five years, including results from research and preventive archaeology fieldwork outside this region, namely in the Côa, Sabor and Vouga Valleys (northern Portugal), as well as in the Guadiana Valley and Algarve regions (southern Portugal). Our chronological boundaries are the Greenland Stadial 2-1b and the 8.2 ka event, from Early Magdalenian to Early Mesolithic. Regarding vegetation, deciduous Quercus underwent expansion during the warm phases of the Tardiglacial and retracted during cold ones, when pines increased. After the Solutrean, the faunal assemblages show a decrease in the variability of the represented species and an increase in fish, birds, small mammals and rabbits (Oryctolagus cuniculus). Concerning the cultural sequence, the Middle Magdalenian remains uncharacterised. After the Upper Magdalenian, and thenceforward, the use of local raw materials and of cores-on-flakes (burin or carinated endscraper type) for bladelet production gradually increased. In terms of lithic armatures typology, a four-stage sequence can be discerned: 1) Upper Magdalenian with axial points rather than backed bladelets, quite common in previous phases; 2) Final Magdalenian with an increase in the diversity of armature types; 3) Azilian with geometric microliths, curved backed points (Azilian points) and Malaurie points, and 4) Early Mesolithic without retouched bladelet tools or at best a persistence of Azilian armature types. There were some changes in the Palaeolithic rock art of the Douro basin between phase 3 (Final Magdalenian) and phase 4 (Late Azilian): figurative animal representations give place to animal depictions characterized by their geometrical bodies, often filled-in, and red deer becomes the best-represented animal.FCT: PTDC/EPH-ARQ/0326/2014info:eu-repo/semantics/publishedVersio

Thymus Inception: Molecular Network in the Early Stages of Thymus Organogenesis

The thymus generates central immune tolerance by producing self-restricted and self-tolerant T-cells as a result of interactions between the developing thymocytes and the stromal microenvironment, mainly formed by the thymic epithelial cells. The thymic epithelium derives from the endoderm of the pharyngeal pouches, embryonic structures that rely on environmental cues from the surrounding mesenchyme for its development. Here, we review the most recent advances in our understanding of the molecular mechanisms involved in early thymic organogenesis at stages preceding the expression of the transcription factor Foxn1, the early marker of thymic epithelial cells identity. Foxn1-independent developmental stages, such as the specification of the pharyngeal endoderm, patterning of the pouches, and thymus fate commitment are discussed, with a special focus on epithelial&ndash;mesenchymal interactions

Linking Oxidative Stress and DNA Damage to Changes in the Expression of Extracellular Matrix Components

&lt;jats:p&gt;Cells are subjected to endogenous [e.g., reactive oxygen species (ROS), replication stress] and exogenous insults (e.g., UV light, ionizing radiation, and certain chemicals), which can affect the synthesis and/or stability of different macromolecules required for cell and tissue function. Oxidative stress, caused by excess ROS, and DNA damage, triggered in response to different sources, are countered and resolved by specific mechanisms, allowing the normal physiological equilibrium of cells and tissues to be restored. One process that is affected by oxidative stress and DNA damage is extracellular matrix (ECM) remodeling, which is a continuous and highly controlled mechanism that allows tissues to readjust in reaction to different challenges. The crosstalk between oxidative stress/DNA damage and ECM remodeling is not unidirectional. Quite on the contrary, mutations in ECM genes have a strong impact on tissue homeostasis and are characterized by increased oxidative stress and potentially also accumulation of DNA damage. In this review, we will discuss how oxidative stress and DNA damage affect the expression and deposition of ECM molecules and conversely how mutations in genes encoding ECM components trigger accumulation of oxidative stress and DNA damage. Both situations hamper the reestablishment of cell and tissue homeostasis, with negative impacts on tissue and organ function, which can be a driver for severe pathological conditions.&lt;/jats:p&gt