Portuguese contributions to the discovery and characterization of the embryonic molecular clock

Embryonic development is strictly regulated both in time and in space. This extraordinary control is clearly evidenced during the process of somitogenesis. In this process, pairs of somites are formed periodically, such that the time required to form a new somite pair is constant and species specific. The tight temporal control underlying somitogenesis has been shown to depend upon a molecular clock, manifested by the cyclic expression of an increasing number of genes in the unsegmented paraxial mesoderm. Portuguese researchers have been intimately connected to the achievements that have been made in this new field of research: the somitogenesis molecular clock. This article intends to report the Portuguese contributions to the discovery and characterization of the molecular clock underlying somite formation and possibly other embryonic processes. This work inspired many scientists around the world and it has been followed in Portugal by teams that keep on pursuing the characterization of the machinery of this molecular oscillator and its function in the acquisition of both temporal and positional information during development.Financial support was provided by FCT/FEDER (POCTI/BCI/42040/2001) and by the EU/FP6-Network of Excellence-Cells into Organs. S.P. (SFRH/BPD/26638/2006) was supported by FCT, Portugal

Novos aspectos na coordenação do desenvolvimento do membro dos vertebrados

Limb development requires precise orchestration of cell proliferation and differentiation in both time and space. Chick limb skeletal elements are laid down as cartilaginous primordia with the same initial size and grow differentially in a proximaldistal (P-D) sequence giving rise to seven skeletal compartments. Two models seek to explain cell fate specification along the P-D limb axis. Although fundamentally different, both models imply the existence of a limb bud distal zone where cells reside until they reach the time to differentiate - progress zone model -, or to expand - early specification model. However, how these cells measure time remains unknown. In 1997, the identification of the somitogenesis molecular clock brought new insight into how embryonic cells measure time. Another interesting discovery made in the somitogenesis system is the observation of a maturation wavefront. When this maturation wavefront is experimentally shifted anteriorly by placing an Fibroblast Growth Factor (FGF)8-coated bead in the mid-presomitic mesoderm (PSM), smaller somites are formed. In the work developed during the course of this PhD thesis, we analysed the expression of the somitogenesis molecular clock components hairy1 and hairy2 during chick forelimb development. We provide the first evidence for a molecular clock operating during limb outgrowth and patterning by showing that the expression of the somitogenesis clock component hairy2 cycles in limb chondrogenic precursor cells with a 6 hour periodicity. We determined the time period required to form an autopod skeletal limb element and propose that the forelimb second phalanx takes 12 hours to be formed, suggesting that an autopod limb skeletal element is formed by cells with “n” and “n+1” hairy2 expression cycles. In analogy to the somitogenesis maturation wavefront, we also hypothesise the existence of a wavefront that could be travelling in limb bud distal mesenchyme. Since FGF8 protein and fgf8 mRNA expressed in the Apical Ectodermal Ridge (AER) is activating MAPK phosphatase 3 (mkp3) expression in the distal limb mesenchyme, we have characterized the expression pattern of this gene and found that mkp3 presents a graded distribution of its transcripts in distal limb bud mesenchyme. We also analysed the expression pattern of intronic mkp3 probe and propose that mkp3 in limb distal mesenchyme acts in a similar way to fgf8 in the PSM. Furthermore, similarly to what occurs during somitogenesis, shorter limb elements are obtained when mkp3 expression under influence of FGF8 is extended proximally. We also demonstrate that AER-derived FGF8 signalling positively regulates hairy2 expression in the distal limb mesenchyme. The work performed in this thesis proposes that somitogenesis and limb bud are two parallel systems. Both have a zone where cells are maintained in an undifferentiated state (PSM and distal limb mesenchyme). In both these zones a molecular clock is operating regulating the periodicity of structure formation. We also suggest that these zones are maintained undifferentiated by the FGF/ Wingless int (WNT) signalling pathways that control the size of the elements formed. Recently, Mitogen Activated Protein Kinase/ Extracellular signal-Regulated Kinase (MAPK/ERK) pathway has been implicated in this process. We moreover propose that retinoic acid (RA) and FGF have mutually inhibitory roles in limb bud development, similarly to what occurs during somitogenesis, playing a function in allocating a certain number of cells to the initiation of the chondrogenic differentiation program, resulting in the regulation of element size. Until now, all studies regarding cyclic gene expression during development have focused exclusively on somitogenesis. However, time control is absolutely required during all embryonic development and may even be considered the fourth developmental dimension. The studies performed in this thesis suggest that temporal control exerted by cyclic gene expression can be a widespread mechanism providing cellular temporal information during vertebrate embryonic development, not being an exclusive propriety of PSM cells, as previously thought.O desenvolvimento dos membros dos vertebrados envolve uma regulação temporal e espacial estrita, quer da proliferação, quer da diferenciação celulares. As células precursoras dos elementos esqueléticos que formam o membro são dispostas sequencialmente ao longo do eixo proximal-distal (P-D). Na asa da galinha, estes elementos esqueléticos são formados com o mesmo tamanho inicial, crescendo depois diferencialmente e dando origem a sete compartimentos esqueléticos. Existem dois modelos que tentam explicar a especificação dos destinos celulares ao longo do eixo PD do membro dos vertebrados. Embora essencialmente diferentes, ambos postulam a existência de uma zona distal no membro onde as células residem até chegar o tempo de diferenciarem – modelo da zona de progresso - , ou de expandirem – modelo da especificação precoce. Contudo, continua ainda por determinar a forma como estas células adquirem a noção de tempo. Em 1997, a identificação de um relógio molecular ligado ao processo da somitogénese proporcionou novos conhecimentos na forma de como é que as células adquirem a noção de tempo. Outra descoberta interessante realizada no processo da somitogénese foi a observação de uma “wavefront” de maturação. Quando esta “wavefront” de maturação é deslocada anteriormente através da introdução de esferas embebidas em proteína Fibroblast Growth Factor (FGF)8 na região mediana da mesoderme presomítica (MPS), formam-se sómitos mais pequenos. Os estudos realizados durante o curso desta tese de doutoramento, permitiramnos mostrar pela primeira vez a existência de um relógio molecular que actua durante o desenvolvimento do membro superior do embrião de galinha. Experiências realizadas nesse sentido permitiram-nos estudar o padrão de expressão dos componentes do relógio molecular da somitogénese, hairy1 e hairy2 durante o desenvolvimento do membro superior da galinha e observar que um dos componentes do relógio, o gene hairy2 apresenta ciclos de expressão com uma periodicidade de 6 horas nas células percursoras condrogénicas do membro. Foram realizados estudos para determinar o período de tempo necessário para a formação de um elemento esquelético do membro, e propomos que cada falange é formada em 12 horas, sugerindo que um elemento esquelético do membro é formado por células com “n” e “n+1” ciclos de expressão do gene hairy2. Em analogia com a “wavefront” descrita no modelo da somitogénese, sugerimos que um processo semelhante possa estar a ocorrer no mesênquima distal do membro dos vertebrados. Devido à expressão da proteína de FGF8 e do RNAm de fgf8 na Apical Ectodermal Ridge (AER) activar a expressão de MAPK phosphatase 3 (mkp3) no mesênquima distal do membro, fomos caracterizar o padrão de expressão deste gene e observámos que existe um gradiente de expressão de mkp3 no mesênquima distal do membro. Analisamos também o padrão de expressão obtido com uma sonda intrónica de mkp3, e sugerimos que mkp3 no mesênquima distal do membro actua de uma maneira semelhante à de fgf8 na MPS. De modo idêntico ao modelo proposto para a somitogénese, demonstramos também que quando a expressão de mkp3 sob a influência de FGF8 é estendida proximalmente, os elementos esqueléticos do membro que se estão a formar nessa altura são menores. Por último, demonstramos que a sinalização de FGF8 proveniente da AER regula positivamente a expressão de hairy2 no mesênquima distal do membro. O trabalho realizado nesta tese sugere a existência de um paralelismo entre o processo da somitogénese e do membro. Ambos apresentam uma zona onde as células são mantidas num estado indiferenciado (MPS e o mesênquima distal do membro) onde está presente o relógio molecular responsável por regular a periodicidade da formação das estruturas. Em ambos os casos, estas zonas são mantidas num estado indiferenciado pelas vias de sinalização FGF/ Wingless int (WNT) que controlam o tamanho dos elementos formados. Recentemente, a via do Mitogen Activated Protein Kinase/ Extracellular signal-Regulated Kinase (MAPK/ERK) também foi implicada neste processo. Outro paralelismo interessante entre estes dois sistemas é o facto do ácido retinóico (AR) e da via de sinalização FGF inibirem-se mutuamente dando origem a que um certo número de células se distribuam para a iniciação do programa de diferenciação condrogénico, resultando na regulação do tamanho dos elementos. Até ao presente momento, todos os estudos relativos à expressão cíclica de genes ao longo do desenvolvimento embrionário, têm-se focado exclusivamente na somitogénese. Porém, o controlo temporal é absolutamente necessário durante todo o desenvolvimento embrionário, podendo mesmo ser considerado uma quarta dimensão do desenvolvimento. Os estudos realizados nesta tese de doutoramento sugerem que o controlo temporal exercido pela expressão cíclica de genes pode ser um mecanismo geral para atribuição de informação temporal celular durante o desenvolvimento embrionário dos vertebrados, e não uma propriedade exclusiva das células da MPS como acreditado até agora.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/8657/2002; POCTI/BCI/42040/2001.Fundo Social Europeu (FSE) - POCTI 2010.Rede Europeia de Excelência, “Cells into Organs” - EU/FP6

Watch-ing out for chick limb development

Time control is a crucial issue during embryonic development. Nevertheless, little is known about how embryonic cells measure time. Until recently, the only molecular clock known to operate during vertebrate embryonic development was the somitogenesis clock, exclusively functioning in coordinating the precise timing of each new pair of somites formed from the presomitic mesoderm. We have recently evidenced that a similar molecular clock also underlies the timing at which autopod chondrogenic precursors are laid down to form a skeletal limb element. In addition, we herein suggest that the molecular clock is not the only parallelism that can be established between somitogenesis and limb-bud development. In an evolutionary perspective, we support the previously proposed idea that the molecular mechanisms involved in the segmentation of the body axis may have been partially reused in the mesoderm of the lateral plate, thereby allowing the emergence of paired appendages.Financial support was provided by FCT/FEDER (POCTI/BCI/42040/2001) and by the EU/FP6-Network of Excellence-Cells into Organs. S.P. (SFRH/BPD/26638/2006) was supported by FCT, Portugal

Additive manufactured stoneware fired in microwave furnace

In the context of ceramic manufacturing, additive manufacturing or 3D printing creates new opportunities and perspectives, allowing the fabrication of parts with complex shapes, which by traditional means would be impossible to produce or would be very expensive [1]. This is the case for dinnerware and artworks (stoneware, porcelain and clay-based products). After piece forming, the greenware is gas or electrically fired at high temperatures to achieve its mechanical strength and aesthetic properties. These conventional firing processes usually require long processing times, in the present case taking 10 h to reach temperatures around 1200 °C [2]. In the search for faster firing processes, small size and cup shaped 3D printed stoneware pieces were fired using microwave radiation as the energy source. As microwave radiation has the potential to penetrate the material to be sintered, volumetric heating can be achieved, and faster firing processes are possible to implement without cracks formation and other thermal related defects. Pieces were fired in 10% of the conventional manufacturing time in a six magnetrons (energy sources) microwave furnace [3]. The microwave, the electrically-fast-fired and conventionally-fired pieces are presented in Figure 1. The conventionally-fired pieces are seen as reference samples. Temperature was controlled through a calibrated pyrometer [3], and using Process Temperature Control Rings (PTCR) the temperature of the pieces of (1207 ± 15) °C was determined. An error of only 1.25% was calculated between the temperature measured by the pyrometer and the PTCR in the piece where the pyrometer is measuring the temperature. The PTCR elements give a better representation of the real heating process at its location, concomitantly of each piece when they are placed inside it. The results show that microwave-fast-fired pieces present comparable mechanical strength to the references (10 h electrically fired) and to the electrically fast-fired pieces (41, 46 and 34 (N/mm2), respectively), and present aesthetic features closer to the reference ones. Porosity quantification does not fully agree with the mechanical strength of the pieces, of ~5% for electrically fast-fired, ~9% for the references and ~4% for microwave-fired ones. Overall, microwave heating can be used as an alternative stoneware firing technology, without compromising its quality and features with gains in the manufacturing time. Another advantage attributed to microwave heating is the reduction in the firing temperature, as claimed by the literature [4,5]. However, this possibility still requires confirmation in 3D-printed stoneware.publishe

Teste de Stroop

Teste de medida da atenção seletiva de acordo com o paradigma Stroop. Avalia a atenção seletiva (capacidade de inibir informações irrelevantes), inibição (capacidade de suprimir uma tendência de resposta comportamental), flexibilidade cognitiva, velocidade de processamento, inteligência fluida e sistema semântico (lezak, Howieson, & Loring, 2004; Strauss, Sherman, & Spreen, 2006). Há mais de 70 anos que o teste Stroop constitui uma referência na avaliação da atenção seletiva (Macleod, 1992). Neste teste nomeia-se a cor de palavras impressas em cores incongruentes, tendo de se selecionar o estfmulo relevante (a cor) e ignorar os estfmulos distratares (as palavras) que podem induzir erros e aumentar o tempo de resposta. A alteração na velocidade de nomeação de palavras coloridas (NP) e de nomeação de cores de palavras incongruentes (NC) é denominada interferência de Stroop (Strauss et ai., 2006). Se se inibir completamente o processamento lexical da palavra na nomeação da cor impressa, então NC deve ser igual a zero. A diminuição da capacidade de inibir seletivamente o processamento lexical leva a aumento de NC com a idade (Boaz, David, & Schneider, 2009). A interferência Stroop tem interesse na área do envelhecimento para explicar os défices associados a esta etapa do desenvolvimento (Verhaeghen & Cerella, 2002), pois quando ocorre alguma perturbação da atenção, as funções cognitivas podem permanecer intactas, mas a produtividade cognitiva geral é afetada (lezak et ai., 2004). O teste destina-se a adultos, letrados e sem dificuldades visuais (Strauss et ai., 2006)

Influence of ultrasound stimulation on the viability, proliferation and protein expression of osteoblasts and periodontal ligament fibroblasts

Among the adjunctive procedures to accelerate orthodontic tooth movement (OTM), ultrasound (US) is a nonsurgical form of mechanical stimulus that has been explored as an alternative to the currently available treatments. This study aimed to clarify the role of US in OTM by exploring different stimulation parameters and their effects on the biological responses of cells involved in OTM. Human fetal osteoblasts and periodontal ligament fibroblasts cell lines were stimulated with US at 1.0 and 1.5 MHz central frequencies and power densities of 30 and 60 mW/cm2 in continuous mode for 5 and 10 min. Cellular proliferation, metabolic activity and protein expression were analyzed. The US parameters that significantly improved the metabolic activity were 1.0 MHz at 30 mW/cm2 for 5 min and 1.0 MHz at 60 mW/cm2 for 5 and 10 min for osteoblasts; and 1.0 MHz at 30 mW/cm2 for 5 min and 1.5 MHz at 60 mW/cm2 for 5 and 10 min for fibroblasts. By stimulating with these parameters, the expression of alkaline phosphatase was maintained, while osteoprotegerin synthesis was induced after three days of US stimulation. The US stimulation improved the biological activity of both osteoblasts and periodontal ligament fibroblasts, inducing their osteogenic differentiation.This in vitro study was supported by FCT (Fundação para a Ciência e Tecnologia) in the scope of the grants UI/BD/150951/2021 and UI/BD/09375/2020, the funding contract 2020.00215.CEECIND (DOI: 10.54499/2020.00215.CEECIND/CP1600/CT0009) and through the projects UIDB/04436/2020, UIDP/04436/2020, FunFibRai—PTDC/EME-EME/4197/2021 (DOI: 10.54499/PTDC/EME-EME/4197/ 2021), BrainStimMap—PTDC/EME-EME/1681/2021 (DOI: 10.54499/PTDC/EME-EME/1681/2021), AlignAgen-GI2-CESPU-2022 and UID/CTM/00264/2021 project of 2C2T under the COMPETE and FCT/MCTES (PIDDAC), co-financed by FEDER through the PT2020 program

Novel cellulose-based composites based on nanofibrillated plant and bacterial cellulose: recent advances at the University of Aveiro - a review

The development of (nano)materials based on the renewable cellulose is a challenge. The present article provides a brief overview of the recent research efforts carried out at the CICECO Laboratory of the University of Aveiro on the development of novel composites based on nanofibrillated plant and bacterial cellulose embedded in natural and synthetic polymeric matrices such as poly(lactic acid), chitosan, starch, and pullulan. These materials have high potential for applications in packaging, paper coating, organic electronics, and biomedical products and devices

High-content drug screening in zebrafish xenografts reveals high efficacy of dual MCL-1/BCL-XL inhibition against Ewing sarcoma

Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application. Keywords: Anti-apoptotic protein inhibitors; Ewing sarcoma; High-content imaging; Phenotypic drug screening; Zebrafish xenograft

Novel transparent nanocomposite films based on chitosan and bacterial cellulose

New nanocomposite films based on different chitosan matrices (two chitosans with different DPs and one water soluble derivative) and bacterial cellulose were prepared by a fully green procedure by casting a water based suspension of chitosan and bacterial cellulose nanofibrils. The films were characterized by several techniques, namely SEM, AFM, X-ray diffraction, TGA, tensile assays and visible spectroscopy. They were highly transparent, flexible and displayed better mechanical properties than the corresponding unfilled chitosan films. These new renewable nanocomposite materials also presented reasonable thermal stability and low O(2) permeability.FCT - SFRH/BD/41388/ 2007FCT - SFRH/BPD/38515/200