Organ Dose Estimates in Thorax CT: Voxel Phantom Organ Matching With Individual Patient Anatomy

Given the continuous usage and spread of computed tomography (CT), the potential harmful e↵ects and the radiation dose to the patient have become high interest topics among the scientific community. The main objective of this investigation was to modify existing three-dimensional (3D) voxel phantom models to resemble real patients as much as possible, trying to progress the concept of a more personalized patient dosimetry. This work focused essentially in one of the biggest and most radiosensitive organs in the thorax, the lungs. Additionally, the variations of organ doses when a standard phantom is used instead were studied. During the course of this work a FORTRAN-based program was developed, which is able to semi-automatically modify the volumetric information of organs of interest in a standard voxel phantom (Female ICRP Adult Reference). The voxel resolution was also altered so the phantom’s diameters match the patient’s ones. Monte Carlo (MC) PENELOPE simulation code was used to mimic CT scan conditions and, therefore, generate 2D projections, used for visual organ matching with clinical patient CT images, and access organ dose in both phantoms (ICRP standard and ICRP modified). The main results reported that matching the voxel phantom’s size and lungs provides organ dose values significantly di↵erent from the ones measured in the ICRP reference phantom. Voxel models matched to patients’ size and overall anatomy allow increased accuracy in organ dose estimation, which, as reported by this study, can su↵er from up to 20% underestimation and 40% overestimation. This study demonstrates that voxel phantoms developed using single patient data provide a better and more precise organ dose assessment by MC methods than a standard phantom. The presented methodology should be of interest for dose optimization studies and quick enough for routine clinical use

A study on regeneration: insights from the zebrafish caudal fin and neural retina

Tese de mestrado em Biologia Evolutiva e do Desenvolvimento, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016A regeneração é a capacidade que um organismo tem de recuperar totalmente, após uma lesão, a estrutura e função de um tecido, órgão ou membro danificado. Dado que os humanos não possuem grande capacidade regenerativa, estudos têm sido feitos no sentido de compreender quais os processos celulares e moleculares na base deste evento em organismos que possuem, naturalmente, capacidade de regenerar. Um dos modelos animais mais usados neste contexto é o peixe-zebra, Danio rerio. Este modelo animal possui a capacidade de regenerar a maioria dos seus órgãos e apêndices, e uma fácil manipulação genética, que permite criar linhas transgénicas e mutantes. O peixe-zebra possui uma extraordinária capacidade de regenerar tecidos como a barbatana caudal e a retina. Após amputação da cauda, inicia-se um processo de cicatrização da ferida, onde esta é coberta por células da epiderme, seguida pela migração de células para o plano de amputação onde vão formar uma estrutura designada blastema, composta por células em proliferação que vão regenerar o tecido perdido. Por fim há uma fase de crescimento que é caracterizada por processos de diferenciação, de modo a restaurar a estrutura e função originais da cauda. Um dos tecidos mais abundantes na cauda é o tecido ósseo. O nosso grupo e outros demonstraram que a regeneração do tecido ósseo ocorre através da desdiferenciação dos osteoblastos maduros, que adquirem capacidade proliferativa e formam osteo-progenitores, capazes de se rediferenciar e regenerar o novo tecido ósseo. Contudo, um estudo recente demonstrou que após ablação dos osteoblastos presentes na cauda, a regeneração do osso progride normalmente, sugerindo a existência de outras fontes celulares capazes de originar novos osteoblastos nesta situação. Um dos possíveis candidatos são os pericitos, células perivasculares associadas aos vasos sanguíneos da barbatana caudal. Estas células partilham vários marcadores com células estaminais do mesênquima humanas e são capazes de originar osteoblastos in vitro. Assim sendo, um dos objetivos deste trabalho é explorar que tecidos, ou tipos celulares, têm a capacidade de originar osteoblasts in vivo durante o processo regenerativo, quando a população de osteoblastos residente está comprometida, com particular enfâse na população de pericitos. A retina é o tecido do olho responsável por converter a luz em sinais químicos e transportá-los para o cérebro. Esta é composta por vários tipos celulares, entre eles as células Müller Glia, que após lesão da retina desdiferenciam, entrando em seguida no ciclo celular. Desta forma, produzem progenitores neurais que migram para a camada danificada e se diferenciam no tipo celular danificado. Contudo, como este processo regenerativo progride na ausência das células Müller Glia nunca foi estudado no peixe-zebra. É igualmente importante descobrir fatores que possam regular as várias etapas da regeneração da retina. Sabe-se que a via de sinalização Hippo está envolvida na regeneração de vários órgãos e estruturas. Dados preliminares do nosso grupo indicam também que o efetor desta via, Yap, está presente nas células Müller Glia durante o desenvolvimento larvar. Deste modo decidimos averiguar se a via de sinalização Hippo e o seu efetor Yap têm alguma função durante a regeneração da retina. Assim, neste trabalho propusemos investigar por um lado como é que os tecidos da barbatana caudal respondem à ablação dos osteoblastos, e qual o papel dos pericitos e a sua contribuição para a regeneração desta estrutura; por outro criar uma linha de ablação de células Müller Glia que nos permitirá investigar como é que a regeneração da retina ocorre na ausência das mesmas; e se o Yap poderá ter alguma função durante o processo normal de regeneração da retina. Observámos que, após amputação, em caudas desprovidas de osteoblastos, as regiões da epiderme e o mesênquima da barbatana caudal adjacentes à matriz óssea, são os primeiros tecidos a responder a este evento aumentando a proliferação celular e possivelmente originando novos progenitores osteogénicos, sugerindo o seu potencial como fontes osteogénicas durante a regeneração. Decidimos também averiguar o papel dos pericitos como uma possível fonte de osteoblastos durante a regeneração. Para isso, tentámos criar uma linha transgénica que permita a ablação específica desta população e outra linha para seguir permanentemente a linhagem celular dos pericitos. Para ambas as construções usámos um promotor específico das células que se pretende analisar, o promotor do gene sdf1α, que foi demonstrado marcar estas células na barbatana caudal. Para gerar a linha de ablação usámos como base o sistema NTR/Mtz, construindo um plasmídeo no qual a enzima NTR, que tem capacidade de induzir morte celular, está sob o controlo do promotor sdf1α. Apesar de duas tentativas diferentes de criar esta linha, nenhuma delas se verificou viável. De futuro teremos de pensar e adaptar estratégias mais eficientes para criar esta linha de ablação. Por outro lado, para criar uma linha para seguir a descendência dos pericitos usámos como base o sistema Cre/Lox. Com esse objetivo, construímos um plasmídeo onde o promotor do sdf1α controla a expressão da CreERT2 recombinase, induzível por tamoxifeno. Foi possível criar com êxito uma linha transgénica estável, Tg(sdf1α:CREERT2; Crya:VENUS), que foi cruzada com a linha Tg(β-actin2:loxP-DsRed-loxP-GFP) que irá permitir a marcação permanente e o seguimento dos pericitos e da sua descendência. Estas duas linhas servem para avaliar se esta população de células perivasculares é essencial e se contribui de alguma forma para o processo regenerativo, principalmente para a formação de novo tecido ósseo. Relativamente ao estudo da regeneração da retina, decidimos criar uma linha de ablação das células Müller Glia, usando a mesma estratégia NTR/Mtz. Para tal, usámos o promotor do gene gfap, marcador de células Müller Glia diferenciadas, e tentámos gerar a linha gfap:GFP-NTR. Estamos atualmente a aguardar que os peixes cresçam para confirmar se algum poderá ser portador do transgene e deste modo estabelecer uma linha estável. Com o intuito de termos um ensaio que nos permitisse induzir regeneração na retina, implementámos no laboratório uma técnica já descrita que permite lesionar especificamente os fotorreceptores através de exposição à luz UV. Aplicámos esta técnica primeiro em peixes-zebra selvagem, onde verificámos a resposta regenerativa esperada, e em seguida à linha transgénica DN-yap na qual, após aplicação de choque térmico, se induz a expressão de uma forma negativa da proteína Yap. Infelizmente, os pontos temporais escolhidos para a recolha de tecido são ainda insuficientes para se conseguir observar uma possível perturbação no processo de regeneração dos fotorreceptores. Contudo, após choque-térmico, pudemos observar redução num marcador específico das células Müller Glia e a progressão no ciclo celular não parece ser afetada, sugerindo assim que o Yap não é necessário para a sua desdiferenciação e proliferação. Contudo, não podemos excluir a hipótese deste fator ser apenas necessário num ponto mais tardio da regeneração, por exemplo, durante a diferenciação dos fotorreceptores. Para isso teremos de fazer recolhas do tecido em períodos mais tardios durante o processo regenerativo. Em conclusão, este trabalho apresenta principalmente resultados preliminares onde nos focámos na utilização e geração de linhas transgénicas, e no estudo de vias de sinalização, numa tentativa de identificar novos candidatos que possam auxiliar na regeneração do sistema esquelético e da retina, não só após lesão, mas também no contexto de patologias associadas a estes órgãos. Em mamíferos, estes sistemas estão desprovidos de capacidade regenerativa, podendo ser danificados e ficar com a sua função comprometida. Desta forma, é importante descobrir novos mecanismos celulares e moleculares que possam contribuir para o estabelecimento de novas terapias capazes de promover e induzir a capacidade regenerativa destas estruturas em mamíferos.Regeneration is the capacity to fully restore the structure and function of an organ or limb, upon damage or injury. One of the most popular animal models used to study the mechanisms underlying tissue regeneration is the zebrafish, Danio rerio. Two tissues that hold an outstanding regenerative capacity are the caudal fin and the retina. Caudal fin skeletal tissue regeneration occurs via dedifferentiation of mature osteoblasts. However, upon osteoblast ablation, the regenerative process is not impaired, suggesting the existence of other cell sources capable of producing new osteoblasts. Possible candidates are the pericytes, shown to be capable of differentiating into osteoblasts in vitro. Upon injury in the neural retina, Müller Glia cells dedifferentiate and produce neuronal progenitors that allow damaged tissue recovery. However, how regeneration progresses in the absence of these cells has never been addressed, and the pathways that can modulate the process of retina regeneration are not fully understood. The Hippo pathway is a possible candidate to mediate retina regeneration, since it has an important role during the regeneration of several organs and recent data indicates that the Hippo pathway effector Yap is localized in the Müller Glia. Our results indicate that during fin regeneration, upon mature osteoblast ablation, the epidermis and the mesenchyme surrounding the bone matrix respond by increasing cell proliferating and by producing osteo-progenitors, suggesting that they could act as potential sources for de novo osteoblasts formation. In addition, to address if pericytes are a possible source of new osteoblasts, we tried to generate a pericyte ablation line, and succeeded in generating a pericyte-lineage tracing line. Regarding retina regeneration, in order to explore the role of Müller Glia during this process, we generated a Müller Glia cell ablation transgenic line, soon to be validated. To assess the contribution of Yap also in the context of retina regeneration, we induced photoreceptor damage in a Dominant Negative Yap transgenic zebrafish and observed no impairment until 6 days post injury, suggesting that Yap does not contribute towards dedifferentiation or proliferation of Müller Glia cells In this work, we focused on establishing transgenic lines and in assessing new pathways that could assist us in better understanding the regeneration of the skeletal tissue and neural retina. When these are damaged in mammals, in the context of osteo-degenerative disorders and retinopathies, both systems fail to regenerate properly, leading to severe impairment of normal tissue functions. It is therefore of major importance to unravel the cellular and molecular mechanism underlying tissue regeneration to promote more efficient therapeutic strategies to improve the regenerative capacity of these tissues in mammalian systems

Yap Regulates Müller Glia Reprogramming in Damaged Zebrafish Retinas

Funding: This work was supported by funding from Fundação para a Ciência e Tecnologia, in the context of a program contract to RL (4, 5, and 6 of article 23.◦ of D.L. no. 57/2016 of August 29, as amended by Law no. 57/2017 of 19 July), UIDB/04462/2020 and PTDC/BIM-MED/0659/2014 in the context of a grant project; SFRH/BPD/93453/2013 to RL, SFRH/BD/51990/2012 to AB, SFRH/BD/131929/2017 to JB, and SFRH/BD/140124/2018 to RG. Zebrafish used as animal models were reproduced and maintained in the CEDOC Fish Facility with support from Congento LISBOA-01-0145-FEDER-022170, co-financed by FCT (Portugal) and Lisboa2020, under the PORTUGAL2020 agreement (European Regional Development Fund).Vertebrates such as zebrafish have the outstanding ability to fully regenerate their retina upon injury, while mammals, including humans, do not. In zebrafish, upon light-induced injury, photoreceptor regeneration is achieved through reprogramming of Müller glia cells, which proliferate and give rise to a self-renewing population of progenitors that migrate to the lesion site to differentiate into the new photoreceptors. The Hippo pathway effector YAP was recently implicated in the response to damage in the retina, but how this transcription coactivator is integrated into the signaling network regulating Müller glia reprogramming has not yet been explored. Here, we show that Yap is required in Müller glia to engage their response to a lesion by regulating their cell cycle reentry and progenitor cell formation, contributing to the differentiation of new photoreceptors. We propose that this regulation is accomplished through a lin28a–ascl1a-dependent mechanism, bona fide Müller glia-reprogramming factors. Overall, this study presents Yap as a key regulator of zebrafish Müller glia reprogramming and consequently retina regeneration upon injury.publishersversionpublishe

Experimental benchmark data for Monte Carlo simulated radiation effects of gold nanoparticles. Part II: comparison of measured and simulated electron spectra from gold nanofoils

Electron emission spectra of a thin gold foil after photon interaction were measured over the energy range between 50 eV and 9500 eV to provide reference data for Monte Carlo radiation-transport simulations. Experiments were performed with the HAXPES spectrometer at the PETRA III high-brilliance beamline P22 at DESY (Hamburg, Germany) for photon energies just below and above each of the gold L-edges, that is, at 11.9 keV, 12.0 keV, 13.7 keV, 13.8 keV, 14.3 keV, and 14.4 keV. The data were analyzed to obtain the absolute values of the particle radiance of the emitted electrons per incident photon flux. Simulations of the experiment were performed using the Penelope and Geant4 Monte Carlo radiation-transport codes. Comparison of the measured and simulated results shows good qualitative agreement. On an absolute scale, the experiments tend to produce higher electron radiance values at the lower photon energies studied as well as at the higher photon energies for electron energies below the energy of the Au L3 photoelectron. This is attributed to the linear polarization of the photon beam in the experiments, something which is not considered in the simulation codes

Experimental benchmark data for Monte Carlo simulated radiation effects of gold nanoparticles. Part II: Comparison of measured and simulated electron spectra from gold nanofoils

Electron emission spectra of a thin gold foil after photon interaction were measured over the energy range between 50 eV and 9500 eV to provide reference data for Monte Carlo radiation-transport simulations. Experiments were performed with the HAXPES spectrometer at the PETRA III high-brilliance beamline P22 at DESY (Hamburg, Germany) for photon energies just below and above each of the gold L-edges, i.e., at 11.9 keV, 12.0 keV, 13.7 keV, 13.8 keV, 14.3 keV, and 14.4 keV. The data were analyzed to obtain the absolute values of the particle radiance of the emitted electrons per incident photon flux. Simulations of the experiment were performed using the Monte Carlo radiation-transport codes Penelope and Geant4. Comparison of the measured and simulated results shows good qualitative agreement. When simulation results are convolved with curves that take into account the effect of lifetime broadening, line shapes of photoelectron and Auger peaks similar to those observed experimentally are obtained. On an absolute scale, the experiments tend to give higher electron radiance values at the lower photon energies studied as well as at the higher photon energies for electron energies below the energy of the Au L3 photoelectron. This is attributed to the linear polarization of the photon beam in the experiments which is not considered in the simulation codes.Comment: Revised manuscript after peer review, 13 pages, 9 figure

The effects of metabolic reprogramming during zebrafish tissue regeneration

Regeneration is the ability to fully restore the structure and function of a lost body part, after damage. While mammals have a very limited regenerative capacity, other vertebrates, such as the zebrafish, have the outstanding capacity to fully regenerate organs, like the heart and liver, and appendages, like the caudal fin. The zebrafish caudal fin is a relatively simple tissue. It has a bi-lobed shape that is composed and supported by a series of skeletal elements, called bony-rays. The bony-rays are covered by a monolayer of osteoblasts, the bone-producing cells, and they encompass an inner mesenchymal compartment, containing nerves, blood vessels and fibroblast-like cells. Externally, a multi-layered epidermis covers the bony-rays. The caudal fin possesses a series of advantages that make it an ideal system to study regeneration, namely easy to amputate, quick to regenerate, amenable to live imaging and its amputation does not compromise fish survival. Its amputation triggers a regenerative program that is divided into three steps: first, the wound healing phase, in which the cells from the epidermis migrate to cover the wound site and form a specialized structure called wound epidermis; second, the blastema formation phase, in which mature cells dedifferentiate and migrate distally to form a mass of proliferative cells, called the blastema, the hallmark of fin regeneration; third, the regenerative outgrowth phase, during which there is a series of differentiation and patterning events to restore the lost tissue organization and function. A major focus of our group are the initial stages of regeneration, which are also the most neglected. Early regeneration events include cell fate transition, like dedifferentiation, cell cycle re-entry, and proliferation. Importantly, preliminary results from our lab indicate that early in regeneration, cell dedifferentiation is preceded by a change in the metabolic profile, increasing their preference for glycolysis. Depending on the cellular function and requirements, glucose can have two main destinations: go through the TCA and be oxidised via OXPHOS or be converted into lactate via glycolysis. Switches of the metabolic profile from OXPHOS to glycolysis have already been described in other contexts but only recently proposed to occur in regeneration. Interestingly, lactate, one of the main products of glycolysis, has recently been proposed to be a substrate for lactylation, a newly identified epigenetic modification. Taking this into account, the main goal of this thesis is to unveil the importance of energy metabolism to the regenerative process and explore a potential link between energy metabolism and epigenetics during regeneration through lactylation. Our hypothesis is that after caudal fin amputation there is an early metabolic reprogramming event from OXPHOS to glycolysis that regulates regenerative events such as dedifferentiation and proliferation, partially through modulation of lactylation and epigenetics.We started by investigating whether the caudal fin tissue responded to an amputation by undergoing a metabolic reprogramming. For that, we performed a thorough characterisation of key enzymes and metabolites using transcriptomic and metabolomic approaches. We observed that during the initial regenerative stages, before blastema formation, between 6 and 24 hour-post amputation, there is a dramatic upregulation of several glycolytic and lactate producing enzymes and an increase in the levels of glucose and lactate. This was accompanied by an increase of mitochondrial fission and possibly biogenesis events, which are associated with high glycolysis and cell division. To address the requirement of this increased glycolytic activity for regeneration, we performed functional assays with inhibitors for glycolysis, lactate formation and OXPHOS. Inhibition of glycolysis and lactate production led to a severe and mild impairment of blastema formation, respectively. On the other hand, inhibition of OXPHOS using different compounds had no effect, or slightly accelerated regeneration. Furthermore, inhibition of glycolysis also led to a decrease of cell proliferation and to discrepancies in osteoblast subtypes. Overall, this suggests that a metabolic reprogramming towards glycolysis is critical for the initial set up of the regenerative process that culminates in blastema assembly, contributing, at least, to maintain cell proliferation and for new osteoblast formation and organization. Next, we decided to investigate additional mechanisms by which metabolic reprogramming could be regulating regeneration. Several metabolites generated from glycolysis and OXPHOS contribute for epigenetic modifications. Given the increase in lactate levels and the reduced regenerative capacity when inhibiting lactate production, we proposed that lactate could be necessary to mediate histone lactylation to modulate chromatin dynamics during regeneration. This epigenetic modification has only been recently described, thus lacking proper tools to allow research. Through proteomic analysis, we successfully identified, for the first time, the existence of histone lactylation in the zebrafish. Additionally, we also demonstrated that the levels of histone lactylation increase during the first 24 hours of regeneration, when cells dedifferentiate and begin to proliferate, concomitantly with changes in metabolism. This increase was impaired when glycolysis was inhibited, confirming that lactylation is dependent on the metabolic reprograming towards glycolysis. To identify possible genomic targets of histone lactylation during regeneration, we performed ChIP-sequencing. We observed that histone lactylation was associated with genes related to actin cytoskeleton, axon guidance, electron transport chain, protein phosphorylation and regulation of transcription, among other processes. From these, we further investigated HDAC4, a histone deacetylase that inhibits runx2 expression and regulates osteogenic differentiation. As expected, we observed that the expression levels of hdac4 decreased during the early event of cell dedifferentiation, suggesting that associated histone lactylation could be inhibiting hdac4 expression. Surprisingly, upon glycolysis inhibition during regeneration, which leads to less lactylation, we also detected a further decrease of hdac4 expression. This suggests that the regulation of hdac4 by lactylation is an intricate system that must be thoroughly explored. Comparing the initial stages of caudal fin regeneration with other biological contexts, such as development and cancer, our results highlight some similarities and differences. Like stem cells and growing tumours, we observed that dedifferentiating cells also change their metabolic profile, increasing glycolysis and histone lactylation levels. However, the enhanced activity upon OXPHOS inhibition seems to be specific of the regenerative process. This indicates that studying the differences between these processes is crucial to understand the unique features controlling regeneration. Our observations are in accordance with recent findings describing the requirement of metabolic reprogramming towards glycolysis during zebrafish adult heart and larva tail regeneration, suggesting that the metabolic reprogramming seems to be a shared featured among regenerative processes. Taken together, this project proposes a series of new contributors for the field of tissue regeneration. We demonstrated that a metabolic reprogramming towards glycolysis is critical for the correct progress of caudal fin regeneration and provided for the first-time evidence of the existence of histone lactylation in zebrafish and in regeneration. Future experiments to better understand the mechanism between these two events would greatly improve our understanding of the regenerative process