Novel culture systems to model testicular function and organization

In vitro systems to model the testicular microenvironment are required to study physiology and pathology of the testis, the gonadotoxic effect of environmental and pharmaceutical chemicals, as well as to explore the mechanisms ruling spermatogonial stem cell (SSC) self-renewal and differentiation. The knowledge produced in in vitro experiments, using animal, but most importantly utilizing human models, will contribute to the discovery of safe medical treatments and might provide translational tools to treat male infertility problems. The current models are vital to study the testicular microenvironment in vitro, but further improvements are required to more closely reconstruct the testicular organization and function found in vivo. The main focus of the research conducted in this thesis was to establish and optimize three-dimensional culture conditions to further improve the current approaches to model testicular architecture and physiology in vitro. For that purpose, we explore three distinct approaches: the three-layer gradient system (3-LGS) to culture rat testicular cells suspended in extracellular matrix (ECM) components; the decellularized testicular extracellular matrix (DTM) to culture human testicular cells in their native ECM; and the organ culture system in the air-liquid interface to culture human testicular cells in their native tissue organization. During our experiments, we found that the 3-LGS promoted the reorganization of rat testicular cells into seminiferous tubule-like organoids with both a functional blood-testis barrier and the capacity to maintain proliferative undifferentiated germ cells. Moreover, the DTM allowed the generation of human testicular organoids that, despite not demonstrating characteristic testicular organization, were able to produce testosterone and inhibin B as well as to maintain spermatogonia proliferating during the entire culture period. Finally, the organ culture system served to maintain human testicular tissue in viable conditions and to demonstrate differences in terms of testicular somatic cell functionality among different patient groups exposed to chemotherapy or treatments against haematological diseases. In perspective, the rat and human organoid systems demonstrate potential to explore aspects of testicular development and toxicology, but also the possibility to, easier than before, manipulate the culture conditions to study the influence of different niche elements on SSC self-renewal and differentiation. Moreover, the organ culture system showed the aptitude to be used as a quality control tool in the assessment of bio-banked human testicular tissue and to help in determining the best-fit fertility preservation strategy for each group of patients

Graphical simulation of numerical algorithms : an approach based on code instrumentation and Java technologies

We want to create a working tool for mathematics teachers and a corresponding learning tool for students, namely a graphical simulator of mathematical algorithms (GraSMa). To achieve it we try two different strategies. We started by annotate manually the original algorithm with inspector functions. Now we are testing a new approach that aims to automatically annotate the original code with inspector functions. To achieve this we are developing a language translator module that enables to comment automatically any code written in Octave language. The run of the annotated code gated by one of these two ways, records in a XML (eXtensible Markup Language) file everything that happened during the execution. Subsequently, the XML file is parsed by a Java application that graphically represents the mathematic objects and their behaviour during execution. The final application will be accessed on-line through a website (WebGraSMa) which is currently under development. In this paper we report and discuss about the procedures followed and present some intermediate results

Testicular organoids: a new model to study the testicular microenvironment in vitro?

BACKGROUND: In recent decades, a broad range of strategies have been applied to model the testicular microenvironment in vitro. These models have been utilized to study testicular physiology and development. However, a system that allows investigations into testicular organogenesis and its impact in the spermatogonial stem-cell (SSC) niche in vitro has not been developed yet. Recently, the creation of tissue-specific organ-like structures called organoids has resurged, helping researchers to answer scientific questions that previous in vitro models could not help to elucidate. So far, a small number of publications have concerned the generation of testicular organoids and their application in the field of reproductive medicine and biology. OBJECTIVE AND RATIONALE: Here, we aim to elucidate whether testicular organoids might be useful in answering current scientific questions about the regulation and function of the SSC niche as well as germ cell proliferation and differentiation, and whether or not the existing in vitro models are already sufficient to address them. Moreover, we would like to discuss how an organoid system can be a better solution to address these prominent scientific problems in our field, by the creation of a rationale parallel to those in other areas where organoid systems have been successfully utilized. SEARCH METHODS: We comprehensively reviewed publications regarding testicular organoids and the methods that most closely led to the formation of these organ-like structures in vitro by searching for the following terms in both PubMed and the Web of Science database: testicular organoid, seminiferous tubule 3D culture, Sertoli cell 3D culture, testicular cord formation in vitro, testicular morphogenesis in vitro, germ cell 3D culture, in vitro spermatogenesis, testicular de novo morphogenesis, seminiferous tubule de novo morphogenesis, seminiferous tubule-like structures, testicular in vitro model and male germ cell niche in vitro, with no restrictions to any publishing year. The inclusion criteria were based on the relation with the main topic (i.e. testicular organoids, testicular- and seminiferous-like structures as in vitro models), methodology applied (i.e. in vitro culture, culture dimensions (2D, 3D), testicular cell suspension or fragments) and outcome of interest (i.e. organization in vitro). Publications about grafting of testicular tissue, germ-cell transplantation and female germ-cell culture were excluded. OUTCOMES: The application of organoid systems is making its first steps in the field of reproductive medicine and biology. A restricted number of publications have reported and characterized testicular organoids and even fewer have denominated such structures by this method. However, we detected that a clear improvement in testicular cell reorganization is recognized when 3D culture conditions are utilized instead of 2D conditions. Depending on the scientific question, testicular organoids might offer a more appropriate in vitro model to investigate testicular development and physiology because of the easy manipulation of cell suspensions (inclusion or exclusion of a specific cell population), the fast reorganization of these structures and the controlled in vitro conditions, to the same extent as with other organoid strategies reported in other fields. WIDER IMPLICATIONS: By way of appropriate research questions, we might use testicular organoids to deepen our basic understanding of testicular development and the SSC niche, leading to new methodologies for male infertility treatment

Influence de l'effeuillage sur l'efficience thermique de l'accumulation des anthocyanes dans la baie

The aim of this work was to analyse the effects of leaf removal on Touriga Nacional berry temperature and consequent thermal efficiency for anthocyanins biosynthesis. The field experiment was located at Dão Wine Research Station, Nelas, Portugal in an adult vineyard planted with North-South oriented rows, with the red grape variety Touriga Nacional grafted on 110R rootstock. The vines were trained on a vertical shoot positioning, spur-pruned on a bilateral Royat cordon system and deficit irrigated (50% ETc). The experimental design was a randomized complete block design with four replications of twelve vines per elemental plot, and the following two treatments: basal leaf removal (LR) and a control non-defoliated (ND). Berry temperature (Tb) was measured continuously during the second half (3rd to 19th September) of the 2009 ripening period using two-junction, fine-wires copper-constantan thermocouples manually inserted into the berries and connected to a data logger. A sample of clusters located in different canopy positions (exposed and internal; facing East and West) of 4 vines per treatment were used. To quantify the effect of Tb on anthocyanins biosynthesis, the berry hourly mean temperatures were converted into normal heat hours (NHH) and accumulated per day (NHHd) and per monitoring period (NHHc). For quantification of thermal requirements for anthocyanins synthesis and accumulation, a minimum of 10°C, a maximum of 35°C, and an optimum of 26°C were used. Meteorological variables were measured at an automatic weather station installed within the experimental plot. For all days of the monitoring period, daily average berry temperature (dTb) of all monitored berries was lower in ND treatment than in LR, being the maximum differences between treatments registered on 11th September. The highest dTb differences between treatments were registered on the clusters located at the west side of the canopy on 7th September while dTb of the clusters located in the centre of the canopy was less affected by leaf removal. The control non-defoliated treatment (ND) presented a significantly higher NHHc than that of LR being the higher differences presented by the clusters located in the west side. The lowest differences in NHHc were obtained in the clusters located in the centre of the canopy. Our results show that the thermal efficiency for berry anthocyanins accumulation was significantly affected by leaf removal and that this effect was dependent of the meteorological conditions, time of the day and berry/cluster location into the vine canopy

Testicular organoid generation by a novel in vitro three-layer gradient system.

A system that models the testicular microenvironment and spermatogonial stem-cell (SSC) niche in vitro has not been produced yet. Here, we developed and characterized a novel three-dimensional multilayer model, the Three-Layer Gradient System (3-LGS), which permits the generation of rat testicular organoids with a functional blood-testis barrier (BTB) and germ cell establishment and proliferation. The model is unique as regards the formation of cellular organizations that more closely represent the in vivo germ-to-somatic cell associations in vitro. Moreover, we also verified the roles of retinoic acid (RA), IL-1α, TNFα and RA inhibitors in germ cell maintenance and BTB organization in vitro. Treatment with RA was beneficial for germ cell maintenance, while IL-1α and TNFα were observed to impair the formation of testicular organoids and germ cell maintenance. Taking in account our characterization and validation results, we propose the 3-LGS as a new platform to investigate the SSC niche in vitro and to search for novel unknown factors involved in germ cell proliferation and differentiation. Moreover, we suggest that this model can be used in other scientific fields to study organogenesis and development by the generation of organoids

Self-organising human gonads generated by a Matrigel-based gradient system.

BACKGROUND: Advances in three-dimensional culture technologies have led to progression in systems used to model the gonadal microenvironment in vitro. Despite demonstrating basic functionality, tissue organisation is often limited. We have previously detailed a three-dimensional culture model termed the three-layer gradient system to generate rat testicular organoids in vitro. Here we extend the model to human first-trimester embryonic gonadal tissue. RESULTS: Testicular cell suspensions reorganised into testis-like organoids with distinct seminiferous-like cords situated within an interstitial environment after 7 days. In contrast, tissue reorganisation failed to occur when mesonephros, which promotes testicular development in vivo, was included in the tissue digest. Organoids generated from dissociated female gonad cell suspensions formed loosely organised cords after 7 days. In addition to displaying testis-specific architecture, testis-like organoids demonstrated evidence of somatic cell differentiation. Within the 3-LGS, we observed the onset of AMH expression in the cytoplasm of SOX9-positive Sertoli cells within reorganised testicular cords. Leydig cell differentiation and onset of steroidogenic capacity was also revealed in the 3-LGS through the expression of key steroidogenic enzymes StAR and CYP17A1 within the interstitial compartment. While the 3-LGS generates a somatic cell environment capable of supporting germ cell survival in ovarian organoids germ cell loss was observed in testicular organoids. CONCLUSION: The 3-LGS can be used to generate organised whole gonadal organoids within 7 days. The 3-LGS brings a new opportunity to explore gonadal organogenesis and contributes to the development of more complex in vitro models in the field of developmental and regenerative medicine

perceptions and choices among rheumatology outpatients

OBJECTIVE: Biobanks for research (BBR) have enormous value for research, including those specifically oriented to chronic diseases. Knowing public attitudes and perceptions is key to design and implement patient-centered BBR. We assessed patient awareness, perception and choices among rheumatology outpatients regarding aging biobanking activities. METHODS: We conducted a cross-sectional survey of patients, aged 50 or older, attending an outpatient rheumatology tertiary department. Demographic data and perceptions about biobanking were collected and statistical analysis was performed. RESULTS: 132 valid questionnaires were obtained (mean age: 63,4; 68,2% female; mean education years: 8,35). 61,7% of respondents did not know the specific term "biobank", 57,7% knew they could donate biological material for BBR, 89,9% agreed with these infrastructures and 88,3% would consider participation Those participants with more years of education were more knowledgeable and prone to biobank participation. Willingness to participate in BBR was mainly related (86,4%) to the advancement of scientific knowledge and not individual gain. Scientific research institutes were indicated as the most adequate institutions to manage BBR. Informed consent, anonymity and confidentiality ranked as top requisites for biobank participation. 61,3% of respondents expressed their agreement with aging biobanks, considering these as a sign of respect for specific problems of people of older ages such as higher disease burdens. CONCLUSION: Knowledge of biobanks was found to be limited. Participants were positive toward the setting up of biobanks in general and patient-centered aging biobanks in particular. Knowledge about biobanks and acceptance were higher among participants with higher education years.publishersversionpublishe

A web-based information system for a regional public mental healthcare service network in Brazil

Acknowledgements We would like to thank all the participating representatives of public mental health services for their invaluable contribution to this system development and implementation and the XIII Regional Health Department of Sao Paulo state for their support. Funding: This study was funded by the ‘Conselho Nacional de Desenvolvimento Científico e Tecnológico’ (CNPq) and ‘Coordenação de Aperfeiçoamento de Pessoal de Nível Superior’ (CAPES)—Science Without Borders Programme.Peer reviewedPublisher PD

Breast cancer surgery with augmented reality

© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Introduction: Innovations in 3D spatial technology and augmented reality imaging driven by digital high-tech industrial science have accelerated experimental advances in breast cancer imaging and the development of medical procedures aimed to reduce invasiveness. Presentation of case: A 57-year-old post-menopausal woman presented with screen-detected left-sided breast cancer. After undergoing all staging and pre-operative studies the patient was proposed for conservative breast surgery with tumor localization. During surgery, an experimental digital and non-invasive intra-operative localization method with augmented reality was compared with the standard pre-operative localization with carbon tattooing (institutional protocol). The breast surgeon wearing an augmented reality headset (Hololens) was able to visualize the tumor location projection inside the patient's left breast in the usual supine position. Discussion: This work describes, to our knowledge, the first experimental test with a digital non-invasive method for intra-operative breast cancer localization using augmented reality to guide breast conservative surgery. In this case, a successful overlap of the previous standard pre-operative marks with carbon tattooing and tumor visualization inside the patient's breast with augmented reality was obtained. Conclusion: Breast cancer conservative guided surgery with augmented reality can pave the way for a digital non-invasive method for intra-operative tumor localization.info:eu-repo/semantics/publishedVersio

O papel da microbiota intestinal na patogênese das doenças inflamatórias intestinais / The role of the intestinal microbiota in the pathogenesis of inflammatory intestinal disease

As doenças inflamatórias intestinais (DIIs) são desordens crônicas de etiopatogenia não esclarecida, cuja incidência tem aumentado globalmente. Evidências apontam que a suscetibilidade genética associada a fatores ambientais são capazes de determinar resposta imune anormal e comprometimento da barreira epitelial do intestino. Inúmeras pesquisas demonstram uma possível conexão fisiopatológica das DIIs com a microbiota intestinal (MI), sobretudo em casos da alteração de sua composição - disbiose. Embora controversa, a continuidade da investigação nesta área faz-se necessária para elucidar tal relação. OBJETIVOS: Apresentar uma revisão sobre a correlação entre as DIIs e MI, vislumbrando um possível papel da disbiose na etiopatogenia dessas doenças e avaliar a aplicabilidade prognóstica e terapêutica dos resultados encontrados. METODOLOGIA DE BUSCA: Utilizou-se pesquisa na plataforma PubMed de revisões literárias com os termos “Inflammatory Bowel Disease”, “Microbiome” e “Microbiota” com os pesquisadores booleanos AND e OR respectivamente entre os termos. DISCUSSÃO: A principal hipótese acerca da patogenia das DIIs consiste na interação da MI com fatores extrínsecos, causando uma quebra ou penetração da barreira epitelial das células intestinais, internalizando antígenos nas camadas submucosas. Em indivíduos geneticamente suscetíveis, uma resposta imune aberrante contra a MI invasora é desencadeada, gerando a inflamação intestinal. A respeito de sua constituição, constatou-se que alguns grupos de bactérias interferem no desenvolvimento das DIIs e em seu grau de complicações, principalmente relacionados a uma redução da diversidade da MI. Tendo em vista este cenário, propostas terapêuticas visam a correção da MI para o tratamento das DIIs. CONSIDERAÇÕES FINAIS: Conhecer o papel da MI nas DII é de extrema importância para seu entendimento fisiopatológico. Futuramente, esclarecer como essa associação ocorre poderá permitir, através da manipulação da MI, o desenvolvimento de novos alvos terapêuticos e a identificação precoce dos pacientes de risco além de prever fenótipos, evoluções e possíveis complicações das doenças