Male infertility diagnostic laboratories during COVID-19 pandemic: development of a novel teaching/learning strategy

[EN] Objective: To design and implement a laboratory teaching/learning strategy applied to male infertility diagnostic during COVID-19 pandemic. Methods: Seventy-five students attending Module 3 (M3): Male Infertility Diagnostic Laboratories, of curricular unit Laboratories in Biomedicine 5 were divided into 8 groups to reduce students’ number in class. M3 was organized in 2 Wet labs (WL; laboratory training) and 2 Dry labs (DL; theoretical-practical classes). At the end of M3, students completed a satisfaction survey. Results: Results showed that most students were satisfied with topics addressed, time dedicated outside the class, evaluation, and overall organization of WL and DL. The protocols provided and general conditions of WL were considered satisfactory. Only positive classifications were attributed to the learning goals, relevance of knowledge acquired, and opportunities to ask about evaluation. Qualitative data suggested that the dynamics, innovation, and organization of classes, allied to the knowledge and motivation of professors, provided the appropriate learning environment. Conclusions: The methodology implemented in M3 during COVID-19 pandemic represents a valid strategy to encourage and motivate students learning, stimulate teamwork, communication, and autonomy in the laboratory. Preparing dynamic, attractive, and motivating classes increases student's general satisfaction, improving the efficiency of the learning/teaching process.The authors thank the DCM, UA for the infrastructure support, and the support from FCT of the Portuguese Ministry of Science and Higher Education to J.S. (SFRH/BD/136896/2018) and P.C. (2020.10111.BD). The authors deeply acknowledge the students of the curricular unit Laboratories of Biomedicine 5 of the Biomedical Sciences degree of the UA (class 2020/2021) for being highly motivated and for motivating the professors.Santiago, J.; Corda, P.; Fardilha, M. (2021). Male infertility diagnostic laboratories during COVID-19 pandemic: development of a novel teaching/learning strategy. En 7th International Conference on Higher Education Advances (HEAd'21). Editorial Universitat Politècnica de València. 1019-1026. https://doi.org/10.4995/HEAd21.2021.12889OCS1019102

First insights on the presence of the unfolded protein response in human spermatozoa

The unfolded protein response (UPR) is involved in protein quality control and is activated in response to several stressors. Although in testis the UPR mechanisms are well described, their presence in spermatozoa is contentious. We aimed to investigate the presence of UPR-related proteins in human sperm and the impact of oxidative stress induction in UPR activation. To identify UPR-related proteins in human sperm, a bioinformatic approach was adopted. To explore the activation of UPR, sperm were exposed to hydrogen peroxide (H2O2) and motility, vitality, and the levels of UPR-related proteins were assessed. We identified 97 UPR-related proteins in human sperm and showed, for the first time, the presence of HSF1, GADD34, and phosphorylated eIF2α. Additionally, the exposure of human sperm to H2O2 resulted in a significant decrease in sperm viability and motility and an increase in the levels of HSF1, HSP90, HSP60, HSP27, and eIF2α; all proteins involved in sensing and response to unfolded proteins. This study gave us a first insight into the presence of UPR mechanisms in the male gamete. However, the belief that sperm are devoid of transcription and translation highlight the need to clarify if these pathways are activated in sperm in the same way as in somatic cells.publishe

Exploring the effect of exercise training on testicular function

Purpose The impact of exercise training on testicular function is relatively ill-defined. To gain new insights into this important topic, published data, deriving from both humans and animal studies, were critically analyzed. Results and conclusions The effects of exercise on the hypothalamus–pituitary–gonadal axis, influenced by the type, intensity and duration of the exercise program, can be evaluated in terms of total and free testosterone and/or luteinizing hormone and follicle-stimulating hormone serum levels and sperm parameters. High-intensity exercise promotes a common decrease in these parameters, and therefore, negatively impacts upon testicular function. However, published data for moderate-intensity exercise training are inconsistent. Conversely, there is consistent evidence to support the benefits of exercise training to prevent and/or counteract the impairment of testis function caused by aging, obesity and doxorubicin treatment. This positive effect is likely the consequence of decreased oxidative stress and inflammatory status. In the future, it will be important to clarify the molecular mechanisms which explain these reported discrepancies and to establish guidelines for an active lifestyle to promote healthy testicular function.publishe

How to motivate students to learn Metabolic Biochemistry in a Biomedical Sciences curricula

[EN] Teaching methodologies used in biochemistry classes at the University level are traditionally dependent on theorical classes. The assessment is usually based on written tests performed at the end of the semester. However, most students who learn metabolism by this traditional method consider the study of metabolic biochemistry a terrifying and unforgettable experience. Understanding biochemical metabolic pathways was the proposed goal of the Medical Biochemistry curricular unit. To this end, the multi-method active learning approach was used in order to increase students’ motivation towards the learning process and to allow the development of skills associated with group conflict resolution, critical thinking and communication skills. Overall, students and learning facilitators were highly motivated by the diversity of learning activities, particularly due to the emphasis on correlating theoretical knowledge with human health and disease. As a quality control exercise, the students were asked to answer a questionnaire on their evaluation of the teaching/learning experience. Thus, the initial analysis of the student’s perception questionnaires permits to conclude that the approach undertaken yields results that surpass the traditional teaching methods. Investing in preparing attractive and motivating classes increases students and teacher’s general satisfaction and the learning/teaching process becomes more efficient.This work was also supported by an individual grant from FCT of the Portuguese Ministry of Science and Higher Education to M.C.H (SFRH/BD/131846/2017).Carvalho Henriques, M.; Fardilha, M. (2019). How to motivate students to learn Metabolic Biochemistry in a Biomedical Sciences curricula. En HEAD'19. 5th International Conference on Higher Education Advances. Editorial Universitat Politècnica de València. 1249-1254. https://doi.org/10.4995/HEAD19.2019.9315OCS1249125

Caracterização do interactoma da PP1 do testículo humano

Doutoramento em BiologiaProtein phosphorylation is a major regulatory mechanism notably of signal transduction cascades in eukaryotic cells. Protein phosphorylation is catalysed by protein kinases and can be reversed by the action of protein phosphatases. Although phosphatases were discovered more than sixty years ago, their importance as central players in multiple cellular mechanisms was only recently recognized. PP1, a Serine/Threonine specific phosphatase, is involved in important cellular mechanisms such as the cell cycle, muscle contraction and apoptosis, among others. Its role in such diverse cellular processes depends on its interactions with targeting/regulatory subunits. To date, more than 50 regulatory subunits have been identified that bind the catalytic subunit of PP1 determining its function in a specific cellular location. Several isoforms of PP1 are known, termed PP1 , PP1 and PP1 . The gamma isoform undergoes alternative splicing to yield a ubiquitously expressed PP1 1 and a testis-specific PP1 2 isoform. Incubation of non-motile immature sperm with phosphatase inhibitors induces sperm motility, and PP1 2 was implicated in this process. This led us to search for PP1 2-specific interactors in human sperm that could be targeted for infertility therapeutics or in male contraception. To achieve this goal the Yeast Two Hybrid system was used to screen a human testis library for new PP1 binding proteins using both PP1 1 (YTH1) and PP1 2 (YTH2) as baits. We recovered 120 positive clones in YTH1 and 155 positive clones in YTH2. Among these were clones encoding “bona fide” PP1 interactors such as Nek2 and NIPP1, and also previously uncharacterized proteins. We undertook a more detailed study of a novel gene encoding a novel protein that we termed SEARP-T. This protein of 93KDa is expressed mainly in testis and fluorescence immunocytochemistry was used to determine its intra sperm localization. Both PP1 2 and SEARP-T proteins are present in the tail and in the equatorial segment of the head.These results provide new insights into PP1 function in human testis and sperm motility, and indicates that the Yeast Two Hybrid System provides a mean to understand the roles PP1 plays in diverse cellular regulatory events.A fosforilação de proteínas é um dos principais mecanismos reguladores de cascatas de transdução de sinais em eucariotas. A fosforilação é catalizada por proteínas cinases e é revertida pela acção de proteínas fosfatases. Embora as proteínas fosfatases tenham sido descobertas à mais de sessenta anos, a sua importância central em múltiplos mecanismos celulares só muito recentemente foi reconhecida. PP1, uma fosfatase específica para serina/treonina, está envolvida em importantes mecanismos celulares, como o ciclo celular, a contracção muscular e a apoptose, entre outros. O seu papel em tão variados processos celulares depende das suas interacções com subunidades reguladoras. Até à data, foram descritas mais de 50 subunidades reguladoras que se ligam à subunidade catalítica da PP1, sendo determinantes para a sua função num local específico da célula. Das três isoformas da PP1 conhecidas, PP1 , PP1 e PP1 , a isoforma gama sofre splicing alternativo, originando a PP1 1, ubíqua e, a PP1 2 específica de testículo. Incubação de espermatozóides imaturos com inibidores de fosfatases induz a sua motilidade, sendo a PP1 2 a fosfatase envolvida. Este facto levou-nos a procurar proteínas de testículo humano capazes de interagir especificamente com a PP1 2 que possam ser alvos terapêuticos no tratamento da infertilidade, ou na contracepção masculina. Para atingir este objectivo utilizou-se o sistema Dois Híbrido de Levedura no rastreio de uma biblioteca de testículo humano, na busca de novas proteínas que se ligam à PP1 usando como isco a PP1 1 (no YTH1) ou a PP1 2 (no YTH2). Obtivemos 120 clones positivos no YTH1 e 155 no YTH2. Entre eles encontravam-se clones que codificam reguladores da PP1 previamente descritos, como a Nek2 e a NIPP1, assim como novas proteínas. Efectuámos um estudo detalhado de um novo gene, codificando uma nova proteína, que denominamos SEARP-T. Esta proteína de 93kDa é expressa maioritariamente em testículo e, a sua localização intracelular foi determinada por imunocitoquímica de fluorescência. Ambas as proteínas, PP1 2 e SEARP-T, estão presentes na cauda e no segmento equatorial da cabeça do espermatozóide. Estes resultados clarificam as funções da PP1 no testículo humano e na motilidade do esperma e, indicam que o sistema Dois Híbrido de Levedura é um bom método para compreender o papel da PP1 em múltiplos eventos de regulação celular

The Role of Endocrine-Disrupting Chemicals in Male Fertility Decline

Endocrine-disrupting chemicals (EDCs) are exogenous compounds with natural or anthropogenic origin omnipresent in the environment. These compounds disrupt endocrine function through interaction with hormone receptor or alteration of hormone synthesis. Humans are environmentally exposed to EDCs through the air, water, food and occupation. During the last decades, there has been a concern that exposure to EDCs may contribute to an impairment of human reproductive function. EDCs affect male fertility at multiple levels, from sperm production and quality to the morphology and histology of the male reproductive system. It has been proposed that exposure to EDCs may contribute to an impairment of sperm motility, concentration, volume and morphology and an increase in the sperm DNA damage. Moreover, EDCs exert reproductive toxicity inducing structural damage on the testis vasculature and blood-testis barrier and cytotoxicity on Sertoli and Leydig cells. This chapter will explore the effects of EDCs in male reproductive system and in the decline of male fertility

In Vivo Interaction of the Hepatitis Delta Virus Small Antigen with the ELAV-Like Protein HuR

The small and large delta antigens (S-HDAg and L-HDAg, respectively) represent two forms of the only protein encoded by the hepatitis delta virus (HDV) RNA genome. Consequently, HDV relies, at a large extent, on the host cell machinery for replication and transcription. Until now, only a limited number of cellular proteins were identified as S-HDAg or L-HDAg partners being involved in the modulation of the virus life cycle. In an attempt to identify cellular S-HDAg-binding proteins we made use of a yeast two-hybrid approach to screen a human liver cDNA library. We were able to identify HuR, a ubiquitously expressed protein involved in RNA stabilization, as an S-HDAg partner both in vitro and in vivo. HuR was found to be overexpressed and colocalize with HDAg in human hepatoma cells. siRNA knockdown of HuR mRNA resulted in inhibition of S-HDAg and L-HDAg expression

Protein phosphorylation: deregulation and oncogenesis

A fosforilação reversível proteica é o principal mecanismo regulador das cascatas de sinalização celular em eucariotas sendo este catalisado por cinases e revertido por fosfatases. A desregulação da fosforilação de proteínas integrantes das vias de sinalização (ex. PI3K, TGFβ e apoptóticas) têm sido associadas ao desenvolvimento de diversos tipos de cancro(ex. carcinomas da mama e próstata, o melanoma e o retinoblastoma). O processo oncogénico é dependente de alterações na maquinaria de fosforilação que permitem à célula a manipulação das vias de sinalização resultando numa promoção da proliferação celular. O intuito desta revisão é apresentar como alterações no mecanismo de fosforilação desempenham um papel fulcral no desenvolvimento do processo oncogénico e como pode este ser manipulado com intuitos terapêuticos

The heterogeneous ribonuclear protein C interacts with the hepatitis delta virus small antigen

Background: Hepatitis delta virus (HDV) is considered to be a satellite virus of the Hepatitis B virus. The genome consists of a 1679 nt ssRNA molecule in which a single ORF was identified. This ORF codes for a unique protein, the Delta antigen (HDAg). During transcription, two forms, small (S-HDAg; p24) and large (L-HDAg; p27) of this antigen are derived as a result of an editing mechanism catalyzed by cellular adenosine deaminase 1. Despite its simplicity, little is still known about the host factors that interact with the virus RNA and antigens being to modulate virus replication. Methods: A yeast two-hybrid screening of a human liver cDNA library, using the hepatitis delta virus (HDV) small antigen (S-HDAg) as bait, was performed. Blot overlay and co-immunoprecipitation assays were used in an attempt to confirm the interaction of hnRNPC and S-HDAg. siRNA knockdown assays of hnRNPC were performed to assess the effect on HDV antigen expression. Results: Thirty known proteins were identified as S-HDAg interactors in the yeast two-hybrid screening. One of the identified proteins, hnRNPC, was found to interact with S-HDAg in vitro and in vivo in human liver cells. The interaction of the two proteins is mediated by the C-terminal half of the S-HDAg which contains a RNA-binding domain (aa 98-195). HDV RNA, S-HDAg, and hnRNPC, were also found to co-localize in the nucleus of human liver cells. Knockdown of hnRNPC mRNA using siRNAs resulted in a marked decreased expression of HDV antigens. Conclusions: S-HDAg was found to interact with human liver proteins previously assigned to different functional categories. Among those involved in nucleic acid metabolism, hnRNPC was found to interact in vitro and in vivo in human liver cells. Similar to other RNA viruses, it seems plausible that hnRNPC may also be involved in HDV replication. However, further investigation is mandatory to clarify this question.publishe

The Impact of Lifestyle on Prostate Cancer: A Road to the Discovery of New Biomarkers

Prostate cancer (PCa) is one of the most common cancers among men, and its incidence has been rising through the years. Several risk factors have been associated with this disease and unhealthy lifestyles and inflammation were appointed as major contributors for PCa development, progression, and severity. Despite the advantages associated with the currently used diagnostic tools [prostate-specific antigen(PSA) serum levels and digital rectal examination (DRE)], the development of effective approaches for PCa diagnosis is still necessary. Finding lifestyle-associated proteins that may predict the development of PCa seems to be a promising strategy to improve PCa diagnosis. In this context, several biomarkers have been identified, including circulating biomarkers (CRP, insulin, C-peptide, TNFα-R2, adiponectin, IL-6, total PSA, free PSA, and p2PSA), urine biomarkers (PCA3, guanidine, phenylacetylglycine, and glycine), proteins expressed in exosomes (afamin, vitamin D-binding protein, and filamin A), and miRNAs expressed in prostate tissue (miRNA-21, miRNA-101, and miRNA-182). In conclusion, exploring the impact of lifestyle and inflammation on PCa development and progression may open doors to the identification of new biomarkers. The discovery of new PCa diagnostic biomarkers should contribute to reduce overdiagnosis and overtreatment