“Soy esposa de dios”. Una etnografia sobre la construcció de la identitat femenina en els convents de vida contemplativa

Treballs Finals de Grau Antropologia Social i Cultural, Facultat de Geografia i Història, Universitat de Barcelona, Curs: 2020-2021, Tutor: Mònica Martinez MauriLes adoratrius perpètues són una comunitat de dones cristianes de vida contemplativa. Es tracta de monges de clausura que renuncien a la seva llibertat individual i lliure circulació per la ciutat com a sacrifici personal per poder adorar a Déu constantment. Des d’una perspectiva etnogràfica, aquest treball de final de grau analitza la construcció de la dona religiosa a partir del treball de camp realitzat a un Convent de Vida Contemplativa a la ciutat de Barcelona. Per tal d’aproximar-me a aquesta realitat, he participat en activitats que realitzen de forma comunitària, i he observat les cures que practiquen quotidianament. Gràcies a aquesta immersió he arribat a la conclusió que les activitats al voltant de les cures són les que encara avui marquen la construcció de la identitat de les adoratrius

Apprehensions and Possible Misunderstandings

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Aplicació de tècniques proteòmiques de quantificació i validació en recerca biomèdica

Les tècniques proteòmiques basades en l'espectrometria de masses (MS) permeten la caracterització de la complexa i dinàmica natura del proteoma. Aquestes eines han contribuit a entendre millor certes funcions biològiques i respostes cel·lulars i a obtenir biomarcadors específics per a l'estudi d'algunes malalties. En aquest treball, es van utilitzar tècniques proteòmiques de quantificació i validació, basades en l'espectrometria de masses (MS), en diferents estudis de recerca biomèdica. En el primer estudi es va comparar el perfil de proteïnes del fluid vitri de pacients diabètics amb retinopatia diabètica proliferativa (PDR) amb el de pacients no diabètics amb forat macular idiopàtic (MH). L'anàlisi proteòmica comparativa es va fer amb el sistema d'electroforesi diferencial en gel bidimensional basat en el marcatge fluorescent (2D-DIGE). Per MS es van identificar 11 proteïnes diferencials entre els pacients amb PDR i els individus no diabètics. Cinc de les proteïnes diferencials és van validar per western blot en fluids vitris i per RT-PCR en retines. L'anàlisi proteòmica 2D-DIGE va servir per a identificar potencials candidats involucrats en la patogènesis de la PDR. En el segon estudi es va estudiar l'efecte del gen REgulador AutoImmune (AIRE) mitjançant la comparació dels proteomes de cèl·lules epitelials transfectades o no amb AIRE. L'anàlisi comparativa es va realitzar combinant dues tècniques de proteòmica quantitativa: la 2D-DIGE i l'etiquetatge isotòpic codificat de proteïna (ICPL) en combinació amb cromatografia líquida acoblada a espectrometria de masses (LC-MS). Els resultats van mostrar un nivell incrementat de varies xaperones en les cèl·lules que expressaven AIRE, mentre que diferents proteïnes de interacció del citoesquelet es van trobar disminuïdes. A més, algunes proteïnes relacionades amb apoptosi tenien abundàncies diferencials entre unes cèl·lules i les altres. Aquests resultats es van confirmar per western blot i citometria de flux. Finalment, assajos d'apoptosi amb annexin V i etopòsid van demostrar que les cèl·lules positives en AIRE patien més apoptosi espontània i eren menys resistents a la inducció d'apoptosi. Els resultats obtinguts van corroborar el paper d'AIRE com a inductor d'apoptosi. En el tercer estudi, amb l'objectiu de identificar possibles proteïnes biomarcadores de l'activitat TGFβ en gliomes es van analitzar les proteïnes secretades en els cultius cel·lulars primaris derivats de tumors (PCTCs), tractats o no amb TGFβ, mitjançant experiments quantitatius de LC-MS sense marcatge i ICPL. Es van identificar varies proteïnes candidates per a les que es van desenvolupar mètodes d'anàlisi de seguiment d'una reacció seleccionada (SRM) per a poder validar-les en mostres de líquid cefaloraquidi (CSF) i plasma de pacients amb glioma. Per l'anàlisi dels CSFs els resultats van mostrar una clara correlació entre les proteïnes candidates i els nivells de TGFβ en aquestes mostres. Igualment, al analitzar les proteïnes candidates per SRM en mostres de plasma de pacients amb glioma en fases alternades de tractament amb un inhibidor de TGFβ es va observar que els nivells de les proteïnes d'interès eren modulades pel tractament. Aquestes proteïnes conformarien una firma proteica que podria ser útil per al diagnòstic i el seguiment del tractament dels pacients. En conjunt, els resultats obtinguts en cada un dels tres estudis demostren la utilitat de l'anàlisi proteòmica en diferents aspectes de la investigació biomèdicaProteomic techniques based on mass spectrometry (MS) allow the characterization of the complex and dynamic nature of the proteome. These tools have contributed to better understand certain biological functions and cellular responses and to obtain specific biomarkers useful for the management of some diseases. In this work, quantitative and validation proteomic techniques, based on mass spectrometry, were applied to different biomedical research projects. In the first study, the protein profile of the vitreous fluid of diabetic patients with proliferative diabetic retinopathy (PDR) and non diabetic patients with macular hole (MH) was compared. Comparative proteomic analysis was performed using differential in gel electrophoresis based on fluorescent labeling (2D_DIGE). Eleven differential proteins between PDR and non diabetic patients were identified by MS. Five differential proteins were validated by western blot analysis of vitreous fluid and by RT-PCR of retinas. 2D-DIGE proteomic analysis allowed the identification of potential candidates involved in the pathogenesis of PDR. In the second study, the effect of AutoImmune Regulator (AIRE) gen was studied by comparison of the proteomic profile of epithelial cells transfected or not with AIRE. The comparative analysis was done using to proteomic techniques: 2D-DIGE and Isotopic Coded Protein Labeling (ICPL) in combination with liquid chromatography coupled to mass spectrometry (LC-MS). Results showed an increased level of some chaperons in the cells expressing AIRE, while some cytoskeleton interacting proteins were decreased. Moreover, proteins related wit apoptosis had differential abundances between samples. These results were confirmed by western blot and flow cytometry. Finally, apoptosis assays with annexin V and etoposide demonstrated that AIRE-positive cells suffer more spontaneous apoptosis and are less resistant to apoptosis induction. These results confirm the role of AIRE as an inducer of apoptosis. In the third study, the main objective was the identification of biomarker proteins of TGFβ activity in gliomas. Secreted proteins from primary cultures of tumor cells (PCTCs), treated or not with TGFβ, were analyzed by label free and ICPL LC-MS quantitative experiments. Some candidate proteins were identified. Selected reaction monitoring (SRM) methods were designed to analyze these candidate proteins in cerebrospinal fluid (CSF) and plasma of glioma patients. CSF analysis showed a clear relationship between the protein levels and the TGFβ concentration. When the SRM methods were applied to the plasma of glioma patients under alternate periods of treatment with a TGFβ inhibitor, the levels of proteins of interest were observed to be modulated by the treatment. These proteins can thus constitute a protein signature useful for diagnostic and treatment monitoring. Overall, the results obtained in each of the projects show the usefulness of the proteomic analysis in different aspects of de biomedical researc

Sweet and Sour Ehrlichia: Glycoproteomics and Phosphoproteomics Reveal New Players in Ehrlichia ruminantium Physiology and Pathogenesis

Ehrlichia ruminantium; N-glycoproteins; O-GlcNAcylated proteinsEhrlichia ruminantium; N-glicoproteïnes; Proteïnes O-GlcNAciladesEhrlichia ruminantium; N-glicoproteínas; Proteínas O-GlcNAciladasUnraveling which proteins and post-translational modifications (PTMs) affect bacterial pathogenesis and physiology in diverse environments is a tough challenge. Herein, we used mass spectrometry-based assays to study protein phosphorylation and glycosylation in Ehrlichia ruminantium Gardel virulent (ERGvir) and attenuated (ERGatt) variants and, how they can modulate Ehrlichia biological processes. The characterization of the S/T/Y phosphoproteome revealed that both strains share the same set of phosphoproteins (n = 58), 36% being overexpressed in ERGvir. The percentage of tyrosine phosphorylation is high (23%) and 66% of the identified peptides are multi-phosphorylated. Glycoproteomics revealed a high percentage of glycoproteins (67% in ERGvir) with a subset of glycoproteins being specific to ERGvir (n = 64/371) and ERGatt (n = 36/343). These glycoproteins are involved in key biological processes such as protein, amino-acid and purine biosynthesis, translation, virulence, DNA repair, and replication. Label-free quantitative analysis revealed over-expression in 31 proteins in ERGvir and 8 in ERGatt. While further PNGase digestion confidently localized 2 and 5 N-glycoproteins in ERGvir and ERGatt, respectively, western blotting suggests that many glycoproteins are O-GlcNAcylated. Twenty-three proteins were detected in both the phospho- and glycoproteome, for the two variants. This work represents the first comprehensive assessment of PTMs on Ehrlichia biology, rising interesting questions regarding ER-host interactions. Phosphoproteome characterization demonstrates an increased versatility of ER phosphoproteins to participate in different mechanisms. The high number of glycoproteins and the lack of glycosyltransferases-coding genes highlight ER dependence on the host and/or vector cellular machinery for its own protein glycosylation. Moreover, these glycoproteins could be crucial to interact and respond to changes in ER environment. PTMs crosstalk between of O-GlcNAcylation and phosphorylation could be used as a major cellular signaling mechanism in ER. As little is known about the Ehrlichia proteins/proteome and its signaling biology, the results presented herein provide a useful resource for further hypothesis-driven exploration of Ehrlichia protein regulation by phosphorylation and glycosylation events. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD012589

Cisplatin-induced peripheral neuropathy is associated with neuronal senescence-like response

Altres ajuts: Departament de Salut, programa CERCA SLT008/18/00028Cisplatin-induced peripheral neuropathy (CIPN) is a frequent serious dose-dependent adverse event that can determine dosage limitations for cancer treatment. CIPN severity correlates with the amount of platinum detected in sensory neurons of the dorsal root ganglia (DRG). However, the exact pathophysiology of CIPN is poorly understood, so the chance of developing neuroprotective treatment is reduced. The aim of this study was to determine the exact mechanisms involved in CIPN development. Methods: By single-cell RNA-sequencing (scRNAseq), we have studied the transcriptomic profile of DRG sensory neurons from a well-characterized neurophysiological mouse model of CIPN. Results :Gene Ontology analysis of the scRNAseq data indicated that cisplatin treatment induces the upregulation of biological pathways related to DNA damage response (DDR) in the DRG neuronal population. Moreover, DRG neurons also upregulated the Cdkn1a gene, confirmed later by the measurement of its protein product p21. While apoptosis activation pathways were not observed in DRG sensory neurons of cisplatin-treated mice, these neurons did express several senescence hallmarks, including senescence-associated β-galactosidase, phospho-H2AX, and nuclear factor kappa B (Nfkb)-p65 proteins. Conclusions:In this study, we determined that after cisplatin-induced DNA damage, p21 appears as the most relevant downstream factor of the DDR in DRG sensory neurons in vivo, which survive in a nonfunctional senescence-like state

Inhibition of the neuronal NFκB pathway attenuates bortezomib-induced neuropathy in a mouse model

© 2016 Elsevier B.V. Bortezomib is a proteasome inhibitor with a remarkable antitumor activity, used in the clinic as first line treatment for multiple myeloma. One hallmark of bortezomib mechanism of action in neoplastic cells is the inhibition of nuclear factor kappa B (NFκB), a transcription factor involved in cell survival and proliferation. Bortezomib-induced peripheral neuropathy is a dose-limiting toxicity that often requires adjustment of treatment and affects patient's prognosis and quality of life. Since disruption of NFκB pathway can also affect neuronal survival, we assessed the role of NFκB in bortezomib-induced neuropathy by using a transgenic mouse that selectively provides blockage of the NFκB pathway in neurons. Interestingly, we observed that animals with impaired NFκB activation developed significantly less severe neuropathy than wild type animals, with particular preservation of large myelinated fibers, thus suggesting that neuronal NFκB activation plays a positive role in bortezomib induced neuropathy and that bortezomib treatment might induce neuropathy by inhibiting NFκΒ in non-neuronal cell types or by targeting other signaling pathways. Therefore, inhibition of NFκB might be a promising strategy for the cotreatment of cancer and neuropathy

Context-dependent roles of cellular senescence in normal, aged, and disease states.

Cellular senescence is a state of irreversible cell cycle arrest that often emerges after tissue damage and in age-related diseases. Through the production of a multicomponent secretory phenotype (SASP), senescent cells can impact the regeneration and function of tissues. However, the effects of senescent cells and their SASP are very heterogeneous and depend on the tissue environment and type as well as the duration of injury, the degree of persistence of senescent cells and the organism's age. While the transient presence of senescent cells is widely believed to be beneficial, recent data suggest that it is detrimental for tissue regeneration after acute damage. Furthermore, although senescent cell persistence is typically associated with the progression of age-related chronic degenerative diseases, it now appears to be also necessary for correct tissue function in the elderly. Here, we discuss what is currently known about the roles of senescent cells and their SASP in tissue regeneration in ageing and age-related diseases, highlighting their (negative and/or positive) contributions. We provide insight for future research, including the possibility of senolytic-based therapies and cellular reprogramming, with aims ranging from enhancing tissue repair to extending a healthy lifespan.Work in the authors’ laboratory is supported by MINECO-Spain (RTI2018-096068), H2020 European Research Council-2016-AdG-741966, LaCaixaHEALTH-HR17-00040, MWRF, French Muscular Dystrophy Association, Muscular Dystrophy Association, Fundacio LaMarató TV3 (80/19-202021 and 137/ 38-202033) and UPGRADE-H2020-825825; and María-de-Maeztu-Program for Units of Excellence to UPF (MDM-2014-0370), Severo Ochoa-Program for Centers of Excellence to CNIC (SEV-2015-0505).S

Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis

PhosphoproteomeFosfoproteomaFosfoproteomaGlobal analysis of protein phosphorylation by mass spectrometry proteomic techniques has emerged in the last decades as a powerful tool in biological and biomedical research. However, there are several factors that make the global study of the phosphoproteome more challenging than measuring non-modified proteins. The low stoichiometry of the phosphorylated species and the need to retrieve residue specific information require particular attention on sample preparation, data acquisition and processing to ensure reproducibility, qualitative and quantitative robustness and ample phosphoproteome coverage in phosphoproteomic workflows. Aiming to investigate the effect of different variables in the performance of proteome wide phosphoprotein analysis protocols, ProteoRed-ISCIII and EuPA launched the Proteomics Multicentric Experiment 11 (PME11). A reference sample consisting of a yeast protein extract spiked in with different amounts of a phosphomix standard (Sigma/Merck) was distributed to 31 laboratories around the globe. Thirty-six datasets from 23 laboratories were analyzed. Our results indicate the suitability of the PME11 reference sample to benchmark and optimize phosphoproteomics strategies, weighing the influence of different factors, as well as to rank intra and inter laboratory performance.ProteoRed, PRB3 is supported by grant PT17/0019/0001, of the PE I+D+i 2013-2016, funded by ISCIII and ERDF

DIAGNOSTIC ACCURACY OF SHOCK INDEX AS SCREENING TOOL FOR PRIMARY POSTPARTUM HAEMORRHAGE AFTER CAESAREAN SECTION AMONG WOMEN AT KAWEMPE NATIONAL REFERRAL HOSPITAL -A CROSS-SECTIONAL STUDY.

Background:  Postpartum haemorrhage is the leading cause of maternal mortality globally and in sub-Saharan Africa. The shock index( ratio of the pulse to systolic pressure) is a quicker and more objective assessment tool for assessing the risk of deterioration as may occur in PPH, before apparent clinical deterioration. The diagnostic accuracy of the shock index in a Ugandan setting is not known. The objective of the study was to determine the sensitivity and specificity, positive and negative predictive values of shock index in the diagnosis of primary postpartum haemorrhage after caesarean section among women delivering at Kawempe National Referral Hospital. Methods:   A cross-sectional study design was used. The study was conducted in Kawempe National Referral Hospital among women who were delivered by a caesarean section between 1st January and 31st May 2021. A sample size of 594 was determined using formulae by Buderer. A consecutive sampling technique was used. The research assistants approached 650 participants; while 591 participants were fit for analysis. All participants were subjected to the determination of shock index at different intervals and the change in haematocrit as the gold standard for post-partum haemorrhage.   Results:  The sensitivity and specificity of the shock index at 2 hours were 40.0 and 82.8 percent respectively at a threshold of 0.8. The sensitivity and specificity were 70.0 and 54.6 percent after 24 hours when the shock index threshold of 0.7 was used. The PPV and NPV were 3.8 and 98.3 percent at 2 hours respectively while 2.6 and 99.1 percent at 24 hours. Conclusion:  The shock index is a poor screening tool for diagnosing primary postpartum haemorrhage after caesarean section. Recommendations : Shock index may not be included in routine care of post-operative mothers for early recognition of those at risk of primary postpartum haemorrhage