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    Bases moleculares de la interacción de la E3 ubiquitina ligasa TRIM7 con glucogenina-1 y su potencial relevancia en el contexto de la glucogenosis tipo XV

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    Tesis (Doctor en Ciencias Qu√≠micas) - - Universidad Nacional de C√≥rdoba. Facultad de Ciencias Qu√≠micas, 2021Resumen: La ubiquitinaci√≥n es un tipo de modificaci√≥n postraduccional caracter√≠stico de organismos eucariotas, que requiere de la acci√≥n conjunta de las enzimas E1 activadora, E2 conjugadora y E3 ligasa para mediar la transferencia de ubiquitina a una prote√≠na sustrato. TRIM7 es una E3 ligasa que recientemente ha adquirido inter√©s por su rol en diversas patolog√≠as, y pertenece a la familia de prote√≠nas TRIM, caracterizadas por un motivo conservado tripartito (TRIM) N-terminal y una regi√≥n C-terminal variable que puede mediar el reconocimiento de sustratos. Inicialmente, fue identificada como una prote√≠na que interacciona a trav√©s de su dominio C-terminal B30.2 (TRIM7B30.2) con glucogenina-1 (GN1). Dicha prote√≠na es responsable de la iniciaci√≥n de la bios√≠ntesis del gluc√≥geno, por lo que las mutaciones que afectan al gen que la codifica se traducen en un trastorno extremadamente raro denominado glucogenosis tipo XV. El presente trabajo se centr√≥ en caracterizar a TRIM7B30.2 por cristalograf√≠a de rayos X, y describir detalles sobre las regiones relevantes para la interacci√≥n con GN1. La estructura de TRIM7B30.2 revel√≥ un plegamiento de tipo s√°ndwich-ő≤ formado por dos l√°minas ő≤ antiparalelas caracter√≠stico de este tipo de dominios, adem√°s de presentar una cavidad con carga positiva en la cara c√≥ncava de dicho plegamiento. Adicionalmente, se logr√≥ identificar a residuos clave que delimitan la zona involucrada en la interacci√≥n con GN1, adem√°s de obtener indicios de que el extremo C-terminal de esta √ļltima es necesario para su reconocimiento por TRIM7B30.2 y la prote√≠na TRIM7 entera. Otra parte de este estudio se dedic√≥ a conocer el impacto de la mutaci√≥n patol√≥gica Ala16Pro sobre GN1 de conejo, que presenta una alta homolog√≠a con la prote√≠na humana, mediante el uso de un amplio conjunto de t√©cnicas bioqu√≠micas y biof√≠sicas. El an√°lisis revel√≥ un cambio estructural significativo que ocasion√≥ una disminuci√≥n de la afinidad por su sustrato y, en consecuencia, de la actividad de la prote√≠na; adem√°s de una marcada p√©rdida de estabilidad. Por √ļltimo, se busc√≥ conocer si GN1 y sus mutantes patol√≥gicas Ala16Pro y Thr83Met, son sustrato de su actividad E3 ligasa; y si esto podr√≠a tener un rol relevante en la glucogenosis tipo XV. Los resultados de la reacci√≥n in vitro mostraron que la transferencia de ubiquitina no ocurre bajo las condiciones empleadas, sin embargo, se encontr√≥ que TRIM7 interacciona con la mutante patol√≥gica GN1 Ala16Pro pese al cambio estructural de la prote√≠na, lo que podr√≠a implicar a TRIM7 en la v√≠a de degradaci√≥n de esta mutante, y por ello el estudio ser√° continuado en cultivos celulares.Abstract : Ubiquitination is a common post translational modification in eukaryotes, which involves an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ligase enzyme; in a process that mediates the transfer of ubiquitin to a substrate protein. TRIM7 is an E3 ligase member of the TRIM protein family, characterized by an N-terminal tripartite motif (TRIM) and a variable C-terminal region that can mediate substrate recognition, that has been recently linked to diverse pathological processes. TRIM7 was initially identified as a glycogenin-1 (GN1) interacting protein, and this interaction involves the C-terminal B30.2 domain of TRIM7 (TRIM7B30.2). GN1 is the protein that initiates glycogen biosynthesis, and mutations in the gene that encodes for this enzyme are involved in the onset of an extremely rare disease known as type XV glycogen storage disease. This work was mainly focused on the characterization of TRIM7B30.2, through the use of X-ray crystallography, and the details of the region involved in the interaction with GN1. The structure of TRIM7B30.2 revealed the characteristic B30.2 domain fold consisting of two ő≤-sheets arranged as a distorted ő≤-sandwich, and a positively charged cavity in the hypervariable region of this domain. Besides, key residues that define the zone of interaction with GN1 were identified, and evidence for the requirement of the latter‚Äôs C-terminal region in the binding was found. This study was also focused on the effect of the pathogenic Ala16Pro mutation on rabbit GN1, which displays a high sequence homology with the human protein; and to this end, a wide range of biophysical and biochemical techniques were applied. The analysis revealed a significant structural change which resulted in a decrease in substrate affinity and protein activity, besides a considerable loss of stability. Finally, the thesis was oriented towards analyzing the potential E3 ligase activity of TRIM7 on GN1 and the pathological mutants Ala16Pro and Thr83Met, and its possible functional role on type XV glycogen storage disease. The results have shown that in vitro, TRIM7 does not transfer ubiquitin to GN1 or the mutants in this experimental setup. However, it was found that despite the structural change caused by the mutation, TRIM7 still interacts with the Ala16Pro mutant of human GN1, which might implicate the intervention of TRIM7 in the degradation pathway of this mutant, and for this reason, this study will be continued in cell cultures.2023-08-30Fil: Mu√Īoz Sosa, Christian Javier. Universidad Nacional de C√≥rdoba. Facultad de Ciencias Qu√≠micas; Argentina.Fil: Carrizo Garcia, Mar√≠a Elena. Universidad Nacional de C√≥rdoba. Facultad de Ciencias Qu√≠micas. Departamento de Qu√≠mica Biol√≥gica; Argentina.Fil: Carrizo Garcia, Mar√≠a Elena. Consejo Nacional de Investigaciones Cient√≠ficas y T√©cnicas. Centro de Investigaciones en Qu√≠mica Biol√≥gica de C√≥rdoba; Argentina.Fil: Genti de Raimondi, Susana Del Valle. Universidad Nacional de C√≥rdoba. Facultad de Ciencias Qu√≠micas. Departamento de Bioqu√≠mica Cl√≠nica; Argentina.Fil: Genti de Raimondi, Susana del Valle. Consejo Nacional de Investigaciones Cient√≠ficas y T√©cnicas. Centro de Investigaciones en Bioqu√≠mica Cl√≠nica e Inmunolog√≠a; Argentina.Fil: Carbonio, Raul Ernesto. Universidad Nacional de C√≥rdoba. Facultad de Ciencias Qu√≠micas. Departamento de Fisicoqu√≠mica; Argentina.Fil: Carbonio, Raul Ernesto. Consejo Nacional de Investigaciones Cient√≠ficas y T√©cnicas. Instituto de Investigaciones en Fisicoqu√≠mica de C√≥rdoba; Argentina.Fil: Romero, Jorge Miguel. Universidad Nacional de C√≥rdoba. Facultad de Ciencias Qu√≠micas; Argentina.Fil: Erm√°cora, Mario Roberto. Consejo Nacional de Investigaciones Cient√≠ficas y T√©cnicas. Instituto Multidisciplinario de Biolog√≠a Celular; Argentina

    What is the importance of sperm subpopulations?

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    .The study of sperm subpopulations spans three decades. The origin, meaning, and practical significance, however, are less clear. Current technology for assessing sperm morphology (CASA-Morph) and motility (CASA-Mot) has enabled the accurate evaluation of these features, and there are many options for data classification. Subpopulations could occur as a result of the stage of development of each spermatozoon in the subpopulation. Spermatogenesis might contribute to the production of these subpopulations. Insights from evolutionary biology and recent molecular research are indicative of the diversity among male gametes that could occur from unequal sharing of transcripts and other elements through cytoplasmic bridges between spermatids. Sperm cohorts exiting the gonads would contain different RNA and protein contents, affecting the spermatozoon physiology and associations with the surrounding environmental milieu. Subsequently, these differences could affect how spermatozoa interact with the environmental milieu (maturation, mixing with seminal plasma, and interacting with the environmental milieu, or female genital tract and female gamete). The emergence of sperm subpopulations as an outcome of evolution, related to the reproductive strategies of the species, genital tract structures, and copulatory and fertilization processes. This kind of approach in determining the importance of sperm subpopulations in fertilization capacity should have a practical impact for conducting reproductive technologies, inspiring and enabling new ways for the more efficient use of spermatozoa in the medical, animal breeding, and conservation fields. This manuscript is a contribution to the Special Issue in memory of Dr. Duane GarnerS

    A new index of resilience applicable to external pulse-disturbances that considers the recovery of communities in the short term

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    .Resilience is a key concept in the study of the recovery of ecosystems affected by disturbances. Currently, there are numerous indices to measure resilience, but many of them do not show the accuracy of the resilience value or the behaviour of ecological parameters in the face of disturbances. New approaches and technologies enable large amounts of information to be obtained, facilitating the proposal of new resilience indices that work consistently and intuitively for a wide variety of ecological response variables under different scenarios after pulse-disturbances. In this study, we propose and verify a new resilience index, comparing its performance with others previously published. We validated the performance of the new index using real data based on field measurements of changes in soil bacterial OTUs diversity and abundance after a wildfire. The new resilience index provided an automatic and robust functional classification of the behaviour of ecosystems after disturbances, supported by a bootstrap analysis. We identified 5 scenarios of ecosystem resilience performance according to their behaviour after a pulse-disturbance: resilient, non-resilient, recovering, rebound, and continuing.S

    Bioinformatic characterization of a triacylglycerol lipase produced by Aspergillus flavus isolated from the decaying seed of Cucumeropsis mannii

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    Lipases are enzymes of industrial importance responsible for the hydrolysis of ester bonds of triglycerides. A lipolytic fungus was isolated and subsequently identified based on the ITS sequence analysis as putative Aspergillus flavus with accession number LC424503. The gene coding for extracellular triacylglycerol lipase was isolated from Aspergillus flavus species, sequenced, and characterised using bioinformatics tools. An open reading frame of 420 amino acid sequence was obtained and designated as Aspergillus flavus lipase (AFL) sequence. Alignment of the amino acid sequence with other lipases revealed the presence GHSLG sequence which is the lipase consensus sequence Gly-X1-Ser-X2-Gly indicating that it a classical lipase. A catalytic active site lid domain composed of TYITDTIIDLS amino acids sequence was also revealed. This lid protects the active site, control the catalytic activity and substrate selectivity in lipases. The 3-Dimensional structural model shared 34.08% sequence identity with a lipase from Yarrowia lipolytica covering 272 amino acid residues of the template model. A search of the lipase engineering database using AFL sequence revealed that it belongs to the class GX-lipase, superfamily abH23 and homologous family abH23.02, molecular weight and isoelectric point values of 46.95‚ÄČKDa and 5.7, respectively. N-glycosylation sites were predicted at residues 164, 236 and 333, with potentials of 0.7250, 0.7037 and 0.7048, respectively. O-glycosylation sites were predicted at residues 355, 358, 360 and 366. A signal sequence of 37 amino acids was revealed at the N-terminal of the polypeptide. This is a short peptide sequence that marks a protein for transport across the cell membrane and indicates that AFL is an extracellular lipase. The findings on the structural and molecular properties of Aspergillus flavus lipase in this work will be crucial in future studies aiming at engineering the enzyme for biotechnology applications

    Uso de las histonas circulantes y sus modificaciones post-traduccionales como biomarcadores en sepsis y shock séptico

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    La sepsis es una afecci√≥n potencialmente mortal causada por una respuesta anormal del hu√©sped a una infecci√≥n, produciendo respuestas fisiol√≥gicas alteradas que da√Īan los propios tejidos del paciente y pueden provocar disfunci√≥n org√°nica e incluso la muerte. Asimismo, algunos pacientes s√©pticos progresan a shock s√©ptico, caracterizado por alteraciones circulatorias, celulares y metab√≥licas sustanciales que aumentan el riesgo de mortalidad. A pesar de que la sepsis se caracteriza por un mal funcionamiento del sistema inmunol√≥gico, lo que a su vez conduce a una respuesta inmune alterada e inmunosupresi√≥n, la alta complejidad de la fisiopatolog√≠a de la sepsis requiere una mayor investigaci√≥n para comprender las respuestas inmunes que ocurren durante la sepsis. Asimismo, las histonas extracelulares circulantes han ganado relevancia como mediadores citot√≥xicos en la sepsis, ya que act√ļan como patrones moleculares asociados a da√Īo, que inducen estr√©s oxidativo y activan el inflamasoma NLRP3. Estos mecanismos median la activaci√≥n de la piroptosis, un mecanismo de muerte celular programada que produce inflamaci√≥n mediante la expresi√≥n de IL-18, IL-1ő≤ and IL-1őĪ. Sin embargo, a pesar de la evidencia de activaci√≥n del inflamasoma en las c√©lulas inmunes durante la sepsis, se desconoce si las histonas extracelulares son capaces de activar los inflamasomas endoteliales y sus consecuencias. En este trabajo destacamos el papel previamente desconocido de las histonas extracelulares, mediando la activaci√≥n del inflamasoma NLRP3 y la piroptosis en las c√©lulas endoteliales, contribuyendo a la disfunci√≥n endotelial y la desregulaci√≥n de la respuesta inmune mediada por el endotelio. Asimismo, tambi√©n demostramos c√≥mo la acetilaci√≥n de histonas disminuye la activaci√≥n de la piroptosis. Adem√°s, demostramos que la piroptosis se produce en pacientes con shock s√©ptico y los niveles de histonas circulantes se correlacionan con la expresi√≥n de citoquinas proinflamatorias y citoquinas piropt√≥ticas, la liberaci√≥n de factores de adhesi√≥n endotelial y la gravedad de la enfermedad. Proponemos la piroptosis mediada por histonas como un nuevo objetivo para desarrollar intervenciones cl√≠nicas. De manera similar, hemos analizado las respuestas inmunorelacionadas que ocurren durante las primeras etapas de la sepsis con el objetivo de proporcionar nuevos datos comparando las cantidades de citoquinas, inmunomoduladores y otros mediadores endoteliales en pacientes cr√≠ticamente enfermos no s√©pticos, s√©pticos y de shock s√©ptico. Nuestro enfoque ayudar√° a caracterizar r√°pidamente las respuestas inmunes alteradas en pacientes s√©pticos y de shock s√©ptico ingresados en la Unidad de Cuidados Intensivos. Finalmente analizamos el papel de la metilaci√≥n del ADN en el control del sistema inmune s√©ptico. Nuestros resultados demostraron el papel central de la metilaci√≥n del ADN modulando la respuesta molecular en los pacientes de shock s√©ptico y contribuyendo a la inmunosupresi√≥n, a trav√©s de la alteraci√≥n de los patrones de metilaci√≥n de los promotores de IL-10 y TREM-2.Sepsis is a life-threatening condition caused by an abnormal host response to an infection that produce altered physiological responses which damages own tissues of the patient and can result in organ dysfunction and in some cases death. Likewise, a subset of septic patients progresses to septic shock, characterized by substantial circulatory, cellular and metabolic abnormalities, which substantially increase the risk of mortality. Sepsis is characterized by a malfunction of the immune system and it can lead to an altered immune response and immunosuppression. Moreover, the high complexity of the pathophysiology of sepsis requires of further investigation to characterize the immune responses in sepsis and septic shock. Likewise, circulating extracellular histones have gained relevance as cytotoxic mediators in sepsis pathophysiology, since they act as damage-associated molecular patterns, which induce oxidative stress and activate NLRP3 inflammasome. Subsequently, inflammasome mediates pyroptosis activation, a programmed cell death mechanism that produces inflammation through the release of IL-18, IL-1ő≤ and IL-1őĪ. However, despite inflammasome activation may occur in immune cells during sepsis, it is unknown if this process also takes place in endothelial cells and particularly whether extracellular histones are capable of activating endothelial inflammasomes and their consequences. In this work we highlight a previously unknown role for extracellular histones, that mediates the activation of NLRP3 inflammasome and pyroptosis in endothelial cells by contributing to endothelial dysfunction and the dysregulation of the immune response mediated by endothelium. Likewise, we demonstrated how histone acetylation decreases pyroptosis activation. Furthermore, we show how pyroptosis occurs in septic shock patients and how circulating histone levels correlate with the expression of pro-inflammatory and pyroptotic cytokines, the release of endothelial adhesion factors and septic shock severity. We propose histone-mediated pyroptosis as a new target to develop clinical interventions. Similarly, we have analyzed the immune-related responses occurring during the early stages of sepsis with the aim of providing new data by comparing the amounts of cytokines, immune modulators and other endothelial mediators in critically-ill non-septic patients, septic and septic shock patients. Our approach will help to rapidly characterize the altered immune responses in septic and septic shock patients admitted in the Intensive Care Unit. Finally, we also analyzed the role of DNA methylation in the control of septic immune system. Our results demonstrated the central role of DNA methylation modulating the molecular response in septic shock patients and contributing to immunosuppression, through the alteration of DNA methylation patterns of IL-10 and TREM2 promoters

    The applied psychology of addictive orientations : studies in a 12-step treatment context.

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    The clinical data for the studies was collected at The PROMIS Recovery Centre, a Minnesota Model treatmentc entre for addictions,w hich encouragesth e membership and use of the 12 step Anonymous Fellowships, and is abstinence based. The area of addiction is contextualised in a review chapter which focuses on research relating to the phenomenon of cross addiction. A study examining the concept of "addictive orientations" in male and female addicts is described, which develops a study conductedb y StephensonM, aggi, Lefever, & Morojele (1995). This presents study found a four factor solution which appeared to be subdivisions of the previously found Hedonism and Nurturance factors. Self orientated nurturance (both food dimensions, shopping and caffeine), Other orientated nurturance (both compulsive helping dimensions and work), Sensation seeking hedonism (Drugs, prescription drugs, nicotine and marginally alcohol), and Power related hedonism (Both relationship dimensions, sex and gambling. This concept of "addictive orientations" is further explored in a non-clinical population, where again a four factor solution was found, very similar to that in the clinical population. This was thought to indicate that in terms of addictive orientation a pattern already exists in this non-clinical population and that consideration should be given to why this is the case. These orientations are examined in terms of gender differences. It is suggested that the differences between genders reflect power-related role relationships between the sexes. In order to further elaborate the significance and meaning behind these orientations, the next two chapters look at the contribution of personality variables and how addictive orientations relate to psychiatric symptomatology. Personality variables were differentially, and to a considerable extent predictably involved with the four factors for both males and females.Conscientiousness as positively associated with "Other orientated Nurturance" and negatively associated with "Sensation seeking hedonism" (particularly for men). Neuroticism had a particularly strong association with the "Self orientated Nurturance" factor in the female population. More than twice the symptomatology variance was explained by the factor scores for females than it was for males. The most important factorial predictors for psychiatric symptomatology were the "Power related hedonism" factor for males, and "Self oriented nurturance" for females. The results are discussed from theoretical and treatment perspectives

    Predicted impact of climate change on the distribution of the Critically Endangered golden mantella (Mantella aurantiaca) in Madagascar

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    The impact of climate change on Malagasy amphibians remains poorly understood. Equally, deforestation, fragmentation, and lack of connectivity between forest patches may leave vulnerable species isolated in habitat that no longer suits their environmental or biological requirements. We assess the predicted impact of climate change by 2085 on the potential distribution of a Critically Endangered frog species, the golden mantella (Mantella aurantiaca), that is confined to a small area of the central rainforest of Madagascar. We identify potential population distributions and climatically stable areas. Results suggest a potential south-eastwardly shift away from the current range and a decrease in suitable habitat from 2110 km2 under current climate to between 112 km2 ‚Äď 138 km2 by the year 2085 ‚Äď less than 7% of currently available suitable habitat. Results also indicate that the amount of golden mantella habitat falling within protected areas decreases by 86% over the same period. We recommend research to ascertain future viability and the feasibility of expanding protection to newly identified potential sites. This information can then be used in future conservation actions such as habitat restoration, translocations, re-introductions or the siting of further wildlife corridors or protected areas

    Food for thought! Inulin-type fructans: does the food matrix matter?

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    Food matrices can be described as the final composition of a food product which results from complex interactions between compounds found within different ingredients and the processing parameters used in production. These factors, not only impact on the final structure of a product, but also have the potential to alter both the structural integrity and bioavailability of potentially beneficial compounds present, for example, dietary fibres. As a result, there is growing curiosity amongst the scientific community on whether the food matrix may impact on the prebiotic efficacy of inulin-type fructans. Therefore, the purpose of this review is to explore previous food-based inulin-type fructan supplementation studies to determine whether the food matrix directly impacts on their prebiotic efficacy. Our working hypothesis is that other potentially prebiotic ingredients and components present within the food may alter inulin-type fructans prebiotic effect
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