Morphometric comparison by the ISAS® CASA-DNAf system of two techniques for the evaluation of DNA fragmentation in human spermatozoa

Artículo científicoDNA fragmentation has been shown to be one of the causes of male infertility, particularly related to repeated abortions, and different methods have been developed to analyze it. In the present study, two commercial kits based on the SCD technique (Halosperm ® and SDFA) were evaluated by the use of the DNA fragmentation module of the ISAS ® v1 CASA system. Seven semen samples from volunteers were analyzed. To compare the results between techniques, the Kruskal-Wallis test was used. Data were used for calculation of Principal Components (two PCs were obtained), and subsequent subpopulations were identified using the Halo, Halo/Core Ratio, and PC data. Results from both kits were significantly different (P < 0.001). In each case, four subpopulations were obtained, independently of the classification method used. The distribution of subpopulations differed depending on the kit used. From the PC data, a discriminant analysis matrix was obtained and a good a posteriori classification was obtained (97.1% for Halosperm and 96.6% for SDFA). The present results are the first approach on morphometric evaluation of DNA fragmentation from the SCD technique. This approach could be used for the future definition of a classification matrix surpassing the current subjective evaluation of this important sperm factor

Iron Regulatory Protein/Iron Responsive Element (IRP/IRE) system: associated diseases and new target mRNAs (PPP1R1B)

Iron is a biometal involved in many physiological processes essential for life. Regulation of both systemic and cellular iron homeostasis is crucial for health. The IRP/IRE post-transcriptional regulatory system is in charge to control iron uptake, utilization, storage and export at cellular level. There are two iron regulatory proteins (IRP1 and IRP2) which recognize and bind conserved mRNA motifs named iron responsive elements (IREs). These IREs are located in the mRNA untranslated regions (UTR) of genes involved in iron metabolism. IRP/IRE binding is produced under cellular iron depletion conditions, modifying the expression of several proteins related with cellular iron acquisition, mobilization and storage. Depending on the location of the IREs in the target mRNA, the binding of the IRPs regulates differentially its expression. When IREs are present in the 3' UTR their binding to the IRPs stabilize the mRNA, protecting it front degradation and increasing its translation. On the other hand, the translation is inhibited when IRPs bind IREs located in the 5’ UTR. IRP/IRE system missregulation leads to human diseases denoting the importance of this post-transcriptional gene regulation. As part of my thesis (objective 1) we describe new cases of patients with Hereditary Hyperferritinemia Cataract Syndrome (HHCS) with mutations in the FTL IRE (Paris1 c.-160A>G and Madrid/Philadelphia c.-167G>T mutations) and we did an exhaustive revision of all literature reported cases. A genome-wide study made by our group, in order to characterize all the mRNAs that interact with the IRPs, identified 263 mRNAs that are potential IRP-target genes (44 mRNAs bind both IRPs, 102 bind specifically IRP1 and 117 bind specifically IRP2). The objective 3 of my thesis is focused in the characterization of one of these novel IRP-target mRNAs: PPP1R1B (DARPP-32 protein). DARPP-32 is a dopaminergic neurotransmission integrator which its main function is to inhibit protein phosphatase 1 (PP1). We identified a functional 5’IRE in mouse Ppp1r1b mRNA that binds in vitro with IRP1. Further, DARPP-32 protein expression is downregulated in two human cell lines under iron deprivation by an iron chelator and upregulated with an iron source, in agreement with an IRP/IRE regulation by a 5’IRE. Finally, in my thesis objective 2, we also contributed to the characterization of a Slc11a2 IRE knock-in mouse model to dissect the Dmt1 3’ IRE function in iron homeostasis

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O Complexo Metamórfico Bonfim é um segmento de crosta continental situado a Oeste da Serra da moeda e a sul da Serra dos Três Irmãos, no Quadrilátero Ferrífero em Minas Gerais. A partir do mapeamento geológico realizado na porção setentrional deste complexo (escala 1:25.000), constatou-se a presença de uma grande variedade de rochas de natureza predominante metamórfica e subordinadamente ígnea. Em continuidade, a realização de estudos petrográficos (241 seções delgadas), geoquímicos em rocha total (67 análises envolvendo elementos maiores, menores, traços e terras raras) e geocronológicos (52 determinações radiométricas em minerais e rochas, pelos métodos U/Pb, Rb/Sr e K/Ar), permitiu agrupar os litotipos mapeados em oito unidades litoestratigráficas. Tais unidades são aqui informalmente designadas de Gnaisses Alberto Flores, Anfíbolitos Paraopeba, Gnaisses Souza Noschese, Tonalitos Samambaia, Anfibolitos Candeias, Granitos Brumadinho, Metadiabásios Conceição de Itaguá e Diabásios Santa Cruz. A evolução geológica deste complexo tem início no Arqueano Médio (3,2 Ga) e foi constatada através da herança isotópica U/PB nos zircões dos Gnaisses Alberto Flores, por meio de uma discórdia envolvendo um núcleo (com 2920 Ma) e o seu sobrecrescimento (com 2772 \'+ OU -\' 6 Ma). Entretanto, esta evolução está particularmente relacionada ao Arqueano Superior, quando foram gerados os Gnaisses Alberto Flores (com idade U/Pb mínima de 2920 Ma) e, principalmente, no decorrer de um grande evento tectôno-termal de fácies anfibolito, de 2,78 Ga atrás, aqui designado informalmente de Rio das Velhas. No decorrer deste evento, a crosta continental preexistente no âmbito do Complexo Metamórfico Bonfim Setentrional (e.g.Gnaisses Alberto Flores, Anfibolitos Paraopeba e Gnaisses Souza Noschese) foi invadida por um magmatismo cálcio alcalino (Tonalitos Samambais) e, provavelmente, por um outro magmatismo de características químicas compatíveis com magmas andesíticos e/ou tholeíticos ( Anfíbolitos Candeias). Nesta mesma época, teve lugar também um vulcanismo ácido, mas que é encontrado particularmente nos domínios do Supergrupo Rio das Velhas. Este cenário geológico é sugestivo de um ambiente tectônico semelhante às margens continentais ativas. Encerrando a evolução geológica do Arqueano Superior tem lugar um outro magmatismo félsico, que foi responsável pela geração dos Granitos Brumadinho há 2703 +24/-20 Ma atrás. Estas rochas truncam a foliação milonítica NS dos Gnaisses Alberto Flores e constituem um importante marco litoestratigráfico da evolução geológica do Complexo Metamórfico Bonfim Setentrional. No decorrer do Proterozóico este complexo foi retomado tectonicamente e invadido por dois novos magmatismos básicos. Esta retomada tectônica ocorreu sob condições de fácies xisto verde essencialmente. Por causa disto, os seus sistemas isotópicos K/Ar e/ou, principalmente, Rb/Sr estão acusando rejuvenescimentos parciais e ou incompletos. Assim sendo, as suas idades aparentes K/Ar e/ou errocrônicas Rb/Sr distribuem-se do Proterozóico Inferior ao Proterozóico Superior. Os dois magmatismos básicos, por sua vez, estão representados pelos Metadiabásios Conceição de Itaguá. Com idades aparentes K/Ar (em hornblenda primária) de 1,0 Ga, e pelos Diabásios Santa Cruz, de idade desconhecida mas correlacionáveis a outros magmatismos do Proterozóico Superior no âmbito do Quadrilátero Ferrífero. De acordo com estes resultados o Complexo Metamórfico Bonfim Setentrional constitue o limite oriental da Província Arqueana Divinópolis com o Cinturão Mineiro, de idade Transamazônica. Nos setores marginais desta província, a exemplo da região estudada, os sistemas isotópicos Rb/Sr e K/Ar foram rejuvenescidos a partir do Arqueano.The Bonfim Metamorphic Complex (BMC) constitutes a segment of continental crust which is limited eastwards and northwards by the Serra da Moeda and Serra dos Três Irmãos, respectively, in Quadrilátero Ferrífero (QF), state of Minas Gerais, Within the northern portion of the BMC, eight lithostratigraphic units (most of them metamorphosed ones) were identified through by means of geological mapping on 1:25,000 scale. All of the above units were also characterized on the basis of petrographical studies (241 thin sections), whole rock geochemistry (67 major, minor, trace and REE analyses), and U/Pb, Rb/Sr and K/Ar geochronology (52 mineral and whole rock data). These units were informally named: Alberto Flores Gneisses, Paraopeba Amphibolites, Souza Noschese Gneisses, Samambaia Tonalites, Candeias Amphibolites, Brumadinho Granites, Conceição de Itaguá metadiabases and Santa Cruz Diabases. Geological evolution of the northern BMC started at c. 3,28 Ga ago, as suggested from one inherited U/Pb zircon component age encountered in the Alberto Flores Gneiss, of trondhjemitic composition. However, the BMC is mostly related to Late Archean during which the above gneiss originated, at 2.92 Ga ago (evidenced from its U/Pb zircon core age). One overgrowth from this zircon yielded and age of 2,772 \'+ OU -\' 6 M.a. Based on the geological inferences, both the Paraopeba Amphibolites and Souza Noschese Gneiss also developed, during such a period of time (2,92 - 2,77 Ga). Further on, the continental crust was intruded by the calc-alkaline Samambaia Tonalites, as evidenced from U/Pb zircon and titanites ages of 2,780 +3/-2 Ma. Also related to this epoch is the 2,772 \'+ OU -\' 6 Ma acid volcanism which is found in the Rio das Velhas Supergroup, in QF. Contemporaneously, andesitic and/or tholeiitic magmatism took place (Candeias Amphibolites), as suggested by their geological association with Samambaia Tonalites. The overall described evolutionary setting is consistent with the occurrence a major 2,78 Ga tectono-thermal amphibolite facies event in the investigated area, here informally named Rio das Velhas. The above geological evolution of BMC together with the geochemical and radiometric data are consistent with a model of active continental margins, during the Late Archean time. This Archean evolution finished by instrusion of the Brumadinho Granites, at 2703 +24/-20 Ma ago (U/Pb zircon age). These granites are here considered as a key-marker for the Late Archean geological evolution of the area, because they truncate the NS shear foliation which is recorded in the Alberto Flores Gneisses. During the Proterozoic, the northern BMC was intruded by two distinct basic magmatism. The basic rocks are represented by Conceição de Itaguá Metadiabases ( K/Ar hornblende ages around 1,0 Ga), and the Santa Cruz Diabases. The latter (not dated) are here tentatively correlated with basic dikes of Late Proterozoic age (c. 0,6 Ga; U/PB age), in QF region. The investigated area was also reactivated, during the Proterozoic, under greenschist metamorphic conditions. Because of this metamorphic overprinting both the isotopic K/Ar and /or RB/Sr rock-systems of the northern BMC yielded uncomplete resenting. Consequently, the investigated rocks exhibited scatteres apparent K/Ar mineral ages and/or Rb/Sr ages, in the range 2,1 - 1,0 Ga. Finally, on paleogeographical basis, the geochronological scenario of the northern BMC may correspond to the eastern sector of the Divinópolis Archean Province in relation to the adjacent Transamazônico Mineiro Belt eastwards, at southern São Francisco Craton. As prior mentioned specially within this marginal sector of the Divinópolis province (i.e. northern BMC) the isotopic Rb/Sr rocks-systems and K/Ar minerals have been resetted due to low-grade metamorphism, since the Late Archean

Hereditary Hyperferritinemia Cataract Syndrome: Ferritin L Gene and Physiopathology behind the Disease—Report of New Cases

Hereditary hyperferritinemia-cataract syndrome (HHCS) is a rare disease characterized by high serum ferritin levels, congenital bilateral cataracts, and the absence of tissue iron overload. This disorder is produced by mutations in the iron responsive element (IRE) located in the 5′ untranslated regions (UTR) of the light ferritin (FTL) gene. A canonical IRE is a mRNA structure that interacts with the iron regulatory proteins (IRP1 and IRP2) to post-transcriptionally regulate the expression of proteins related to iron metabolism. Ferritin L and H are the proteins responsible for iron storage and intracellular distribution. Mutations in the FTL IRE abrogate the interaction of FTL mRNA with the IRPs, and de-repress the expression of FTL protein. Subsequently, there is an overproduction of ferritin that accumulates in serum (hyperferritinemia) and excess ferritin precipitates in the lens, producing cataracts. To illustrate this disease, we report two new families affected with hereditary hyperferritinemia-cataract syndrome with previous known mutations. In the diagnosis of congenital bilateral cataracts, HHCS should be taken into consideration and, therefore, it is important to test serum ferritin levels in patients with cataracts