Calcium oxalate modulation of tubular epithelial cell mitochondria: oxidative vulnerability due to restricted glutathione homeostasis.

Calcium oxalate (COM) crystals are the commonest component of kidney stones. These arise mainly in the distal tubules and collecting ducts. To gain further insight for the cellular damage in terms of oxidative stress caused by COM deposition, in vitro and in vivo model studies were performed. In vitro In renal distal tubule cells, COM and free oxalate treatment caused a 3- and 2-fold increase respectively in superoxide (O2*") formation, originating from mitochondria. This was measured by lucigenin chemiluminescence in digitonin permeabilised cells. However, hydroxyapatite produced a much lower but significant enhancement of 02*", whilst other micro-particles, uric acid crystals, brushite, zymosan, and latex beads had no effect. When EDTA was omitted during O2*" monitoring, COM induced mitochondrial 02*" was ablated indicating a requirement for the release of free oxalate. Mitochondrial oxalate uptake was studied by employing different oxalate transport inhibitors. Omitting phosphate from the media or using mersalyl both of which block dicarboxylate transport, caused a significant decrease in the 02*" formation evoked by COM treatments. Using the membrane potential sensitive-probe tetramethylrhodamine methyl ester (TMRM) together with confocal microscopy, evidence is presented that in cells where COM binding had occurred a marked change in the mitochondrial membrane potential (Aij/m) occurred. COM also modulated intracellular Ca2+ signalling as demonstrated using the Ca2- sensitive dye Fura-2 AM, and this was via a non-mitochondrial mechanism. In Vivo Using a rat model of crystalluria and renal stones initiated by treatment with ethylene glycol (EG) and 1, 25-dihydroxycholecalciferol (DHC), nephrolithiasis arose in kidneys and this was linked to oxidative stress. In the EG + DHC treated animals where crystalluria was evident, this oxidative insult was manifest by a decrease in total and mitochondrial glutathione concentration, as well as an increased activity of glucose-6-phosphate dehydrogenase. Severe kidney damage at the mitochondria level was a further observation, indicated by the diminished O2 consumption resulting in a lowered O2 production. In addition, histopathological analysis revealed increased renal tubular pathology characterised by obstruction, distension and interstitial inflammation. The above findings were not observed in hyperoxaluria (EG) or calciuria (DHC) and are therefore a direct effect of crystal formation in kidney distal tubules that have implications in kidney stone disease which are discussed

Microsphere-based binding assays for organic pollutants

In this thesis the “proof of principle” of a preliminary “prototype kit” for the screening of dioxin-like PCBs, PBDEs and PAHs in fish combined with two different simplified generic cleanup procedures, according to the fish fat content, is presented. Toxicants such as the PCBs, PBDEs and PAHs and POPs mixtures (such as the technical mixtures of PCBs (Aroclors)) can be detected at relative low levels (with IC50 values 55±15, 2±0.4 and 4±0.5 ppb for the PCB77, PBDE47 and BaP, respectively) in fish samples in a multiplex, simple and inexpensive manner compared to existing techniques. The 3-plex immunoassay can be performed in two different detection platforms: the traditional flow cytometer- and the new imaging-based bead analyzers. The latter system offers a cheaper analysis and it is an easier transportable platform than the flow cytometer.</p

Heat shock induces rapid resorption of primary cilia

Primary cilia are involved in important developmental and disease pathways, such as the regulation of neurogenesis and tumorigenesis. They function as sensory antennae and are essential in the regulation of key extracellular signalling systems. We have investigated the effects of cell stress on primary cilia. Exposure of mammalian cells in vitro, and zebrafish cells in vivo, to elevated temperature resulted in the rapid loss of cilia by resorption. In mammalian cells loss of cilia correlated with a reduction in hedgehog signalling. Heat-shock-dependent loss of cilia was decreased in cells where histone deacetylases (HDACs) were inhibited, suggesting resorption is mediated by the axoneme-localised tubulin deacetylase HDAC6. In thermotolerant cells the rate of ciliary resorption was reduced. This implies a role for molecular chaperones in the maintenance of primary cilia. The cytosolic chaperone Hsp90 localises to the ciliary axoneme and its inhibition resulted in cilia loss. In the cytoplasm of unstressed cells, Hsp90 is known to exist in a complex with HDAC6. Moreover, immediately after heat shock Hsp90 levels were reduced in the remaining cilia. We hypothesise that ciliary resorption serves to attenuate cilia-mediated signalling pathways in response to extracellular stress, and that this mechanism is regulated in part by HDAC6 and Hsp90

Adapted qPCR methodology to detect telomere oxidative damage (TOD) associated with telomere attrition (TA) in murine tissue

Numerous Studies suggest that oxidative stress (OS) is associated with telomere attrition (TA) but they rarely directly assay telomeric oxidative damage (TOD). TOD can alter shelterin binding, induce replication fork stalling and inhibit telomerase activity. We used a previously published method 1 to measure TOD and adapted it for use in murine tissue. Our model is a C57BL/6J mouse strain and we used qPCR and a formamidopyrimidine DNA glycosylase (FPG) enzyme-based qPCR method to directly determine TOD. FPG analysis compares reaction efficiencies before and after treatment. FPG is normally involved in base excision repairs pathways, removing oxidized Purines (notably 8-Oxo Guanine). A reduction in the efficiency of the qPCR indicates the presence of 8-oxo guanine in the telomeric tract ie TOD

Loss of Nnt Increases Expression of Oxidative Phosphorylation Complexes in C57BL/6J Hearts

Nicotinamide nucleotide transhydrogenase (NNT) is a proton pump in the inner mitochondrial membrane that generates reducing equivalents in the form of NAPDH, which can be used for anabolic pathways or to remove reactive oxygen species (ROS). A number of studies have linked NNT dysfunction to cardiomyopathies and increased risk of atherosclerosis; however, biallelic mutations in humans commonly cause a phenotype of adrenal insufficiency, with rare occurrences of cardiac dysfunction and testicular tumours. Here, we compare the transcriptomes of the hearts, adrenals and testes from three mouse models: the C57BL/6N, which expresses NNT; the C57BL/6J, which lacks NNT; and a third mouse, expressing the wild-type NNT sequence on the C57BL/6J background. We saw enrichment of oxidative phosphorylation genes in the C57BL/B6J in the heart and adrenal, possibly indicative of an evolved response in this substrain to loss of Nnt. However, differential gene expression was mainly driven by mouse background with some changes seen in all three tissues, perhaps reflecting underlying genetic differences between the C57BL/B6J and -6N substrains

A APLICABILIDADE E A EFICIÊNCIA DA MATRIZ SWOT PARA O GERENCIAMENTO DA QUALIDADE DO PROJETO ARQUITETÔNICO E URBANÍSTICO

Este estudo possui o objetivo que demonstrar a aplicabilidade e a eficiência da matriz SWOT/ FOFA, no campo da arquitetura e do urbanismo, para o gerenciamento da qualidade do projeto. Esta análise justifica-se pela possibilidade de utilizar o SWOT na avaliação pré-projeto (APP) e na avaliação pós-ocupação (APO). Através do método correlacional foram estudadas as relações existentes entre a matriz FOFA e os processos arquitetônicos, para verificar as congruências entre ambos. Pela revisão sistemática, buscou-se demonstrar como a análise SWOT contribui para o processo de projeto. Ao identificar as forças, oportunidades, fraquezas e ameaças, de um empreendimento, são gerados dados que permitem embasar, justificar e consolidar as soluções propostas. Assim, através dos exemplos analisados, nota-se que a matriz FOFA é uma ferramenta adequada para o planejamento estratégico de projetos. Isto, pois, permite estruturar um processo produtivo que corrobora para qualidade dos ambientes construídos e para a satisfação dos usuários

Mylk3 null C57BL/6N mice develop cardiomyopathy, whereas Nnt null C57BL/6J mice do not.

The C57BL/6J and C57BL/6N mice have well-documented phenotypic and genotypic differences, including the infamous nicotinamide nucleotide transhydrogenase (Nnt) null mutation in the C57BL/6J substrain, which has been linked to cardiovascular traits in mice and cardiomyopathy in humans. To assess whether Nnt loss alone causes a cardiovascular phenotype, we investigated the C57BL/6N, C57BL/6J mice and a C57BL/6J-BAC transgenic rescuing NNT expression, at 3, 12, and 18 mo. We identified a modest dilated cardiomyopathy in the C57BL/6N mice, absent in the two B6J substrains. Immunofluorescent staining of cardiomyocytes revealed eccentric hypertrophy in these mice, with defects in sarcomere organisation. RNAseq analysis identified differential expression of a number of cardiac remodelling genes commonly associated with cardiac disease segregating with the phenotype. Variant calling from RNAseq data identified a myosin light chain kinase 3 (Mylk3) mutation in C57BL/6N mice, which abolishes MYLK3 protein expression. These results indicate the C57BL/6J Nnt-null mice do not develop cardiomyopathy; however, we identified a null mutation in Mylk3 as a credible cause of the cardiomyopathy phenotype in the C57BL/6N

School architecture from the perspective of autism: a reflection on visual perception

Este estudo busca compreender como as particularidades do processamento sensorial, de pessoas com Transtorno do Espectro do Autismo (TEA), impactam a maneira como estes usuários extraem as informações visuais do ambiente construído e como tais características influenciam o projeto de arquitetura escolar. A relevância desta investigação baseia-se no grande número de crianças e adolescentes que são diagnosticados com TEA no Brasil e no mundo a cada ano. A partir do método correlacional, os temas chaves da pesquisa, que são oriundos de diferentes campos de conhecimentos como: arquitetura, educação e psicologia, foram relacionados a partir de revisões bibliográficas. A metodologia qualitativa, através da lógica argumentativa, possibilitou a apropriação de informações do âmbito do neurodesenvolvimento, a fim de construir respostas arquitetônicas para as questões observadas. Assim, a partir da compreensão do processamento sensorial atípico, dos autistas, foram estruturadas observações projetuais, que visam tornar os projetos arquitetônicos escolares, com foco no conforto visual dos educandos com TEA, sensível às necessidades destes usuários.This study seeks to understand how the particularities of sensory processing, of people with Autism Spectrum Disorder (ASD), impact the way these users extract visual information from the built environment and how these characteristics influence the school architecture project. The relevance of this investigation is based on the large number of children and adolescents who are diagnosed with ASD in Brazil and worldwide each year. From the correlational method, the key themes of the research, which come from different fields of knowledge such as: architecture, education and psychology, were related from bibliographic reviews. The qualitative methodology, through argumentative logic, enabled the appropriation of information from the scope of neurodevelopment, in order to build architectural answers to the observed questions. Thus, from the understanding of the atypical sensory processing of autistic people, design observations were structured, which aim to make school architectural projects, focusing on the visual comfort of students with ASD, sensitive to the needs of these users

Choice of gDNA isolation method has a significant impact on average murine Telomere Length estimates.

Telomere Length (TL) and integrity is significantly associated with age-related disease, multiple genetic and environmental factors. We observe mouse genomic DNA (gDNA) isolation methods to have a significant impact on average TL estimates. The canonical qPCR method does not measure TL directly but via the ratio of telomere repeats to a single copy gene (SCG) generating a T/S ratio. We use a monochromatic-multiplex-qPCR (mmqPCR) method which multiplexes the PCR and enables quantification of the target and the single copy gene within the same qPCR reaction. We demonstrate that TL measurements, from murine gDNA, isolated via Spin Columns (SC) and Magnetic Beads (MB), generate significantly smaller T/S ratios compared to gDNA isolated via traditional phenol/chloroform methods. The former methods may impede correct TL estimation by producing non representative fragment sets and reducing qPCR efficacy. This work highlights discrepancies in TL measurements due to different extraction techniques. We recommend the use of gDNA isolation methods that are shown to preserve DNA length and integrity, such as phenol/chloroform isolation. We propose that widely used high throughput DNA isolation methodologies can create spurious associations within a sample set, thus creating misleading data. We suggest that published TL associations should be revisited in the light of these data

Quality engineering applied to the architectural and urban process: a focus on SWOT analysis

Este estudo possui o objetivo que demonstrar a aplicabilidade e a eficiência da matriz SWOT/ FOFA, no campo da arquitetura e do urbanismo, para o gerenciamento da qualidade do projeto. Esta análise justifica-se pela possibilidade de utilizar o SWOT na avaliação pré-projeto (APP) e na avaliação pósocupação (APO). Através do método correlacional foram estudadas as relações existentes entre a matriz FOFA e os processos arquitetônicos, para verificar as congruências entre ambos. Pela revisão sistemática, buscou-se demonstrar como a análise SWOT contribui para o processo de projeto. Ao identificar as forças, oportunidades, fraquezas e ameaças, de um empreendimento, são gerados dados que permitem embasar, justificar e consolidar as soluções propostas. Assim, através dos exemplos analisados, nota-se que a matriz FOFA é uma ferramenta adequada para o planejamento estratégico de projetos. Isto, pois, permite estruturar um processo produtivo que corrobora para qualidade dos ambientes construídos e para a satisfação dos usuários.This study aims to demonstrate the applicability and efficiency of the SWOT/FOFA matrix, in the field of architecture and urbanism, for project quality management. This analysis is justified by the possibility of using the SWOT in the pre-project evaluation (APP) and in the post-occupancy evaluation (APO). Through the correlational method, the existing relationships between the SWOT matrix and the architectural processes were studied, in order to verify the congruence between them. Through the systematic review, we sought to demonstrate how the SWOT analysis contributes to the design process. By identifying the strengths, opportunities, weaknesses and threats of an enterprise, data are generated that allow to support, justify and consolidate the proposed solutions. Thus, through the analyzed examples, it is noted that the FOFA matrix is an adequate tool for the strategic planning of projects. This, therefore, allows structuring a production process that corroborates the quality of built environments and user satisfaction