Caspase-3-independent photoreceptor degeneration by N-methyl-N-nitrosourea (MNU) induces morphological and functional changes in the mouse retina

Retinal degeneration is followed by significant changes in the structure and function of photoreceptors in humans and several genetic animal models. However, it is not clear whether similar changes occur when the degeneration is induced pharmacologically. Therefore, our aim was to investigate the influence of retinotoxic N-methyl-N-nitrosourea (MNU) on the function, morphology and underlying molecular pathways of programmed cell death.

Comparative Ultrastructure and Molecular Phylogeny of Selenidium melongena n. sp and S. terebellae Ray 1930 Demonstrate Niche Partitioning in Marine Gregarine Parasites (Apicomplexa)

Gregarine apicomplexans are a diverse group of single-celled parasites that have feeding stages (trophozoites) and gamonts that generally inhabit the extracellular spaces of invertebrate hosts living in marine, freshwater, and terrestrial environments. Inferences about the evolutionary morphology of gregarine apicomplexans are being incrementally refined by molecular phylogenetic data, which suggest that several traits associated with the feeding cells of gregarines arose by convergent evolution. The study reported here supports these inferences by showing how molecular data reveals traits that are phylogenetically misleading within the context of comparative morphology alone. We examined the ultrastructure and molecular phylogenetic positions of two gregarine species isolated from the spaghetti worm Thelepus japonicus: Selenidium terebellae Ray 1930 and S. melongena n. sp. The ultrastructural traits of S. terebellae were very similar to other species of Selenidium sensu stricto, such as having vermiform trophozoites with an apical complex, few epicytic folds, and a dense array of microtubules underlying the trilayered pellicle. By contrast, S. melongena n. sp. lacked a comparably discrete assembly of subpellicular microtubules, instead employing a system of fibrils beneath the cell surface that supported a relatively dense array of helically arranged epicytic folds. Molecular phylogenetic analyses of small subunit rDNA sequences derived from single-cell PCR unexpectedly demonstrated that these two gregarines are close sister species. The ultrastructural differences between these two species were consistent with the fact that S. terebellae infects the inner lining of the host intestines, and S. melongena n. sp. primarily inhabits the coelom, infecting the outside wall of the host intestine. Altogether, these data demonstrate a compelling case of niche partitioning and associated morphological divergence in marine gregarine apicomplexans. (C) 2014 Elsevier GmbH. All rights reserved.

Neuropathological and molecular comparison between clinical and asymptomatic bovine spongiform encephalopathy cases

Interest in the proper neuropathological and molecular characterization of bovine spongiform encephalopathy (BSE) has increased since asymptomatic and atypical cases were detected in the cattle population by active disease surveillance. In this respect we investigated a total of 95 confirmed BSE cases originating from different active and passive surveillance categories (clinical suspects, emergency-slaughter, fallen stock and routinely slaughter) in Switzerland for their neuropathological and molecular phenotype. We looked for measurable differences between these categories in lesion profile, severity of spongiform change, degree of astrocytosis as well as immunohistochemical and molecular patterns of the disease-associated isoform of the prion protein (PrPd) in the caudal brainstem. Our results indicate significantly higher intensities of spongiform change in clinically affected compared to asymptomatic BSE cases. Similar effects were in trend observed for the intensities of PrPd deposition and astrocytosis, whereas the frequencies of morphological PrPd types and the molecular patterns in Western immunoblot were not different. Importantly, none of the animals included in this study revealed features of atypical BSE. Taken together, this study suggests that both clinically affected as well as asymptomatic Swiss BSE cases in cattle share the neuropathological and molecular phenotype of classical BSE and that asymptomatic classical BSE cases are at a pre-clinical stage of the disease rather than representing a true sub-clinical form of BSE.

Morphological and immunocytochemical analysis of Escherichia coli-specific surface antigens in wildtype strains and in recombinant Vibrio cholerae

Adhesion is the first step in the pathogenesis of enterotoxigenic Escherichia coli infections. The genes encoding the most prevalent adhesion factors CFA/I, CS3 and CS6 were cloned into Vibrio cholerae strain CVD 103-HgR and expression of fimbriae was investigated in wildtype and recombinant strains by transmission electron microscopy in conjunction with immunolabelling and negative staining. Negative staining was effective in revealing CFA/I and CS3, but not CS6. Although morphology of fimbriae differed between wildtype and recombinant strains, corresponding surface antigens were recognized by specific antibodies. The present study provides evidence that ETEC-specific fimbriae can adequately be expressed in an attenuated V. cholerae vaccine strain and that immunoelectron microscopy is a critical tool to validate the surface expression of antigens in view of their possible suitability for recombinant vaccines.

The cellular and molecular involvement of glutathione in cisplatin-induced apoptosis

The goal of this study was to investigate the cellular and molecular mechanisms by which glutathione (GSH) is involved in the process of apoptosis induced by cisplatin [cis-diamminedichloroplatinum(II), cis-DDP] in the HL60 human promyelocytic leukemia cell line. The data show that during the onset or induction of apoptosis, GSH levels in cisplatin-treated cells increased 50% compared to control cells. The increase in intracellular GSH was associated with enhanced expression of γ-glutamylcysteine synthetase (γ-GCS), the enzyme that catalyzes the rate- limiting step in the biosynthesis of glutathione. After depletion of intracellular GSH with D,L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of γ-GCS, biochemical and morphological analysis revealed that the mechanism of cell death had switched from apoptosis to necrosis. In contrast, when intracellular GSH was elevated by exposure of cells to a GSH-ethyl-ester and then treatment with cisplatin, no change in the induction and kinetics of apoptosis were observed. However, when cells were exposed to cisplatin before intracellular GSH levels were increased, apoptosis was observed to occur 6 hours earlier compared to cells without GSH elevation. To further examine the molecular aspects of these effects of GSH on the apoptotic process, changes in the expression of bcl-2 and bax, were investigated in cells with depleted and elevated GSH. Using reverse transcription polymerase chain reaction, no significant change in the expression of bcl-2 gene transcripts was observed in cells in either the GSH depleted or elevated state; however, a 75% reduction in GSH resulted in a 40% decrease in the expression of bax gene transcripts. In contrast, a 6-fold increase in GSH increased the expression of bax by 3-fold relative to controls. Similar results were obtained for bax gene expression and protein synthesis by northern analysis and immunoprecipitation, respectively. These results suggest that GSH serves a dual role in the apoptotic process. The first role which is indirect, involves the protection of the cell from extensive damage following exposure to a specific toxicant so as to prevent death by necrosis, possibly by interacting with the DNA damaging agent and/or its active metabolites. The second role involves a direct involvement of GSH in the apoptotic process that includes upregulation of bax expression. ^

The adaptive response of humans to exercise: Impact of exercise intensity, fibre-type specific responses and molecular patterns of response

In an attempt to improve the current understanding of the adaptive response to exercise in humans, this dissertation performed a series of studies designed to examine the impact of training intensity and mode on aerobic capacity and performance, fibre-type specific adaptations to training, and individual patterns of response across molecular, morphological and genetic factors. Project #1 determined that training intensity, session dose, baseline VO2max and total training volume do not influence the magnitude of change in VO2max by performing a meta-regression, and meta-analysis of 28 different studies. The intensity of training had no effect on the magnitude of increase in maximal oxygen uptake in young healthy participants, but similar adaptations were achieved with lower training doses following high intensity training. Project # 2 determined the acute molecular response, and training-induced adaptations in aerobic performance, aerobic capacity and muscle phenotype following high-intensity interval training (HIT) or endurance exercise (END). The acute molecular response (fibre recruitment and signal activation) and training-induced adaptations in aerobic capacity, aerobic performance, and muscle phenotype were similar following HIT and END. Project # 3 examined the impact of baseline muscle morphology and molecular characteristics on the training response, and if muscle adaptations are coordinated. The muscle phenotype of individuals who experience the largest improvements (high responders) were lower before training for some muscle characteristics and molecular adaptations were coordinated within individual participants. Project # 4 examined the impact of 2 different intensities of HIT on the expression of nuclear and mitochondrial encoded genes targeted by PGC-1α. A systematic upregulation of nuclear and mitochondrial encoded genes was not present in the early recovery period following acute HIT, but the expression of mitochondrial genes were coordinated at an individual level. Collectively, results from the current dissertation contribute to our understanding of the molecular mechanisms influencing skeletal muscle and whole-body adaptive responses to acute exercise and training in humans.

Phylogeny, molecular and fossil dating, and biogeographic history of Annonaceae and Myristicaceae (Magnoliales)

Annonaceae and Myristicaceae, the two largest families of Magnoliales, are pantropical groups of uncertain geographic history. The most recent morphological and molecular phylogenetic analyses identify the Asian-American genus Anaxagorea as sister to all other Annonaceae and the ambavioids, consisting of small genera endemic to South America, Africa, Madagascar, and Asia, as a second branch. However, most genera form a large clade in which the basal lines are African, and South American and Asian taxa are more deeply nested. Although it has been suggested that Anaxagorea was an ancient Laurasian line, present data indicate that this genus is basically South American. These considerations may mean that the family as a whole began its radiation in Africa and South America in the Late Cretaceous, when the South Atlantic was narrower, and several lines dispersed from Africa-Madagascar into Laurasia as the Tethys closed in the Tertiary. This scenario is consistent with the occurrence of annonaceous seeds in the latest Cretaceous of Nigeria and the Eocene of England and with molecular dating of the family. Based on distribution of putatively primitive taxa in Madagascar and derived taxa in Asia, it has been suggested that Myristicaceae had a similar history. Phylogenetic analyses of Myristicaceae, using morphology and several plastid regions, confirm that the ancestral area was Africa-Madagascar and that Asian taxa are derived. However, Myristicaceae as a whole show strikingly lower molecular divergence than Annonaceae, indicating either a much younger age or a marked slowdown in molecular evolution. The fact that the oldest diagnostic fossils of Myristicaceae are Miocene seeds might be taken as evidence that Myristicaceae are much younger than Annonaceae, but this is implausible in requiring transoceanic dispersal of their large, animal-dispersed seeds.

Imaging, anatomical, and molecular analysis of callosal formation in the developing human fetal brain

A complex set of axonal guidance mechanisms are utilized by axons to locate and innervate their targets. In the developing mouse forebrain, we previously described several midline glial populations as well as various guidance molecules that regulate the formation of the corpus callosum. Since agenesis of the corpus callosum is associated with over 50 different human congenital syndromes, we wanted to investigate whether these same mechanisms also operate during human callosal development. Here we analyze midline glial and commissural development in human fetal brains ranging from 13 to 20 weeks of gestation using both diffusion tensor magnetic resonance imaging and immunohistochemistry. Through our combined radiological and histological studies, we demonstrate the morphological development of multiple forebrain commissures/decussations, including the corpus callosum, anterior commissure, hippocampal commissure, and the optic chiasm. Histological analyses demonstrated that all the midline glial populations previously described in mouse, as well as structures analogous to the subcallosal sling and cingulate pioneering axons, that mediate callosal axon guidance in mouse, are also present during human brain development. Finally, by Northern blot analysis, we have identified that molecules involved in mouse callosal development, including Slit, Robo, Netrin1, DCC, Nfia, Emx1, and GAP-43, are all expressed in human fetal brain. These data suggest that similar mechanisms and molecules required for midline commissure formation operate during both mouse and human brain development. Thus, the mouse is an excellent model system for studying normal and pathological commissural formation in human brain development. (c) 2006 Wiley-Liss, Inc.

Filogenia molecular do gênero Morganella Zeller (Fungi, Basidiomycota) utilizando marcadores ITS

With the development and improvement of techniques for molecular studies and their subsequent application to the systematic, significant changes occurred in the classification of gasteroid fungi. The genus Morganella belongs to the family Lycoperdaceae, and is characterized mainly by lignicolous habit and presence of paracapilicium. Recent data demonstrate the discovery of new species for the group and the existence of a wide variety of species occurring in tropical ecosystems. However, the phylogenetic relationships of the genus, as well as the taxonomic classification, still require revisions to be better understood, the literature studies that address this issue are still very scarce. Thus, the objective of this study was to conduct studies of molecular phylogeny with species of the genus Morganella, to enhance understanding of the phylogeny of the group by including tropical species data. For this, the specimens used both for DNA extractions as for morphological review were obtained from Brazilian and foreign herbaria. For morphological analysis were observed characters relevant to the group's taxonomy. For phylogenetic analysis the Maximum Parsimony and Bayesian Analyzes were used, using the internal transcribed spacer (ITS) of nuclear ribosomal DNA. In phylogenetic analyzes, representatives from Morganella form a monophyletic clade with good support value and based on these results the genus should not be included as subgenus of Lycoperdon. The analysis indicated that M. pyriformis was not grouped with other representatives of Morganella, and therefore should not be included in the group as representative of Apioperdon subgenus because it is a Lycoperdon representative. Moreover, M. fuliginea, M. nuda, M. albostipitata, M. velutina, M. subincarnata are grouped with high support values within the genus Morganella. Morganella arenicola based on morphological and molecular studies does not aggregate in Morganella. Morganella nuda was grouped with M. fuliginea giving indications that can be treated as an intraspecific variation. The results of the analyzes favor to a better understanding of the species of Morganella. However, additional studies using a greater number of species, as well as other molecular markers are needed for a better understanding of the phylogenetic of Morganella.

The adaptive response of humans to exercise: Impact of exercise intensity, fibre-type specific responses and molecular patterns of response

In an attempt to improve the current understanding of the adaptive response to exercise in humans, this dissertation performed a series of studies designed to examine the impact of training intensity and mode on aerobic capacity and performance, fibre-type specific adaptations to training, and individual patterns of response across molecular, morphological and genetic factors. Project #1 determined that training intensity, session dose, baseline VO2max and total training volume do not influence the magnitude of change in VO2max by performing a meta-regression, and meta-analysis of 28 different studies. The intensity of training had no effect on the magnitude of increase in maximal oxygen uptake in young healthy participants, but similar adaptations were achieved with lower training doses following high intensity training. Project # 2 determined the acute molecular response, and training-induced adaptations in aerobic performance, aerobic capacity and muscle phenotype following high-intensity interval training (HIT) or endurance exercise (END). The acute molecular response (fibre recruitment and signal activation) and training-induced adaptations in aerobic capacity, aerobic performance, and muscle phenotype were similar following HIT and END. Project # 3 examined the impact of baseline muscle morphology and molecular characteristics on the training response, and if muscle adaptations are coordinated. The muscle phenotype of individuals who experience the largest improvements (high responders) were lower before training for some muscle characteristics and molecular adaptations were coordinated within individual participants. Project # 4 examined the impact of 2 different intensities of HIT on the expression of nuclear and mitochondrial encoded genes targeted by PGC-1α. A systematic upregulation of nuclear and mitochondrial encoded genes was not present in the early recovery period following acute HIT, but the expression of mitochondrial genes were coordinated at an individual level. Collectively, results from the current dissertation contribute to our understanding of the molecular mechanisms influencing skeletal muscle and whole-body adaptive responses to acute exercise and training in humans.

Development of a novel molecular tool for the rapid assessment of the biodiversity changes of benthic nematodes assemblages. Mares Conference Marine Ecosystems Helath and conservation

The molecular profiling system was developed using directed terminal-restriction fragment length polymorphism (dT-RFLP) to characterize soil nematode assemblages by relative abundance of feeding guilds and validation by comparison to traditional morphological method. The good performance of these molecular tools applied to soil nematodes assemblages create an opportunity to develop a novel approach for rapid assessment of the biodiversity changes of benthic nematodes assemblages of marine and estuarine sediments. The main aim of this research is to combine morphological and molecular analysis of estuarine nematodes assemblages, to establish a tool for fast assessment of the biodiversity changes within habitat recovery of Zostera noltii seagrass beds; and validate the dT-RFLP as a high-throughput tool to assess the system recovery. It was also proposed to develop a database of sequences related to individuals identified at species level to develop a new taxonomic reference system. A molecular phylogenetic analysis of the estuarine nematodes has being performed. After morphological identification, barcoding of 18S rDNA are being determined for each nematode species and the results have shown a good degree of concordance between traditional morphology-based identification and DNA sequences. The digest strategy developed for soil nematodes is not suitable for marine nematodes. Then five samples were cloned and sequenced and the sequence data was used to design a new dT-RFLP strategy to adapt this tool to marine assemblages. Several solutions were presented by DRAT and tested empirically to select the solution that cuts most efficiently, separating the different clusters. The results of quantitative PCR showed differences in nematode density between two sampling stations according the abundance of the nematode density obtained by the traditional methods. These results suggest that qPCR could be a robust tool for enumeration of nematode abundance, saving time.

Development of molecular approaches to assess the diversity and abundance of marine nematodes assemblages for assessment of Good Environmental Status of the estuarine and marine ecosystems

In Europe, the concerns with the status of marine ecosystems have increased, and the Marine Directive has as main goal the achievement of Good Environmental Status (GES) of EU marine waters by 2020. Molecular tools are seen as promising and emerging approaches to improve ecosystem monitoring, and have led ecology into a new era, representing perhaps the most source of innovation in marine monitoring techniques. Benthic nematodes are considered ideal organisms to be used as biological indicator of natural and anthropogenic disturbances in aquatic ecosystems underpinning monitoring programmes on the ecological quality of marine ecosystems, very useful to assess the GES of the marine environment. dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows to assess the diversity of nematode communities, but also allows studying the functioning of the ecosystem, and combined with relative real-time PCR (qPCR), provides a high-throughput semi-quantitative characterization of nematode communities. These characteristics make the two molecular tools good descriptors for the good environmental status assessment. The main aim of this study is to develop and optimize the dT-RFLP and qPCR in Mira estuary (SW coast, Portugal). A molecular phylogenetic analysis of marine and estuarine nematodes is being performed combining morphological and molecular analysis to evaluate the diversity of free-living marine nematodes in Mira estuary. After morphological identification, barcoding of 18S rDNA and COI genes are being determined for each nematode species morphologically identified. So far we generated 40 new sequences belonging to 32 different genus and 17 families, and the study has shown a good degree of concordance between traditional morphology-based identification and DNA sequences. These results will improve the assessment of marine nematode diversity and contribute to a more robust nematode taxonomy. The DNA sequences are being used to develop the dT-RFLP with the ability to easily process large sample numbers (hundreds and thousands), rather than typical of classical taxonomic or low throughput molecular analyses. A preliminary study showed that the digest enzymes used in dT-RFLP for terrestrial assemblages separated poorly the marine nematodes at taxonomic level for functional group analysis. A new digest combination was designed using the software tool DRAT (Directed Terminal Restriction Analysis Tool) to distinguished marine nematode taxa. Several solutions were provided by DRAT and tested empirically to select the solution that cuts most efficiently. A combination of three enzymes and a single digest showed to be the best solution to separate the different clusters. Parallel to this, another tool is being developed to estimate the population size (qPCR). An improvement in qPCR estimation of gene copy number using an artificial reference is being performed for marine nematodes communities to quantify the abundance. Once developed, it is proposed to validate both methodologies by determining the spatial and temporal variability of benthic nematodes assemblages across different environments. The application of these high-throughput molecular approaches for benthic nematodes will improve sample throughput and their implementation more efficient and faster as indicator of ecological status of marine ecosystems.

Genetic diversity of mango accessions (Mangifera indica) using new microsatellite markers and morphological descriptors.

Genetic diversity estimates based on morphological and molecular data can provide different information on the relationship between cultivars of a species. This study aimed to develop new microsatellite markers as additional tools in genetic studies on mangoes (Mangifera indica L.), and to analyze the genetic variability of 20 mango cultivars based on morphological descriptors and microsatellite markers. We aimed to better understand the cultivars enhanced breeding histories and to support crossbreeding planning. Positive clones were selected from a DNA library enriched for microsatellite regions for sequencing and primer design. Four plants of each of the 20 accessions were used for observations, based on 48 morphological descriptors. Twenty accessions were analyzed using 27 microsatellite markers, of which 16 were developed during this study. The clusters, based on the morphological descriptors by Ward - MLM strategy and the microsatellite markers, suggested that Brazilian mango cultivars have extensive genetic diversity and are related to cultivars with different provenances, demonstrating their different enhanced breeding histories.

Effect Of Aloe Vera Application On The Content And Molecular Arrangement Of Glycosaminoglycans During Calcaneal Tendon Healing.

Although several treatments for tendon lesions have been proposed, successful tendon repair remains a great challenge for orthopedics, especially considering the high incidence of re-rupture of injured tendons. Our aim was to evaluate the pharmacological potential of Aloe vera on the content and arrangement of glycosaminoglycans (GAGs) during tendon healing, which was based on the effectiveness of A. vera on collagen organization previously observed by our group. In rats, a partial calcaneal tendon transection was performed with subsequent topical A. vera application at the injury site. The tendons were treated with A. vera ointment for 7 days and excised on the 7(th) , 14(th) , or 21(st) day post-surgery. Control rats received ointment without A. vera. A higher content of GAGs and a lower amount of dermatan sulfate were detected in the A. vera-treated group on the 14(th) day compared with the control. Also at 14 days post-surgery, a lower dichroic ratio in toluidine blue stained sections was observed in A. vera-treated tendons compared with the control. No differences were observed in the chondroitin-6-sulfate and TGF-β1 levels between the groups, and higher amount of non-collagenous proteins was detected in the A. vera-treated group on the 21(st) day, compared with the control group. No differences were observed in the number of fibroblasts, inflammatory cells and blood vessels between the groups. The application of A. vera during tendon healing modified the arrangement of GAGs and increased the content of GAGs and non-collagenous proteins.

Solid Lipid Nanoparticles As Nucleic Acid Delivery System: Properties And Molecular Mechanisms.

Solid lipid nanoparticles (SLNs) have been proposed in the 1990s as appropriate drug delivery systems, and ever since they have been applied in a wide variety of cosmetic and pharmaceutical applications. In addition, SLNs are considered suitable alternatives as carriers in gene delivery. Although important advances have been made in this particular field, fundamental knowledge of the underlying mechanisms of SLN-mediated gene delivery is conspicuously lacking, an imperative requirement in efforts aimed at further improving their efficiency. Here, we address recent advances in the use of SLNs as platform for delivery of nucleic acids as therapeutic agents. In addition, we will discuss available technology for conveniently producing SLNs. In particular, we will focus on underlying molecular mechanisms by which SLNs and nucleic acids assemble into complexes and how the nucleic acid cargo may be released intracellularly. In discussing underlying mechanisms, we will, when appropriate, refer to analogous studies carried out with systems based on cationic lipids and polymers, that have proven useful in the assessment of structure-function relationships. Finally, we will give suggestions for improving SLN-based gene delivery systems, by pointing to alternative methods for SLNplex assembly, focusing on the realization of a sustained nucleic acid release.