Alternative splicing and genetic diversity: silencers are more frequently modified by SNVs associated with alternative exon/intron borders

With the availability of a large amount of genomic data it is expected that the influence of single nucleotide variations (SNVs) in many biological phenomena will be elucidated. Here, we approached the problem of how SNVs affect alternative splicing. First, we observed that SNVs and exonic splicing regulators (ESRs) independently show a biased distribution in alternative exons. More importantly, SNVs map more frequently in ESRs located in alternative exons than in ESRs located in constitutive exons. By looking at SNVs associated with alternative exon/intron borders (by their common presence in the same cDNA molecule), we observed that a specific type of ESR, the exonic splicing silencers (ESSs), are more frequently modified by SNVs. Our results establish a clear association between genetic diversity and alternative splicing involving ESSs.

Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis

Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short. (C) 2009 Elsevier Inc. All rights reserved.

Absence of Melatonin Induces Night-Time Hepatic Insulin Resistance and Increased Gluconeogenesis Due to Stimulation of Nocturnal Unfolded Protein Response

It is known that the circadian rhythm in hepatic phosphoenolpyruvate carboxykinase expression (a limiting catalytic step of gluconeogenesis) and hepatic glucose production is maintained by both daily oscillation in autonomic inputs to the liver and night feeding behavior. However, increased glycemia and reduced melatonin (Mel) levels have been recently shown to coexist in diabetic patients at the end of the night period. In parallel, pinealectomy (PINX) is known to cause glucose intolerance with increased basal glycemia exclusively at the end of the night. The mechanisms that underlie this metabolic feature are not completely understood. Here, we demonstrate that PINX rats show night-time hepatic insulin resistance characterized by reduced insulin-stimulated RAC-alpha serine/threonine-protein kinase phosphorylation and increased phosphoenolpyruvate carboxykinase expression. In addition, PINX rats display increased conversion of pyruvate into glucose at the end of the night. The regulatory mechanism suggests the participation of unfolded protein response (UPR), because PINX induces night-time increase in activating transcription factor 6 expression and prompts a circadian fashion of immunoglobulin heavy chain-binding protein, activating transcription factor 4, and CCAAT/enhancer-binding protein-homologous protein expression with Zenith values at the dark period. PINX also caused a night-time increase in Tribble 3 and regulatory-associated protein of mammalian target of rapamycin; both were reduced in liver of PINX rats treated with Mel. Treatment of PINX rats with 4-phenyl butyric acid, an inhibitor of UPR, restored night-time hepatic insulin sensitivity and abrogated gluconeogenesis in PINX rats. Altogether, the present data show that a circadian oscillation of UPR occurs in the liver due to the absence of Mel. The nocturnal UPR activation is related with night-time hepatic insulin resistance and increased gluconeogenesis in PINX rats. (Endocrinology 152: 1253-1263, 2011)

POLYGLYCOLIC ACID TUBE ASSOCIATED WITH GM1 IN REGENERATION OF PERIPHERAL NERVES

Introduction: Nerve allografting is regarded as a treatment of choice in large neural tissue losses preventing repair by primary anastomosis. In these cases, a synthetic polyglycolic acid tube is an alternative for nerve grafting. On the other hand, several studies have emphasized the importance of neurotrophic factors on neural regeneration, including substances with potential to optimize neural regeneration, especially the GM1, an neurotrophic enhancer factor. Objective: to compare, in rats, the neural regeneration degree using histological analysis, regenerated myelinized axons count, and functional analysis with the use of neurotube and GM1. Methods: This assessment was performed by interposing allograft (group A), polyglycolic acid tube (group B) and polyglycolic acid tube associated to GM1 (group C) on 5-mm sciatic nerve defects. Results: Neuroma formation was found only on group A. Groups A and C showed similar histological patterns, except for the regenerated axons on group C, which were shown to be better organized and myelinized than in group A. Conclusion: on functional recovery, no statistically significant difference was found for the three groups, despite of qualitative and quantitative histological differences found.

BCR-ABL-mediated upregulation of PRAME is responsible for knocking down TRAIL in CML patients

Tumor necrosis factor-related apoptosis-inducing ligand-TNFSF10 (TRAIL), a member of the TNF-alpha family and a death receptor ligand, was shown to selectively kill tumor cells. Not surprisingly, TRAIL is downregulated in a variety of tumor cells, including BCR-ABL-positive leukemia. Although we know much about the molecular basis of TRAIL-mediated cell killing, the mechanism responsible for TRAIL inhibition in tumors remains elusive because (a) TRAIL can be regulated by retinoic acid (RA); (b) the tumor antigen preferentially expressed antigen of melanoma (PRAME) was shown to inhibit transcription of RA receptor target genes through the polycomb protein, enhancer of zeste homolog 2 (EZH2); and (c) we have found that TRAIL is inversely correlated with BCR-ABL in chronic myeloid leukemia (CML) patients. Thus, we decided to investigate the association of PRAME, EZH2 and TRAIL in BCR-ABL-positive leukemia. Here, we demonstrate that PRAME, but not EZH2, is upregulated in BCR-ABL cells and is associated with the progression of disease in CML patients. There is a positive correlation between PRAME and BCR-ABL and an inverse correlation between PRAME and TRAIL in these patients. Importantly, knocking down PRAME or EZH2 by RNA interference in a BCR-ABL-positive cell line restores TRAIL expression. Moreover, there is an enrichment of EZH2 binding on the promoter region of TRAIL in a CML cell line. This binding is lost after PRAME knockdown. Finally, knocking down PRAME or EZH2, and consequently induction of TRAIL expression, enhances Imatinib sensibility. Taken together, our data reveal a novel regulatory mechanism responsible for lowering TRAIL expression and provide the basis of alternative targets for combined therapeutic strategies for CML. Oncogene (2011) 30, 223-233; doi:10.1038/onc.2010.409; published online 13 September 2010

Analysis of the genome of Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV-19) and of the high genomic heterogeneity in group II nucleopolyhedroviruses

The genome of the most virulent among 22 Brazilian geographical isolates of Spodoptera frugiperda nucleopolyhedrovirus, isolate 19 (SfMNPV-1 9), was completely sequenced and shown to comprise 132 565 bp and 141 open reading frames (ORFs). A total of 11 ORFs with no homology to genes in the GenBank database were found. Of those, four had typical baculovirus; promoter motifs and polyadenylation sites. Computer-simulated restriction enzyme cleavage patterns of SfMNPV-1 9 were compared with published physical maps of other SfMNPV isolates. Differences were observed in terms of the restriction profiles and genome size. Comparison of SfMNPV-1 9 with the sequence of the SfMNPV isolate 3AP2 indicated that they differed due to a 1427 bp deletion, as well as by a series of smaller deletions and point mutations. The majority of genes of SfMNPV-1 9 were conserved in the closely related Spodoptera exigua NPV (SeMNPV) and Agrotis segetum NPV (AgseMNPV-A), but a few regions experienced major changes and rearrangements. Synthenic maps for the genomes of group 11 NPVs revealed that gene collinearity was observed only within certain clusters. Analysis of the dynamics of gene gain and loss along the phylogenetic tree of the NPVs showed that group 11 had only five defining genes and supported the hypothesis that these viruses form ten highly divergent ancient lineages. Crucially, more than 60% of the gene gain events followed a power-law relation to genetic distance among baculoviruses, indicative of temporal organization in the gene accretion process.

Functional interferences in host inflammatory immune response by airway allergic inflammation restrain experimental periodontitis development in mice

Aims Periodontal disease (PD) and airway allergic inflammation (AL) present opposing inflammatory immunological features and clinically present an inverse correlation. However, the putative mechanisms underlying such opposite association are unknown. Material and Methods Balb/C mice were submitted to the co-induction of experimental PD (induced by Actinobacillus actinomycetemcomitans oral inoculation) and AL [induced by sensitization with ovalbumin (OVA) and the subsequent OVA challenges], and evaluated regarding PD and AL severity, immune response [cytokine production at periodontal tissues, and T-helper transcription factors in submandibular lymph nodes (LNs)] and infection parameters. Results PD/AL co-induction decreased PD alveolar bone loss and periodontal inflammation while experimental AL parameters were unaltered. An active functional interference was verified, because independent OVA sensitization and challenge not modulate PD outcome. PD+AL group presented decreased tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, -gamma, IL-17A, receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cells ligand and matrix metalloproteinase (MMP)-13 levels in periodontal tissues, while IL-4 and IL-10 levels were unaltered by AL co-induction. AL co-induction also resulted in upregulated T-bet and related orphan receptor gamma and downregulated GATA3 levels expression in submandibular LNs when compared with PD group. Conclusion Our results demonstrate that the interaction between experimental periodontitis and allergy involves functional immunological interferences, which restrains experimental periodontitis development by means of a skewed immune response.

Development of an adenoviral vector with robust expression driven by p53

Here we introduce a new adenoviral vector where transgene expression is driven by p53. We first developed a synthetic promoter, referred to as PGTx beta containing a p53-responsive element, a minimal promoter and the first intron of the rabbit P-globin gene. Initial assays using plasmid-based vectors indicated that expression was tightly controlled by p53 and was 5-fold stronger than the constitutive CMV immediate early promoter/enhancer. The adenoviral vector, AdPG, was also shown to offer p53-responsive expression in prostate carcinoma cells LNCaP (wt p53), DU-145 (temperature sensitive mutant of p53) and PC3 (p53-null, but engineered to express temperature-sensitive p53 mutants). AdPG served as a sensor of p53 activity in LNCaP cells treated with chemotherapeutic agents. Since p53 can be induced by radiotherapy and chemotherapy, this new vector could be further developed for use in combination with conventional therapies to bring about cooperation between the genetic and pharmacologic treatment modalities. (c) 2007 Elsevier Inc. All rights reserved.

Lipid hydroperoxide-induced and hemoglobin-enhanced oxidative damage to colon cancer cells

Epidemiological studies have indicated that Western diets are related to an increase in a series of malignancies. Among the compounds that are credited for this toxic effect are heme and lipid peroxides. We evaluated the effects of hemoglobin (Hb) and linoleic acid hydroperoxides (LAOOH) on a series of toxicological endpoints, such as cytotoxicity, redox status, lipid peroxidation, and DNA damage. We demonstrated that the preincubation of SW480 cells with Hb and its subsequent exposure to LAOOH (Hb + LAOOH) led to an increase in cell death, DCFH oxidation, malonaldehyde formation, and DNA fragmentation and that these effects were related to the peroxide group and the heme present in Hb. Furthermore, Hb and LAOOH alone exerted a toxic effect on the endpoints assayed only at concentrations higher than 100 mu M. We were also able to show that SW480 cells presented a higher level of the modified DNA bases 8-oxo-7,8-dihydro-2`-deoxyguanosine and 1,N(2)-etheno-2`-deoxyguanosine compared to the control. Furthermore, incubations with Hb led to an increase in intracellular iron levels, and this high level of iron correlated with DNA oxidation, as measured as EndoIII- and Fpg-sensitive sites. Thus, Hb from either red meat or bowel bleeding could act as an enhancer of fatty acid hydroperoxide genotoxicity, which contributes to the accumulation of DNA lesions in colon cancer cells. (C) 2011 Elsevier Inc. All rights reserved.

Konstruktion och design av två glassförpackningar i kartongkvaliteten Frövi Light

This degree project was made in cooperation with AssiDomän Frövi and Charlotte Andersson and the main objectivehas been to create to ice cream pacbges from 250 gsm Frövi Light board. The paekages are intended to be easilyresealable while decreasing in size together with the ice cream. The project also describes the food packaging labelsand symbols currently present in the European common market.Both capsules utilize a separate lid for resealing. One of the paekages (Capsuie A) is indended to be cut along withthe ice cream and thereby decrease in size. The other one (Capsuie B) uses a series of flaps for contracting and expandingwhich enab1es adjusting of the size without cutting the package up.The design for Capsuie A is created both as a series of flavours with a super-hero theme intended for children andwith a colfee flavour for a maturer audience. For Capsule B, a design was created for a sorbet.

A Influência do atributo de segurança na segmentação dos consumidores de carne suína da região fronteira noroeste do Rio Grande do Sul

This document constitutes itself a thesis for a master¿s, partial fulfillment for getting a master¿s degree in Public and Business Management. The subject is a research on the influence of the attribute of safety in the segmentation of pork in the northwest border of the State of Rio Grande do Sul, Brazil, involving a bibliographic review related to the theme, and also an explorative research in order to identify the characteristics of the sector. From this information, a descriptive research is carried out from data collected in stores wich commercialize pork products in Santa Rosa, reaching a comprehensive undestanding on the subject. The main results of the research are the theoretical building of the thematic support concerning the safety of the food; the use of interviews aiming at verifying the importance of different attributes of pork which the consumer consider important and also the identification of segments based on these assessments. The conclusions from the research show that the consumers in general consider of high importance the safety of the food, and it is due to the fact that pork is still considered a product which offers a high risk at consumption. Consumers still have a preconceived idea about pork, because years ago, hog breeding was conducted with precarious standards of hygiene. It is imperative that the related business and producers divulge the advances of the sector in order to destroy this negative concept. The study identified three segments of market to the consumer of in natura pork: the first emphasized attributes are the inspection and the presentation of the product (cluster 1), flavour and softness stand out as the second ones. Finally, the third most important attributes that consumers consider when picking a pork product are its price and its aspect. It also favours further studies wich can provide the formulation of new marketing strategies to order products that form part of this agrolimentary system, giving this segment opportunity to boost its business and improve its approach to its consumer.

Modulação diferencial de fatores de transcrição adipogênicos, por indometacina e retinol, durante a conversão fenotípica de uma linhagem representativa de céculas estreladas hepáticas

A modulação fenotípica da célula estrelada hepática (CEH) é um dos eventos primários do processo de fibrose hepática. Durante este evento, a CEH modifica seu fenótipo lipocítico (quiescente) para miofibroblástico (ativado), fenômeno chamado de ativação. Estudos recentes sugerem que fatores de transcrição adipogênicos atuam no processo de ativação das CEH, e conseqüentemente teriam um papel chave na evolução do processo fibrótico. Neste estudo analisamos a expressão gênica de vários fatores de transcrição adipogênicos, como os receptores ativados por proliferadores de peroxissomos (RAPP), receptor X do fígadoα (RXFα), proteína ligante de CCAAT/enhancerα (PLCEα) e proteína ligante do elemento regulatório de esteróis-1 (PLERE-1), em uma linhagem representativa de CEH, a GRX. A linhagem GRX, é capaz de ser induzida in vitro a expressar o fenótipo quiescente através do tratamento com indometacina ou retinol. Após 24 horas ou 7 dias de tratamento, a expressão gênica foi analisada através de PCR em tempo real. Tratamento com indometacina aumentou a expressão de todos os genes avaliados, exceto PLCEα e RAPPβ. O mesmo resultado obtido com desmetil indometacina (LM 4511) sobre RAPPα e γ sugere que a ação da indometacina sobre a diferenciação fenotípica da GRX é independente de sua ação inibitória sobre ciclooxigenase, já que a LM 4511 não possui esta atividade. Tratamento com retinol levou à modulação de um número menor de genes, diminuindo a expressão de PLCEα e aumentando a de RAPPγ, e RAPPβ. Adipsina, gene característico de adipócitos, teve sua expressão induzida apenas nas células tratadas com indometacina. Pex16 e catalase, genes modulados pelos RAPPs, foram diferencialmente expressos, dependendo do tratamento. Os resultados apresentados sugerem que a linhagem representativa de CEH, GRX, tem sua indução fenotípica modulada por fatores de transcrição adipogênicos, que são diferencialmente expressos dependendo do tratamento de conversão.

Effect of the estufagem process on the chemical constituents of Madeira wines

Madeira wine is a product of well-established reputation, whose aroma and flavour is the result of unique combinations. Particularly, its maturation may include estufagem, wherein wine is usually heated at 45 °C for three months. During this period, several chemical changes may occur, so it is essential to assess its impact on the wine. In this sense, the main objective of the thesis was to evaluate the effect estufagem on the chemical constituents of Madeira wine, specifically on those molecules potentially important in the development of its typical features. Firstly, analytical methodologies capable of determining the target compounds, combining precision and reproducibility to execution effectiveness, were developed. Then various monovarietal Madeira wines were analysed during estufagem under standard and overheating conditions in order to assess its effect. The following compounds were evaluated: furans, amino acids, biogenic amines, polyphenols, organic acids and volatile compounds. In addition, the total polyphenolic composition, the antioxidant potential and the colour of these wines were also evaluated. The results show that most constituents change due to the heating process. Particularly, the heating promotes the development of 5-hydroxymethylfurfural (HMF) in sweet wines submitted to estufagem at higher temperatures. Moreover, estufagem provides the decrease of most amino acids, suggesting their involvement in the formation of the bouquet of these wines. Regarding the total polyphenol content and antioxidant potential of these wines they do not seem to be greatly affected by the heating step, however most monomeric polyphenols decrease during this process. The thermal processing of Madeira wines favours the development of the volatile composition, especially of volatiles usually reported as typical aromas of Madeira wines. Finally, it was demonstrated that the thermal degradation of sugars, especially of fructose, promotes the emergence of volatile compounds identified in baked wines.

Olfactory perception threshold assessment of volatile acidity in Madeira wines

Madeira wine is a fortified wine with impact in the Madeira Island’s economy. Similarly to other wines, its acidity should be well controlled in order to ensure Madeira wine quality, mostly the volatile acidity. Due to Madeira wine complex flavour, it is crucial to get a better knowledge about the volatile acidity impact in its features, namely determine the perception limit of acetic acid and ethyl acetate, as both are the main contributors for volatile acidity. Firstly, the olfactory perception threshold of volatile acidity was assessed by a trained and an untrained panel, using 5 and 10 years-old Sercial and Malvasia wines. Moreover, the current work also presents the evolution of organic acids, acetic acid and ethyl acetate during 540 days of ageing of Madeira wines (Malvasia, Bual, Verdelho and Sercial), comparing the same wines aged by both traditional ageing processes: canteiro and estufagem. Other wine samples, aged in wood casks (canteiro) for at least 5 years, were also evaluated. HS-SPME followed by GC-MS analysis was used to determine ethyl acetate concentration and IEC-HPLC-DAD was used for the organic acids determination, including acetic acid. The results indicated that acetic acid and ethyl acetate olfactory perception threshold depends essentially on wine’s age. Concerning acetic acid, the untrained panel was in average 5.45 g/L (5 years-old) and 6.22 g/L (10 years-old). Training the expert panel to recognize acetic acid odour, the values decreased for 1.44 g/L (5 years-old) and 1.87 g/L (10 years-old), but still remained higher than the established volatile acidity legal limits. Ethyl acetate threshold was similar for both panels (in average 327.97 mg/L). Both compounds tend to increase exponentially with age, being more evident in sweet wines. Organic acids in young Madeira wines depend mostly on the nature of grape varieties, but this difference is minimized with wine ageing.

Heterocyclic acetals in Madeira wines

The maturation of Madeira wines usually involves exposure to relatively high temperatures which affect the aroma and flavour composition leading to the formation of the typical and characteristic bouquet of these wines. The formation of heterocyclic acetals (1,3-dioxanes and 1,3-dioxolanes) was investigated in order to determine levels and for possible use as indicators of wine age. The results show a linear correlation of the investigated acetals with wine age but suggest that the acetalization reaction is not particularly affected by the drastic oxidative conditions observed during maturation.