C/EBP delta and C/EBP gamma bind the CCAAT-box in the human beta-globin promoter and modulate the activity of the CACC-box binding protein, EKLF

Developmental- and tissue-specific expression of globin genes is mediated by a few key elements within the proximal promoter of each gene. DNA-binding assays previously identified NF-Y, GATA-1, C/EBP beta and C/EBP gamma as candidate regulators of beta-globin transcription via the CCAAT-box, a promoter element situated between CACC- and TATA-boxes. We have identified C/EBP delta as an additional beta-globin CCAAT-box binding protein. In reporter assays, we show that C/EBP delta can co-operate with EKLF, a CACC-box binding protein, to activate the beta-globin promoter, whereas C/EBP gamma inhibits the transcriptional activity of EKLF in this assay. (c) 2005 Elsevier B.V. All rights reserved.

Genomic organization of human arylamine N-acetyltransferase Type I reveals alternative promoters that generate different 5'-UTR splice variants with altered translational activities

In humans, a polymorphic gene encodes the drug-metabolizing enzyme NATI (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NATI is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5'non-coding region of NATI. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NATI expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9-11-fold in a luciferase reporter assay, while the other isoforrns were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NATI transcripts may contribute to the variation in NATI activity in vivo.

The mismatch negativity (MMN) response to complex tones and spoken words in individuals with aphasia

Background: The mismatch negativity (MMN) is a fronto-centrally distributed event-related potential (ERP) that is elicited by any discriminable auditory change. It is an ideal neurophysiological tool for measuring the auditory processing skills of individuals with aphasia because it can be elicited even in the absence of attention. Previous MMN studies have shown that acoustic processing of tone or pitch deviance is relatively preserved in aphasia, whereas the basic acoustic processing of speech stimuli can be impaired (e.g., auditory discrimination). However, no MMN study has yet investigated the higher levels of auditory processing, such as language-specific phonological and/or lexical processing, in individuals with aphasia. Aims: The aim of the current study was to investigate the MMN response of normal and language-disordered subjects to tone stimuli and speech stimuli that incorporate the basic auditory processing (acoustic, acoustic-phonetic) levels of non-speech and speech sound processing, and also the language-specific phonological and lexical levels of spoken word processing. Furthermore, this study aimed to correlate the aphasic MMN data with language performance on a variety of tasks specifically targeted at the different levels of spoken word processing. Methods M Procedures: Six adults with aphasia (71.7 years +/- 3.0) and six healthy age-, gender-, and education-matched controls (72.2 years +/- 5.4) participated in the study. All subjects were right-handed and native speakers of English. Each subject was presented with complex harmonic tone stimuli, differing in pitch or duration, and consonant-vowel (CV) speech stimuli (non-word /de:/versus real world/deI/). The probability of the deviant for each tone or speech contrast was 10%. The subjects were also presented with the same stimuli in behavioural discrimination tasks, and were administered a language assessment battery to measure their auditory comprehension skills. Outcomes O Results: The aphasic subjects demonstrated attenuated MMN responses to complex tone duration deviance and to speech stimuli (words and non-words), and their responses to the frequency, duration, and real word deviant stimuli were found to strongly correlate with performance on the auditory comprehension section of the Western Aphasia Battery (WAB). Furthermore, deficits in attentional lexical decision skills demonstrated by the aphasic subjects correlated with a word-related enhancement demonstrated during the automatic MMN paradigm, providing evidence to support the word advantage effect, thought to reflect the activation of language-specific memory traces in the brain for words. Conclusions: These results indicate that the MMN may be used as a technique for investigating general and more specific auditory comprehension skills of individuals with aphasia, using speech and/or non-speech stimuli, independent of the individual's attention. The combined use of the objective MMN technique and current clinical language assessments may result in improved rehabilitative management of aphasic individuals.

Transdermal drug delivery: Basic principles for the veterinarian

The use of topical pharmaceutical formulations is increasingly popular in veterinary medicine. A potential concern is that not all formulations are registered for the intended species, yet current knowledge strongly suggests that simple extrapolation of transdermal drug pharmacokinetics and pharmacodynamics between species, including humans, cannot be done. In this review, an overview is provided of the underlying basic principles determining the movement of topically applied molecules into and through the skin. Various factors that may affect transdermal drug penetration between species, between individuals of a particular species and regional differences in an individual are also discussed. A good understanding of the basic principles of transdermal drug delivery is critical to avoid adverse effects or lack of efficacy when applying topical formulations in veterinary medicine. (c) 2005 Elsevier Ltd. All rights reserved.

Gene expression in human alcoholism: Microarray analysis of frontal cortex

Background: Changes in brain gene expression are thought to be responsible for the tolerance, dependence, and neurotoxicity produced by chronic alcohol abuse, but there has been no large scale study of gene expression in human alcoholism. Methods: RNA was extracted from postmortem samples of superior frontal cortex of alcoholics and nonalcoholics. Relative levels of RNA were determined by array techniques. We used both cDNA and oligonucleotide microarrays to provide coverage of a large number of genes and to allow cross-validation for those genes represented on both types of arrays. Results: Expression levels were determined for over 4000 genes and 163 of these were found to differ by 40% or more between alcoholics and nonalcoholics. Analysis of these changes revealed a selective reprogramming of gene expression in this brain region, particularly for myelin-related genes which were downregulated in the alcoholic samples. In addition, cell cycle genes and several neuronal genes were changed in expression. Conclusions: These gene expression changes suggest a mechanism for the loss of cerebral white matter in alcoholics as well as alterations that may lead to the neurotoxic actions of ethanol.

S. cerevisiae as a model for studying mutations in the human gene OPA1 associated with dominant optic atrophy and for drug discovery

L’atrofia ottica dominante (ADOA) è una malattia mitocondriale caratterizzata da difetti visivi, che si manifestano durante l’infanzia, causati da progressiva degenerazione delle cellule gangliari della retina (RGC). ADOA è una malattia genetica associata, nella maggior parte dei casi, a mutazioni nel gene OPA1 che codifica per la GTPasi mitocondriale OPA1, appartenente alla famiglia delle dinamine, principalmente coinvolta nel processo di fusione mitocondriale e nel mantenimento del mtDNA. Finora sono state identificate più di 300 mutazioni patologiche nel gene OPA1. Circa il 50% di queste sono mutazioni missenso, localizzate nel dominio GTPasico, che si pensa agiscano come dominanti negative. Questa classe di mutazioni è associata ad una sindrome più grave nota come “ADOA-plus”. Nel lievito Saccharomyces cerevisiae MGM1 è l’ortologo del gene OPA1: nonostante i due geni abbiano domini funzionali identici le sequenze amminoacidiche sono scarsamente conservate. Questo costituisce una limitazione all’uso del lievito per lo studio e la validazione di mutazioni patologiche nel gene OPA1, infatti solo poche sostituzioni possono essere introdotte e studiate nelle corrispettive posizioni del gene di lievito. Per superare questo ostacolo è stato pertanto costruito un nuovo modello di S. cerevisiae, contenente il gene chimerico MGM1/OPA1, in grado di complementare i difetti OXPHOS del mutante mgm1Δ. Questo gene di fusione contiene una larga parte di sequenza corrispondente al gene OPA1, nella quale è stato inserito un set di nuove mutazioni trovate in pazienti affetti da ADOA e ADOA-plus. La patogenicità di queste mutazioni è stata validata sia caratterizzando i difetti fenotipici associati agli alleli mutati, sia la loro dominanza/recessività nel modello di lievito. A tutt’oggi non è stato identificato alcun trattamento farmacologico per la cura di ADOA e ADOA-plus. Per questa ragione abbiamo utilizzato il nostro modello di lievito per la ricerca di molecole che agiscono come soppressori chimici, ossia composti in grado di ripristinare i difetti fenotipici indotti da mutazioni nel gene OPA1. Attraverso uno screening fenotipico high throughput sono state testate due differenti librerie di composti chimici. Questo approccio, noto con il nome di drug discovery, ha permesso l’identificazione di 23 potenziali molecole attive.

Reformulation of a thermostable broadly protective recombinant vaccine against human papilloma virus

The causal relationship between Human Papilloma Virus (HPV) infection and cervical cancer has motivated the development, and further improvement, of prophylactic vaccines against this virus. 70% of cervical cancers, 80% of which in low-resources countries, are associated to HPV16 and HPV18 infection, with 13 additional HPV types, classified as high-risk, responsible for the remaining 30% of tumors. Current vaccines, Cervarix® (GlaxoSmithKline) and Gardasil®(Merk), are based on virus-like particles (VLP) obtained by self-assembly of the major capsid protein L1. Despite their undisputable immunogenicity and safety, the fact that protection afforded by these vaccines is largely limited to the cognate serotypes included in the vaccine (HPV 16 and 18, plus five additional viral types incorporated into a newly licensed nonavalent vaccine) along with high production costs and reduced thermal stability, are pushing the development of 2nd generation HPV vaccines based on minor capsid protein L2. The increase in protection broadness afforded by the use of L2 cross-neutralizing epitopes, plus a marked reduction of production costs due to bacterial expression of the antigens and a considerable increase in thermal stability could strongly enhance vaccine distribution and usage in low-resource countries. Previous studies from our group identified three tandem repeats of the L2 aa. 20-38 peptide as a strongly immunogenic epitope if exposed on the scaffold protein thioredoxin (Trx). The aim of this thesis work is the improvement of the Trx-L2 vaccine formulation with regard to cross-protection and thermostability, in order to identify an antigen suitable for a phase I clinical trial. By testing Trx from different microorganisms, we selected P. furiosus thioredoxin (PfTrx) as the optimal scaffold because of its sustained peptide epitope constraining capacity and striking thermal stability (24 hours at 100°C). Alternative production systems, such as secretory Trx-L2 expression in the yeast P. pastoris, have also been set-up and evaluated as possible means to further increase production yields, with a concomitant reduction of production costs. Limitations in immune-responsiveness caused by MHC class II polymorphisms –as observed, for example, in different mouse strains- have been overcome by introducing promiscuous T-helper (Th) epitopes, e.g., PADRE (Pan DR Epitope), at both ends of PfTrx. This allowed us to obtain fairly strong immune responses even in mice (C57BL/6) normally unresponsive to the basic Trx-L2 vaccine. Cross-protection was not increased, however. I thus designed, produced and tested a novel multi-epitope formulation consisting of 8 and 11 L2(20-38) epitopes derived from different HPV types, tandemly joined into a single thioredoxin molecule (“concatemers”). To try to further increase immunogenicity, I also fused our 8X and 11X PfTrx-L2 concatemers to the N-terminus of an engineered complement-binding protein (C4bp), capable to spontaneously assemble into ordered hepatmeric structures, previously validated as a molecular adjuvant. Fusion to C4bp indeed improved antigen presentation, with a fairly significant increase in both immunogenicity and cross-protection. Another important issue I addressed, is the reduction of vaccine doses/treatment, which can be achieved by increasing immunogenicity, while also allowing for a delayed release of the antigen. I obtained preliminary, yet quite encouraging results in this direction with the use of a novel, solid-phase vaccine formulation, consisting of the basic PfTrx-L2 vaccine and its C4bp fusion derivative adsorbed to mesoporus silica-rods (MSR).

EDUCAÇÃO INFANTIL COMO DIREITO FUNDAMENTAL DA CRIANÇA CEGA CONGÊNITA

Esta dissertação se refere a uma pesquisa exploratória que tem como objetivo a Educação Infantil como direito fundamental da criança cega congênita de zero a cinco anos, ou que tenha ficado cega até os 12 meses de vida. Buscou-se compreender os benefícios da integração nos espaços educativos infantis públicos, as políticas públicas federais e as do município de São Paulo, bem como a relação do direito à educação na Modalidade Educação Especial. Para tanto, aborda o que é a cegueira, relacionando aspectos históricos da educação das pessoas com deficiência visual e de políticas públicas com o direito à educação. Embora a educação tenha despertado o interesse de muitos órgãos da sociedade e de agências da ONU, envolvendo documentos jurídicos como a Declaração Universal de Direitos Humanos (1948), a Constituição Federal do Brasil (1988), o Estatuto da Criança e do Adolescente (1990), a Declaração de Salamanca (1994), que se constitui em um marco da Educação Especial, a Lei de Diretrizes e Bases da Educação Nacional (1996) lei esta que garante a educação como direito de todos, conforme o enunciado constitucional e, ainda que a Educação Infantil seja essencial como alicerce da educação básica, ela ainda não é vista como direito fundamental. Esta pesquisa mostra que tanto crianças cegas congênitas quanto seus pais enfrentam obstáculos quando procuram as escolas: falta de vagas nas Creches e EMEIs, formação insuficiente dos pedagogos para trabalhar com a inclusão do aluno com necessidades educacionais especiais, estigmas e falta de estrutura física para a acessibilidade e autonomia do discente. A falta de salas de apoio à inclusão e de equipamentos de educação infantil, bem como de pessoal especializado, são alguns dos exemplos da situação evidenciada, que necessita de um olhar de caráter interventivo no município de São Paulo, sob pena de responsabilização das autoridades responsáveis por sua oferta, por ferir um direito que é fundamental pelas leis nacionais e internacionais.

A INSERÇÃO DE BEBÊS EM CRECHES: UM OLHAR PARA AS POLÍTICAS PÚBLICAS

O presente trabalho propõe um estudo sobre a inserção de bebês em creches públicas no município de São Paulo. De acordo com Rosemberg (2010), a infância constitui fase importantíssima na formação da criança e embora a duração da primeira infância seja de curta duração, considerando-se a expectativa de vida de 70 anos, ela constitui a vida inteira dos bebês e das crianças pequenas. Nos dias atuais os bebês ingressam na creche a partir dos quatro meses de idade e lá permanecem por até dez horas. Nesse sentido, esta pesquisa buscou compreender as políticas públicas para esse atendimento, o qual, com a Constituição de 1988, foi considerado a primeira etapa da Educação Básica compondo a Educação Infantil brasileira, de oferta obrigatória e direito das crianças, garantindo, em complementação à família, o desenvolvimento integral da criança pequena. Desse contexto, alguns questionamentos foram trazidos para a discussão: quais as propostas de atendimento de bebês na creche e como funcionam as instituições que os recebem? Qual o olhar das políticas públicas para esse segmento de educação? A Constituição garante o ingresso dos bebês na creche, mas e seu desenvolvimento integral, está garantido? Recentemente atrelada à esfera educacional, a creche tem o desafio de compreender seu papel com essas crianças, desvinculando-se de práticas apenas assistencialistas e higienistas, e de construir novas concepções acerca desse atendimento. Tais concepções ficam explícitas não nas politicas públicas, mas efetivam-se na prática da creche, nas atividades desenvolvidas, nos espaços e processos pedagógicos pensados para receber o bebê. Em face do exposto, esta pesquisa possibilitou inferir que, apesar dos avanços acerca do atendimento educacional ofertado a primeira infância, falta ainda clareza por parte da sociedade em geral, sobre a importância de uma educação de qualidade para as crianças pequenas e seu impacto na formação humana. Essa lacuna merece o olhar das políticas públicas, uma vez que demanda ações nas diversas instâncias da creche, desde a formação e a valorização do professor de Educação Infantil, até a estrutura física e a escassez das vagas. Os poucos estudos que discutem tais políticas para a educação de bebês nas creches, justificam a realização deste trabalho.