Multiple domains of repressor activator protein 1 contribute to facilitated binding of glycolysis regulatory protein 1

The function of repressor activator protein 1 (Rap1p) at glycolytic enzyme gene upstream activating sequence (UAS) elements in Saccharomyces cerevisiae is to facilitate binding of glycolysis regulatory protein 1 (Gcr1p) at adjacent sites. Rap1p has a modular domain structure. In its amino terminus there is an asymmetric DNA-bending domain, which is distinct from its DNA-binding domain, which resides in the middle of the protein. In the carboxyl terminus of Rap1p lie its silencing and putative activation domains. We carried out a molecular dissection of Rap1p to identify domains contributing to its ability to facilitate binding of Gcr1p. We prepared full-length and three truncated versions of Rap1p and tested their ability to facilitate binding of Gcr1p by gel shift assay. The ability to detect ternary complexes containing Rap1p⋅DNA⋅Gcr1p depended on the presence of binding sites for both proteins in the probe DNA. The DNA-binding domain of Rap1p, although competent to bind DNA, was unable to facilitate binding of Gcr1p. Full-length Rap1p and the amino- and carboxyl-truncated versions of Rap1p were each able to facilitate binding of Gcr1p at an appropriately spaced binding site. Under these conditions, Gcr1p displayed an approximately 4-fold greater affinity for Rap1p-bound DNA than for otherwise identical free DNA. When spacing between Rap1p- and Gcr1p-binding sites was altered by insertion of five nucleotides, the ability to form ternary Rap1p⋅DNA⋅Gcr1p complexes was inhibited by all but the DNA-binding domain of Rap1p itself; however, the ability of each individual protein to bind the DNA probe was unaffected.

Cooperative functions of the reaper and head involution defective genes in the programmed cell death of Drosophila central nervous system midline cells

In Drosophila, the chromosomal region 75C1–2 contains at least three genes, reaper (rpr), head involution defective (hid), and grim, that have important functions in the activation of programmed cell death. To better understand how cells are killed by these genes, we have utilized a well defined set of embryonic central nervous system midline cells that normally exhibit a specific pattern of glial cell death. In this study we show that both rpr and hid are expressed in dying midline cells and that the normal pattern of midline cell death requires the function of multiple genes in the 75C1–2 interval. We also utilized the P[UAS]/P[Gal4] system to target expression of rpr and hid to midline cells. Targeted expression of rpr or hid alone was not sufficient to induce ectopic midline cell death. However, expression of both rpr and hid together rapidly induced ectopic midline cell death that resulted in axon scaffold defects characteristic of mutants with abnormal midline cell development. Midline-targeted expression of the baculovirus p35 protein, a caspase inhibitor, blocked both normal and ectopic rpr- and hid-induced cell death. Taken together, our results suggest that rpr and hid are expressed together and cooperate to induce programmed cell death during development of the central nervous system midline.

Genomic and genetic dissection of an archaeal regulon

The extremely halophilic archaeon Halobacterium sp. NRC-1 can grow phototrophically by means of light-driven proton pumping by bacteriorhodopsin in the purple membrane. Here, we show by genetic analysis of the wild type, and insertion and double-frame shift mutants of Bat that this transcriptional regulator coordinates synthesis of a structural protein and a chromophore for purple membrane biogenesis in response to both light and oxygen. Analysis of the complete Halobacterium sp. NRC-1 genome sequence showed that the regulatory site, upstream activator sequence (UAS), the putative binding site for Bat upstream of the bacterio-opsin gene (bop), is also present upstream to the other Bat-regulated genes. The transcription regulator Bat contains a photoresponsive cGMP-binding (GAF) domain, and a bacterial AraC type helix–turn–helix DNA binding motif. We also provide evidence for involvement of the PAS/PAC domain of Bat in redox-sensing activity by genetic analysis of a purple membrane overproducer. Five additional Bat-like putative regulatory genes were found, which together are likely to be responsible for orchestrating the complex response of this archaeon to light and oxygen. Similarities of the bop-like UAS and transcription factors in diverse organisms, including a plant and a γ-proteobacterium, suggest an ancient origin for this regulon capable of coordinating light and oxygen responses in the three major branches of the evolutionary tree of life. Finally, sensitivity of four of five regulon genes to DNA supercoiling is demonstrated and correlated to presence of alternating purine–pyrimidine sequences (RY boxes) near the regulated promoters.

Integration host factor suppresses promiscuous activation of the sigma 54-dependent promoter Pu of Pseudomonas putida.

In the presence of m-xylene, the Pu promoter of the TOL plasmid of Pseudomonas putida is activated by the prokaryotic enhancer-binding protein XylR. The intervening DNA segment between the upstream activating sequences (UASs) and those for RNA polymerase binding contains an integration host factor (IHF) attachment site that is required for full transcriptional activity. In the absence of IHF, the Pu promoter can be cross-activated by other members of the sigma 54-dependent family of regulatory proteins. Such illegitimate activation does not require the binding of the heterologous regulators to DNA and it is suppressed by bent DNA structures, either static or protein induced, between the promoter core elements (UAS and RNA polymerase recognition sequence). The role of IHF in some sigma 54 promoters is, therefore, not only a structural aid for assembling a correct promoter geometry but also that of an active suppressor (restrictor) of promiscuous activation by heterologous regulators for increased promoter specificity.

Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes.

Regulation of Synaptogenesis by the miRNA Pathway and FMR/P Bodies

Post-transcriptional regulation of mRNA is facilitated by different mechanisms, such as microRNA (miRNA) induced gene silencing or fragile X mental retardation protein (FMRP) mediated repression either independent of or acting through cytoplasmic RNA Processing bodies (P bodies). DPTP99A, Lar, and Wg have known functions during synaptogenesis and may be targets of miR-8. Here, we provide evidence that miR-8 regulates DPTP99A in vitro. Non-endogenous miR-8 expressed using an UAS driver regulates Lar. Endogenous miR-8 may regulate DPTP99A in vivo. Here we show that FMRP is capable of colocalizing with the P body components: DCP1, HPat, and Me31B, but not CCR4. We also show that RNAi against HPat and Me31B but not CCR4 and DCP1 are required for FMRP’s repression of a translational reporter in vivo. This functional analysis provides additional insight into another aspect of FMRP’s and P bodies’ ability to cooperatively control repression of mRNA targets.

Let´s go the University Laboratory: a successful collaborative experience

Paper submitted to ICERI2013, the 6th International Conference of Education, Research and Innovation, Seville (Spain), November 18-20, 2013.

2C-Cas9: a versatile tool for clonal analysis of gene function

CRISPR/Cas9-mediated targeted mutagenesis allows efficient generation of loss-of-function alleles in zebrafish. To date this technology has been primarily used to generate genetic knockout animals. Nevertheless, the study of the function of certain loci might require tight spatiotemporal control of gene inactivation. Here, we show that tissue-specific gene disruption can be achieved by driving Cas9 expression with the Gal4/UAS system. Furthermore, by combining the Gal4/UAS and Cre/loxP systems, we establish a versatile tool to genetically label mutant cell clones, enabling their phenotypic analysis. Our technique has the potential to be applied to diverse model organisms, enabling tissue-specific loss-of-function and phenotypic characterization of live and fixed tissues.

Lyra Gesang Verein (Ann Arbor, Mich.), organized 1885

On mat: Reuben Kempf born 1859 - Ann Arbor; Died 1945 - Ann Arbor. Graduate Royal Conservatory of Music 1879, Stuttgart, Germany. On verso: Other identification in UAs Kempf, Reuben H.

Two NextGen Air Safety Tools: An ADS-B Equipped UAV and a Wake Turbulence Estimator

Thesis (Master's)--University of Washington, 2016-06

Functional dissection of the Suppressor of UnderReplication protein of Drosophila melanogaster: identification of domains influencing chromosome binding and DNA replication

The Suppressor of UnderReplication (SuUR) gene controls the DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster salivary gland polytene chromosomes. In the present work, we investigate the functional importance of different regions of the SUUR protein by expressing truncations of the protein in an UAS-GAL4 system. We find that SUUR has at least two separate chromosome-binding regions that are able to recognize intercalary and pericentric heterochromatin specifically. The C-terminal part controls DNA underreplication in intercalary heterochromatin and partially in pericentric heterochromatin regions. The C-terminal half of SUUR suppresses endoreplication when ectopically expressed in the salivary gland. Ectopic expression of the N-terminal fragments of SUUR depletes endogenous SUUR from polytene chromosomes, causes the SuUR(-) stopphenotype and induces specific swellings in heterochromatin.

Towards platform-independent real-time systems

Real-time software systems are rarely developed once and left to run. They are subject to changes of requirements as the applications they support expand, and they commonly outlive the platforms they were designed to run on. A successful real-time system is duplicated and adapted to a variety of applications - it becomes a product line. Current methods for real-time software development are commonly based on low-level programming languages and involve considerable duplication of effort when a similar system is to be developed or the hardware platform changes. To provide more dependable, flexible and maintainable real-time systems at a lower cost what is needed is a platform-independent approach to real-time systems development. The development process is composed of two phases: a platform-independent phase, that defines the desired system behaviour and develops a platform-independent design and implementation, and a platform-dependent phase that maps the implementation onto the target platform. The last phase should be highly automated. For critical systems, assessing dependability is crucial. The partitioning into platform dependent and independent phases has to support verification of system properties through both phases.

BASES DE PODER SOCIAL, ENGAJAMENTO NO TRABALHO E RESILIÊNCIA: UM ESTUDO COM SERVIDORES PÚBLICOS MUNICIPAIS DE DIADEMA - SP

Este estudo teve a intenção de analisar até que ponto o uso de diferentes bases de poder dos superiores hierárquicos predizem os níveis de engajamento no trabalho e de resiliência dos trabalhadores, colaborando para aumentar o conhecimento sobre o comportamento dos servidores públicos municipais, quanto aos níveis de engajamento no trabalho e resiliência apresentados. Partiu-se da definição de poder de French e Raven (1959): poder é a influência potencial que o agente O poderia causar no sujeito P; adotou-se o conceito de engajamento no trabalho de Schaufeli e Bakker (2003), que definem engajamento no trabalho como um construto motivacional positivo, caracterizado por vigor, dedicação e absorção, sempre relacionado ao trabalho, o qual implica sentimento de realização, envolve estado cognitivo positivo, é persistente no tempo, apresentando, assim, natureza motivacional e social e por fim utilizou-se o conceito de Grotberg (2005) que define resiliência como a capacidade humana para enfrentar, vencer e ser fortalecido ou transformado por experiências de adversidades . Para isto, definiu-se como objetivo geral testar a capacidade preditiva das bases de poder social dos chefes sobre a resiliência e o engajamento no trabalho, em servidores públicos municipais de Diadema - SP. Participaram deste estudo 95 servidores públicos municipais do município de Diadema, SP, com pequena maioria de indivíduos do sexo feminino (51,6%), com maior percentual de idade entre 25 e 40 anos (38,9%). A maioria dos participantes (60%) declarou possuir nível superior completo (35,8%) ou pós-graduação (24,2%). Utilizou-se os instrumentos: Escala de Bases de Poder do Supervisor (EBPS), escala desenvolvida por Martins e Guimarães (2007); Escala de Avaliação da Resiliência (EAR), escala construída por Martins, Siqueira e Emilio (2011) e a Escala de Engajamento no Trabalho de UTRECHT (UWES) que tiveram seus indicadores de validade e fidedignidade apurados neste estudo. Como resultado constatou-se parcialmente a existência de associação entre engajamento no trabalho e resiliência, pois engajamento no trabalho correlacionou-se com três dos cinco fatores de resiliência: adaptação positiva à mudanças, competência pessoal e persistência diante de dificuldade. Verificou-se que as dimensões que compõem a variável resiliência obtiveram médias ao redor do ponto quatro da escala de resiliência (frequentemente é verdade), indicando que os participantes frequentemente percebem a si mesmos como capazes de enfrentar as adversidades da vida em função da sua alta percepção de persistência, capazes de adaptar-se às mudanças, com bom nível de competência pessoal e de espiritualidade. Constatou-se que as médias nas dimensões que compõem a variável engajamento no trabalho ficaram muito próximas do ponto quatro da escala de engajamento (algumas vezes na semana), indicando que eles percebem em si um alto grau engajamento no trabalho, ou seja, que possuem vigor, são dedicados e deixam-se absorver pelo trabalho. Verificou-se ainda que os trabalhadores percebem o poder de perícia como o mais empregado pelos seus superiores hierárquicos com média de 4,46 (DP= 0,71). Por fim, os resultados obtidos apontaram que o papel e o posicionamento da chefia não provocaram impacto significativo em nenhuma das variáveis consequentes, portanto, bases de poder não explicam resiliência para os trabalhadores participantes desta pesquisa como também não predisseram engajamento no trabalho.

Speech recognition use in healthcare applications

Speech recognition technology is regarded as a key enabler for increasing the usability of applications deployed on mobile devices -- devices which are becoming increasingly prevalent in modern hospital-based healthcare. Although the use of speech recognition is not new to the hospital-based healthcare domain, its use with mobile devices has thus far been limited. This paper presents the results of a literature review we conducted in order to observe the manner in which speech recognition technology has been used in hospital-based healthcare and to gain an understanding of how this technology is being evaluated, in terms of its dependability and reliability, in healthcare settings. Our intent is that this review will help identify scope for future uses of speech recognition technologies in the healthcare domain, as well as to identify implications for the meaningful evaluation of such technologies given the specific context of use.

Speech recognition use in healthcare applications

Speech recognition technology is regarded as a key enabler for increasing the usability of applications deployed on mobile devices -- devices which are becoming increasingly prevalent in modern hospital-based healthcare. Although the use of speech recognition is not new to the hospital-based healthcare domain, its use with mobile devices has thus far been limited. This paper presents the results of a literature review we conducted in order to observe the manner in which speech recognition technology has been used in hospital-based healthcare and to gain an understanding of how this technology is being evaluated, in terms of its dependability and reliability, in healthcare settings. Our intent is that this review will help identify scope for future uses of speech recognition technologies in the healthcare domain, as well as to identify implications for the meaningful evaluation of such technologies given the specific context of use.