Characterization of the role of Drosophila JAK/STAT signalling in cellular proliferation

Der Janus Kinase / signal transducer and activator of transcription (JAK/STAT) Signal- transduktionsweg wird für viele Entwicklungsvorgänge benötigt und spielt eine zentrale Rolle bei der Hämatopoese und bei der Immunantwort. Obwohl der JAK/STAT-Signalweg in den vergangenen Jahren Gegenstand intensiver Forschung war, erschwert die Redundanz des Signalwegs bei Wirbeltieren genetische Untersuchungen zur Identifizierung derjenigen Mechanismen, die den JAK/STAT-Signalweg regulieren. Der JAK/STAT-Signaltransduktionsweg ist evolutionär konserviert und ebenfalls bei der Taufliege Drosophila melanogaster vorhanden. Im Gegensatz zu Wirbeltieren ist der Signaltransduktionsweg von Drosophila weniger redundant und beinhaltet folgende Hauptkomponenten: den Liganden Unpaired (Upd), den Transmembranrezeptor Domeless (Dome), die einzige JAK-Tyrosinkinase Hopscotch (hop), sowie den Transkriptionsfaktor STAT92E. In der vorliegenden Arbeit wird die Rolle des JAK/STAT-Signalwegs bei der zellulären Proliferation mithilfe der Modellsysteme der Flügel- und der Augen-Imaginalscheiben von Drosophila charakterisiert. "Loss-of-function"- und "Gain-of-function"-Experimente zur Verminderung beziehungs-weise Erhöhung der Signalaktivität zeigten, dass der JAK/STAT-Signalweg eine Rolle bei der zellulären Proliferation der Flügel-Imaginalscheiben spielte, ohne die Zellgröße oder Apoptose zu verändern. Bei der Flügelentwicklung während des zweiten und des frühen dritten Larvalstadiums war die Aktivität des JAK/STAT-Signalwegs sowohl notwendig für die zelluläre Proliferation als auch hinreichend, um Überproliferation anzutreiben. Allerdings änderte sich während der späten dritten Larvalstadien die JAK/STAT-Signalaktivität, sodass endogene STAT92E-Mengen einen anti-proliferativen Effekt im gleichen Gewebe aufwiesen. Weiterhin reichte die ektopische Aktivierung des JAK/STAT-Signalwegs zu diesem späten Entwicklungszeitpunkt aus, um die Mitose zu inhibieren und die Zellen in der Phase G2 des Zellzyklus zu arretieren. Diese Ergebnisse legen den Schluss nahe, dass der JAK/STAT-Signalweg sowohl pro-proliferativ in frühen Flügelscheiben als auch anti-proliferativ zu späten Stadien der Flügelscheiben-Entwicklung wirken kann. Dieser späte anti-proliferative Effekt wurde durch einen nicht-kanonischen Mechanismus der STAT92E-Aktivierung vermittelt, da späte hop defiziente Zellverbände im Vergleich zu Wildtyp-Zellen keine Veränderungen im Ausmaß der zellulären Proliferation aufwiesen. Ferner konnte gezeigt werden, dass eine während der Larvalstadien exprimierte dominant-negative und im N-Terminus deletierte Form von STAT92E (?NSTAT92E) nicht für den anti-proliferativen Effekt verantwortlich ist. Diese Tatsache ist ein weiteres Indiz dafür, dass das vollständige STAT92E den späten anti-proliferativen Effekt verursacht. Um Modulatoren für die von JAK/STAT vermittelte zelluläre Proliferation zu identifieren, wurde ein P-Element-basierter genetischer Interaktions-Screen in einem sensibilisierten genetischen Hintergrund durchgeführt. Insgesamt wurden dazu 2267 unabhängige P-Element-Insertionen auf ihre Wechselwirkung mit der JAK/STAT-Signalaktivität untersucht und 24 interagierende Loci identifiziert. Diese Kandidaten können in folgende Gruppen eingeordnet werden: Zellzyklusproteine, Transkriptionsfaktoren, DNA und RNA bindende Proteine, ein Mikro-RNA-Gen, Komponenten anderer Signaltransduktionswege und Zelladhäsionsproteine. In den meisten Fällen wurden mehrere Allele der interagierenden Kandidatengene getestet. 18 Kandidatengene mit übereinstimmend interagierenden Allelen wurden dann zur weiteren Analyse ausgewählt. Von diesen 18 Kandidaten-Loci wurden 7 mögliche JAK/STAT-Signalwegskomponenten und 6 neue Zielgene des Signalwegs gefunden. Zusammenfassend wurde das Verständnis um STAT92E verbessert. Dieses Protein hat die gleiche Funktion wie das STAT3-Protein der Wirbeltiere und treibt die zelluläre Proliferation voran. Analog zu STAT1 hat STAT92E aber auch einen anti-proliferativen Effekt. Ferner wurden 24 mögliche Modulatoren der JAK/STAT-Signalaktivität identifiziert. Die Charakterisierung dieser Wechselwirkungen eröffnet vielversprechende Wege zu dem Verständnis, wie JAK/STAT die zelluläre Proliferation reguliert und könnte bei der Entwicklung von neuartigen therapeutischen Targets zur Behandlung von Krebskrankheiten und Entwicklungsstörungen beitragen.

Accidentes con riesgo biológico en una población afiliada a una ARL, 2014, Colombia

Introducción: Existen condiciones que definen la gravedad de accidentes laborales de tipo riesgo biológico como el contacto con una fuente conocida o desconocida, tipo de exposición según el fluido de contacto, el elemento causante y el tipo de lesión que causa. Objetivo: Describir e identificar la ocurrencia de los accidentes de trabajo con riesgo biológico que tienen la población trabajadora del sector salud y afines, afiliados a una ARL reportados durante el año 2014, suministradas en una base de datos de la IPS encargada del manejo de los eventos, con el fin de destacar su impacto y métodos de prevención de los mismos a través de la ARL. Materiales y métodos: Se realizó un estudio de corte transversal en el que se analizaron datos secundarios procedentes de una base de datos que incluyo los eventos sobre riesgo biológico reportados a una ARL entre el 1 de febrero del 2014 y el 31 de diciembre de 2014, se reportaron 1806 accidentes de tipo riesgo biológico donde se analizaron características sociodemográficas, tipo de fluido, tipo de fuente, tipo de accidente y tipo de elemento. Resultados: Se evidencio que los auxiliares de la salud fueron el grupo con mayor accidentalidad (65%) y los técnicos (49.9%). El sitio de mayor frecuencia de ocurrencia de los accidentes fue el intrahospitalario con 89.5%. La mayor accidentalidad se relacionó con el contacto con sangre y se presentó en trabajadores con nivel universitario (89.8%), seguido por los técnicos (84.6%) y auxiliares (67.3%). Se observó una ocurrencia de accidentes entre trabajadores con nivel primaria y secundaria con un porcentaje de 95.7% y 91.6% respectivamente, quienes además tienen mayor riesgo a tener accidentes con fuentes desconocidas, mientras que los técnicos (68.5%) y los universitarios (80.1%) fueron con fuente conocida. Dentro del grupo de auxiliares se destaca accidentes con fuente desconocida con 53.4%. Debido a la manipulación de elementos cortopunzantes con las manos, los dedos fueron la parte anatómica más afectada (84.1%) con elementos de alto riesgo y específicamente con sangre (77.1%). Conclusiones: El personal del área de la salud fue el más afectado dado el contacto directo con pacientes. Las auxiliares de servicios de salud fueron las más afectadas por tener contacto directo en la manipulación de elementos cortopunzantes con fluidos de alto riesgo como la sangre, y los dedos la parte anatómica más afectada con fuente conocida.

Fitness of transgenic Anopheles stephensi mosquitoes expressing the SM1 peptide under the control of a vitellogenin promoter

Three transgenic Anopheles stephensi lines were established that strongly inhibit transmission of the mouse malaria parasite Plasmodium berghei. Fitness of the transgenic mosquitoes was assessed based on life table analysis and competition experiments between transgenic and wild-type mosquitoes. Life table analysis indicated low fitness load for the 2 single-insertion transgenic mosquito lines VD35 and VD26 and no load for the double-insertion transgenic mosquito line VD9. However, in cage experiments, where each of the 3 homozygous transgenic mosquitoes was mixed with nontransgenic mosquitoes, transgene frequency of all 3 lines decreased with time. Further experiments suggested that reduction of transgene frequency is a consequence of reduced mating success, reduced reproductive capacity, and/or insertional mutagenesis, rather than expression of the transgene itself. Thus, for transgenic mosquitoes released in the field to be effective in reducing malaria transmission, a driving mechanism will be required.

Characterization of two full-sized P elements from Drosophila sturtevanti and Drosophila prosaltans

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The distribution of the P-M hybrid dysgenesis system in Drosophila melanogaster strains from Brazil

Wild-caught flies of Drosophila melanogaster from seven natural populations of extreme regions of Brazil (Sao Luis, MA; Teresina, PI; Rio Cipo, MG; Maringa, PR; Sao Jose do Rio Preto, SP; Joinville, SC; and Porto Alegre, RS) were studied with the purpose of evaluating hybrid dysgenesis due to mobilization of P elements and the regulatory capacity of the strains' cytotypes. Diagnostic crosses were made and the strains classified according to their P-M phenotypes. Four strains were classified as moderate P (MA, MG, PI, and SP), two as Q (PR and RS) and one as M' (SC). Females of southern strains (PR, SC, and RS) presented in A crosses lower degrees of gonadal dysgenesis scores than those from northern strains (MA and PI).

P elements in the saltans group of Drosophila: a new evaluation of their distribution and number of genomic insertion sites

Few are studies on P elements that have addressed the saltans group. These studies had shown that species from the cordata and elliptica subgroups were devoid of any discernible P homologous sequences, while species from the parasaltans, sturtevanti, and saltans subgroups all contain P element sequences. Our analyses showed the presence of one to 15 P element insertion sites in species of the saltans group, including Drosophila neocordata and Drosophila emarginata (cordata and elliptica subgroups, respectively). From these species, only those from the parasaltans, sturtevanti, and saltans subgroups harbor canonical P elements and, only those of the last two subgroups seem to harbor putative full-sized elements. Due to the low similarity of the sequences found in D. neocordata and D. emarginata to those earlier described, we suggest that these sequences might be rudimental P element derivatives that were present in the ancestral of the subgenus Sophophora. (C) 2004 Elsevier B.V. All rights reserved.

Drosophila melanogaster P transposable elements: mechanisms of transposition and regulation

The molecular mechanisms that control P element transposition and determine its tissue specificity remain incompletely understood, although much information has been compiled about this element in the last decade. This review summarizes the currently available information about P element transposition, P-M hybrid dysgenesis and P cytotype features, P element-encoded repressors, and regulation of transposition.

Canonical P elements are transcriptionally active in the saltans group of Drosophila

Up to now, investigations of expression and regulation of P transposable element have been almost exclusively carried out with the Drosophila melanogaster canonical P element. Analyzing eight species of the saltans group, we detected transposase mRNA in germline tissues of D. saltans and D. prosaltans and repressor mRNA in somatic tissues of D. saltans and D. sturtevanti. Sequencing analysis suggested that these transcripts might belong to the canonical subfamily and that they can be transpositionally active only in D. saltans. dN and dS values of Adh and the P element suggested that the sequences found in D. saltans and D. prosaltans might have been present in the ancestor of the saltans subgroup and that the sequence found in D. sturtevanti might have been horizontally transferred from D. saltans.

Influence of brush type as a carrier of adhesive solutions and paper points as an adhesive-excess remover on the resin bond to root dentin

Purpose: To evaluate the influence of the brush type as a earner of priming adhesive solutions and the use of paper points as a remover of the excess of these solutions on the push-out bond strength of resin cement to bovine root dentin. The null hypotheses were that brush type and the use of paper points do not affect the bond strength. Materials and Methods: The canals of 80 single-root bovine roots (16 mm in length) were prepared at 12 mm using the preparation drill (FRC Postec Plus, Ivoclar). Half of each root was embedded in acrylic resin and the specimens were divided into 8 groups, considering the factors brush type (4 levels) and paper point (2 levels) (n = 10): Gr 1: small microbrush (Cavi-Tip, SDI); Gr 2: Microbrush (Dentsply); Gr 3: Endobrush (Bisco); Gr 4: conventional brush (Bisco); Gr 5: Cavi-Tip (SDI) + paper points; Gr 6: Microbrush (Dentsply) + paper points; Gr 7: Endobrush (Bisco) + paper points; Gr 8: conventional brush (Bisco) + paper points. The root dentin was treated with a multistep total-etch adhesive system (All Bond 2). The adhesive system was applied using each microbrush, with and without using paper points. One fiber post was molded with addition silicon and 80 posts were made of resin cement (Duolink), The resin posts were luted (Duolink resin cement), and the specimens were stored for 24 h in water at 37°C. Each specimen was cut into 4 disk-shaped samples (1.8 mm in thickness), which were submitted to the push-out test. Results: The brush type (p < 0.0001) (small microbrush > microbrush = endobrush = conventional brush) and the use of paper points (p = 0.0001) (with > without) influenced the bond strength significantly (two-way ANOVA). The null hypotheses were rejected. Conclusion: The smallest brush (Cavi-Tip) and the use of paper points significantly improved the resin bond to bovine root dentin.

Repair bond strength of microhybrid, nanohybrid and nanofilled resin composites: Effect of substrate resin type, surface conditioning and ageing

Objectives: This study evaluated the microtensile bond strength (MTBS) of non-aged and aged resin-based composites (RBC) (nanohybrid and nanofilled) after two surface conditioning methods, repaired using the composite of the same kind or a microhybrid composite. Materials and methods: Nanohybrid (Tetric EvoCeram-TE) and nanofilled (Filtek Supreme-FS) RBC blocks (5 × 5 × 6 mm) (N = 128) were fabricated and randomly divided into two groups: (a) no ageing (control group) and (b) ageing (5.000 thermocycling, 5-55 °C). RBC surfaces were polished by up to 1,200-grit silicone carbide papers and conditioned with either (a) air abrasion with 30-μm SiO2 particles (CoJet Sand) for 4 s + silane coupling agent (ESPE-Sil) + adhesive resin (VisioBond) (n = 16) or (b) adhesive application only (Multilink A+B for TE; Adper ScotchBond 1XT for FS) (n = 16). In half of the groups, repair resin of the same kind with the RBC and, in the other half, a different kind of composite (microhybrid, Quadrant Anterior Shine-AS) with its corresponding adhesive (Quadrant UniBond) was used. The specimens were submitted to MTBS test (0.5 mm/min). Data were analysed using three-way ANOVA and Tukey's tests. Degree of conversion (DC) of non-aged and aged resin composites (TE, FS) (n = 3 per group) was measured by micro-Raman analyses. Results: RBC type (p = 0.001) and ageing affected the MTBS results significantly (p = 0.001). Surface conditioning type did not show significant difference (p = 0.726), but less number of pre-test failures was experienced with the CoJet system compared to adhesive resin application only. Repair strength on aged TE showed significantly less (p < 0.05) MTBS than for FS. FS repaired with the same kind of RBC and adhesive resin presented the highest cohesive failures (43 %). DC was higher for TE (71 %) than for FS (58 %) before ageing. Conclusion: On the aged RBCs, less favourable repair strength could be expected especially for nanohybrid composite. For repair actions, RBC surface conditioning could be accomplished with either adhesive resin application only or with CoJet system, providing that the latter resulted in less pre-test failures. Clinical relevance: Clinicians could condition the resin surface prior to repair or relayering with either CoJet system or adhesive resin application only, depending on the availability of the system. © 2012 Springer-Verlag Berlin Heidelberg.

Effect of root canal preparation, type of endodontic post and mechanical cycling on root fracture strength

Objective: To evaluate the impact of the type of root canal preparation, intraradicular post and mechanical cycling on the fracture strength of roots. Material and Methods: Eighty human single rooted teeth were divided into 8 groups according to the instruments used for root canal preparation (manual or rotary instruments), the type of intraradicular post (fiber posts-FRC and cast post and core-CPC) and the use of mechanical cycling (MC) as follows: Manual and FRC; Manual, FRC and MC; Manual and CPC; Manual, CPC and MC; Rotary and FRC; Rotary, FRC and MC; Rotary and CPC; Rotary, CPC and MC. The filling was performed by lateral compactation. All root canals were prepared for a post with a 10 mm length, using the custom # 2 bur of the glass fiber post system. For mechanical cycling, the protocol was applied as follows: an angle of incidence of 45 degrees, 37 degrees C, 88 N, 4 Hz, 2 million pulses. All groups were submitted to fracture strength test in a 45 degrees device with 1 mm/min cross-head speed until failure occurred. Results: The 3-way ANOVA showed that the root canal preparation strategy (p<0.03) and post type (p<0.0001) affected the fracture strength results, while mechanical cycling (p=0.29) did not. Conclusion: The root canal preparation strategy only influenced the root fracture strength when restoring with a fiber post and mechanical cycling, so it does not seem to be an important factor in this scenario.

Effect of Resin Cement Type on the Microtensile Bond Strength to Lithium Disilicate Ceramic and Dentin Using Different Test Assemblies

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The study of a novel zinc finger gene cluster TZF and a genomic region flanking the histone H 4 replacement gene H 4 r of Drosophila melanogaster

Part I : A zinc finger gene Tzf1 was cloned in the earlier work of the lab by screening a ë-DASH2 cDNA expression library with an anti-Rat SC antibody. A ë-DASH2 genomic DNA library and cosmid lawrist 4 genomic DNA library were screened with the cDNA fragment of Tzf1 to determine the genomic organization of Tzf1. Another putative zinc finger gene Tzf2 was found about 700 bp upstream of Tzf1.RACE experiment was carried out for both genes to establish the whole length cDNA. The cDNA sequences of Tzf and Tzf2 were used to search the Flybase (Version Nov, 2000). They correspond to two genes found in the Flybase, CG4413 and CG4936. The CG4413 transcript seems to be a splicing variant of Tzf transcripts. Another two zinc finger genes Tzf3 and Tzf4 were discovered in silico. They are located 300 bp away from Tzf and Tzf2, and a non-tandem cluster was formed by the four genes. All four genes encode proteins with a very similar modular structure, since they all have five C2H2 type zinc fingers at their c-terminal ends. This is the most compact zinc finger protein gene cluster found in Drosophila melanogaster.Part II: 34,056 bp insert of the cosmid 19G11

A novel mutation in the steroidogenic acute regulatory protein gene promoter leading to reduced promoter activity

We have identified a novel cytosine/thymidine polymorphism of the human steroidogenic acute regulatory (StAR) gene promoter located 3 bp downstream of the steroidogenic factor-1 (SF-1)-binding site and 9 bp upstream of the TATA box (ATTTAAG). Carriers of this mutation have a high prevalence of primary aldosteronism. In transfection experiments, basal StAR promoter activity was unaltered by the mutation in murine Y-1 cells and human H295R cells. In Y-1 cells, forskolin (25 microM, 6 h) significantly increased wild-type promoter activity to 230+/-33% (P<0.05, n=4). In contrast, forskolin increased mutated promoter activity only to 150+/-27%, with a significant 35% reduction compared to wild type (P<0.05, n=3). In H295R cells, angiotensin II (AngII; 10 nM) increased wild-type StAR promoter activity to 265+/-22% (P<0.01, n=3), while mutated StAR promoter activity in response to AngII only reached 180+/-29% of controls (P< 0.01, n=3). Gel mobility shift assays show the formation of two additional complexes with the mutated promoter: one with the transcription repressor DAX-1 and another with a yet unidentified factor, which strongly binds the SF-1 response element. Thus, this novel mutation in the human StAR promoter is critically involved in the regulation of StAR gene expression and is associated with reduced promoter activity, a finding relevant for adrenal steroid response to physiological stimulators.

Evaluation of Hay-Type and Grazing Tolerant Alfalfa Hybrids in Season-Long or Complementary Rotational Stocking Systems for Beef Cows

Pastures containing hay-type and grazing tolerant alfalfa hybrids were grazed in a season-long or complementary rotational stocking system with Nfertilized smooth bromegrass. The pastures were stocked at a seasonal density of .8 cow-calf pairs per acre for 120 days in 1998 and 141 days in 1999. Pastures were intensively managed by daily stripstocking with the assumptions that 50% of live forage was available and daily live dry matter consumption of each cow-calf pair was 3.5% of the cow’s body weight. First-cutting forage was harvested as hay from 40% of the pasture acres to remove excess forage growth early in the grazing season. Grazing occurred on the remaining 60% of each pasture for the first 44 and 54 days and 100% of each pasture after days 45 and 55 in 1998 and 1999, respectively. Proportions of ‘Amerigraze’ and ‘Affinity’ alfalfa in the live forage dry matter decreased by 70% and 55% in pastures stocked season-long and by 60% and 42% in pastures used for complementary stocking (alfalfa type, p<.05; grazing management, p<.05) in 1998, but decreased by a mean of 72% and was unaffected by hybrid or stocking system in 1999. Cows grazing either alfalfa hybrid by either grazing system had greater weight gains during the breeding and overall grazing seasons and greater increases in body condition score pre-breeding and during the breeding season than the cows that grazed smooth bromegrass for the entire season in 1998. Also, cows grazing either alfalfa hybrid in the season-long system had greater breeding season increases in body condition score than cows grazing alfalfa in the complementary system with smooth bromegrass in 1998. Cows grazing in the season-long alfalfa system had greater prebreeding season weight (p<.10) increases and condition score (p<.05) increases than cows grazing alfalfa in the complementary system in 1999. Daily and seasonal body weight gains of calves were not affected (p>.10) by the presence of alfalfa in 1998 or by alfalfa type and grazing management in 1998 and 1999. Total animal production (cow and calf) in 1998 was greater (p<.10) from the season-long alfalfa pastures compared with the complementary stocked pastures. Total (p<.10) and live (p<.05) forage masses, estimated by monthly clippings, were greater in September of 1998 from the season-long alfalfa pastures than pastures using alfalfa for complementary stocking. Total (p<.10) and live (p<.05) forage masses were greater in August of 1999 from season-long alfalfa pastures than pastures using alfalfa for complementary stocking.