Identification of direct transcriptional targets of V600EBRAF/MEK signalling in melanoma

Oncogenic mutations in BRAF are common in melanoma and drive constitutive activation of the MEK/ERK pathway. To elucidate the transcriptional events downstream of V600EBRAF/MEK signalling we performed gene expression profiling of A375 melanoma cells treated with potent and selective inhibitors of V600EBRAF and MEK (PLX4720 and PD184352 respectively). Using a stringent Bayesian approach, we identified 69 transcripts that appear to be direct transcriptional targets of this pathway and whose expression changed after 6 h of pathway inhibition. We also identified several additional genes whose expression changed after 24 h of pathway inhibition and which are likely to be indirect transcriptional targets of the pathway. Several of these were confirmed by demonstrating their expression to be similarly regulated when BRAF was depleted using RNA interference, and by using qRT-PCR in other BRAF mutated melanoma lines. Many of these genes are transcription factors and feedback inhibitors of the ERK pathway and are also regulated by MEK signalling in NRAS mutant cells. This study provides a basis for understanding the molecular processes that are regulated by V600EBRAF/MEK signalling in melanoma cells.

Making the fun stop : youth justice reform in Queensland

In 2013 the newly elected conservative Liberal National Party government instigated amendments to the Youth Justice Act 1992 (Qld). Boot camps replaced court ordered youth justice conferencing. In 2014 there were more drastic changes, including opening the Children’s Court proceedings to the public, permitting publication of identifying information of repeat offenders, removing the principle of ‘detention as a last resort’, facilitating prompt transferral of 17 year olds to adult prisons and instigating new bail offences and mandatory boot camp orders for recidivist motor vehicle offenders in Townsville. This article compares these amendments to the legislative frameworks in other jurisdictions and current social research. It argues that these amendments are out of step with national and international best practice benchmarks for youth justice. Early indications are that Indigenous children are now experiencing increased rates of unsentenced remand. The article argues that the government’s policy initiatives are resulting in negative outcomes and that early and extensive evaluations of these changes are essential.

An elegy for Greg Ham: Copyright law, the Kookaburra case, and remix culture

The ‘Kookaburra’ case was a tragic and controversial copyright dispute, highlighting the need for copyright law reform by the Australian Parliament. In the Kookaburra case, a copyright action was brought by Larrikin Records against Men at Work’s song ‘Down Under’, alleging copyright infringement of the ‘Kookaburra’ song composed by Marion Sinclair. The dispute raised a host of doctrinal matters. There was disquiet over the length of the copyright term. There were fierce contests as to the copyright ownership of the ‘Kookaburra’ song. The litigation raised questions about copyright infringement and substantiality – particularly in relation to musical works. The ‘Kookaburra’ case highlighted frailties in Australia’s regime of copyright exceptions. The litigation should spur the Australian Law Reform Commission to make recommendations for law reform in its inquiry Copyright and the Digital Economy. This article provides a critical evaluation of the options of a defence for transformative use; a defence for fair use; and statutory licensing. The ‘Kookaburra’ case also examines the question of appropriate remedies in respect of copyright infringement. The conclusion considers the implications of the Kookaburra case for other forms of musical works – including digital sampling, mash-ups, and creative remixes. It finishes with an elegy for Greg Ham – paying tribute to the multi-instrumentalist for Men at Work.

Litigiousness in Australia: Lessons from comparative law

How litigious are Australians? Although quantitative studies have comprehensively debunked the fear of an Australian civil justice system in crisis, the literature has yet to address the qualitative public policy question of whether Australians are under- or over-using the legal system to resolve their disputes. On one view, expressed by the insurance industry, the mass media and prominent members of the judiciary, Australia is moving towards an American-style hyper-litigiousness. By contrast, Australian popular culture paints the typical Australian as culturally averse to formal rights assertion. This article explores the comparative law literature on litigiousness in two jurisdictions that have attracted significant scholarly attention — the United States and Japan. More specifically, it seeks to draw lessons from this literature for both understanding litigiousness in modern Australia and framing future research projects on the issue.

miR-514a regulates the tumour suppressor NF1 and modulates BRAFi sensitivity in melanoma

To identify ‘melanoma-specific’ microRNAs (miRNAs) we used an unbiased microRNA profiling approach to comprehensively study cutaneous melanoma in relation to other solid malignancies, which revealed 233 differentially expressed (≥ 2 fold, p < 0.05) miRNAs. Among the top 20 most significantly different miRNAs was hsa-miR-514a-3p. miR-514a is a member of a cluster of miRNAs (miR-506-514) involved in initiating melanocyte transformation and promotion of melanoma growth. We found miR-514a was expressed in 38/55 (69%) melanoma cell lines but in only 1/34 (3%) other solid cancers. To identify miR-514a regulated targets we conducted a miR-514a-mRNA ‘pull-down’ experiment, which revealed hundreds of genes, including: CTNNB1, CDK2, MC1R, and NF1, previously associated with melanoma. NF1 was selected for functional validation because of its recent implication inacquired resistance to BRAFV600E-targeted therapy. Luciferase-reporter assays confirmed NF1 as a direct target of miR-514a and over-expression of miR-514a in melanoma cell lines inhibited NF1 expression, which correlated with increased survival of BRAFV600E cells treated with PLX4032. These data provide another mechanism for the dysregulation of the MAPK pathway which may contribute to the profound resistance associated with current RAF-targeted therapies.

Activator Protein-1 and Epidermal Growth Factor Receptor Interplay : In Vivo & In Vitro Studies

Critical cellular decisions such as should the cell proliferate, migrate or differentiate, are regulated by stimulatory signals from the extracellular environment, like growth factors. These signals are transformed to cellular responses through their binding to specific receptors present at the surface of the recipient cell. The epidermal growth factor receptor (EGF-R/ErbB) pathway plays key roles in governing these signals to intracellular events and cell-to-cell communication. The EGF-R forms a signaling network that participates in the specification of cell fate and coordinates cell proliferation. Ligand binding triggers receptor dimerization leading to the recruitment of kinases and adaptor proteins. This step simultaneously initiates multiple signal transduction pathways, which result in activation of transcription factors and other target proteins, leading to cellular alterations. It is known that mutations of EGF-R or in the components of these pathways, such as Ras and Raf, are commonly involved in human cancer. The four best characterized signaling pathways induced by EGF-R are the mitogen-activated protein kinase cascades (MAPKs), the lipid kinase phosphatidylinositol 3 kinase (PI3K), a group of transcription factors called Signal Transducers and Activator of Transcription (STAT), and the phospholipase Cγ; (PLCγ) pathways. The activation of each cascade culminates in kinase translocation to the nucleus to stimulate various transcription factors including activator protein 1 (AP-1). AP-1 family proteins are basic leucine zipper (bZIP) transcription factors that are implicated in the regulation of a variety of cellular processes (proliferation and survival, growth, differentiation, apoptosis, cell migration, transformation). Therefore, the regulation of AP-1 activity is critical for the decision of cell fate and their deregulated expression is widely associated with many types of cancers, such as breast and prostate cancers. The aims of this study were to characterize the roles of EGF-R signaling during normal development and malignant growth in vitro and in vivo using different cell lines and tissue samples. We show here that EGF-R regulates cell proliferation but is also required for regulation of AP-1 target gene expression in fibroblasts in a MAP-kinase mediated manner. Furthermore, EGF-R signaling is essential for enterocyte proliferation and migration during intestinal maturation. EGF-R signaling network, especially PI3-K-Akt pathway mediated AP-1 activity is involved in cellular survival in response to ionizing radiation. Taken together, these results elucidate the connection of EGF-R and AP-1 in various cellular contexts and show their importance in the regulation of cellular behaviour presenting new treatment cues for intestinal perforations and cancer therapy.

KRAS variation and risk of endometriosis

Endometriosis is a common gynaecological disease with symptoms of pelvic pain and infertility which affects 7-10% of women in their reproductive years. Activation of an oncogenic allele of Kirsten rat sarcoma viral oncogene homologue (KRAS) in the reproductive tract of mice resulted in the development of endometriosis. We hypothesized that variation in KRAS may influence risk of endometriosis in humans. Thirty tagSNPs spanning a region of 60.7 kb across the KRAS locus were genotyped using iPLEX chemistry on a MALDI-TOF MassARRAY platform in 959 endometriosis cases and 959 unrelated controls, and data were analysed for association with endometriosis. Genotypes were obtained for most individuals with a mean completion rate of 99.1%. We identified six haplotype blocks across the KRAS locus in our sample. There were no significant differences between cases and controls in the frequencies of individual single-nucleotide polymorphisms (SNPs) or haplotypes. We also developed a rapid method to screen for 11 common KRAS and BRAF mutations on the Sequenom MassARRAY system. The assay detected all mutations previously identified by direct sequencing in a panel of positive controls. No germline variants for KRAS or BRAF were detected. Our results demonstrate that any risk of endometriosis in women because of common variation in KRAS must be very small.

Synthesis and Antileukemic Activity of 1-((S)-2-Amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-3-(substituted phenyl)urea Derivatives

Heterocyclic urea derivatives play an important role as anticancer agents because of their good inhibitory activity against receptor tyrosine kinases (RTKs), raf kinases, protein tyrosine kinases (PTKs), and NADH oxidase, which play critical roles in many aspects of tumorigenesis. Benzothiazole moiety constitutes an important scaffold of drugs, possessing several pharmacological functions, mainly the anticancer activity. Based on these interesting properties of benzothiazoles and urea moiety to obtain new biologically active agents, we synthesized a series of novel 1-((S)-2-amino-4,5,6.7-tetrahydrobenzo[d]thiazol-6-yl)-3-(substituted phenyl)urea derivatives and evaluated for their efficacy as antileukemic agents against two human leukemic cell lines (K562 and Reh). These compounds showed good and moderate cytotoxic effect to cancer cell lines tested. Compounds with electron-withdrawing chloro and fluoro substituents on phenyl ring showed good activity and compounds with electron-donating methoxy group showed moderate activity. Compound with electron-withdrawing dichloro substitution on phenyl ring of aryl urea showed good activity. Further, lactate dehydrogenase (LDH) assay, flow cytometric analysis of annexin V-FITC/propidium iodide (PI) double staining and DNA fragmentation studies showed that compound with dichloro substitution on phenyl ring of aryl urea can induce apoptosis.

Genome-wide analysis and experimentation of plant serine/threonine/tyrosine-specific protein kinases

Protein tyrosine phosphorylation plays an important role in cell growth, development and oncogenesis. No classical protein tyrosine kinase has hitherto been cloned from plants. Does protein tyrosine kinase exist in plants? To address this, we have performed a genomic survey of protein tyrosine kinase motifs in plants using the delineated tyrosine phosphorylation motifs from the animal system. The Arabidopsis thaliana genome encodes 57 different protein kinases that have tyrosine kinase motifs. Animal non-receptor tyrosine kinases, SRC, ABL, LYN, FES, SEK, KIN and RAS have structural relationship with putative plant tyrosine kinases. In an extended analysis, animal receptor and non-receptor kinases, Raf and Ras kinases, mixed lineage kinases and plant serine/threonine/tyrosine (STY) protein kinases, form a well-supported group sharing a common origin within the superfamily of STY kinases. We report that plants lack bona fide tyrosine kinases, which raise an intriguing possibility that tyrosine phosphorylation is carried out by dual-specificity STY protein kinases in plants. The distribution pattern of STY protein kinase families on Arabidopsis chromosomes indicates that this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. Genome-wide analysis is supported by the functional expression and characterization of At2g24360 and phosphoproteomics of Arabidopsis. Evidence for tyrosine phosphorylated proteins is provided by alkaline hydrolysis, anti-phosphotyrosine immunoblotting, phosphoamino acid analysis and peptide mass fingerprinting. These results report the first comprehensive survey of genome-wide and tyrosine phosphoproteome analysis of plant STY protein kinases.

Opiskelijaliikkeen äärilaidalta populaarikulttuurin hahmoiksi ja 2000-luvun alussa Rote Armee Fraktion Der Spiegel -lehden sivuilla 1970-luvulla

Työ käsittelee vasemmistoradikalismia ilmiönä ja erityisesti kyseisen ilmiön eri vaiheita. Tarkempana tarkastelukohtana on Rote Armee Fraktion (RAF) -niminen vasemmistovallankumouksellinen ryhmä, joka toimi Saksan liittotasavallassa vuosina 1970-1998. Ryhmää tarkastellaan siltä kannalta, kuinka uutislehti Der Spiegel on en toimintaa eri aikoina tulkinnut. Alkuperäislähteenä on käytetty pääasiassa saksalaisessa Der Spiegel - viikkolehdessä ilmestyneitä ryhmää käsitteleviä artikkeleita. Erityistä painoa on laitettu sille, miten ryhmän toiminnassa ja toimintaympäristössä tapahtuneet muutokset ovat vaikuttaneet eri ajankohtina julkaistuissa artikkeleissa näkyvissä oleviin tapoihin käsitellä ryhmää ilmiönä. Tarkastelu on rajattu koskemaan lähinnä vuosia 1970. 1977. 2007 ja 2008. Lisäksi mukaan on valikoitu vuosilta 1968. 1972 ja 1975 muutamia yksittäisiä artikkeleita. Aihe liittyy osaltaan keskusteluun sekä opiskelijaliikkeen radikalisoitumisesta 1960-ja 1970-lukujen vaihteessa että terrorismista laajempana ilmiönä. Tutkimustavoitetta lähestytään kolmen eri teeman kautta. Ensimmäisenä teemana tarkastellaan RAF:iin liittyviä artikkeleita siltä kannalta, mitä nimitystä ryhmästä on milloinkin käytetty. Toisena sitä. missä vaiheessa Der Spiegelissä alettiin pitää ryhmän toimintaa terrorismina ja toimijoita terroristeina ia kolmantena temaattisena aiheena analysoidaan sitä. millaista kirjoitustyyliä on ryhmää koskevissa artikkeleissa eri aikoina käytetty. Vuoden 1977 vaikutus siihen, kuinka ryhmästä kirjoitetaan vielä 2000-luvun alussa, on ollut suuri. Kun vielä RAF:n terroriaallon ollessa kovimmillaan syksyllä 1977 kerrottiin Der Spiegelissä ryhmän pitäneen Länsi-Saksaa polvillaan edeltäneet kaksi vuotta, puhuttiin vuonna 2007 seitsemästä vuodesta. Samanaikaisesti kun 2000-luvulla kirjoitetuissa artikkeleissa annetaan ryhmälle koko sen toiminnan ajaksi se asema ja nimi, jotka se oikeastaan lopulta sai vasta syksyn 1977 myötä ja vain muutamaksi kuukaudeksi, on artikkeleissa tyylillisesti kuitenkin palattu lähemmäs 1970-luvun alkua. Heti ryhmän perustamisen jälkeen ei sitä otettu vakavana yhteiskunnallisena toimijana ja tämä näkyi Der Spiegelin uutisoinnissa viihteellisenä kerrontatyylinä. Terroriaallon ollessa pahimmillaan ei tällainen kerrontatyyli ollut käytössä vaan RAF liitettiin osaksi laajempaa terrorismi-ilmiötä. Sen sijaan vuonna 2007 ilmestyneessä artikkelisarjassa jälleen palattiin samanlaiseen suorastaan vallankumousromantiikalla leikittelevään kerrontatyyliin. Näin Der Spiegelin tuli piirtäneeksi lähes neljässäkymmenessä vuodessa pitkän kaaren opiskelijaliikkeen äärilaidan radikalisoitumisesta 1960- ja 1970-lukujen vaihteessa aina Saksan liittotasavallan tuomiseen polvilleen 1977 ja yli kolmekymmentä vuotta aikaisemmin selleissään kuolleiden RAF-johtohahmojen muodostumiseen osaksi populaarikulttuuria 2000-luvun alussa.

Cell fate specification during Caenorhabditis elegans male tail development

The cells of the specialized mating structures of the nematode Caenorhabditis elegans adult male tail develop from sex-specific divisions of postembryonic blast cells. One male-specific blast cell, B, is the precursor to all the cells of the copulatory spicules. Both cell interactions and autonomous fate specification mechanisms are utilized in the B lineage to specify fate.

During development the anterior daughter of B, B.a, generates four distinct pairs of cells. Cell ablation experiments indicate that the cells of each pair respond to positional cues provided by other male-specific blast cells. F and U promote anterior fates, Y.p promotes some posterior fates, and the B.a progeny promote posterior fates. The cells within each pair may also interact.

The lin-3/let-23 signalling pathway, identified for its function in C. elegans hermaphrodite vulval induction, mediates the signal from F and U. Reduction-of-function mutations in lin-3 (EGF-like signal), let-23 (receptor), sem-5 (adaptor), let-60 (ras), or lin-45 (raf) disrupt the fates of the anterior cells, and mimic F and U ablation. In addition, ectopically expressed lin-3 disrupts the fates of the posterior cells, and can promote anterior fates even in the absence of F and U.

A genetic screen of over 9000 mutagenized gametes recovered 22 mutations in 20 loci that disrupt fate specification in male tail lineages. Seven of these mutations may represent new genes that play a role in male tail development.

The first division of the B cell is asymmetric. The gene vab-3 is required for specification of B.a fates, and it may represent a factor whose activity is localized to the B.a cell via the gene lin-17. lin-17 acts both at the first division of the B cell and at specific other cell divisions in the lineage.

Acordo Aeroespacial Teuto-Brasileiro (1969-1989; 1990-2001 e 2002-2011): uma cooperação complementar

Este projeto de dissertação se propõe a analisar a cooperação aeroespacial entre o Brasil e a Alemanha de 1969 a 2011 em três recortes temporais: 1969 a 1989, 1990 a 2001 e 2002 a 2011. Seguindo as bases teóricas de classificação da CID (Cooperação Internacional para o Desenvolvimento), e apoiada em pesquisa de campo conduzida em ambos os países, este trabalho apresenta um novo conceito de cooperação que, até onde a pesquisa bibliográfica aqui conduzida avaliou, constitui uma contribuição original deste trabalho: a Cooperação Complementar. A cooperação aeroespacial teuto-brasileira é pouco conhecida e divulgada, embora tenha completado vigorosas quatro décadas de exitosa existência. A conclusão de êxito desta cooperação encontrou lastro em pesquisa de campo conduzida pela autora no Brasil (IAE Instituto de Aeronáutica e Espaço) e na Alemanha (DLR Deutsche Zentrum für Luft- und Raumfahrt), consubstanciada por quatro entrevistas (SILVA, 2011a), (SILVA, 2011c), (SILVA, 2011d) e (SILVA, 2011e) realizadas junto a importantes representantes destes dois centros. Os conhecimentos extraídos por meio destas entrevistas agregaram, no entender desta autora, importantes informações à bibliografia específica e relativamente escassa disponível em ambos os países.O êxito defendido nesta dissertação fundamenta-se não apenas na longevidade advinda dos 40 anos de existência desta Cooperação, na sua capacidade de renovação e na complementaridade atingida, mas sobretudo pela consecução dos diversos objetivos técnico-científicos integrantes do escopo do referido Tratado, muitos dos quais responsáveis por importantes desdobramentos de tecnologias em outras áreas do saber, tais como o projeto DEBRA 25 (SCHUSTER, 2011), de energia eólica, e o projeto VLS (Veículo Lançador de Satélites), que utiliza como seus motores os foguetes desenvolvidos no escopo desta Cooperação.