Compositional bisimulation metric reasoning with Probabilistic Process Calculi

We study which standard operators of probabilistic process calculi allow for compositional reasoning with respect to bisimulation metric semantics. We argue that uniform continuity (generalizing the earlier proposed property of non-expansiveness) captures the essential nature of compositional reasoning and allows now also to reason compositionally about recursive processes. We characterize the distance between probabilistic processes composed by standard process algebra operators. Combining these results, we demonstrate how compositional reasoning about systems specified by continuous process algebra operators allows for metric assume-guarantee like performance validation

Recomendações para a construção, manutenção e segurança de pontos de abastecimento de pulverizadores para a Produção Integrada de Maçãs no Brasil.

bitstream/item/123254/1/cot052a.pdfNova edição com alterações a primeira foi em 200

SOS rule formats for convex and abstract probabilistic bisimulations

Probabilistic transition system specifications (PTSSs) in the $nt \mu f\theta / nt\mu x\theta$ format provide structural operational semantics for Segala-type systems that exhibit both probabilistic and nondeterministic behavior and guarantee that bisimilarity is a congruence for all operator defined in such format. Starting from the $nt \mu f\theta / nt\mu x\theta$ format, we obtain restricted formats that guarantee that three coarser bisimulation equivalences are congruences. We focus on (i) Segala's variant of bisimulation that considers combined transitions, which we call here "convex bisimulation"; (ii) the bisimulation equivalence resulting from considering Park & Milner's bisimulation on the usual stripped probabilistic transition system (translated into a labelled transition system), which we call here "probability obliterated bisimulation"; and (iii) a "probability abstracted bisimulation", which, like bisimulation, preserves the structure of the distributions but instead, it ignores the probability values. In addition, we compare these bisimulation equivalences and provide a logic characterization for each of them.Comment: In Proceedings EXPRESS/SOS 2015, arXiv:1508.0634

Avaliação impactos ambientais e uso do Ambitec na Rede AP: uma proposta de aplicação.

A aferição da importância dos impactos causados por uma pesquisa ou desenvolvimento de atividade científica é extremamente importante para auxiliar na orientação e eventuais correções de rumo da pesquisa. Mesmo com o desenvolvimento do sistema AMBITEC para medir o impacto das tecnologias da Embrapa, ainda há poucas ferramentas que permitam a aferição de descobertas pré-tecnológicas, sem as quais a geração da tecnologia não ocorre. É sobre este problema que este artigo pretende se debruçar rapidamente, buscando, durante a execução do projeto de Agricultura de Precisão, estabelecer, no mínimo, uma metodologia que permita aferir tais progressos

Improving a joint inversion of GRACE, GPS and modelled ocean bottom pressure by using in-situ data.

To investigate the changes in ocean bottom pressure (OBP) and ocean mass Rietbroek et al. (2009) performed a joint least square inversion of weekly GRACE solutions, patterns of large-scale deformation measured by a network of GPS stations and modelled OBP from the Finite Element Sea ice Ocean Model (FESOM). The correlation of this inversion with in-situ OBP ranges between 0.7 and 0.8 in some regions but for example in the tropical Atlantic the correlation is below 0.4. To improve the agreement of the inversion with in-situ data, a part of the in-situ data is included directly into the inversion. The in-situ OBP data was taken from the global OBP data base of Macrander et al. (2010) and averaged to weekly means. Depending on the weight put on the in-situ data, the correlation and regression increases significantly to a value larger than 0.9. The variance of the system is locally reduced by almost 50% at the locations included into the inversion while the difference of the global ocean mean is on average below 10%. Furthermore the global ocean mean is used to compute a bias term for correcting the global ocean mean obtained by the FESOM model

Banco de informações ambientais e toxicológicas dos agrotóxicos utilizados até a safra 2002/2003 na Produção Integrada de Maças no Brasil.

bitstream/CNPUV/8125/1/cir048.pd

Concealed Revealment

http://deepblue.lib.umich.edu/bitstream/2027.42/90989/1/msgebler_1334773022.pd

Pontos de abastecimento de pulverizadores agrícolas: uma revisão comparando os modelos em uso.

Pontos de abastecimento de pulverizadores são locais destinados ao preparo das caldas agroquímicas e carregamento de equipamentos de pulverização agrícola, classificados pela legislação ambiental e trabalhista como equipamento de segurança ambiental. Há suficiente bibliografia técnica que recomenda e descreve os processos construtivos e de manejo de tais locais, mas há pouca clareza sobre como se chegou a esta forma consensual, aliado ao pouco embasamento científico de seus benefícios e riscos agregados. Alguns aspectos como impermeabilização, constituição do material, manejo do piso e dos resíduos de agroquímicos, interferem na capacidade de evitar que os possíveis contaminantes atinjam o solo e as águas, interferindo no descarte final. Aspectos funcionais ainda carecem de respostas, como a possibilidade do piso reter e armazenar o contaminante e se tornar um emissor a longo prazo, a definição do tempo de vida útil do equipamento, dentre outros. Recentemente houve avanços na filosofia construtiva dos pontos de abastecimento de pulverizadores, com a introdução da remediação de resíduos in situ, através da biodegradação, ganhando denominações, como biobeds, dentre outras. O objetivo desta revisão é discutir as diferentes formas de pisos e manejo nestes locais, seus riscos e a possibilidade de se tornarem um passivo ambiental

Developing the CRISPR/Cas-system for Inactivation of Proto-oncogenes in Human Cancer Cells

Numerous mutations contribute to tumorigenesis of cancer cells. For most of them it remains unclear whether they are driver or passenger mutations. A classic knock-out to study their function in cancer cells used to take a lot of effort. The CRISPR/Cas-system can be used as a programmable “genome editing” tool. In this work, oncogenes have been inactivated with the CRISPR/Cas-system. Considering off-targets, Streptococcus pyogenes sgRNAs can be designed for 88% of the known cancer mutations. The activity of 15 sgRNAs, targeting 13 mutations in proto-oncogenes (deletions, insertions and point mutations), has been tested with a RFP-GFP-reporter plasmid. For 13 sgRNAs, activity prediction scores correlated with measured activity. Furthermore, sgRNAs have shown preferential binding to mutated versions of targeted proto-oncogene sequence and did not induce double strand breaks in the wild type sequence. For 10 sgRNAs, the activity against their target sequence has been more than 4 times higher than against the wild type sequence. Most of those sgRNAs target insertions or deletions and fewer target point mutations. Permanent knock-out of three mutated proto-oncogenes NPM1, BRAF and PIK3CA has been achieved with a lentiviral expression of CRISPR/Cas. Accordingly, effects on proliferation and phenotype have been studied. Knock-out of NPM1 c.863_864insTCTG mutation has been studied in heterozygous mutated OCI AML3 cell line. Proliferation was strongly inhibited by the corresponding sgRNA. Cells arrested in G0/1-phase of cell cycle (77%) compared to control cells (56%), although no difference was observed for sub-G1 phase, indicating no induction of apoptosis. Cells treated with NPM1 sgRNA had 88% reduced expression of NPM1 c.863_864insTCTG mRNA as well as less cytoplasmic localization of nucleophosmin as assessed by immunostaining. The activity of sgRNA has been confirmed by deep sequencing, showing a shift of wild type to mutated allele ratio from 51:49 to 68:32. This effect was enhanced by the additional treatment with the NHEJ inhibitor SCR7. A BRAFV600E sgRNA was tested in homozygously mutated melanoma cell lines A-375 and SK MEL-28. No differences were detected in comparison to controls. However, in the CRC cell line RKO, heterozygous for BRAFV600E and PIK3CAH1047R, proliferation was inhibited through sgRNAs against either BRAF or PIK3CA. A combination of both had no synergistic effect on proliferation. Activity and specificity of the sgRNA targeting BRAF were confirmed by deep sequencing, while the PIK3CA sgRNA showed a moderate induction of double strand breaks also in the wild type allele. The relation of wild type to mutated allele of BRAF was changed from 32:68 before treatment to 51:49 afterwards. This effect can be explained by a “re mutation” to the wild type after DSB via HDR with wild type sister chromatid as template. This effect was observed for PIK3CA sgRNA to a lesser extent. In conclusion, these results show the applicability of the CRISPR/Cas-system for the inactivation of mutated proto-oncogenes.:List of tables III List of figures IV List of abbreviations V 1 Introduction 1 1.1 Cancer 1 1.2 Oncogenes 2 1.2.1 Role in cancer 2 1.2.2 Targeted therapies 3 1.2.3 NPM1 5 1.2.4 BRAF 6 1.2.5 PIK3CA 7 1.3 CRISPR/Cas-system 7 1.4 Aim and motivation 10 2 Material and Methods 11 2.1 Design of sgRNAs 11 2.2 Plasmids 11 2.3 Cell culture 12 2.4 FACS analysis 14 2.5 T7 assay 14 2.6 Cell cycle analysis 15 2.7 Immunostaining 15 2.8 Apoptosis assay 16 2.9 Quantification of mutant NPM1 transcripts 16 2.10 Deep sequencing 16 2.11 Statistical Analysis 19 3 Results 20 3.1 Design of sgRNAs targeting oncogenes 20 3.2 Evaluation of sgRNA efficacy and selectivity 23 3.3 Effects of oncogene knock-out in cancer cell lines 27 3.3.1 Targeting NPM1 in AML cells 27 3.3.2 Targeting BRAF in melanoma cells 30 3.3.3 Targeting BRAF and PIK3CA in colorectal carcinoma cells 31 4 Discussion 37 4.1 The design of sgRNAs is possible for most cancer mutations 37 4.2 sgRNAs targeting oncogenes have to be tested 37 4.3 Oncogenes can be knocked out with the CRISPR/Cas-system 37 4.3.1 NPM1 in AML cells 37 4.3.2 BRAF in melanoma cells 38 4.3.3 BRAF and PIK3CA in CRC cells 38 4.4 Advantages and disadvantages to target oncogenes with the CRISPR/Cas-system 40 4.5 Concluding remarks 41 5 Original Article 43 6 Summary 47 7 Zusammenfassung 49 List of references 51 Appendix VIIIIn Krebszellen tritt eine Vielzahl von Mutationen auf. Für den Großteil der Mutationen ist ungeklärt, ob es sich um krebsverursachende oder passagere Mutationen handelt. Ein gezieltes Ausschalten (Knock-out) dieser Gene zur Untersuchung ihrer Funktion in Krebszellen war bisher mit großem Aufwand verbunden. Das CRISPR/Cas-System lässt sich als programmierbares „Genome-editing“ Werkzeug einsetzen und wurde in der vorliegenden Arbeit verwendet, um gezielt mutierte Protoonkogene zu inaktivieren. Für 88% der bekannten, in Krebszellen auftretenden Mutationen lassen sich, unter Berücksichtigung von off-targets, Streptococcus pyogenes sgRNAs entwerfen. Mit Hilfe eines RFP-GFP-Reporter-Plasmides wurde die Aktivität von 15 sgRNAs gegen 13 Mutationen (Deletionen, Insertionen und Punktmutationen) in Protoonkogenen überprüft. Für 13 der sgRNAs zeigte sich eine Aktivität, die mit der Vorhersage durch den Algorithmus korrelierte. Außerdem wurde gezeigt, dass die sgRNAs spezifisch genug binden, um zwar bei der mutierten Sequenz eines Protoonkogens, jedoch nicht bei der Wildtyp-Sequenz Doppelstrangbrüche zu erzeugen. Unter den sgRNAs waren 10 mit mehr als 4-fach höherer Aktivität bei komplett übereinstimmender Zielsequenz gegenüber der Wildtyp-Sequenz. Diese spezifischen sgRNAs waren vor allem gegen Insertions- oder Deletionsmutationen gerichtet, einige auch gegen Punktmutationen. Durch permanente, lentivirale Expression von CRISPR/Cas wurden die Effekte eines Knock-out von drei mutierten Protoonkogenen, NPM1, BRAF und PIK3CA, auf das Wachstum und phänotypische Aspekte humaner Krebszelllinien untersucht. Ein Knock-out der NPM1 c.863_864insTCTG Mutation wurde in heterozygot mutierten OCI AML3 Zellen untersucht, es zeigte sich eine starke Proliferationshemmung. In der Zellzyklusanalyse trat ein G0/1-Arrest dieser Zellen (77%) im Vergleich mit Kontroll-Zellen (56%) auf, jedoch keine Unterschiede in der sub-G1-Analyse, sodass nicht von einer vermehrten Apoptose auszugehen ist. Die mit sgRNA behandelten OCI-AML3 Zellen zeigten sowohl eine um 88% verminderte NPM1 c.863_864insTCTG mRNA-Expression als auch verminderte zytoplasmatische Sublokalisation des Nucleophosmins in der Immunfärbung. Die hohe Aktivität der gRNA gegen mutiertes NPM1 wurde durch Deep Sequencing bestätigt, außerdem hat sich das Verhältnis vom Wildtyp- zu mutiertem Allel von 51:49 zu 68:32 verschoben. Dieser Effekt wurde durch Zugabe des NHEJ-Hemmstoffes SCR7 noch verstärkt. Die sgRNA gegen BRAFV600E wurde in den homozygot mutierten Melanom-Zelllinien A-375 und SK-MEL-28 getestet. Bei Proliferationsversuchen zeigten sich keine Unterschiede im Vergleich zu Kontrollzellen. In der kolorektalen Krebszelllinie RKO, die heterozygot BRAFV600E und PIK3CAH1047R ist, zeigte sich bei der Testung von sgRNAs gegen BRAF, PIK3CA und Kombination beider sgRNAs eine Wachstumshemmung. Jedoch lag kein synergistischer Effekt bei sgRNA-Kombination vor. Zudem bestätigten sich Aktivität und Spezifität der sgRNA gegen BRAF im Deep Sequencing, während die sgRNA gegen PIK3CA in mäßigem Umfang Doppelstrangbrüche im Wildtyp-Allel verursachte. Das Verhältnis vom Wildtyp- zu mutiertem BRAF Allel verschob sich von 32:68 ohne sgRNA zu 51:49 nach sgRNA-Behandlung. Eine mögliche Erklärung dieser Beobachtung ist die Rückmutation zum Wildtyp-Allel nach Doppelstrangbruch mit Hilfe homologer Rekombination durch das Wildtyp-Schwesterchromatid. Für PIK3CA konnte dieser Effekt in schwächerem Ausmaß ebenfalls beobachtet werden. Zusammengefasst zeigen diese Ergebnisse, dass das CRISPR/Cas-System zur Inaktivierung mutierter Protoonkogene genutzt werden kann.:List of tables III List of figures IV List of abbreviations V 1 Introduction 1 1.1 Cancer 1 1.2 Oncogenes 2 1.2.1 Role in cancer 2 1.2.2 Targeted therapies 3 1.2.3 NPM1 5 1.2.4 BRAF 6 1.2.5 PIK3CA 7 1.3 CRISPR/Cas-system 7 1.4 Aim and motivation 10 2 Material and Methods 11 2.1 Design of sgRNAs 11 2.2 Plasmids 11 2.3 Cell culture 12 2.4 FACS analysis 14 2.5 T7 assay 14 2.6 Cell cycle analysis 15 2.7 Immunostaining 15 2.8 Apoptosis assay 16 2.9 Quantification of mutant NPM1 transcripts 16 2.10 Deep sequencing 16 2.11 Statistical Analysis 19 3 Results 20 3.1 Design of sgRNAs targeting oncogenes 20 3.2 Evaluation of sgRNA efficacy and selectivity 23 3.3 Effects of oncogene knock-out in cancer cell lines 27 3.3.1 Targeting NPM1 in AML cells 27 3.3.2 Targeting BRAF in melanoma cells 30 3.3.3 Targeting BRAF and PIK3CA in colorectal carcinoma cells 31 4 Discussion 37 4.1 The design of sgRNAs is possible for most cancer mutations 37 4.2 sgRNAs targeting oncogenes have to be tested 37 4.3 Oncogenes can be knocked out with the CRISPR/Cas-system 37 4.3.1 NPM1 in AML cells 37 4.3.2 BRAF in melanoma cells 38 4.3.3 BRAF and PIK3CA in CRC cells 38 4.4 Advantages and disadvantages to target oncogenes with the CRISPR/Cas-system 40 4.5 Concluding remarks 41 5 Original Article 43 6 Summary 47 7 Zusammenfassung 49 List of references 51 Appendix VII