No description available.

The public sector in Australia, as in other western countries, has been accused in recent times of being too costly, too rigid, inefficient and ineffective. What is apparently needed is a public sector that is smaller, less costly, more efficient and more effective. The search for alternative and better ways to organise and undertake work to meet these reform objectives is at the heart of the rapid expansion of Competitive Tendering and Contracting (CTC) within the public sector in the last two decades. But increased reliance on government contracting does not always lead to outsourcing. Some government agencies allow, indeed encourage, their current employees to also bid for the work on offer by including an In-House Option (IHO) within their CTC processes. In a number of cases these IHOs have been selected ahead of their commercial competitors. IHOs are effectively internal tenders that, if selected, must be implemented by work areas within the confines of the policies and practices of their parent organisation. The reasons commonly expressed in support of IHOs are to do with addressing the potentially problematic aspects of organisational review and possible outsourcing, and to assist the parent organisation achieve its reform intentions in the most effective and least disruptive manner possible. This research examined the achievements of six IHOs within the Australian Defence Organisation. It also asked what can be learned from their experiences? The findings show that IHOs can contribute to reform and enhance the effectiveness of CTC processes but that these achievements come at a price�borne primarily by the staff who work within selected IHOs. IHOs add to the competition of CTC exercises. They also act as an insurance policy against being caught with no reasonable bids and offer a benchmark against which to assess unknown bids. But competition can also focus bidders on doing what is necessary to win rather than what is best for an organisation or its staff. Having IHOs increases the uncertainty for staff about their future employment while at the same times raising expectations that if they can be successful they will be able to make changes and improve their work areas. This research has shown that this does not always occur and staff can find the whole experience frustrating and demoralising. Organisations that include IHOs within their CTC methodologies need to assist them if they are to have the best opportunity to propose new and innovative ways of working. And they must be prepared for the possibility that their IHOs could win. Selected IHOs need support to successfully implement changes, and as the IHOs examined here have shown, they can make significant improvements in work practices and more efficient use of resources if given the chance.

[Truncated abstract] The design of effective immunotherapies, using tumour antigens to stimulate a functional effector cytotoxic T cell (CTL) response in a tumour bearing host, requires an understanding of the 'real time' in vivo relationship between the host immune system and antigens expressed by the developing tumour. However, effector function of endogenous anti-tumour CTLs generated during tumour progression has largely been assessed by indirect ex vivo assays and often focused on a single antigen. Therefore, studies in this thesis evaluated the endogenous in vivo CTL response to multiple tumour antigenic epitopes in murine tumour models using Lewis lung carcinoma cells transfected with ovalbumin (an antigen that contains several intra-molecular MHC class I epitopes with a defined hierarchy) or a polyepitope (that contains a string of immunodominant MHC class I epitopes). Potent effector CTLs were generated to multiple dominant tumour antigenic epioptes early in tumour progression. However, in general, these CTL effectors only transiently retarded tumour growth, and at the later time points of tumour growth they were no longer generated in tumour draining lymph nodes. This coincided with diminished tumour antigen presentation in the same nodes which was found to be due to antigen loss. In both models antigen loss was the result of two processes; immuno-editing of the tumour by the host immune response and genetic instability resulting in antigen loss variants that could evade immune surveillance. A third model was generated that maintained low level tumour antigen expression throughout tumour progression. ... The impact of pre-existing endogenous dominant-epitope specific CTLs on tumour expressing the same epitope was also assessed, and resulted in a reduced tumour incidence and a CTL response restricted to a single antigen of the same MHC allele. Finally, the effects of two different immunotherapy regimens were examined. Intratumoural IL-2 treatment enhanced pre-existing CTL responses to the dominant epitopes leading to tumour regression. In addition, use of a multiple peptide vaccination regimen that avoided T cells competing for peptide-MHC complexes on APC was far more likely to be effective than one that did not. These results demonstrate that immunotherapies targeting tumours that express several dominant neo antigenic epitopes can be effective. The caveat for this approach is that it will only be effective in tumours that have generated an endogenous CTL response and must be used before antigen loss variants emerge.

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2018-05-17T17:27:26Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Roberto Carlos Doring Wolter.pdf: 2034054 bytes, checksum: d53dff149845c79db461dd69fc6b8806 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-05-18T20:34:58Z (GMT) No. of bitstreams: 2 Tese_Roberto Carlos Doring Wolter.pdf: 2034054 bytes, checksum: d53dff149845c79db461dd69fc6b8806 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-05-18T20:34:58Z (GMT). No. of bitstreams: 2 Tese_Roberto Carlos Doring Wolter.pdf: 2034054 bytes, checksum: d53dff149845c79db461dd69fc6b8806 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-08-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A toxidez por ferro é um dos mais importantes estresses abióticos em solos de várzea. Depois do surgimento dos sintomas de toxidez na lavoura, apenas medidas paliativas podem ser tomadas para diminuir o efeito do problema. Os objetivos do trabalho foram verificar se os critérios de interpretação para prognóstico do risco de ocorrência da toxidez por ferro em arroz irrigado por alagamento baseado na porcentagem de saturação da CTC por Fe2+ (PSFe2+), que é calculada a partir da estimativa do ferro trocável, proveniente da determinação do ferro extraído por oxalato de amônio pH 6,0 de uma amostra coletada anterior ao alagamento, são válidos para um grupo de solos de várzea do Rio Grande do Sul. Além disso, se a inclusão de outras variáveis melhora a estimação e o prognóstico, e ainda se a relação das classes de riscos está de acordo com a ocorrência da toxidez nas plantas. Para isso foram conduzidos quatro experimentos com plantas usando diferentes solos em casa de vegetação e um experimento em solução nutritiva em laboratório. Os resultados do experimento em solução nutritiva mostram que o surgimento dos sintomas de toxidez por ferro nas plantas de arroz apenas ocorreram a partir da fração entre 0,45 e 0,60 de ferro pelos cátions divalentes, diferente do valor de 0,40 da fração de ferro na solução definido como crítico para a toxidez pelo método, no entanto, não foi detectada mudança nos teores de ferro no tecido das plantas quando se variou a fração molar de ferro na solução nutritiva. Com os resultados dos experimentos com plantas em solos verificou-se que a alta PSFe2+ de uma amostra de solo está relacionada a uma maior probabilidade de ocorrência de toxidez por ferro, e em amostra com baixa PSFe2+ quase não ocorrem sintomas de toxidez por ferro, indicando que o método foi eficiente para prever a ocorrência de sintomas de toxidez por ferro nas plantas de arroz, para o grupo de solos. Também foi constatado que o uso das variáveis: C orgânico, ferro e manganês extraídos por oxalato de amônio pH 6 melhora a exatidão da estimação do ferro trocável comparado a estimação apenas pela extração de ferro, porém, isso não se refletiu em melhores prognósticos do risco de ocorrência de toxidez por ferro nas plantas de arroz do grupo de solos estudados, observou-se que as correlações com as duas formas de estimativas de ferro trocável com os atributos relacionados com a toxidez por ferro tiveram coeficientes bem próximos, impossibilitando a definição do melhor método. Pelo menor número de variáveis a serem determinadas, ou seja, pela maior facilidade de execução, a estimação do ferro trocável obtido apenas pelo ferro extraído por oxalato de amônio pH 6,0 é o método mais eficiente para prognosticar o risco de ocorrência de toxidez por ferro para o arroz irrigado. / The iron toxicity is one of the most important abiotic stress in lowland soils. After the appearance of toxicity symptoms in the field, only palliative measures can be taken to lessen the effect of the problem. The aims of this work were to verify if the interpretation criteria for predicting the risk of iron toxicity occurrence in rice plants by flooding based on the percentage saturation of CEC by Fe2+ (PSFe2+), which is calculated from the estimated exchangeable iron from the determination of iron extracted by ammonium oxalate pH 6.0 in a sample collected prior to flooding, are valid for a group of lowland soils from of Rio Grande do Sul. Furthermore, if the inclusion of other variables improves the estimation and prognosis, and if the relation of the classes of risks is consistent with the occurrence of toxicity in plants. For this, four experiments were conducted with plants, using different soils in a greenhouse and an experiment in nutrient solution in laboratory. The results of the experiment in nutrient solution have shown that the emergence of symptoms of iron toxicity in rice plants occurred only for the 0.45 and 0.60 molar ratio of iron to divalent cations, different from the 0.40 molar ratio of iron in solution defined as critical to the toxicity by the method. However, no change was detected for the iron levels on plant tissue when varying the molar ratio of iron in the nutrient solution. With the results of experiments with plants in soil one could verify that the higher the PSFe2+ of a soil sample the greater the probability of toxicity by iron, and for samples with low PSFe2+ almost no symptoms of toxicity occurred, indicating that the method was efficient in predicting the occurrence of iron toxicity symptoms in rice plants, for the group of soils. It was also found that the use of the variables: organic C, iron and manganese extracted by ammonium oxalate pH 6 improve the accuracy of the exchangeable iron estimation, when compared to estimating only by the iron extraction. However, it was not reflected in better prognostic of the risk of iron toxicity occurrence in rice plants, for the group of soils. It was observed that the correlations with the two forms of estimating exchangeable iron-related attributes of toxic iron coefficients were very close, making impossible the definition of the best method. By the small number of variables to be determined, ie, by the easiness of implementation, the estimation of exchangeable iron obtained only by the iron extracted by ammonium oxalate pH 6.0 is the most efficient method to predict the risk of iron toxicity for rice crop.

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2018-11-23T15:19:44Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Roberto Carlos Doring Wolter.pdf: 2034054 bytes, checksum: d53dff149845c79db461dd69fc6b8806 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-11-23T18:52:35Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Roberto Carlos Doring Wolter.pdf: 2034054 bytes, checksum: d53dff149845c79db461dd69fc6b8806 (MD5) / Made available in DSpace on 2018-11-23T18:52:35Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Roberto Carlos Doring Wolter.pdf: 2034054 bytes, checksum: d53dff149845c79db461dd69fc6b8806 (MD5) Previous issue date: 2014-08-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / A toxidez por ferro é um dos mais importantes estresses abióticos em solos de várzea. Depois do surgimento dos sintomas de toxidez na lavoura, apenas medidas paliativas podem ser tomadas para diminuir o efeito do problema. Os objetivos do trabalho foram verificar se os critérios de interpretação para prognóstico do risco de ocorrência da toxidez por ferro em arroz irrigado por alagamento baseado na porcentagem de saturação da CTC por Fe2+ (PSFe2+), que é calculada a partir da estimativa do ferro trocável, proveniente da determinação do ferro extraído por oxalato de amônio pH 6,0 de uma amostra coletada anterior ao alagamento, são válidos para um grupo de solos de várzea do Rio Grande do Sul. Além disso, se a inclusão de outras variáveis melhora a estimação e o prognóstico, e ainda se a relação das classes de riscos está de acordo com a ocorrência da toxidez nas plantas. Para isso foram conduzidos quatro experimentos com plantas usando diferentes solos em casa de vegetação e um experimento em solução nutritiva em laboratório. Os resultados do experimento em solução nutritiva mostram que o surgimento dos sintomas de toxidez por ferro nas plantas de arroz apenas ocorreram a partir da fração entre 0,45 e 0,60 de ferro pelos cátions divalentes, diferente do valor de 0,40 da fração de ferro na solução definido como crítico para a toxidez pelo método, no entanto, não foi detectada mudança nos teores de ferro no tecido das plantas quando se variou a fração molar de ferro na solução nutritiva. Com os resultados dos experimentos com plantas em solos verificou-se que a alta PSFe2+ de uma amostra de solo está relacionada a uma maior probabilidade de ocorrência de toxidez por ferro, e em amostra com baixa PSFe2+ quase não ocorrem sintomas de toxidez por ferro, indicando que o método foi eficiente para prever a ocorrência de sintomas de toxidez por ferro nas plantas de arroz, para o grupo de solos. Também foi constatado que o uso das variáveis: C orgânico, ferro e manganês extraídos por oxalato de amônio pH 6 melhora a exatidão da estimação do ferro trocável comparado a estimação apenas pela extração de ferro, porém, isso não se refletiu em melhores prognósticos do risco de ocorrência de toxidez por ferro nas plantas de arroz do grupo de solos estudados, observou-se que as correlações com as duas formas de estimativas de ferro trocável com os atributos relacionados com a toxidez por ferro tiveram coeficientes bem próximos, impossibilitando a definição do melhor método. Pelo menor número de variáveis a serem determinadas, ou seja, pela maior facilidade de execução, a estimação do ferro trocável obtido apenas pelo ferro extraído por oxalato de amônio pH 6,0 é o método mais eficiente para prognosticar o risco de ocorrência de toxidez por ferro para o arroz irrigado. / The iron toxicity is one of the most important abiotic stress in lowland soils. After the appearance of toxicity symptoms in the field, only palliative measures can be taken to lessen the effect of the problem. The aims of this work were to verify if the interpretation criteria for predicting the risk of iron toxicity occurrence in rice plants by flooding based on the percentage saturation of CEC by Fe2+ (PSFe2+), which is calculated from the estimated exchangeable iron from the determination of iron extracted by ammonium oxalate pH 6.0 in a sample collected prior to flooding, are valid for a group of lowland soils from of Rio Grande do Sul. Furthermore, if the inclusion of other variables improves the estimation and prognosis, and if the relation of the classes of risks is consistent with the occurrence of toxicity in plants. For this, four experiments were conducted with plants, using different soils in a greenhouse and an experiment in nutrient solution in laboratory. The results of the experiment in nutrient solution have shown that the emergence of symptoms of iron toxicity in rice plants occurred only for the 0.45 and 0.60 molar ratio of iron to divalent cations, different from the 0.40 molar ratio of iron in solution defined as critical to the toxicity by the method. However, no change was detected for the iron levels on plant tissue when varying the molar ratio of iron in the nutrient solution. With the results of experiments with plants in soil one could verify that the higher the PSFe2+ of a soil sample the greater the probability of toxicity by iron, and for samples with low PSFe2+ almost no symptoms of toxicity occurred, indicating that the method was efficient in predicting the occurrence of iron toxicity symptoms in rice plants, for the group of soils. It was also found that the use of the variables: organic C, iron and manganese extracted by ammonium oxalate pH 6 improve the accuracy of the exchangeable iron estimation, when compared to estimating only by the iron extraction. However, it was not reflected in better prognostic of the risk of iron toxicity occurrence in rice plants, for the group of soils. It was observed that the correlations with the two forms of estimating exchangeable iron-related attributes of toxic iron coefficients were very close, making impossible the definition of the best method. By the small number of variables to be determined, ie, by the easiness of implementation, the estimation of exchangeable iron obtained only by the iron extracted by ammonium oxalate pH 6.0 is the most efficient method to predict the risk of iron toxicity for rice crop. Keywords: symptoms of iron toxicity, molar ratio of iron, iron oxalate, percentage of CEC saturation by Fe2+.

No description available.

Epigenetic modifications, such as DNA methylation and histone modifications, play important roles in gene expression and regulation, and are highly involved in cellular processes such as stem cell pluripotency/differentiation and tumorigenesis. Chromatin immunoprecipitation (ChIP) is the technique of choice for examining in vivo DNA-protein interactions and has been a great tool for studying epigenetic mechanisms. However, conventional ChIP assays require millions of cells for tests and are not practical for examination of samples from lab animals and patients. Automated microfluidic chips offer the advantage to handle small sample sizes and facilitate rapid reaction. They also eliminate cumbersome manual handling. In this report, I will talk about three different projects that utilized microfluidic immunoprecipitation followed by next genereation sequencing technologies to enable low input and high through epigenomics profiling. First, I examined RNA polymerase II transcriptional regulation with microfluidic chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) assays. Second, I probed the temporal dynamics in the DNA methylome during cancer development using a transgenic mouse model with microfluidic methylated DNA immunoprecipitation followed by next generation sequencing (MeDIP-seq) assays. Third, I explored negative enrichment of circulating tumor cells (CTCs) followed by microfluidic ChIP-seq technology for studying temporal dynamic histone modification (H3K4me3) of patient-derived tumor xenograft on an immunodeficient mouse model during the course of cancer metastasis. In the first study, I adapted microfluidic ChIP-seq devices to achieve ultrahigh sensitivity to study Pol2 transcriptional regulation from scarce cell samples. I dramatically increased the assay sensitivity to an unprecedented level (~50 K cells for pol2 ChIP-seq). Importantly, this is three orders of magnitude more sensitive than the prevailing pol2 ChIP-seq assays. I showed that MNase digestion provided better ChIP-seq signal than sonication, and two-steps fixation with MNase digestion provided the best ChIP-seq quality followed by one-step fixation with MNase digestion, and lastly, no fixation with MNase digestion. In the second study, I probed dynamic epigenomic changes during tumorigenesis using mice often require profiling epigenomes using a tiny quantity of tissue samples. Conventional epigenomic tests do not support such analysis due to the large amount of materials required by these assays. In this study, I developed an ultrasensitive microfluidics-based methylated DNA immunoprecipitation followed by next-generation sequencing (MeDIP-seq) technology for profiling methylomes using as little as 0.5 ng DNA (or ~100 cells) with 1.5 h on-chip process for immunoprecipitation. This technology enabled me to examine genome-wide DNA methylation in a C3(1)/SV40 T-antigen transgenic mouse model during different stages of mammary cancer development. Using this data, I identified differentially methylated regions and their associated genes in different periods of cancer development. Interestingly, the results showed that methylomic features are dynamic and change with tumor developmental stage. In the last study, I developed a negative enrichment of CTCs followed by ultrasensitive microfluidic ChIP-seq technology for profiling histone modification (H3K4Me3) of CTCs to resolve the technical challenges associated with CTC isolation and difficulties related with tools for profiling whole genome histone modification on tiny cell samples. / Ph. D.

Objective: The aim of this study was to develop an immunomagnetic/real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay and assess its clinical value for the molecular detection of circulating tumor cells (CTCs) in peripheral blood of pancreatic cancer patients. Methods: The presence of CTCs was evaluated in 34 pancreatic cancer patients before systemic therapy and in 40 healthy controls, through immunomagnetic enrichment, using the antibodies BM7 and VU1D9 [targeting mucin 1 and epithelial cell adhesion molecule (EpCAM), respectively], followed by real-time RT-PCR analysis of the genes KRT19, MUC1, EPCAM, CEACAM5 and BIRC5. Results: The developed assay showed high specificity, as none of the healthy controls were found to be positive for the multimarker gene panel. CTCs were detected in 47.1% of the pancreatic cancer patients before the beginning of systemic treatment. Shorter median progression-free survival (PFS) was observed for patients who had at least one detectable tumor-associated transcript, compared with patients who were CTC negative. Median PFS time was 66.0 days [95% confidence interval (CI) 44.8–87.2] for patients with baseline CTC positivity and 138.0 days (95% CI 124.1–151.9) for CTC-negative patients (p = 0.01, log-rank test). Conclusion: Our results suggest that in addition to the current prognostic methods, CTC analysis represents a potential complementary tool for prediction of outcome in pancreatic cancer patients. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Le cancer est l'une des principales causes de décès dans le monde. D'après la société américaine contre le cancer; en 2015, un quart des décès aux Etats-Unis est du au cancer du poumon avant même les maladies cardiaques. Cette situation nous incite et bien d'autres scientifiques dans le monde à développer des moyens plus efficaces de traitement, le diagnostic et le dépistage de la maladie. Parce que près de 90% des décès par cancer sont dus à des métastases, de nombreuses études se sont concentrées sur le mécanisme de métastases et sur son impact clinique. Les cellules tumorales circulantes (CTC) sont les cellules s’échappent de tumeurs primaires ou métastatiques pour rejoindre le flux sanguin périphérique, ces cellules sont un élément de transition dans le processus métastatique et portent ainsi des informations cruciales sur ce mécanisme encore mal compris. Les CTCs ont déjà montré leur potentiel comme biomarqueur de pronostic de la progression de la maladie et de l'indicateur de l'efficacité du traitement en fonction l’augmentation ou de la diminution de leur nombre. Leur caractérisation moléculaire peut également donner des informations vis à vis de cibles thérapeutiques possibles et des mécanismes de progression de la maladie ou de la résistance aux médicaments. Leur comptage au cours du traitement combiné avec leur caractérisation moléculaire devrait améliorer la prise en charge des patients dans le cadre de la médecine personnalisée. Cependant CTCs sont extrêmement rares, 1 à 10 cellules / ml de sang parmi les 106 globules blancs et 109 globules rouges, leur capture à partir du sang reste donc un challenge analytique. Dans les dernières décennies, Une grande variété de techniques d'enrichissement et de capture a été mise au point et l'approche microfluidique est l'une des méthodes efficaces, flexibles et à haut débit. Au sein de notre équipe, un dispositif microfluidique (système Ephesia) puissant pour la capture et l'analyse des cellules tumorales circulantes a déjà été mis au point précédemment. Le principe de capture est basé sur l'auto-assemblage de billes magnétiques greffées par des anticorps, grâce aux quelles les cellules sont enrichies via l’interaction Ab- l'antigène de surface EpCAM que l'on trouve communément dans les cellules cancéreuses d'origine épithéliale. Ce système a déjà été validé avec des lignées cellulaires et des échantillons de patients. Cependant, le système n'a pas permis l'isolement / détection des sous-populations de CTCs ou d'effectuer une caractérisation moléculaire très poussée. Par conséquent, mon projet de thèse vise à améliorer encore les capacités du système sur les deux principaux aspects: le ciblage sous-populations de CTC et à l'étude des interactions des protéines à la surface des CTCs dans le Système Ephesia... / Metastasis is the advanced stage of cancer progression and is the cause of 90% of deaths in cancer disease. During metastatic cascade, it is suggested that the successful metastatic initiation depends on the survival of circulating tumor cells (CTCs). CTCs are the cells that shed from the primary or secondary tumor sites into the blood circulation. it is now widely recognized as potential biomarker for companion diagnostics in which high number of CTCs in blood can indicate association with poor survival or high risk of disease progression. Besides, following the number of CTCs during the course of treatment can help to adapt the selected therapy and predict the treatment efficacy. On the other hand molecular characterization can provide patient stratification and identifying the therapeutic targets. However they are extremely rare in the bloodstream, estimated between 1-10 CTC among 6×106 leukocytes, 2×108 platelets and 4×109 erythrocytes per one mL of blood which makes their isolation very challenging. A very attractive way of isolation of CTCs is to integrate microfluidics. Microfluidics offers great advantages such as low volume of reagent consumption and short analysis times with automation as well as isolation and detection analysis can be integrated resulting in highly efficient biomedical devices for diagnostics. As parallel to state of the art, a powerful microfluidic device for circulating tumor cells capture and analysis had already been developed previously in our laboratory. The principle of capture is based on self-assembly of antibody-coated (EpCAM) magnetic beads in which the cells are enriched by EpCAM surface antigen which is found commonly in epithelial origin cancer cells. This system was already validated with cell lines and patients samples. However, the system did not allow isolation/detection of subpopulations of CTCs or performing high content molecular characterization. Therefore, my PhD project aimed at further improving the capabilities of the system on the main two aspects: targeting subpopulations of CTC and studying of protein interactions of CTCs in Ephesia System...

No description available.