Dissertationen zum Thema „Mpny“

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Les néoplasies myéloprolifératives (MPN) Ph-négatifs sont des anomalies clonales des cellules souches hématopoïétiques provoquant une prolifération massive de cellules sanguines et plusieurs manifestations cliniques: la Polyglobulie de Vaquez (PV) et la Thrombocytémie essentielle (TE) affectent respectivement les lignées érythrocytaire et mégacaryocytaire ; la myélofibrose primitive consiste en un enrichissement de la moelle osseuse en collagène. Des troubles de l'hémostase ont été rapportés avec en particulier des risques de thromboses élevés qui sont la première cause de mortalité chez les patients MPN. Des facteurs de risques de thrombose sont identifiés mais l'étude de la pathogénie est limitée par l'hétérogénéité des patients et les traitements prophylactiques. Notre objectif principal a été de déterminer l'effet de la mutation JAK2v617F, retrouvée fréquemment chez les patients MPN, sur l'hémostase et la thrombose. Dans des modèles murins mimant une PV, nous avons observés des dysfonctions complexes de l'hémostase non directement liés à l'expression de JAK2v6I7F. Cette étude nous a permis d'élaborer une stratégie pour étudier l'impact des traitements sur l'activation plaquettaire et endothéliale, sur une cohorte de patients MPN. Nos résultats préliminaires suggèrent que l'interféron alpha a un impact sur l'hémostase. Ces résultats, intégrés à l'ensemble de données récentes, ouvrent de nouvelles perspectives sur le mécanisme des thromboses
Myeloproliferative neoplasms (MPN) Ph-negative are clonai disorders of hematopoietic stem cells characterized by the predominant proliferation of the erythrocyte lineage in PV and of the megakaryocyte lineage in ET. In primary myelofibrosis the bone marrow is invaded by collagen leading to pancytopenia. Thrombosis episodes are frequent in MPN and represent the major cause of morbidity and mortality. Some risks factors have been identified but the pathophysiology is difficult to establish due to the heterogeneity of MPN patients. The aim of this work was to determine the effect of a mutation frequently found in MPN (JAK2v617F) on hemostasis and thrombosis. In mice models of PV, we evidenced complex and opposite hemostasis defects that were not directly linked to the expression of the mutation. This study allowed us to develop a strategy to conduct a clinical study to analyze the impact of treatments on piatelet and endothelial activation in a cohort of MPN patients. Our preliminary data suggest that alpha-interferon may impact hemostasis in MPN patients. These resuits and other recent data open new perspectives on mechanisms leading to thrombosis

Le compostage est une technique de valorisation des déchets organiques en un produit stable et riche en matières humiques. Certains composts « verts » se sont révélés être de 2 à 3 fois plus efficaces sur la croissance des plantes que les composts classiques. Notre étude réalisée sur un compost fabriqué à partir de déchets végétaux a permis de suivre l’évolution de la densité et de la diversité de la microflore (bactéries, champignons) au cours du processus de maturation puis de tester l’impact de ce compost sur la diversité et l’activité de la microflore tellurique. Cette analyse a été effectuée par des techniques complémentaires : biochimiques (dosages enzymatiques), microbiologiques (cultures in vitro) et de biologie moléculaire (PCR-DGGE, Séquençage). Les résultats montrent qu’au cours de sa maturation, le compost étudié présente une baisse significative de son taux d’humidité et une augmentation sensible de son pH. Sa microflore subit une complète restructuration avec apparition de souches bactériennes susceptibles de dégrader des composés polluants comme les plastiques, les pesticides et les hydrocarbures. L’ajout de ce compost à deux types de sol présentant des propriétés physico-chimiques différentes, n’a pas montré de modifications importantes et durables de la diversité microbienne et fonctionnelle de celui-ci. Les causes de l’effet remarquable de ce compost sur la croissance végétale sont discutées
Composting is a technique of transformation organic waste in a stable product rich in organic materials. Some "green" compost proved to be from 2 to 3 times more benefit on the growth of the plants than traditional composts. The main of this study is to follow the evolution of density and diversity of the microflora (bacteria, fungi) during the process of maturation of green compost manufactured from vegetable wastes, and to investigate the impact of this compost on the diversity and the activity of the telluric microflora. This analysis was carried out by complementary techniques: biochemical (enzymatic activity), microbiological (in vitro cultures) and molecular biology (PCR-DGGE, DNA sequencing). The results show that during its maturation, the studied compost presents a significant decrease of its water content and an appreciable increase in its pH. The microflora undergoes a complete reorganization with appearance of bacterial strain suitable for degrade polluting compounds like the plastics, the pesticides and hydrocarbons. The addition of this compost with two types of soil presenting of the different physicochemical properties, did not show significant and durable modifications of the microbial and functional diversity of this one. The causes of the remarkable effect of this compost on the vegetable growth are discussed

One of the most serious and economically important defects caused by clostridia in milk products is the late blowing of semi-hard cheeses.

Clostridia occur naturally in soil and can contaminate milk through crops contaminated by dung and soil followed by a less successful silage process, that give them opportunity to grow unaerobically. When anaerobic conditions occur, such as storage of semi-hard cheese, they ferment lactic acid to butyric acid and the gases CO₂ and H₂.

At the fusion of Arla and MD Foods, a series of changes were conducted on the MPN method for lactic acid fermentation for clostridia in milk. These changes resulted in an increased accuracy due to an increased number of test tubes and the change of media from MRCM to BBB, Bryant & Burkey Broth, that was thought to be more selective for Cl. tyrobutyricum, the organism mostly found in hard cheese. When the number of dairy farmers that were given quality reduction fines increased, the new method was suspected and a validation was conducted.

The validation included inoculation of different clostridia and bacillus strains into BBB substrate and enzymatic testing of positive samples with Rapid ID 32A. The inoculation result showed that almost all tested different clostridia strains could grow in BBB substrate.

Test on BBB positive tubes with Rapid ID 32A resulted in 95% clostridia of which 70% was Cl. tyrobutyricum. These results correlated well with earlier studies on MRCM substrate and the increase in quality reduction fines probably depended on the larger number of test tubes used in the new method rather than the change of substrate.

Clostridier eller främst Cl. tyrobutyricum är den art som i de flesta fall orsakar feljästa ellersönderjästa ostar, vilket är ett välkänt problem inom mejeriindustrin. Clostridiesporer finns naturligt i jord och hamnar i mjölkråvaran via gröda som kontaminerats med gödsel och jord och som tillsammans med en mindre lyckad ensileringsprocess gör att clostridierna växer till. När anaeroba förhållanden uppstår, såsom vid lagring av hårdost, förjäser clostridierna laktat och smörsyra varvid vätgas och koldioxid bildas.

I samband med fusionen av Arla/MD Foods, genomfördes metodförändringar för MPN-metoden för laktatjäsande clostridier i mjölk, som används inom mjölkbedömningen. Dessa förändringar innebar en skärpning i noggrannheten genom att man ökade antal rör, samt ett substratbyte från MRCM till BBB, Bryant & Burkey Broth, som ansågs mer gynnsamt för Cl. tyrobutyricum.

Då antalet mjölkproducenter som fick kvalitetsavdrag ökade, riktades misstankar mot den nya metoden och en validering genomfördes.

Resultatet av valideringen, som innebar ympning av renkulturer, utodling av positiva rör med efterföljande typning, visade att de flesta av de tillsatta stammarna av clostridier hade förmågan att ge ett positivt utslag vid tillsats av renkulturer. Utodling och typning av positiva rör visade att ca 70 % av de positiva utslagen innehöll Cl. tyrobutyricum och hela 95 % någon clostridieart. Dessa resultat korrelerar med tidigare studier på MRCM-substrat och ökningen av positiva utslag berodde troligtvis på det ökade antalet rör som den nya metoden innebar.

The breakdown of contaminants in soil resulting from microbial activity that is enhanced in the presence of the plant root zone, rhizosphere, has been termed rhizoremediation. To date, Australian native plants have not been assessed for their hydrocarbon rhizoremediation potential. The use of native plants offers an economically feasible and environmentally sustainable cleanup option for the rehabilitation and restoration of hydrocarbon contaminated sites in Australia. The aim of the study was to evaluate the potential of Australian native grass species for the rhizoremediation of aliphatic hydrocarbon contaminated soil from a mine site. Candidate Australian native grass species Poaceae were selected following the development of essential and desirable growth criteria. Nine perennial Australian grasses were evaluated for seedling emergence in sandy loam soil sourced from a mine site which was artificially contaminated with a 60:40 diesel/oil mix at concentrations of 30 000 mg/kg, 10 000 mg/kg, 5 000 mg/kg and 0 mg/kg control. Seedling emergence was not adversely affected by the presence of hydrocarbon contamination at the exposed concentrations for eight of the nine species studied p > 0.05. Three promising species were assessed for relative growth performance in diesel/oil contaminated 10 000 mg/kg, 5 000 mg/kg and uncontaminated control soils in greenhouse studies to assess their tolerance of aliphatic hydrocarbon contaminated soil. Cymbopogon ambiguus Lemon Scented grass is a summer growing perennial with widespread distribution throughout Australia including the region where the mine site is situated. Brachiaria decumbens Signal grass – naturalised - is adapted to humid tropical areas of Australia and is native to the site and sourced from seed banks. Microlaena stipoides Weeping grass var. Griffin is a cool season grass, widely distributed throughout Australia in moister regions. The three evaluated species survived for 120 days in the diesel/oil contaminated soil at the exposed concentrations without adverse growth affect p > 0.05. In some instances e.g. C. ambiguus growth stimulation occurred in the presence of contamination producing significantly more root biomass compared with the control p < 0.0001. Most hydrocarbon degradation is believed to occur through microbial processes, and so the plant-associated microbial community was examined in the three tolerant species. The assessment of the influence of grass on the abundance and activity of microorganisms in the rhizosphere revealed species-specific plant-induced changes in the soil microbial community. Selective enrichment of hydrocarbon degrading microorganisms was demonstrated in the rhizosphere soil of the Australian grasses tested, to varying degrees. C. ambiguus appeared to have the greatest influence on stimulation of hydrocarbon degrading microorganisms, followed by the cool season grass M. stipoides. B. decumbens showed consistently lower numbers of hydrocarbon degrading microorganisms in rhizosphere soil over time compared to the other two species p < 0.01. The influence of grasses on microbial community structure - defined as community DNA fingerprint - in diesel/oil contaminated soil suggested no new microbial population was favoured by the grasses - qualitative shift - rather there were relative quantitative changes in existing members of the microbial population. Soil lipase activity did not appear to be an optimal bioindicator of rhizoremediation and may encompass total soil microbial activity not exclusively the hydrocarbon degrading microorganisms of interest. The assessment of biodegradation of hydrocarbons in soil is essential to characterise the effectiveness of plant species in rhizoremediation. Residual diesel and oil concentrations as total petroleum hydrocarbons, TPH were measured using Gas Chromatography. The presence of single species successfully enhanced the removal of hydrocarbons from soil for all species. All showed significantly lower residual hydrocarbon concentrations than those in unplanted soil after 100 days p < 0.01. Significantly, it was not necessary to add N and P to achieve up to 90% reduction in hydrocarbon concentrations in the soil. The relative performance of each grass species varied. In soil planted with C. ambiguus hydrocarbon concentrations were reduced faster and to a greater extent than the other species studied, from 10 000 mg/kg to approximately 1 100 mg/kg TPH, 88% removal. Similar endpoint success was recorded for M. stipoides which facilitated 80% reduction in hydrocarbon concentrations. Interestingly, B. decumbens, the only naturalised species, did not perform as well as the other species, although still significantly better compared to unplanted controls, with hydrocarbon concentrations reduced to approximately 4 500 mg/kg, 49%. Hydrocarbon concentrations in unplanted control soil were reduced by 45% through natural biodegradation processes. Plant root and shoot tissue was periodically assessed for hydrocarbon accumulation and was shown to be negligible. A multispecies planted trial using C. ambiguus plus B. decumbens had no additional influence on total TPH removal. The final TPH removal efficiency in the multispecies trial was not significantly different p > 0.05 from that of the best single species performer of the two i.e. C. ambiguus. In a field application the planting of multiple species may still be desirable in order to preserve site biodiversity and assist rehabilitation of the area. A strong relationship between abundance of hydrocarbon degrading microorganisms in the rhizosphere and hydrocarbon biodegradation was demonstrated for all species p < 0.01. Those species which showed greatest stimulation of the microbial population resulted in enhanced TPH removal from soil. These species were the summer grass C. ambiguus and the winter species M. stipoides. This may allow for broader application both seasonally and geographically across Australia. B. decumbens showed successful rhizoremediation to a lesser degree, but may still be an option in multiple planting strategies. This investigation identified three Australian grass species from the nine evaluated that are candidates for further investigation for in situ rhizoremediation potential at field scale.

The past decade has witnessed significant progress in the understanding of the molecular pathogenesis of myeloproliferative neoplasms (MPN). Mutations in a large number of genes have now been implicated in the pathogenesis of MPN but their relative importance, the mechanisms by which they cause different cells to predominate and differentiate into the separate MPN syndromes and their implications for prognosis remain unknown. I hypothesized that other genes and epigenetic mechanisms may contribute to the pathogenesis of the different disease subtypes at a cell-type specific level. This study focused on four areas: (1) Clinical phenotype - The demographic and clinical landscape of MPN in Malaysia showed that MPN is predominantly found in older Malay men. Compared to other studies, there is a higher incidence of JAK2 V617F positivity, with a more severe clinical phenotype and complications. (2) MPN panel - JAK2 V617F is the commonest somatic driver mutation detected in MPN patients using this 26-gene MPN targeted sequencing panel. The highest allelic frequencies were found in polymorphonuclear (PMN) and mononuclear cells (MNC). Several novel variants were found. Primary myelofibrosis patients who harbor the JAK2 V617F in combination with ASXL1, DNMT3A, RUNX1, TET2 and U2AF1 putatively pathogenic variants were found to have more severe clinical phenotypes and poor prognosis. (3) Gene Expression and (4) DNA methylation - Using microarray and next generation sequencing techniques, cell type-specific differentially expressed genes and differentially methylated CpG sites were found in PMN and MNC in the different disease subtypes. The lack of differential expression and methylation in T cells validated the approach and indicated they are not part of the neoplastic clone. The differentially expressed genes and differentially methylated CpG sites with associated genes are candidate loci to explain the pathogenesis of MPN and its different forms. These loci also represent targets for further investigation and disease-specific therapeutic targets.

Bifenilas policloradas (PCBs) são compostos de difícil degradação presentes na composição de ascarel, muito utilizado como fluidos dielétricos e isolantes. Neste contexto, a presente pesquisa teve como objetivo avaliar a diversidade de microrganismos em biofilmes de reatores anaeróbios na presença de PCB empregando Métodos de Microbiologia de Anaeróbios Estritos e de Biologia Molecular. Em reator anaeróbio horizontal de leito fixo (RAHLF), alimentado com etanol, formiato, Triton X-100 (0,1%) e ascarel (1 mL/L), operado com tempo de detenção hidráulica (TDH) de 24 horas, foi retirado a comunidade microbiana do biofilme da espuma de poliuretano. Os grupos microbianos encontrados por meio da clonagem e sequenciamento do gene RNAr 16S para o domínio Bacteria foram relacionados aos filos Thermogae, Proteobacteria (Brachymonas petroleovorans, 100% de similaridade e Methylobacillus, 98% de similaridade), Firmicutes (Clostridium, 97% de similaridade, Syntrophomonas, 100% de similaridade e Sporomusa com 100% de similaridade), Synegistetes (Synergistes, 98% de similaridade), Spirochaetes (Leptonema illini, 98% de similaridade), Aminanaerobia, Deferribacteres, Chlorobi, Chloroflexi e Armatimonadetes. Além disso, como nesse biofilme foram identificadas bactérias redutoras de ferro, procedeu-se a sua quantificação por meio da técnica de tubos múltiplos (NMP, Número Mais Provável) obtendo 5,26 x \'10 POT.12\' NMP/g STV de bactérias redutoras de ferro. Ensaio em batelada foi realizado separadamente sob duas condições: (1) metanogênica e (2) ferro redutora. Em ambas as condições foram adicionadas aroclor 1260 (PCB). Os reatores, sob condição metanogênica, foram alimentados com meio de cultivo Angelidaki e substratos orgânicos (formiato e etanol), além de aroclor 1260 (0,2 \'mü\'g/L). Para simular a condição redutora de ferro foi acrescido ao meio de cultura Angelidaki, EDTA férrico (1,86 g/L). A produção de metano, na presença de aroclor 1260 foi de 3,8 x \'10 POT.-4\' mmol \'CH IND.4\'/g STV. A presença de bactérias ferro redutoras foi confirmada indiretamente pela taxa média de redução férrica (90%) nos reatores em batelada, após 60 dias de operação. Por meio de PCR/DGGE, elaborou-se um dendograma das amostras deste ensaio em batelada (metanogênico e redutor de ferro) comparativamente com as do reator RAHLF (biofilme presente na parede do reator e no material suporte). Os reatores em batelada apresentaram similaridade entre si de 79% e 92% para os domínios Bacteria e Archaea, respectivamente. As amostras do reator RAHLF foram 80% (Bacteria) e 96% (Archaea) similares. A existência de bactérias degradadoras de PCB, bem como, bactérias redutoras de ferro no biofilme anaeróbio contribuiu com informações sobre o consórcio microbiano e sua diversidade.
Polychlorinated biphenyls (PCBs) are compounds of difficult degradation, a component of askarel, which were used widely as coolants and lubricants. Hence, this study evaluated the diversity of microorganisms in the presence of PCBs in anaerobic reactors. For such, methods as Strict Anaerobic Microbiology and Molecular Biology were employed. The microbial community of the biofilm, developed in a fixed horizontal bed anaerobic reactor (RAHLF), was studied using the technique of cloning and sequencing of RNAr 16S gene for the Bacteria domain. The reactor had immobilized cells in polyurethane foam with ethanol and formate as a carbon source, Triton X-100 (0.1%) and polychlorinated biphenyls (1 mL/L), and operated with 24 hours HRT. The microbial groups found in this biofilm were related to phyla Thermogae, Proteobacteria (Brachymonas petroleovorans, 100% similarity and Methylobacillus, 98% similarity), Firmicutes (Clostridium, 97% similarity Syntrophomonas, and 100% similarity with Sporomusa 100% similarity), Synegistetes (Synergistes, 98% similarity), Spirochaetes (Leptonema Illini, 98% similarity), Aminanaerobia, Deferribacteres, Chlorobi, Chloroflexi and Armatimonadetes. Furthermore, as bacteria that reduce iron were found, we proceeded the quantification by the multiple tube method (MPN) for this group, obtaining 5.26 x \'10 POT.12\' MPN/g STV of iron-reducing bacteria. The batch reactors evaluated the growth of microorganisms in two condictions: (1) methanogenic e (2) iron reduction, both had the presence of PCBs (Aroclor 1260). The reactor, under methanogenic condition, was fed with synthetic substrate Angelidaki, ethanol and formate, used as carbon source, and aroclor 1260 (0.2 \'mü\'g /L). To simulate the condition of iron reducing, the same synthetic substrate was supplemented with ferric EDTA (1.86 g/L). The production of methane in the presence of aroclor 1260, was 3.8 x \'10 POT.-4\' mmol \'CH IND.4\'/g STV. The presence of iron reducing bacteria, after 60 days, was confirmed indirectly by the average rate of iron ferric reduction (90%). Filogenetics analysis (PCR/DGGE) compared the samples of this batch reactor - methanogenic and reduction of iron ferric -, with the samples of RAHLF - the biofilm in the reactor wall and the support material. The two condictions in batch reactors showed similarity of 79% and 92% respectively for the Bacteria and Archaea domain. Therefore, both samples of RAHLF showed 80% (Bacteria) and 96% (Archaea) of similarity. In other words, more similarity were presented due configuration of the reactor as well as the type of PCB added. As a result, the existence of PCBs degrading bacteria and iron-reducing bacteria in anaerobic biofilm, provided informations about the microbial consortium and its diversity in the presence of PCB.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O manejo agrícola altera, em muito, as características físicas, químicas e biológicas dos solos. A cultura e as condições climáticas, assim como o tipo de solo propriamente dito, afetam a decomposição da matéria orgânica do solo e, consequentemente, a biogeociclagem dos nutrientes. A matéria orgânica é uma característica importante em relação à fertilidade do solo, de modo que o impacto do uso do solo precisa ser avaliado principalmente em agroecossistemas. O conhecimento dos efeitos do uso da terra e das práticas agrícolas sobre a comunidade microbiana é de fundamental importância, em vista das importantes funções que os microorganismos desempenham no solo e que irão se refletir na produtividade agrícola. Neste sentido, uma avaliação da biomassa microbiana e de microorganismos do solo pode evidenciar diversas alterações no ecossistema do solo que estão associadas ao teor de argila e/ou sistemas de plantio. No presente estudo, caracterizou-se a camada superficial do perfil de solos sob plantio direto e plantio convencional em quatro fazendas no Brasil em relação a alguns de seus componentes microbiológicos. Para tanto, analisou-se, mensalmente, a respiração do solo, a biomassa microbiana e grupos de microorganismos. A avaliação da biomassa microbiana foi feita através da técnica da fumigação-incubação (FI), utilizando-se a Equação: [(C-CO2 liberado pelo solo fumigado, no período de 0-10 dias de incubação) - (C-CO2 liberado pelo solo não-fumigado, ao longo de 10-20 dias de incubação)]/0,45. Os cálculos indicaram um conteúdo de carbono da biomassa microbiana significativamente maior nos solos sob sistema de plantio direto em relação àqueles sob plantio convencional, na camada amostrada (0-10 cm de profundidade). Quantidades significativamente maiores...
The crop management changes greatly the physical, chemical and biological soil properties. Furthermore, the crop and soil types, and the climatic conditions would affect on soil organic matter decomposition and on nutrients biogeociclying. Soil organic matter is a important characteristic in relationship soil fertility. The knowledge about effects on soil using and agriculture practices on soil microbial communities is very important, due to the function that microorganisms have in soil and it was going to in soil fertility. In this sense, evaluation of the soil microbial biomass and micro-organisms greatly aids predictions several changes in the soil ecosystems are associated with reduced tillage as compared with conventional tillage. Surface soils from long-term no-till and conventional tillage plots at four Brazil farms were characterised for microbial components. Soil respiration, microbial biomass carbon and counts of microorganisms were measured at intervals monthly. The evaluation of microbial biomass carbon was done by fumigation-incubation technique (FI). For calculating the soil microbial biomass carbon, the equation used was: Equation = [(CO2-C evolved by fumigated soil, 0-10 days) - (CO2-C evolved by unfumigated soil, 10-20 days)]/0,45. Significantly greater amounts of CO2-C were released from no-till than from conventional tilled soils. qCO2 values were not significantly different between tillage systems. This observation confirms that the tillage affected biological activity in those soils, further that qCO2 values didn't have significantily different which two tillage systems, in studied soils. Number both fungi and bacteria were assayed by Most Probable Number (MPN) by the agar drop counting technique and the microorganisms groups were calculated using by traditional Most Probable Number (MPN) method...(Complete abstract, click electronic access below)

The small village Chonyonyo, in the district Karagwe in the northwest of Tanzania has a shortage of safe drinking water. Women and children spend several hours a day fetching water and gathering firewood to boil the water to make it more suitable for drinking. The need of new sustainable water supply solutions is fundamental for providing more people with safe drinking water.   Two water distribution alternatives was suggested by Engineers Without Borders and MAVUNO as possible solutions to supply the community with water.  Alternative 1 consisted of a 10 km distribution system from an already existing groundwater well at the MAVUNO office to Chonyonyo. Alternative 2 consisted of a 3.5 km distribution system from the most neighboring valley to Chonyonyo, where no groundwater well exists today. Both alternatives would be powered by solar panels and operated six hours a day. The most sustainable distribution solution was chosen by modeling the distribution alternatives in the modeling software EPANET. Input parameters to simulate the model were position, elevation and dimension of storage tanks and pipes. Other required input parameters were absolute roughness, water withdrawal, operation hours, description of the withdrawal pattern for the water outlet and other modelling conditions such as a suitable simulation time. The selection of water distribution system was based on minimum requirements of energy used for operation weighed with lowest possible water residence time in the storage tank. Water quality analyses of the raw water source for distribution alternative 1 were performed in order to classify the water and select suitable water treatment solutions. The analyses consisted of microbiological and metal/metalloid analyses, and measurements of EC and pH. The result of the simulation showed that neither of the distribution alternatives met all the pipe design criteria. The main reason is that the system can not be constantly operated. If this criterion is excluded the optimal solution is distribution alternative 1 with an outer pipe diameter of 110 mm throughout the whole distribution system and a water residence time in the storage tank of 57.1 hours. The water quality analysis showed that the ground water source for distribution alternative 1 was affected by surface water and is thus classified as unusable because of high levels of harmful bacteria and lead. The most suitable water treatment solution due to the aspects of sustainable water supply are the microbiological barriers ultrafiltration and UV-light in combination with a treatment method to remove lead from the water.
I samhället Chonyonyo i distriktet Karagwe i nordvästra Tanzania råder brist på säkert dricksvatten. Kvinnor och barn spenderar flera timmar om dagen för att hämta vatten och samla ved för att koka vattnet så att det blir säkrare att dricka. Behovet av nya hållbara vattenlösningar är fundamental för att kunna försörja fler människor med säkert dricksvatten.   Två vattendistributionsalternativ lades fram av Ingenjörer utan gränser och MAVUNO som möjliga lösningar för att försörja invånarna i Chonyonyo med dricksvatten. Alternativ 1 bestod av ett 10 km distributionssystem från en befintlig grundvattenbrunn vid MAVUNO:s kontor. Alternativ 2 bestod av ett 3.5 km distributionssystem från den närmaste dalen till samhället Chonyonyo, där det inte finns någon befintlig grundvattenbrunn. Båda alternativen kommer att drivas av solpaneler och vara under drift sex timmar per dygn. Det lämpligaste distributionsalternativet valdes ut genom simulering i mjukvaran EPANET. Ingångsparametrar för simuleringen var bl.a. position, höjd och dimension på reservoarer och ledningar. Ytterligare nödvändiga parametrar var skrovlighet på ledningar, storlek på vattenuttag, antal driftstimmar, uttagmönster från vattenkranar i systemet samt andra modelleringsförhållanden såsom en lämplig simuleringstid. Valet av distributionssystem grundades på lägsta möjliga energibehov för drift viktat mot lägsta möjliga uppehållstid i vattenreservoarerna.   Kvalitetsanalyser av råvattnet för distributionsalternativ 1 genomfördes för att klassificera vattnet och göra lämpliga val av vattenreningslösningar. Analyserna omfattade mätning av ett antal mikrobiologiska parametrar, metaller/metalloider samt EC och pH. Simuleringen visade att ingen av alternativen kunde uppnå alla designkriterierna. Huvudorsaken till det är att systemet endast är i drift periodvis. Bortsett från dessa kriterier var det optimala lösningen distributionsalternativ 1 med en yttre rördiameter på 110 mm genom hela systemet med en maximal uppehållstid i vattenreservoaren på ca 57 timmar.   Analyserna visade att grundvattnet var ytvattenpåverkat och klassificeras som otjänligt med höga nivåer av skadliga bakterier och bly. De lämpligaste vattenreningslösningarna i förhållande till hållbarhetsaspekterna var de mikrobiologiska barriärerna ultrafiltrering och behandling med UV-ljus kombinerat med en reningsmetod för att avskilja bly från vattnet.

Orientador: Marli Teixeira de Almeida Minhoni
Resumo: O manejo agrícola altera, em muito, as características físicas, químicas e biológicas dos solos. A cultura e as condições climáticas, assim como o tipo de solo propriamente dito, afetam a decomposição da matéria orgânica do solo e, consequentemente, a biogeociclagem dos nutrientes. A matéria orgânica é uma característica importante em relação à fertilidade do solo, de modo que o impacto do uso do solo precisa ser avaliado principalmente em agroecossistemas. O conhecimento dos efeitos do uso da terra e das práticas agrícolas sobre a comunidade microbiana é de fundamental importância, em vista das importantes funções que os microorganismos desempenham no solo e que irão se refletir na produtividade agrícola. Neste sentido, uma avaliação da biomassa microbiana e de microorganismos do solo pode evidenciar diversas alterações no ecossistema do solo que estão associadas ao teor de argila e/ou sistemas de plantio. No presente estudo, caracterizou-se a camada superficial do perfil de solos sob plantio direto e plantio convencional em quatro fazendas no Brasil em relação a alguns de seus componentes microbiológicos. Para tanto, analisou-se, mensalmente, a respiração do solo, a biomassa microbiana e grupos de microorganismos. A avaliação da biomassa microbiana foi feita através da técnica da fumigação-incubação (FI), utilizando-se a Equação: [(C-CO2 liberado pelo solo fumigado, no período de 0-10 dias de incubação) - (C-CO2 liberado pelo solo não-fumigado, ao longo de 10-20 dias de incubação)]/0,45. Os cálculos indicaram um conteúdo de carbono da biomassa microbiana significativamente maior nos solos sob sistema de plantio direto em relação àqueles sob plantio convencional, na camada amostrada (0-10 cm de profundidade). Quantidades significativamente maiores ...(Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The crop management changes greatly the physical, chemical and biological soil properties. Furthermore, the crop and soil types, and the climatic conditions would affect on soil organic matter decomposition and on nutrients biogeociclying. Soil organic matter is a important characteristic in relationship soil fertility. The knowledge about effects on soil using and agriculture practices on soil microbial communities is very important, due to the function that microorganisms have in soil and it was going to in soil fertility. In this sense, evaluation of the soil microbial biomass and micro-organisms greatly aids predictions several changes in the soil ecosystems are associated with reduced tillage as compared with conventional tillage. Surface soils from long-term no-till and conventional tillage plots at four Brazil farms were characterised for microbial components. Soil respiration, microbial biomass carbon and counts of microorganisms were measured at intervals monthly. The evaluation of microbial biomass carbon was done by fumigation-incubation technique (FI). For calculating the soil microbial biomass carbon, the equation used was: Equation = [(CO2-C evolved by fumigated soil, 0-10 days) - (CO2-C evolved by unfumigated soil, 10-20 days)]/0,45. Significantly greater amounts of CO2-C were released from no-till than from conventional tilled soils. qCO2 values were not significantly different between tillage systems. This observation confirms that the tillage affected biological activity in those soils, further that qCO2 values didn't have significantily different which two tillage systems, in studied soils. Number both fungi and bacteria were assayed by Most Probable Number (MPN) by the agar drop counting technique and the microorganisms groups were calculated using by traditional Most Probable Number (MPN) method...(Complete abstract, click electronic access below)
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Este trabalho apresenta a descrição e análise das possibilidades e limitações da aplicação do Balanced Scorecard (BSc), com foco na sua face ferramental enquanto sistema de gestão estratégica, no agronegócio. Nesse trabalho foi realizada a pesquisa-ação de caráter qualitativo e a metodologia escolhida foi o estudo de caso, utilizando-se para tal de pesquisa documental e entrevistas semi-estruturadas. Como resultado, tivemos a percepção da importância do contexto na indicação de qual ou quais perspectivas deveriam ser utilizadas como eixo no processo de desenvolvimento e implementação da metodologia-ferramenta, bem como a possibilidade de reconfiguração de perspectivas, em especial em relação aos aspectos de responsabilidade social pelo desenvolvimento no contexto em questão, o meio rural em um país emergente. O trabalho indica que a aplicação da “metodologia-ferramenta” BSc requer sua face ferramental associada a um sistema de gestão estratégica em linguagem computacional, sendo útil o seu desenvolvimento com o uso de software em nível de usuário, como o Excel, de forma a constituir-se em ferramenta de uso aplicado imediato na gestão e, ao mesmo tempo, em fase de modelagem de processos de negócio (MPN) para uso da área de TI (Tecnologia de Informação) no desenvolvimento de sistemas integrados ERP (Enterprise Resource Planning) e ou SEM (Strategic Enterprise Planning).
Salvador

Foram estudados três procedimentos para o tratamento prévio de amostras provenientes de três sistemas de biodigestão anaeróbia operados com resíduos sólidos urbanos padronizados (RSUDp), com o fim de promover o desprendimento de células microbianas de fibras e outros materiais, e com isso obter um inóculo mais homogêneo para contagens celulares. Os tratamentos foram os seguintes: (a) suspensão de amostras em tampão fosfato e processamento em liqüidificador; (b) homogeneização mecânica e manual de amostras em solução mineral anaeróbia; (c) amostras submetidas a banho de ultra-som. O método do Número Mais Provável (NMP) foi utilizado para avaliar o número de bactérias celulolíticas e arqueas metanogênicas, em meio mineral contendo celulose em pó. Não foram constatadas diferenças entre os três tratamentos empregados, considerando-se os valores encontrados para ambos os grupos, da ordem de 102 células/mL. O tratamento com ultra-som foi escolhido para outras determinações em função da simplicidade quanto à manipulação. Assim, foram operados dois biorreatores anaeróbios com RSUDp para avaliação da reprodutibilidade dos valores das amostras após tratamento com ultra-som, para a determinação do número de microrganismos (celulolíticos, acetogênicos e metanogênicos) e avaliação da atividade microbiana anaeróbia na degradação do RSUDp. Os resultados das contagens de bactérias celulolíticas e arqueas metanogênicas pelo método do NMP em meio contendo celulose não foram superiores a 103 células/mL, e as réplicas foram bastante semelhantes. O aumento do número do grupo metanogênico pôde ser correlacionado com o aumento dos teores de metano no biorreator amostrado. As contagens de organismos acetogênicos e metanogênicos em meio mineral com fontes específicas revelou valores até 103 células/mL. Os tipos morfológicos predominantes nos exames microscópicos dos sistemas de biodigestão foram bacilos curvos, retos, sarcinas e cistos de sarcinas e as amostras dos tubos de contagem mostraram para celulolíticas, bacilos, cocos e sarcinas, para acetogênicas bacilos, bacilos espessos, cocos e cistos de sarcinas e para as arqueas metanogênicas bacilos fluorescentes e sarcinas. A evolução do processo de biodigestão anaeróbia nos dois últimos sistemas operados mostrou-se bastante próxima a proposta por BARLAZ et al. (1989b), em que o número de bactérias celulolíticas e de arqueas metanogênicas aumenta na fase considerada metânica acelerada. O conteúdo de sólidos totais diminuiu em 50% ao longo do processo e os teores de ácidos orgânicos voláteis diminuíram de 27,5 para 8, 79 g de ác. acético/L.
Three different treatments were applied to samples from three anaerobic biodigestion systems operated with standardized municipal solid waste to promote the release of microbial cells adhered to fibers and other materials, resulting in a more homogeneous inoculum to cellular counting. The following treatments were applied to the samples: a) suspension in phosphate buffer followed by blending; b) mechanical and hand homogenization in anaerobic mineral solution; c) sonication in ultrasound bath. The Most Probable Number (MPN) technique was employed to evaluate the populations of cellulolytic bacteria and methanogenic Archaea, in mineral medium containing powdered cellulose. The populations of both groups of microorganisms were close to 102 cells/mL independently of the treatment applied to release the cells and no clear distinction among them could be made concerning their efficiencies. In view of this fact the ultrasound treatment was employed in all other determinations due to its simple execution. Two anaerobic bioreactors operated with standardized municipal solid waste were monitored to evaluate the reliability of sonication as a procedure for cell release, to determine microorganism populations (cellulolytic, acetogenic bacteria and methanogenic Archaea) and to evaluate the microbial anaerobic activity concerning the biodegradation of the standardized municipal solid waste. Cellulolytic bacteria and methanogenic Archaea had similar populations (lower than 103 cells/mL) as well as acetogenic and methanogenic microorganisms (up to 103 cells/mL). The increase in the methanogenic population could be directly related to the increase of methane production in the studied reactor. The morphological types which predominate in the microscopic examinations of the biodigestion systems were curved and straight rods and sarcina. The tubes for MPN countings showed the presence of rods, coccus and sarcina for cellulolytic bacteria; rods, tick rods, coccus and sarcina for acetogenic bacteria and fluorescent rods and sarcina for methanogenic Archaea. The evolution of the anaerobic biodigestion process in the latter two monitored systems was very similar to the one proposed by BARLAZ et al. ( 1989b) where the number of cellulolytic bacteria and methanogenic Archaea increase during the accelerated methane phase. The total content of solids decreased 50% during the process and the volatile acidity decreased from 27,5 g acetic acid/L to 8,79 g acetic acid/L.

O presente estudo teve como objetivo avaliar a comunidade envolvida na desnitrificação autotrófica pela oxidação de sulfetos, aplicada ao pós-tratamento de efluentes anaeróbios. O enriquecimento da comunidade bacteriana e da comunidade desnitrificante autotrófica foi realizado a partir de amostras da biomassa coletadas de três reatores verticais de leito fixo operados em condições distintas, sendo, redução autotrófica de nitrato, redução autotrófica de nitrito e redução autotrófica de nitrato com excesso de sulfeto. Após a determinação da melhor condição de enriquecimento, a cultura foi purificada, identificada por meio de ferramentas da biologia molecular e caracterizada quanto às melhores condições de crescimento. O enriquecimento foi bem sucedido com a biomassa dos três reatores. No entanto, a condição de redução de nitrato com relação \'N\'/\'S\' igual a 0,8 foi a que apresentou maior concentração de microrganismos desnitrificantes autotróficos. A bactéria isolada foi identificada como Pseudomonas stutzeri. A velocidade específica máxima de crescimento da cultura (\'mü\'máx) foi de 0,037/h, com tempo de duplicação de 18,7 horas. O rendimento celular (Y) do composto nitrogenado foi de 0,15 gSSV.g/\'N\' e a velocidade de desnitrificação foi de aproximadamente 0,24 g\'N\'/gSSV.h. Os dados obtidos indicaram a viabilidade da aplicação da espécie isolada no processo de desnitrificação autotrófica utilizando sulfeto como doador de elétrons.
The aim of this study was to evaluate the community involved on autotrophic denitrification by sulfide oxidation applied to the post-treatment of anaerobic effluents. The enrichment of the bacterial community and autotrophic denitrifier community was accessed in three immobilized bed reactors operated at the conditions of autotrophic reduction of nitrate, autotrophic reduction of nitrite and autotrophic reduction of nitrate under excess of sulfide. Following the determination of the best enrichment conditions, the culture was purified, identified by molecular biology tools and the best growth conditions were characterized. Enriched cultures were obtained for the three operational conditions, but the best condition for the growth of autotrophic denitrifiers microorganisms was at \'N\'/\'S\' ratio of 0,8. The isolated microorganism was identified as Pseudomonas stutzeri. The maximum specific growth rate (\'mü\'máx) was 0.037/h, with a doubling time of 18.7 hours. The growth yield (Y) of nitrogen compound was 0.15 gSSV/g\'N\' and the specific rate of nitrogen utilization was approximately 0.24 g\'N\'/gSSV.h. The results indicated the viability of application of this microorganism for autotrophic denitrification using sulfide as electron donor.

碩士
國立交通大學
應用數學系
89
For a fixed graph $G$, we define the smallest integer $r=R(G)$ to be the order of a complete graph $K_{r}$ such that no matter how we assign two colors to the edges of $K_{r}$, there exists a monochromatic subgraph which is isomorphic to $G$. In this thesis, we show that for $2 \leq n \leq 7$, $R(mP_{n})=m(n+[n/2])-1$ for any $m$.

碩士
東吳大學
微生物學系
92
Nitrification is the first process of the nitrogen cycle. Ammonium-oxidizing bacteria (AOB) are the major organisms responsible for the first step in nitrification. To our environment, they are truly important. However, the growth rate of AOB is much slower than other organisms and their own metabolic products (i. e., nitrite) will damage themselves. So it’s difficult and time consuming to enumerate AOB using the traditional methods. So far, it do exist dispute about the biomass of AOB in the environment. Many ecologists try to use molecular biological methods (i. e., PCR) to improve this situation. Ammonia monooxygenase is the first enzyme involves in ammonia oxidation. It contains three genes amoA, amoB and amoC. Lots of studies using amoA to detect and enumerate AOB according to it’s low similarity to other genes and well known sequences, and each AOB contain two to three copies of this gene. So, amoA is perfect for research. In this study, we designed a pair of primers and a probe based on amoA and used polymerase chain reaction enzyme-linked immunosorbent assay（PCR-ELISA）, it is a kind of quantitative polymerase chain reaction (Q-PCR), to detect and enumerate AOB in soil samples. At first, we have to confirm the specificity of our primers. We amplified AOB and 19 known bacteria with our primers using Booster PCR, only AOB are amplified and the size, sequences of the amplified product are just what we expected. Then we confirmed that with southern hybridization, there’s no signal except AOB. Second, we set up a standard curve about AOB’s cell numbers and the luminosity units of PCR ELISA. We obtained a standard curve (Y = 0.0025X+0.8209) with the correlation coefficient (R2 value) about 0.9768. The range of cell numbers could be detected is 103 to 2.5x105 cells per milliliter. Then, we calculated the ammonium oxidation rates of the pure culture of AOB that we isolated from soil. The rate is 7.85×10-7 μg/ml·h per cell. A final goal of this study was like to find out the nitrification rates per cell of AOB in soil sample. We have calculated the ammonium oxidation rates of the soil and the rate is 11.87μg/ml·h per gram of soil. We enumerated the numbers of AOB in soil with MPN and there are 3.3×104 cells per gram of soil. We also tried to enumerated AOB in soil with PCR ELISA, this part is still proceeded.

碩士
國立臺北科技大學
化學工程與生物科技系化學工程碩士班
107
This thesis contains two research themes, mainly discussing the synthesis, electrochemical, spectroelectrochemical and other properties of microporous polymer networks (MPNs) and hyperbranched polymers, which derived from a tetra-amine monomer with the ether-linked bis-triphenylamine unit. In the first part, two microporous polymer networks (MPNs) films were successfully electrodeposited on ITO-glass from the electroactive monomers with an ether-linked bis(triphenylamine) core linked via amide groups to four triphenylamine (TPA) or N-phenylcarbazole (NPC) terminal groups. These microporous polymer films exhibit high transparency, reversible electrochemical redox behavior, and strong color change upon electro-oxidation. The P-OT(NHCO-TPA) film exhibits excellent electrochromic properties, changing color from colorless neutral state to deep blue oxidized state with the optical contrast of up to 91%. Their electrochromic properties were clearly explained by the spectroelectrochemical experiments. In addition, during the cyclic voltammetry study, the oxidation process of the amide group linked to the triphenylamine unit was observed. A possible oxidation pathway of the amide group was proposed, and the spectroelectrochemistry concerning the amide oxidation was also studied. In the second part, a series of A4 + B2 hyperbranched polyamides and polyimides were synthesized by polycondensation of a tetra-amine monomer, namely N,N,N',N'-tetrakis(4-aminophenyl)4,4'-oxydianiline (TAPODA), with the commercially available dicarboxylic acids and aromatic tetracarboxylic dianhydride, respectively. The electrochemical and spectroelectrochemical properties of the polymers are investigated. These hyperbranched polymers have low viscosity and good solubility and thus can be easily processed into polymer films. They show high thermal stability, exhibiting char yields more than 45% when heated to 800 oC under nitrogen atmosphere. Furthermore, these hyperbranched polymers also reveal reversible electrochemical and electrochromic properties due to the triphenylamine units.

Indiana University-Purdue University Indianapolis (IUPUI)
Hematopoiesis proceeds through three developmental phases, each with a unique and indispensable function. The individual roles of these phases in the pathogenesis of blood disorders is unknown. We have adapted murine lineage trace models to identify the relative contributions of embryonic, fetal, and adult hematopoietic phases to the origin of Juvenile Myelomonocytic Leukemia. We hypothesized that the fetal phase would have the most pronounced contribution to the development of JMML, a pediatric myeloproliferative disorder whose disease-initiating somatic mutations occur in utero. Progenitors expressing PTPN11E76K from all three waves were growth hypersensitive to GM-CSF due to hyperactive RAS-ERK signaling. However, fulminant myeloproliferation was only seen in fetal and adult cohorts. We observed equal disease severity in FLT3Cre; PTPN11E76K; ROSA26mTmG and CSF1R-MCM; PTPN11E76K; ROSA26YFP cohorts, which had high and low mutant allele frequencies, respectively. This led to the revelation that all progenitors in the BM niche of mutant animals have equal growth hypersensitivity and RAS-ERK hyperactivation due to non-cell autonomous effects of PTPN11E76K. We further identified that FLT3Cre has hematopoietic-restricted expression, and thereby circumvented morbidity from PTPN11E76K expression in endothelial and stromal cells. This led us to hypothesize that FLT3Cre; KrasG12D; ROSA26mTmG would be the first faithful model of JMML to express this disease-initiating mutation. Indeed, FLT3Cre; KrasG12D mice were born at expected Mendelian ratio and showed normal weight gain to 2 weeks of age. Thereafter, they acquired defining features of JMML including monocytosis, anaemia, thrombocytopenia, and hepatosplenomegaly. All FLT3Cre; KrasG12D mice succumb to a JMML-like disease, which was propagated following transplantation. This is in contrast with CSF1R-MCM; KrasG12D; ROSA26YFP mice, in which low mutant allele frequencies in either fetal or adult HSCs uniformly resulted in T-ALL. Our models reveal previously underappreciated features of JMML including an expansion of dendritic cells and a pronounced defect in T-lymphocyte development. We are the first to demonstrate non-cell autonomous effects of hematopoietic-restricted PTPN11E76K expression. Most importantly, we have shown that both the spatial and the temporal origin of JMML-initiating mutations will affect disease manifestations. Each of our findings suggest novel strategies to treat this intractable disease.

RESUMO: As neoplasias mieloproliferativas (NMPs) constituem um grupo de doenças hematopoéticas clonais resultantes da transformação de células estaminais hematopoiéticas resultando na anormal amplificação e proliferação de uma ou mais linhagens mielóides. As NMPs são doenças raras, com uma incidência estimada de 6-10/100.000 indivíduos por ano, apresentando maior incidência em indivíduos do sexo masculino, caucasianos e idosos. Neste grupo de neoplasias estão incluídas as NMPs Philadelphia negativas (NMPs-PN), que englobam três entidades principais, a Policitémia Vera (PV), a Trombocitémia Essencial (TE) e a Mielofibrose Primária (MFP). Nestas, as mutações V617F (exão 14) e no exão 12 do gene Janus Kinase 2 (JAK2), alterações nos genes que codificam para o recetor da trombopoitina (MPL) e da calriticulina (CALR) foram identificadas como driver mutations. Este estudo pretende avaliar o nível de expressão de miRNAs numa população de doentes caucasianos com NMPs-PN e determinar a validade de miRNAs específicos como um possível biomarcador minimamente invasivo no seu diagnóstico. Os miRNAsforam selecionados com base em dados publicados e na regulação das vias de expressão relacionada ao gene da caspase 9 (miR-182-5p e miR-301a-3p), via JAK/STAT (miR-125b-5p e miR-375-3p) e resistência terapêutica (miR- 203a-3p e 203b-3p). Um total de 76 amostras de medula óssea fixada em formalina e embebida em parafina (FFEP) de doentes caucasianos portugueses com NMPs-PN e 36 controlos saudáveis foram avaliados por RT-PCR. De acordo com os nossos resultados, a doença predominante foi a Trombocitémia Essencial (TE). No que diz respeito à expressão de miRNAs, não foram identificadas diferenças entre as duas populações: os miR182-5p e miR-301a-3p estão sobreexpressos, o miRNA-375-3p está subexpressos e os miR-125b-5p e miR203a-3p não apresentam tendência. Deste modo, concluímos que não podemos assumir que esses microRNAs são biomarcadores das NMPs-PN, uma vez que existe expressão simultânea em doentes e controlos. No entanto, não podemos excluir essa possibilidade, pois uma caracterização genética mais abrangente poderá contribuir para um melhor entendimento da patogénese da doença.
ABSTRACT: Myeloproliferative neoplasms (MPNs) are a group of clonal hematopoietic diseases resulting from the transformation of hematopoietic stem cells that lead to abnormal amplification and proliferation of one or more myeloid lineages. MPNs are rare diseases, with an estimated incidence of 6-10/100,000 individuals per year, with a higher incidence in males, Caucasians and the elderly. This group of neoplasms includes Philadelphia negative MPNs (PN-MPNs), which encompass three main entities, namely Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). In these, mutations V617F (exon 14) and exon 12 of the Janus Kinase 2 gene (JAK2), Myeloproliferative leukaemia virus oncogene (MPL) and Calreticulin gene (CALR) were identified as driver mutations. The present study aims to evaluate the level of expression of miRNAs in a population of Caucasian patients with PN-MPNs and to determine the validity of specific miRNAs as a possible minimally invasive biomarker in the diagnosis of this group of pathologies. The miRNAs studied were selected and grouped into: relative expression of the caspase 9 gene (miR-182-5p and miR-301a-3p), via JAK/STAT (miR-125b5p and miR-375-3p) and therapeutic resistance (miR-203a-3p and 203b-3p). A total of 76 bone marrow samples fixed in paraffin-embedded formalin (FFPE) from Portuguese Caucasian patients with PN-MPNs and 36 healthy controls were assessed by RT-PCR. According to our results, the predominant disease in the patient population studied was Essential Thrombocythemia (ET). With regard to miRNA expression the miRNAs selected were not differently expressed in both populations: miR-182-5p and miR-301a-3p are overexpressed, miR-375-3p is underexpressed and miR125b-5p and miR-203a-3p do not show any specific tendency. Thus, we conclude that it is not possible to assume that these miRNAs are biomarkers of PNMPNs, since our results indicate that there is simultaneous expression in patients and controls. However, this possibility cannot be excluded, since a more comprehensive genetic characterization may contribute to a better understanding of the pathogenesis of the disease.

Στα πλαίσια αυτής της διδακτορικής εργασίας μελετήθηκαν οι εμπορικά σημαντικότερες περιοχές καλλιέργειας και συγκομιδής οστρακοειδών του Ελλαδικού χώρου. Κατά τη διάρκεια περιόδου 18 μηνών πραγματοποιήθηκε μηνιαία συλλογή δειγμάτων στρειδιών (Οstrea edulis) και μυδιών (Mytilus galloprovincialis), τα οποία συλλέχθηκαν από έξι (6) διαφορετικά σημεία του Ελλαδικού χώρου και αναλύθηκαν για τους εντεροϊούς (EV), τους αδενοϊούς (Adv), τον ιό της ηπατίτιδας Α (HAV), τους ιούς Noro I και II (NLVI και NLVII), για το βακτήριο Ε. coli, καθώς και για σωματικούς κολιφάγους, τους F-sperific RNA βακτηριοφάγους και τους βακτηριοφάγους του Β. fragilis. Επιπλέον αναπτύχθηκαν μέθοδοι τόσο για την ανίχνευση παθογόνων ιών ανθρώπινης προέλευσης στα οστρακοειδή, όσο και για την ανίχνευση των "πιθανών δεικτών" αυτών των ιών. Οι μέθοδοι εξετάστηκαν προκειμένου να αξιολογηθεί η απόδοση καλής ποιότητας από όλα τα εργαστήρια μέσω διεργαστηριακών αναλύσεων. Η μέθοδος που εφαρμόστηκε σε αυτή τη μελέτη για την ανίχνευση των ιών στα οστρακοειδή βασίζεται στην εξαγωγή και την ομογενοποίηση του πεπτικού αδένα με χρήση διαλύματος γλυκίνης, pH 10, απομόνωση των νουκλεϊνικών οξέων και ενίσχυση του γονιδιώματος των ιών που αναλύονται. Για την ανίχνευση του βακτηρίου E. coli χρησιμοποιήθηκε η μέθοδος των πολλαπλών σωλήνων, ενώ για την ανίχνευση των βακτηριοφάγων χρησιμοποιήθηκε η μέθοδος καλλιέργειας διπλοστιβάδας. Για το βακτήριο E. coli, σε σύνολο 138 δειγμάτων, 110 δείγματα (ποσοστό 79,7%) βρέθηκαν να ανήκουν στην κατηγορία Α (MPN/100g σάρκας = <20 έως 220), δηλαδή χαρακτηρίζονται σαν δείγματα χαμηλής μόλυνσης, 25 δείγματα (ποσοστό 18,1%) βρέθηκαν να ανήκουν στην κατηγορία Β (MPN/100g σάρκας = 220 έως 3500), οπότε χαρακτηρίζονται σαν δείγματα μεσαίας μόλυνσης, ακατάλληλα προς κατανάλωση χωρίς να προηγηθεί διαδικασία εξυγίανσης, ενώ μόνο 3 δείγματα (ποσοστό 2,2%) βρέθηκαν να ανήκουν στη κατηγορία C (MPN/100g σάρκας =3500 έως >18000), δηλαδή είναι δείγματα υψηλής μόλυνσης. Οι ιοί που εμφανίζονται με μεγαλύτερη συχνότητα στα οστρακοειδή της Ανατολικής Μεσογείου είναι οι αδενοϊοί (34% των δειγμάτων βρέθηκαν θετικά για τους αδενοϊούς) και ακολουθούν οι εντεροϊοί (16,7% των δειγμάτων βρέθηκαν θετικά για τους εντεροϊούς). Αντίθετα, ο ιός της ηπατίτιδας Α (ποσοστό θετικών δειγμάτων = 4,34%), καθώς και οι ιοί Noro I (ποσοστό θετικών δειγμάτων = 2,1%) και Noro II (ποσοστό θετικών δειγμάτων = 1,47%%) εμφανίζονται σε μικρό ποσοστό δειγμάτων. Τέλος, 80 δείγματα (58%) βρέθηκαν θετικά (παρουσία πλακών βακτηριοφάγων) για τους σωματικούς κολιφάγους, με τον αριθμό των πλακών να κυμαίνεται από 71,4 έως 584800 pfp/100g, 52 δείγματα (37,7%) βρέθηκαν θετικά για τους F-specific RNA βακτηριοφάγους (αριθμός των πλακών από 76,2 έως 17051 p100g) και 33 δείγματα (24%) βρέθηκαν θετικά για τους βακτηριοφάγους του Bacteroides fragilis (αριθμός των πλακών από 194.5 έως 5266,25 pfp/100g). Τόσο για το βακτήριο E. coli όσο και για τους βακτηριοφάγους πραγματοποιήθηκαν διεργαστηριακές αναλύσεις προτύπων, οι οποίες οδήγησαν στο συμπέρασμα ότι οι αντίστοιχες μέθοδοι χαρακτηρίζονται ως αξιόπιστες. Η στατιστική ανάλυση έδειξε ότι το βακτήριο E. coli παρουσιάζει θετική συσχέτιση με τους σωματικούς κολιφάγους, αλλά δεν δείχνει στατιστικά σημαντική συσχέτιση ούτε με τους F-specific RNA βακτηριοφάγους, ούτε με κανέναν από τους ιούς εντερικής προέλευσης. Επίσης, θετική συσχέτιση παρουσίασαν οι αδενοϊοί με τους εντεροϊούς, καθώς και οι σωματικοί κολιφάγοι με τους βακτηριοφάγους του B. fragilis. Η μοναδική συσχέτιση μεταξύ ιών εντερικής προέλευσης και βακτηριοφάγων βρέθηκε για τους αδενοϊούς και τους βακτηριοφάγους του B. fragilis. Εάν αυτό επιβεβαιωθεί σε περαιτέρω μελέτες, τότε η συγκεκριμένη κατηγορία βακτηριοφάγων θα μπορούσε να αποτελέσει έναν καλό δείκτη πρόβλεψης της παρουσίας αδενοϊών σε δείγματα οστρακοειδών. Επιπλέον μελετήθηκε η σχέση που μπορεί να υπάρχει μεταξύ των φυσικοχημικών παραμέτρων και των μικροοργανισμών που εξετάστηκαν. Η επεξεργασία αυτή οδήγησε στο συμπέρασμα ότι το βακτήριο E. coli ανιχνεύεται σε μεγαλύτερα ποσά όταν το διαλυμένο οξυγόνο και η περιεκτικότητα σε άλας του ύδατος είναι αυξημένα. Αντίθετα, αύξηση της θερμοκρασίας οδηγεί σε μείωση της ανίχνευσης του βακτηρίου. Επίσης, η περιεκτικότητα σε άλας φαίνεται να επηρεάζει θετικά και τον ιό της ηπατίτιδας Α, αν και ο μικρός αριθμός θετικών δειγμάτων γι’αυτόν τον ιό δεν μπορεί να επιτρέψει την εξαγωγή ασφαλών συμπερασμάτων. Το pH και το διαλυμένο οξυγόνο των υδάτων οδηγεί σε αύξηση της ανίχνευσης των βακτηριοφάγων του B. fragilis, χωρίς όμως να μπορούμε να ισχυριστούμε ότι κάτι τέτοιο ισχύει, λόγω του μικρού αριθμού θετικών δειγμάτων γι’αυτούς τους βακτηριοφάγους. Τέλος, η αύξηση της θερμοκρασίας των υδάτων φαίνεται να οδηγεί και σε αύξηση της παρουσίας των F-specific RNA βακτηριοφάγων, και το ίδιο παρατηρήθηκε και με την αύξηση του διαλυμένου οξυγόνου στο νερό. Η παρούσα μελέτη αποτελεί την πρώτη διεξοδική έρευνα για την ιογενή κοπρανώδη μόλυνση τον οστρακοειδών στην Ελλάδα. Επιπλέον, αντιπροσωπεύει την πρώτη μελέτη σχετικά με τη αποτελεσματικότητα των οργανισμών - δεικτών ιϊκής μόλυνσης, καθώς και για τη συσχέτιση της μικροβιολογικής επιβάρυνσης των οστρακοειδών με τις φυσικοχημικές παραμέτρους του περιβάλλοντος ύδατος. Η μελέτη κατάλληλων δεικτών που σχετίζονται με την παρουσία εντερικών ιών στα οστρακοειδή οδήγησε σε χρήσιμα συμπεράσματα για τη χρήση της ανίχνευσης των βακτηριοφάγων ως δεικτών ιϊκής μόλυνσης. Εντούτοις, απαιτείται περαιτέρω μελέτη προκειμένου να προσδιοριστεί και η χρήση των βακτηριοφάγων ως δεικτών που θα μαρτυρούν την προέλευση (ανθρώπινη ή ζωική) των εντερικών ιών που ανιχνεύονται στα οστρακοειδή.
In this doctorate investigation, important shellfish growing areas of Greece have been defined and studied. Oysters (Ostrea edulis) and mussels (Mytilus galloprovincialis) were obtained on a monthly basis over an 18 month sampling period. These samples were collected by six (6) different points of Greece and were analyzed for enteroviruses (EV), adenoviruses (Adv), virus of hepatitis A (HAV), Noro viruses I and II (NLVI and NLVII ), bacterium E. coli, as well as for somatic coliphages, F-sperific RNA bacteriophages and bacteriophages of B. fragilis. Moreover, methods were developed for the detection of pathogenic viruses of human origin in the shellfish, as well as for the detection of potential "viral indicators". The methods were examined in order to validate the good quality performance from all the laboratories via interlaboratory analyses. The method that used in this study for the detection of human enteric viruses in the shellfish is based on the export and homogenisation of digestive gland with glycine buffer at pH 10, viral nucleic acid extraction and amplification of the genomes of the analysed human viruses. The procedure applied for detection of E. coli consists on a five tube, three dilution most probable number (MPN) method, while the method for the detection of bacteriophages was the double-agar-layer method. For E. coli analysis, in a total number of 138 samples, 110 samples (79,7%) were found to belong in category A (MPN / 100 g of flesh = < 20 until 220), that it means these samples are characterized as samples of low pollution, 25 samples (18,1%) were found to belong in category B (MPN / 100 g of flesh = 220 until 3500), therefore are characterized as samples of intermediate pollution, inadequate to consumption without precedes process of cleansing, while only 3 samples (2,2%) were found to belong in category C (MPN / 100 g of flesh = 3500 until > 18000), that it means they are samples of high pollution. The viruses that are presented with higher frequency in the shellfish of Eastern Mediterranean are the adenoviruses (34% of samples were found positive for adenoviruses) and follow enteroviruses (16,7% samples they were found positive for enteroviruses). On the contrary, virus of hepatitis A (percentage of positive samples = 4,34%), as well as the Noro I viruses (percentage of positive samples = 2,1%) and Noro II viruses (percentage of positive samples = 1,47%%) are presented in small number of samples. Finally, 80 samples (58%) were found positive (presence of plaques of bacteriophages) for somatic coliphages, with the number of plaques between 71,4 and 584800 pfp / 100 g, 52 samples (37,7%) were found positive for F - specific RNA bacteriophages (number of plaques from 76,2 to 17051 pfp/ 100 g) and 33 samples (24 %) were found positive for the bacteriophages of B. fragilis (number of plaques from 194,5 to 5266,25 pfp / 100 g). Interlaboratory studies involved the testing of reference materials of E. coli and bacteriophages were used as part of the good quality performance assessment program to be applied all over the study, and led to the conclusion that the corresponding methods are characterized by good reliability. According to the statistical analysis of the results, the presence of E. coli seems to be significantly related with the presence of somatic coliphages. However, E. coli do not present significant statistical relation neither with F - specific RNA bacteriophages, nor with all of the viruses of intestinal origin. Also, adenoviruses were significantly related with enteroviruses, as well as somatic coliphages with the bacteriophages of B. fragilis. The unique significant relation between viruses of intestinal origin and bacteriophages was found for the adenoviruses and bacteriophages of B. fragilis. If this is confirmed in further studies, then this category of bacteriophages could constitute a good indicator of forecast of presence adenoviruses in samples of shellfish. Moreover, we studied the relation that can exist between the physic-chemical parameters and the micro-organisms that were examined. This analysis led to the conclusion that E. coli is detected in higher levels when the dissolved oxygen and the salinity of water are increased. On the contrary, increase of temperature leads to reduction of detection of E. coli. Also, the salinity appears to influence positively also virus of hepatitis A, even if the small number of positive samples of this virus cannot allow the export of sure conclusions. The pH and the dissolved oxygen of waters lead to increase of detection of bacteriophages of B. fragilis, but the small number of positive samples for these bacteriophages can’t give safe conclusions. Finally, the increase of temperature of waters appears to lead also to increase of presence of F - specific RNA of bacteriophages, and the same was also observed with the increase of dissolved oxygen in water. This study constitutes the first extensive research for the fecal viral pollution of shellfish in Greece. Moreover, it represents the first study with regard to the effectiveness viral indicators, as well as for the correlation of microbiological parameters of shellfish with the physical-chemical parameters of water. The study of suitable indicators that are related with the presence of enteric viruses in the shellfish led to useful conclusions on the use of detection of bacteriophages as indicators of viral pollution. Nevertheless, further study is required in order to determine also the use of bacteriophages as indicators that will testify the origin (human or animal) of the enteric viruses that are detected in the shellfish.

Hematopoiesis is the continual process of blood cell generation that primarily occurs in the bone marrow of adult animals. Hematologic neoplasms can also occur in the bone marrow and often result from dysregulation of signal transduction pathways. One example is the activation of the Ras oncogene, which has been linked to a variety of different cancers, including hematologic neoplasms. Ras is located proximal to the cell membrane and can activate many downstream effector pathways, thus it is difficult to determine which downstream pathway is mediating oncogenic Ras function. My thesis work focused on the effect of inappropriate activation of MEK/ERK, a downstream Ras effector pathway, in the hematopoietic system.

Using a retroviral transduction system, we expressed a constitutively active form of MEK1 in hematopoietic stem cells (HSCs). Mice transplanted with HSCs expressing active MEK developed a lethal myelodysplastic syndrome/myeloproliferative disease (MDS/MPN) characterized by the expansion of granulocytes/macrophages (GM) at the expense of lymphoid cell development. Transplantation of active MEK-induced MDS/MPNs into naïve mice did not result in further disease, suggesting that the MDS/MPN is not a frank leukemia.

Bcl-2 is an anti-apoptotic molecule that has been shown to play a role in leukemia development and maintenance. Coupling expression of active MEK and Bcl-2 resulted in MDS/MPNs that were phenotypically identical and had very similar disease onset compared to active MEK-induced MDS/MPNs. However, transplantation of Bcl-2/active MEK-induced MDS/MPNs did not result in a myeloid disease; rather, it resulted in the development of T-acute lymphoblastic leukemia (T-ALL) that was marked by activated Notch signaling.

These results led us to conclude that activation of MEK/ERK was sufficient to cause a pre-leukemic myeloid disease; however, additional oncogenic factors, such as Bcl-2 and Notch, were necessary for frank leukemia development. Moreover, additional oncogenic factors can alter the disease phenotype and disease course. Future analysis of the interplay between oncogenic factors will help shed light on disease development and aid in the development of more effective cancer treatments.

Dissertation