Dissertations / Theses on the topic 'EC50'

The incidence of cancer worldwide has increased over the years, and gastrointestinal cancers (G.I.) are amongst the most common forms of cancer. Nevertheless, there is still no curative treatments for this group of tumors. Nucleoside analogues are widely used in cancer treatment. The prevailing compounds are Gemcitabine (used for pancreatic cancer and other carcinomas), 5-Fluorouracil (used in breast, colon, and other cancers), Cytarabine and Clofarabine (used in leukemias). Gemcitabine, the current standard of care for various forms of solid tumors, has a limited efficacy against pancreatic cancer. The objective of this project was the development of effective drugs against pancreatic cancer. We focused on a novel class of nucleoside analogues designed to bypass the most common cellular road blocks and resistance mechanisms. After an extensive screen for cell killing activity, two lead molecules were exclusively studied: LCB2151 and LCB2132. These two molecules showed high efficacy in killing human cancer cells from three different human G.I. cell lines: BxPC3 and Capan-2, two pancreatic cell lines representative of K-Ras positive and negative tumors, as well as the liver cell line HepG2. LCB2151 showed high efficacy in killing Gemcitabine-resistant cancer cells, and a low toxicity in normal cells. Interestingly, results show that these prodrugs can efficiently bypass key resistance mechanisms developed by cancer cells. The results obtained in this project are promising and could pave the way for a more effective treatment of pancreatic cancer.

The ethanol extract of A. phylicoides was investigated for its antioxidant activity using the DPPH scavenging method. The extract showed good antioxidant results with a EC50 value of 10.64 ± 0.0842 µ/ml. The extract was also tested for antibacterial activity against microorganisms (Staphylococcus aureus, Enterococus faecalis, Bacillus cereus, Bacillus subtilis, Bacillus pumilus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia) commonly known to pose a threat in the wellbeing of man. All tested microorganisms were significantly inhibited by the extract with the MIC values ranging from 3.13 µg/ml to 6.25 µg/ml. Folin-Ciocalteu’s reagent method was used to determine total phenolic content of dried and freshly prepared crude extract of A. phylicoides. Higher total phenolic content (28.28 ± 0.019 mg GAC/100g) and antioxidant activity (EC50, 10.64 ± 0.084 µg/ml) was observed in the dried extract compared to the fresh extract with a TPC value of 23.04 ± 0.003 mg GAC/100g and EC50 of 13.97 ± 0.066 µg/ml. Bioassay-guided fractionation of ethanolic extract from aerial parts of Athrixia phylicoides using silica and sephadex column chromatography led to the isolation of four known flavanoids, 5-hydroxy-6,7,8,3’,4’,5’-hexamethoxyflavon-3-ol (1), 3-0- demethyldigicitrin (2), 5,6,7,8,3’,4’-hexamethoxyflavone (3) and Quecertin (4). Due to the low yield, no further tests were done on compound 3. A DPPH-scavenging assay was performed to evaluate the antioxidant activity of the isolated compounds. All the tested compounds showed potent antioxidant activity with EC50 values ranging from 1.27 to 3.41 µg/ml. Compound 4 showed a higher antioxidant activity (EC50, 1.27 µg/ml) than vitamin C (EC50, 2.66 µg/ml) used as a control. The MIC values of the isolated compounds against tested microorganisms varied from 20 to more than 40 µg/ml. All the tested compounds showed no activity against S. aureus, B. pumilus, K. pneumonia and P. aeruginosa at the highest concentration tested (40 µg/ml). These compounds together with the extract were further analyzed by XTT assay on Vero cells. The extract showed a low toxicity effect on the cells at lower concentrations exhibiting EC50 value of 107.8 ± 0.129 µg/ml. Compound 4 showed minimal toxicity effect on the cells with a EC50 value of 81.38±0.331 µg/ml, compared to Compound 1 and 2 which exhibited EC50 values of 27.91 ± 0.181 µg/ml and 28.92 ± 0.118 µg/ml respectively. The results obtained from this study provide a clear rationale for the medicinal uses of Athrixia phylicoides.<br>Dissertation (MSc)--University of Pretoria, 2011.<br>Plant Science<br>unrestricted

Scopo di questa tesi è stato di valutare se le attività biologiche possono essere utilizzate come indicatori di adattamento microbico. Le prove condotte sinora in letteratura si sono concentrate unicamente sull’attività di nitrificazione. Pertanto si è voluto valutare se altre attività biologiche possono essere utilizzate come indicatori d’adattamento. Allo scopo si è prima valutata la sensibilità di alcune importanti proprietà biologiche del suolo (nitrification, fluorescein diacetate (FDA)) e attività enzimatiche (urease, nitrate reductase, phosphatase, arylsulfatase, β-galactosidase, phenol-oxidase and dehydrogenase) allo Zn mediante l’impiego di test ecotossicologici Per le attività più sensibili sono state condotte delle prove di adattamento secondo il protocollo di Rusk. Questo esclude le interferenze dovuto all’ aging. Questo protocollo è basato sul reinoculo di un suolo sterile (contaminato con concentrazioni crescenti di Zn) con sottocampioni di suolo che sono stati precedentemente incubati per un periodo di 4 mesi con e senza Zn Il confronto tra i valori di EC50 delle attività biologiche dei suoli reinoculati ci ha permesso di dimostrare un significativo recupero della β-galactosidase, mentre per il nitrate reductase e la nitrificazione potenziale è stato trovato un chiaro e significativo shift delle curve di dose e risposta, anche se con parziale sovrapposizione del range dell’EC50 stimato.<br>The purpose of this thesis was to assess whether the biological activities can be used as indicators of the microbial adaptation. The tests conducted so far in the literature have focused only nitrification activity. Therefore we wanted to assess whether other biological activities can be used as indicators of adaptation. To do this you first evaluated the sensitivity of some important biological properties of the soil (nitrification, fluorescein diacetate (FDA)) and enzymatic activities (urease, nitrate reductase, phosphatase, arylsulfatase, β-galactosidase, phenol-oxidase and dehydrogenase) Zn through the use of toxicological test. Then the activities most sensitive were tests to evaluate to adapt according to the protocol by Rusk This method to exclude interferences to two chemical aging in soil, This protocol is based on the reinoculation of sterilized soil (contaminated with increasing Zn concentrations) with sub-samples of soil which have been incubated for 4 months with or without Zn. The comparison between the EC50 of the biological properties of reinoculated soils allow us to demonstrate a significant restoration was found for β-galactosidase, while for nitrate reductase and potential nitrification there was a clear and significant shift of dose response curves but with partial overlap of the EC50 ranges estimation.

Scopo di questa tesi è stato di valutare se le attività biologiche possono essere utilizzate come indicatori di adattamento microbico. Le prove condotte sinora in letteratura si sono concentrate unicamente sull’attività di nitrificazione. Pertanto si è voluto valutare se altre attività biologiche possono essere utilizzate come indicatori d’adattamento. Allo scopo si è prima valutata la sensibilità di alcune importanti proprietà biologiche del suolo (nitrification, fluorescein diacetate (FDA)) e attività enzimatiche (urease, nitrate reductase, phosphatase, arylsulfatase, β-galactosidase, phenol-oxidase and dehydrogenase) allo Zn mediante l’impiego di test ecotossicologici Per le attività più sensibili sono state condotte delle prove di adattamento secondo il protocollo di Rusk. Questo esclude le interferenze dovuto all’ aging. Questo protocollo è basato sul reinoculo di un suolo sterile (contaminato con concentrazioni crescenti di Zn) con sottocampioni di suolo che sono stati precedentemente incubati per un periodo di 4 mesi con e senza Zn Il confronto tra i valori di EC50 delle attività biologiche dei suoli reinoculati ci ha permesso di dimostrare un significativo recupero della β-galactosidase, mentre per il nitrate reductase e la nitrificazione potenziale è stato trovato un chiaro e significativo shift delle curve di dose e risposta, anche se con parziale sovrapposizione del range dell’EC50 stimato.<br>The purpose of this thesis was to assess whether the biological activities can be used as indicators of the microbial adaptation. The tests conducted so far in the literature have focused only nitrification activity. Therefore we wanted to assess whether other biological activities can be used as indicators of adaptation. To do this you first evaluated the sensitivity of some important biological properties of the soil (nitrification, fluorescein diacetate (FDA)) and enzymatic activities (urease, nitrate reductase, phosphatase, arylsulfatase, β-galactosidase, phenol-oxidase and dehydrogenase) Zn through the use of toxicological test. Then the activities most sensitive were tests to evaluate to adapt according to the protocol by Rusk This method to exclude interferences to two chemical aging in soil, This protocol is based on the reinoculation of sterilized soil (contaminated with increasing Zn concentrations) with sub-samples of soil which have been incubated for 4 months with or without Zn. The comparison between the EC50 of the biological properties of reinoculated soils allow us to demonstrate a significant restoration was found for β-galactosidase, while for nitrate reductase and potential nitrification there was a clear and significant shift of dose response curves but with partial overlap of the EC50 ranges estimation.

Biologiska toxicitetstester utförs genom att exponera en testorganism för olika koncentrationer av kemikalier under en bestämd period. Det finns akuta toxicitetstester och kroniska. Resultat från akuta studier presenteras som EC50-värde (Effect Concentration, affecting 50 % of the population). Tester som används som underlag för riskbedömningar ska utföras på kvalitetsmässigt acceptabelt sätt som grundas på International Organization for Standardization (ISO) och Good Labaratory Practice (GLP). Syftet med studien var att studera toxisk inverkan av kaliumdikromat på encellig sötvattenalg, R. subcapitata, genom att räkna cellantal med två olika instrument: flödescytometer och automatisk cellräknare. Vidare att jämföra testernas EC50 medelvärde mot ISO 8692 angivet värde i precisions syfte och att utvärdera de båda räknemetoderna gällande precision och analystid. Kaliumdikromat användes för tillväxtinhibering vid toxicitetstest. EC50-resultaten visade ingen statistiskt signifikant skillnad mellan de båda instrumenten (p=0,47). Den akuta toxicitetsanalysens precision kunde bekräftas som giltig då båda mätmetoders EC50 medelvärden vid jämförelse med ISO 8692 värde befanns inom bestämda 95% konfidensintervallet. Vid jämförelse av studiens två mätningsmetoder, observerades större spridning kring medelvärdet i cellräknarens resultat, där tre EC50 värden hamnade utanför 95% CI. Däremot visade resultatet från flödescytometern mindre spridning och högre noggrannhet jämfört med cellräknarens. Studien visade att flödescytometer skulle kunna användas i framtida toxikologiska tester med encelliga alger, men det krävs flera upprepade försök för att bekräfta fördelar med analysen i flödescytometern.<br>Biological toxicity tests are performed by exposing a test organism to different concentrations of chemicals over a certain period of time. Results from acute studies are presented as EC50 (Effect Concentration, affecting 50% of the population). Tests used as a basis for risk assessments shall be performed in a quality acceptable way based on the International Organization for Standardization (ISO) and Good Laboratory Practice (GLP). The aim of this study was to study the toxic effect of potassium dichromate on unicellular green algae R. subcapitata, by counting cells with two different apparatus: flow cytometer and automatic cell counter. Additionally, to compare the EC50 mean values against the ISO 8692 value for control of test precision and to compare accuracy and analytical time of two methods. Potassium dichromate was used for growth inhibition in toxicity tests. The EC50 results showed no statistically significant difference between the two instruments (p = 0.47). The accuracy of acute toxicity analysis was confirmed as valid as both EC50 average measurement values compared to ISO 8692 value were found within the 95% confidence interval. When comparing the two methods of the study, greater spread was observed around the mean value in the cell count's results, where three EC50 values were outside 95% CI. The result of the flow cytometer had less spread and higher accuracy compared to the cell count. The study showed that flow cytometers could be used in future toxicological tests with algae, but several repeated tests are required to confirm the benefits of analysis with the flow cytometer.

Submitted by Nadia Maria Poloni null (nadiapoloni@gmail.com) on 2016-04-16T00:04:34Z No. of bitstreams: 1 Dissertação - Nadia Poloni - completa.pdf: 1931593 bytes, checksum: e8e92f6dc3b3aa46033959f97f62b795 (MD5)<br>Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-04-18T12:53:57Z (GMT) No. of bitstreams: 1 polani_nm_me_ilha.pdf: 1931593 bytes, checksum: e8e92f6dc3b3aa46033959f97f62b795 (MD5)<br>Made available in DSpace on 2016-04-18T12:53:57Z (GMT). No. of bitstreams: 1 polani_nm_me_ilha.pdf: 1931593 bytes, checksum: e8e92f6dc3b3aa46033959f97f62b795 (MD5) Previous issue date: 2016-02-22<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)<br>O fungo ascomiceto, Pyricularia oryzae, tem distribuição mundial como patógeno do arroz, mas recentemente emergiu como patógeno causando a brusone do trigo no Brasil. Os grupos de fungicidas estrobirulinas e triazóis tem sido intensivamente utilizados para manejo de doenças do trigo há cerca de três décadas. Há relato de resistência de P. oryzae do trigo a estrobirulinas no Brasil e a ineficácia dos fungicidas triazóis aponta para um cenário semelhante. Os triazóis, inibidores da desmetilação de esteróis (DMI), inibem a biossíntese de ergosterol e possuem como alvo a enzima 14α-demetilase, codificada pelo gene cyp51. Em nosso estudo, determinamos as distribuições de frequência da sensibilidade de 180 isolados de P. oryzae do trigo amostrados de sete estados do Centro-Sul do Brasil, com base na EC50 a tebuconazol e epoxiconazol. Descrevemos, também, a presença de mutações no gene cyp51A e reconstruímos sua filogenia reticulada para esclarecer a relação evolutiva entre haplotipos. Relatamos que todas as populações de P. oryzae amostradas se mostraram insensíveis aos fungicidas testados, comparando-se com as doses recomendadas para o manejo da doença no campo. Com base na sequência do gene cyp51A de P. oryzae do trigo, foram detectados oito haplotipos distintos. Foram encontradas sete mutações não-sinônimas, que podem estar correlacionadas com a resistência à DMI's.<br>The fungus ascomycete Pyricularia oryzae has worldwide distribution as rice pathogen, but it has recently emerged as pathogen causing wheat blast in Brazil. The strobirulin and azole fungicides have been intensively used for management of wheat diseases in the last three decades. The report of resistance of P. oryzae from wheat to strobirulins in Brazil and the inefficiency of azole fungicides indicates a similar scenario. The triazoles represented sterols demethylation inhibitor fungicides group (DMI), characterized by inhibiting the biosynthesis of ergosterol and their target is the 14α-demethylase enzyme, encoded by the cyp51 gene. We determined the azole sensitivity frequency distributions of 180 isolates of P. oryzae of wheat sampled in seven states of the south-central Brazil, based on the EC50 of tebuconazole and epoxiconazole. We described the presence of mutations in the cyp51A gene and built their reticulate phylogeny to identify the evolutionary relationship among haplotypes. We reported that all P. oryzae populations sampled were insensitive to the azoles when compared to the recommended doses for the management of the disease in the field. Based on the sequence of cyp51A gene from P. oryzae of wheat, eight distinct haplotypes were detected. We found seven non-synonymous mutations that could be correlated with resistance to DMI's.

Orientador: Paulo Cezar Ceresini<br>Resumo: O fungo ascomiceto, Pyricularia oryzae, tem distribuição mundial como patógeno doarroz, mas recentemente emergiu como patógeno causando a brusone do trigo noBrasil. Os grupos de fungicidas estrobirulinas e triazóis tem sido intensivamenteutilizados para manejo de doenças do trigo há cerca de três décadas. Há relato deresistência de P. oryzae do trigo a estrobirulinas no Brasil e a ineficácia dos fungicidastriazóis aponta para um cenário semelhante. Os triazóis, inibidores da desmetilaçãode esteróis (DMI), inibem a biossíntese de ergosterol e possuem como alvo a enzima14α-demetilase, codificada pelo gene cyp51. Em nosso estudo, determinamos asdistribuições de frequência da sensibilidade de 180 isolados de P. oryzae do trigoamostrados de sete estados do Centro-Sul do Brasil, com base na EC50 a tebuconazole epoxiconazol. Descrevemos, também, a presença de mutações no gene cyp51A ereconstruímos sua filogenia reticulada para esclarecer a relação evolutiva entrehaplotipos. Relatamos que todas as populações de P. oryzae amostradas semostraram insensíveis aos fungicidas testados, comparando-se com as dosesrecomendadas para o manejo da doença no campo. Com base na sequência do genecyp51A de P. oryzae do trigo, foram detectados oito haplotipos distintos. Foramencontradas sete mutações não-sinônimas, que podem estar correlacionadas com aresistência à DMI's.<br>Abstract: The fungus ascomycete Pyricularia oryzae has worldwide distribution as rice pathogen, but it has recently emerged as pathogen causing wheat blast in Brazil. The strobirulin and azole fungicides have been intensively used for management of wheat diseases in the last three decades. The report of resistance of P. oryzae from wheat to strobirulins in Brazil and the inefficiency of azole fungicides indicates a similar scenario. The triazoles represented sterols demethylation inhibitor fungicides group (DMI), characterized by inhibiting the biosynthesis of ergosterol and their target is the 14α-demethylase enzyme, encoded by the cyp51 gene. We determined the azole sensitivity frequency distributions of 180 isolates of P. oryzae of wheat sampled in seven states of the south-central Brazil, based on the EC50 of tebuconazole and epoxiconazole. We described the presence of mutations in the cyp51A gene and built their reticulate phylogeny to identify the evolutionary relationship among haplotypes. We reported that all P. oryzae populations sampled were insensitive to the azoles when compared to the recommended doses for the management of the disease in the field. Based on the sequence of cyp51A gene from P. oryzae of wheat, eight distinct haplotypes were detected. We found seven non-synonymous mutations that could be correlated with resistance to DMI's.<br>Mestre

This thesis focuses on solution antimicrobial effectiveness for copolyoxetanes with quaternary ammonium and PEG-like side chains. Ring opening copolymerization of 3-((4-bromobutoxy)methyl)-3-methyloxetane (BBOx) and 3-((2-(2-methoxyethoxy) ethoxy) methyl)-3-methyloxetane (ME2Ox) yielded random copolymers with 14-100 (m) mole% BBOx designated P[(BBOx-m)(ME2Ox)]. Reaction of P[(BBOx-m)(ME2Ox)] with dodecyl dimethylamine gave the corresponding quaternary P[(C12-m)(ME2Ox)] polycation salts, designated C12-m. Mole ratios and molecular weights were obtained from 1H-NMR and end group analysis. Differential scanning calorimetry (DSC) studies showed Tg’s between 69 and -34 °C. Minimum inhibitory concentrations (MIC) against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa showed MIC decreasing with increasing C12 mole% reaching a minimum between C12-43 and C12-60. C12-43 had the lowest MIC for all strains. At 5× MIC (challenge:108 cfu/ml), C12 43 kills ≥ 99% of the tested strains within 1 hr. C12-m copolyoxetane cytotoxicity toward human red blood cells, HFF (Human Foreskin Fibroblast) and HDF (Human Dermal Fibroblast) was low, indicating good prospects for biocompatibility. Cx-m copolyoxetane antimicrobial efficacy, hemolytic activity and cytotoxicity were further explored by changing quaternary alkyl chain length. Copolyoxetanes are represented as Cx-50, where 50 is the mole percent quaternary repeat units and ‘x’ is quaternary alkyl chain length (2 to 16 carbons). Reaction of P[(BBOx-m)(ME2Ox)] with a series of tertiary amines yielded the desired quaternary ammonium segment. DSC studies showed Tg’s between -40 °C and -60 °C and melting endotherms for C14-50 and C16-50. A systematic dependence of alkyl chain length on MIC was found with C8-50 being the most effective antimicrobial. Kill kinetics for C8-50 (5× MIC, challenge: 108 cfu/ml) effected >99% kill in 1 hour for S. aureus (7 log reduction). C8-50 efficacy on biomass and cell viability of P. aeruginosa biofilms was investigated. Crystal violet (CV) staining assays demonstrate that C8-50 had no effect on adhesion of already established P. aeruginosa biofilms, but reduced biofilm formation by killing cells prior to attachment. For anti-adhesion assays, noticeable reduction in biofilm mass occurred at concentrations greater than 2× MIC. Viability studies show a substantial log reduction of 2.1 at MIC. The low cytotoxicity of Cx-m copolyoxetanes coupled with low MICs and favorable biofilm results indicate good prospects for therapeutic applications.

The Alzheimer's Disease Life Events study examines whether there is a relationship between life events and Alzheimer's disease (AD). The ADLE study uses a mixed methods approach to answer the central research question:Are life events a risk factor for Alzheimer's disease? The central research question uses the following theory questions to examine:1. Is there a difference between the number of life events between patients and controls, using the Life Events and Difficulties Schedule (LEDS)(Brown and Harris, 1978) as a measurement tool?2. Is there a difference in the way (i.e. positive, neutral and negative) life events are discussed and in the range of emotions expressed when discussing life events between the patients and controls? 3. Are there any differences in the narrative constructions of life events, as interpreted by the Biographic Narrative Interpretive Method (BNIM)(Wengraf, 2001, 2008) between the patient and control groups? 4. Can the differences, between the patient and control groups, in the narratives be developed into a diagnostic marker? 5. Can the Emotion Word Coding (EWC)(Danner et aI., 2000) be used as a diagnostic marker by being applied to text collected from patients and controls over a period of decades? The ADLE study found that the patient group had experienced more life events in comparison with the control group as defined by the LEOS (Brown and Harris, 1978), and that the patient group had experienced more bereavements under the age of 51 years. The evidence supports the association between life events and AD.Even though there were significantly more life events experienced by the patients, the EWC (Danner et aI., 2001) found significantly fewer discussions expressing emotion bythe patients, particularly the negatively described ones. The range of negative and positive words used to describe the life events was significantly fewer too. This implies that the ways the patients express emotions about life events is substantially different from the controls. This finding was mirrored in the thematic field analysis of the BNIM interviews (Wengraf, 2001, 2008), which found differences in the content and structure of the narratives, and the emotional expression in the narratives about life events. A tool has been constructed using the differences between patients and controls to contribute to the early diagnosis of AD. In addition, the ADLE study has contributed to a gap in the knowledge about life events and AD.

This diploma thesis deals with a use of algal test in ecotoxicology. A freshwater algae, Desmodesmus subspicatus, was used to asses the ecotoxicity of selected chemical substances. A spectrophotometry method, of VIS spectrum, was used in evaluation of the EC50. A correlation, between a number of algal cells and the wavelength absorbance at 683 nm, has been determined for this purpose. Following chemicals were tested, 2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetic acid, (RS)-2-[4-(2-methylpropyl)phenyl]propanoic acid and N-(4-hydro-xyphenyl)acetamide. An EC50 value was determined for all these substances and also their ecotoxicity has been evaluated.

A citricultura brasileira se destaca no mercado global de citros, apresentando altos valores de produção e produtividade. Dentre as diversas doenças que afetam a cultura, a podridão floral dos citros (PFC) vem ganhando destaque com o deslocamento de áreas produtoras para regiões mais favoráveis à ocorrência desta doença. A PFC, causada por espécies dos complexos Colletotrichum acutatum e C. gloeosporioides, é especialmente problemática em anos de temperaturas amenas e alta umidade. Os citricultores realizam pulverizações preventivas para o controle da PFC todos os anos, embora condições climáticas favoráveis à doença ocorram apenas ocasionalmente. Além do impacto econômico, as frequentes pulverizações com fungicidas aumentam a pressão de seleção sobre isolados resistentes, o que pode interferir na eficiência do controle químico. A utilização de sistemas de previsão de epidemias pode evitar que pulverizações desnecessárias de fungicidas sejam realizadas caso não haja condições favoráveis à ocorrência da doença. Para o desenvolvimento destes sistemas, é imprescindível determinar um limiar de ação para a aplicação de fungicidas nos pomares. Dessa forma, o presente trabalho teve por objetivos: (i) estabelecer um limiar de ação para a aplicação de fungicidas com base na germinação de conídios de C. acutatum que permita o controle a doença igualmente ou de forma mais eficiente do que o sistema de pulverização adotado pelos citricultores do sudoeste paulista; (ii) caracterizar a sensibilidade de isolados dos complexos C. gloeosporioides e C. acutatum à trifloxistrobina e ao tebuconazol in vitro e molecularmente, para identificar possíveis mudanças de sensibilidade das espécies a estes fungicidas. Para a definição do limiar de ação, tratamentos baseados em índices de risco foram comparados ao tratamento testemunha e ao calendário fixo de aplicações, usualmente adotado pelos produtores. A aplicação de fungicidas quando limiar de 15% de germinação de conídios era atingido, foi eficiente em controlar a doença e reduzir o número de aplicações de fungicidas. Nos ensaios de sensibilidade a fungicidas dois métodos foram utilizados para a determinação da CE50: o da diluição em gradiente espiral para trifloxistrobina e tebuconazol e o da inibição da germinação de conídios para a trifloxistrobina. Foram utilizados isolados coletados entre 1999 e 2016. A CE50 média da coleção de isolados de acordo com o método da diluição em gradiente espiral variou de 0,158 a 0,297 &mu;g/ml e 0,1 a 0,182 &mu;g/ml para trifloxistrobina e tebuconazol, respectivamente. Para a trifloxistrobina, de acordo com o método da inibição da germinação, a CE50 média foi de 0,002 &mu;g/ml. Não foram verificadas características moleculares nem valores de CE50 atrelados à mudança de sensibilidade dos isolados.<br>Brazilian citrus industry represents a significant share in the global citrus market. Amongst several diseases that affect the crop, postbloom fruit drop (PFD) has been gaining prominence in Sao Paulo with the displacement of citrus areas to regions in which weather conditions are more favorable to the occurrence of this disease. PFD, caused by species of the complexes Colletotrichum acutatum and C. gloeosporioides, is especially problematic in years of mild temperatures and high humidity. Citrus growers spray the orchards preventively for PFD control every year, although favorable climatic conditions do not occur regularly. In addition to the economic impact, this practice increases the selection pressure of fungicide resistant isolates, and may decrease the efficiency of chemical control in a long term. The use of disease forecasting systems is able to prevent unnecessary fungicide sprays. For the development of such systems, it is essential to determine an action threshold for the application of fungicides in the orchards. The objectives of this work were: (i) to establish an action threshold for fungicide sprays based on the germination of C. acutatum conidia; (ii) to characterize the sensitivity of C. gloeosporioides and C. acutatum isolates to trifloxystrobin and tebuconazole in vitro and molecularly. Regarding the definition of the action threshold, treatments based on risk indices were compared to the control treatments and calendar based sprays, usually adopted by growers. The 15% conidia germination threshold was efficient in controlling the disease and reducing the number of fungicide applications. In the fungicide sensitivity tests, two methods were used to determine the EC50, the spiral gradient dilution for trifloxystrobin and tebuconazole, and the method of conidial germination inhibition for trifloxystrobin. Isolates collected between 1999 and 2016 were used. The mean EC50 of the isolate collection determined by the spiral gradient dilution method ranged from 0.158 to 0.297 &mu;g/ml and from 0.1 to 0.182 &mu;g/ml for trifloxystrobin and tebuconazole, respectively. Mean EC50 of trifloxystrobin estimated by the conidial germination inhibition method was 0.002 &mu;g/ml. No mutations or EC50 values indicated shifts of fungicide sensitivity on the isolates.

Rhizoctonia blight (sensu lato) is a common and serious disease of many turfgrass species. The most widespread causal agent R. solani consists of several genetically different anastomosis groups (AGs) and subgroups. Though anastomosis or hyphal fusion reactions have been used to group Rhizoctonia species, they are time consuming and sometimes difficult to interpret. Anastomosis reactions are incapable of identifying isolates belonging to different AG subgroups within an AG. This study evaluated molecular techniques in comparison with traditional anastomosis grouping (AG) to identify and group isolates of Rhizoctonia. More than 400 Rhizoctonia isolates were collected from diseased turfgrass leaves from eight geographic areas in Virginia and Maryland. A random sample of 86 isolates was selected and initially characterized by colony morphology, nuclei staining and anastomosis grouping. Molecular identification was performed by analysis of rDNA-ITS region and DNA fingerprinting techniques universally primed PCR (UP-PCR) and amplified fragment length polymorphism (AFLP). The cladistic analysis of ITS sequences and UP-PCR fragments supported seven clusters. Isolates of R. solani AG 1-IB (n=18), AG 2-2IIIB (n=30) and AG 5 (n=1) clustered separately. Waitea circinata var. zeae (n=11), and var. circinata (n=4) grouped separately. A cluster of six isolates (UWC) did not fall into any known Waitea group. Most of the binucleate Rhizoctonia-like fungi (BNR) (n=16) grouped separately. AFLP grouping also largely agreed with the above results. However, UWC isolates clustered into two groups. Molecular analyses corresponded well with traditional anastomosis grouping by clustering isolates within an AG or AG subgroup together. UP-PCR cross-hybridization could distinguish closely related Rhizoctonia isolates to their infraspecies level. Genetically related isolates belonging to the same AG subgroups cross-hybridized strongly, while isolates of different AGs did not cross-hybridize or did so weakly. Sequence-characterized amplified region (SCAR) markers were generated from UP-PCR products to identify isolates of major pathogenic groups AG 1-IB and AG 2-2IIIB. Specific primer pairs successfully distinguished isolates of AG 1-IB and AG 2-2IIIB from isolates of other AGs. Sensitivity of Rhizoctonia species and AGs was tested in vitro to commercial formulations of iprodione, triticonazole and pyraclostrobin. W. circinata isolates were moderately sensitive to iprodione while isolates of R. solani and BNR were extremely sensitive. Isolates of AG 2-2IIIB showed less sensitivity to triticonazole than other Rhizoctonia isolates. W. circinata var. zeae isolates were moderately sensitive to pyraclostrobin while most of the other isolates were extremely sensitive.<br>Ph. D.

The amphibian decline phenomenon now involves in excess of a third of the roughly 6000 species of amphibians on the planet. The problems that drive the declines are diverse with no end in sight. The Amphibian Conservation Action Plan (ACAP) aims to stem amphibian decline through four recommended actions by researchers and conservation biologists: (1) Expand scientific understanding of amphibian declines and extinctions; (2) continue to document amphibian diversity and ecology and how they are changing; (3) develop and implement long-term conservation programs; (4) prepare emergency response actions for eminent crises. This Dissertation focused on two of those recommendations: expanding scientific understanding of amphibian declines and extinctions and continuing to document amphibian diversity and ecology and how they are changing. The first chapter is a review of the amphibian decline phenomenon. The second, third, and fourth chapters focus on expanding scientific understanding of amphibian diversity and ecology with the description of a formerly unknown species (chapter 2), and ecological papers on two poorly known species (chapters 3 and 4). Chapter five focuses on the first ACAP recommendation in improving scientific understanding of the causes behind amphibian decline. The chapter is an experimental examination of two related species and their developmental reactions to common heavy metal contaminants. The goal of this Dissertation is to contribute toward the general amphibian knowledge base relative to the recommendations of ACAP.

In industries today the demand for reading the state of industrial equipment and thus prevent machine breakdown, is increasing. The company HMS Industrial Networks AB has a product on the market, Netbiter EC350 that is used to read sensors and thus find out the condition of industrial equipment. By reporting scanned data to users through a cloud service, users can keep track of their equipment.   When developing Netbiter EC350 a slot was made for a Bluetooth module to offer clients a wireless reading in future developments. In this project a prototype was made to show how this Bluetooth communication can be implemented.   The goal of the project was to create a Bluetooth communication between a Bluetooth sensor and Netbiter EC350. A user interface was made to allow the user to read sensor values.   The result of the project shows how a Bluetooth communication can be implemented to read sensors wireless and therefore it fulfills its purpose and goal. The user can find connectable Bluetooth devices, connect to a device and read measured values through a user interface.   The prototype demonstrates how a Bluetooth communication with a Netbiter EC350 can be implemented and the project is therefore considered to be a good basis for future development.

Mefenoxam has been a premier compound for Phytophthora disease control in the nursery industry for 30 years. The primary objectives of this research were to examine whether Phytophthora species have developed resistance to this compound and to investigate fungicide resistance management strategies. Phytophthora nicotianae, a destructive pathogen of numerous herbaceous and some woody ornamental plants, was used as a model system. P. cinnamomi, a major pathogen of a wide range of tree species and shrub plants, was also included for comparison. Twenty-six isolates of P. nicotianae were highly resistant to mefenoxam with a mean EC50 value of 326.5 µg/ml while the remaining 70 were sensitive with an EC50 of <0.01 µg/ml (Label rate: 0.08µg/ml). All resistant isolates were recovered from herbaceous annuals and irrigation water in 3 Virginia nurseries. Resistant isolates were compared with sensitive ones using seedlings of Lupinus â Russell Hybridsâ in the absence of mefenoxam for relative competitive ability. Resistant isolates out-competed sensitive ones within 3 to 6 sporulation cycles. Resistant isolates exhibited greater infection rate and higher sporulation ability than sensitive ones. No mefenoxam resistant isolates were identified in P. cinnamomi. All 65 isolates of P. cinnamomi were sensitive to mefenoxam with an EC50 of < 0.04 ï ­g/ml. Attempts to generate mutants with high resistance to mefenoxam through UV mutagenesis and mycelial adaptation were not successful. However, there were significant reductions in sensitivity to mefenoxam; those slightly resistant mutants carried fitness penalties, which may explain why P. cinnamomi remains sensitive to mefenoxam. The effect of propamocarb hydrochloride on different growth stages of Phytophthora nicotianae was evaluated in search for an alternative fungicide. Propamocarb greatly inhibited sporangium production, zoospore motility, germination and infection. However, it has little inhibition of mycelial growth and infections. Propamocarb can be used as an alternative fungicide to mefenoxam where mefenoxam resistance has become problematic. However, it must be used preventively; i.e. before infections occur. The genetic inheritance of mefenoxam resistance in P. nicotianae was studied using F1 progenies of a cross between resistant and sensitive isolates. The F1 progenies segregated for mefenoxam resistance in ratio of 1R:1S, indicating the mefenoxam resistance is controlled by a single dominant gene. One RAPD marker putatively linked to resistant locus in repulsion phase was obtained by bulked segregant analysis and was converted to the SCAR marker. This marker is capable of differentiating mefenoxam resistant populations from sensitive populations included in this study.<br>Ph. D.

This thesis attempts to account for the appearance, persistence, and eventual decline of an architectural motif, derived from ancient pagan temples, widely used as the principal feature on an increasing variety of building types in Britain, during the period 1630 to 1850. The thesis seeks to do this by defining both the word 'portico' and the architectural forms to which, historically, it was applied, and by examining the religious, political, social, and stylistic contexts in which the portico, as a metaphor for the temple, was utilized. The rationalization within the Vitruvian-Christian tradition of the ancient temple's pagan connotations; the portico's intrinsic capacity to symbolize virtue, distinction, and authority; the changing perceptions of the idea of the temple; and the different nature and sources of both the authority and the architectural style which the portico expressed, are investigated. Architecturally, the portico expressed grandeur, centrality, and an entry; it controlled, defined, and gave focus to urban space. Introduced to Britain by Inigo Jones, and based on classical Roman and Palladian models but with Salomonic overtones, the portico initially symbolized Stuart dynastic claims to divine kingship. As political and economic power shifted to an aristocratic oligarchy, the temple that was Britain, Rome's heir, symbolized a church and state united, and the secular virtues of the Augustan age. Palladio's fusion of Roman temple and villa provided the model for the oligarchy's power base, the porticoed country house. Archaeology and politics combined, first to project mercantile opulence through imperial Roman-inspired neo-classicism, then the more fundamental qualities of the Greek temple. The Pantheon gave way to the Parthenon; the temple of private wealth to the imagined temple of democracy. After epitomizing the characteristic early nineteenth-century public style, the too-pagan Greek portico succumbed - as did the classical ideal - in the anarchy of styles, to the Gothic.

The Communist Party of Great Britain (CPGB) is largely absent from the narrative of Welsh devolution, in particular from the left’s engagement with the national question, as it is from much of the discussion of Welsh politics in general, a surprising omission considering its significant role within the labour movement in Wales and its commitment from the 1930s to legislative devolution for Wales. This study offers, for the first time, an in-depth analysis of the party’s engagement with the national question in Wales, from its formation in 1920 to its dissolution in 1991. The party’s engagement with the national question is approached through a study of its policy on Welsh self-government, its attitude to the Welsh language and Welsh culture, its relationship with the nationalist movement, and its broader policy programme for Wales. Despite its early neglect of the issue, the CPGB developed, from the mid-1930s onwards, a policy on the national question in Wales based on support for a legislative Welsh parliament within a federal governmental system, alongside support for the equal status for the Welsh language. While the level of the CPGB’s engagement with the national question was linked to the fortunes of the Welsh nationalist movement, its policy on the issue was guided by its need to accommodate both the right of national self-determination and its commitment to the unity of the British working class movement, its federalism derived from its commitment to this left-wing form of British unionism. Policy on these issues was developed primarily in Wales, the party’s prolonged, if at times intermittent, engagement with the issue, marking the Welsh party out as having, alongside its internationalism and its commitment to the British working class movement, a distinct Welsh identity.

Observations of the transient radio sky provide an insight into the most dynamic and energetic events in the Universe. The exploration of transient and variable sources, for a long time primarily focused on higher frequencies, has recently been extended to the long wavelength regime, with the development of facilities such as the Low Frequency Array (LOFAR). In this work I describe the general design of LOFAR as well as details of the commissioning work performed during the early stages of operation. Secondly, I perform an analysis of the first dedicated LOFAR transient survey - the Radio Sky Monitor. The investigation is focused on exploring 30 seconds time-scale images and allows us to place an upper limit on the expected areal densities of fast radio transients in the low frequency sky. It also provides spectral index constraints on the previously found, first LOFAR transient event. I also investigate the relation between the radio luminosities and variability time-scales across a wide range of sources - from nearby flare stars to distant supermassive black holes. The results are discussed in context of the potential use as a classification method, as well as in comparison with the expected brightness temperature of the events. The relation is used as a base for building a tool which classifies (with a given probability) an observed event, depending on the measured rise/decline rates of the flare. It includes the automatic selection of flares and takes into account the estimated areal densities of different classes of objects. Lastly, I discuss the results of the luminosity - time-scale correlation in context of the estimation of the kinetic power, as well as future prospects of implementing the presented classification method into an automated transient detection pipeline for instruments such as MeerKAT and the Square Kilometer Array.

JMJD6 or PSR (phosphatidyl serine receptor) was initially proposed to be a membrane receptor involved in apoptotic cell clearance by recognition of apoptotic cells. However, sequence analyses implied the presence of a jelly roll or double stranded beta helix (DSBH) structural domain in PSR/JMJD6 and similarity with JmjC family of enzymes which are involved in chromatin regulation. Subsequently, PSR was renamed as JMJD6 and was reported to be a histone arginine demethylase. Previous work from our group has shown that JMJD6 is a lysine hydroxylase that interacts with nuclear proteins including CROP and U2AF65 which are involved in mRNA splicing. Peptide screening and cell based assays led to the conclusion that JMJD6 catalyses lysine hydroxylation of splicing regulatory proteins containing arginine serine rich domains (SR proteins) including U2AF65 and Luc7like-2. Studies were carried out to investigate the putative arginine demethylation activity of JMJD6 using MS analysis of histone peptides and luminescence-based assays. New substrates from SR proteins were identified by immunoprecipitation of JMJD6 expressed in human cell lines followed by LC-MS/MS analysis and MALDI-MS based assays of synthesised peptide substrates. Work then focussed on studying the mechanism of lysyl-hydroxylation from substrate and enzyme perspective. A crystal structure of seleno-methionine labelled JMJD6 was obtained and it provided insights into the JMJD6 active site and its substrate interactions. Based on this data, single point variants of JMJD6 were prepared and their substrate binding properties were studied by MALDI-MS and 2OG turnover assays. Collagen lysyl-hydroxylases are also 2OG dependent oxygenases. Efforts to investigate the stereochemistry of JMJD6 catalysed hydroxylation, employing NMR and amino acid analyses were carried out. These studies led to the interesting finding that the C-5 stereochemistry of hydroxylysine in LUC7L2 peptide is opposite (2S,5S-hydroxylysine) to that present in collagen (2S,5R-hydroxylysine). It was found that JMJD6 undergoes autocatalytic self-hydroxylation. Lysine residues from both recombinant JMJD6 and that from HeLa cells at endogenous level were identified to be hydroxylated by amino acid and LC-MS/MS analyses. JMJD6 has a strong tendency to form aggregates and gel electrophoresis always reveals multimeric bands of various JMJD6 constructs. Characterisation and identification of oligomeric states of JMJD6 was carried out using Electron Microscopy. Studies were initiated to identify possible inhibitors by screening a set of 2OG analogues. The results from this preliminary inhibition studies have identified the tricarboxylic acid (TCA) cycle intermediates, succinate and fumarate to be JMJD6 inhibitors and form a basis of further studies aimed at identifying selective inhibitors.

This thesis deals with gender, the body and political power in the seventeenth century through readings of selected nonliterary texts set against readings of the writings of Milton and seventeenth-century women. Milton's poems and even his prose have most often been contextualized with reference to the higher reaches of literary history; here they are placed in more unfamiliar contexts: certain seventeenth-century women's writings, the emerging discourses of feminine conduct, and the discourses of 'news' produced in and around the Civil War. I show how both Milton and women writers struggle to come to terms with the implications of the new and powerful ideological formulations which result from the explosion of print culture which produces these texts.

The aim of the current study is to develop an efficient model which can predict low-energy compatible microstructures in ferroelectric bulks and film devices and their dynamic behaviour. The results are expected to assist in the interpretation of microstructure observations and provide a knowledge of the possible domain arrangements that can be used to design future materials with optimum performance. Several recent models of ferroelectric crystals assume low energy domain configurations. They are mainly based on the idea of fine phase mixtures and average compatibility, and can require intensive computation resulting in complex domain configurations which rarely occur in nature. In this research, criteria for the exact compatibility of domain structure in the form of a periodic multi-rank laminate are developed. Exactly compatible structure is expected to be energetically favourable and does not require the concept of a fine mixture to eliminate incompatibilities. The resulting method is a rapid and systematic procedure for finding exactly compatible microstructures. This is then used to explore minimum rank compatible microstructure in various crystal systems and devices. The results reveal routes in polarization and strain spaces along which microstructure can continuously evolve, including poling paths for ferro- electric single crystals. Also, the method is capable to generate all possible exactly compatible laminate configurations for given boundary conditions. It is found that simple configurations are often energetically favourable in conditions where previous approaches would predict more complex domain patterns. Laminate domain patterns in ferroelectrics are classified and corre- lated with observations of domains in single crystals, showing good agreement. The evolution of microstructures under applied mechanical and electrical loads is studied. A variational method, which minimises the overall energy of the crystal is developed. A new concept of transitional “pivot states” is introduced which allows the model to capture the feature that the microstructure in ferroelectric crystal switches between possible domain patterns that are energetically favourable, rather than assuming one particular domain pattern throughout. This model is applied to study the hysteresis responses of barium titanate (BaTiO3) single crystals subjected to a variety of loads. The results have good agreement with experimental data in the literature. The relationship between domain patterns and ferroelectric hysteresis responses is discussed.

This thesis presents a series of algorithmic innovations in Combinatorial Compressed Sensing and Persistent Homology. The unifying strategy across these contributions is in translating structural patterns in the underlying data into specific algorithmic designs in order to achieve: better guarantees in computational complexity, the ability to operate on more complex data, highly efficient parallelisations, or any combination of these.

Cardiovascular magnetic resonance imaging (MRI) is a well established technique in clinical cardiology. Different MRI sequences are routinely used to assess cardiac anatomy, function, viability and other parameters that aid diagnosing cardiac disease. Conversely, cardiac magnetic resonance spectroscopy (MRS), the only available method for a non-invasive study of human cardiac metabolism, has not evolved into a clinical tool yet. The combination of both techniques holds great potential to gain insight into the causality of cardiomyopathy diseases or other medical conditions with high cardiovascular risk profile, like diabetes or obesity and improve the clinical management of cardiac diseases. Nowadays, high field clinical MR systems have the great potential of improving the low spatial and temporal resolution and reproducibility of MRS. The aim of this thesis was to develop and implement a cardiac 1H-MRS method at 3 T that can be applied in clinical routine for the assessment of creatine and lipid levels in the human myocardium. The methodological developments to advance cardiac MRS are presented first. A robust 1H-MRS method comprising an optimized single-voxel technique, phased-array coil combination routine, optimized water suppression, breath-hold averaging and post-processing methods were developed. First, reproducibility and feasibility of the method were validated in vivo by acquiring 1H-MRS of the liver in almost one hundred healthy subjects. Subsequently, myocardial lipids levels were obtained in healthy volunteers by single breath-hold 1H-MRS triggered to mid-diastole, showing good reproducibility in an acquisition time less than 12 s. The good spectral resolution achieved using this method was demonstrated by the ability to differentiate for the first time two pools of myocardial lipids in spectra from the septum of patients with suspected myocardial lipid excess. Finally, creatine levels for healthy volunteers were investigated using multiple breath-hold acquisitions. Thus, this study shows the practicality and feasibility to incorporate this rapid cardiac 1H-MRS method into clinical studies of the human myocardium.

Magister Scientiae (Medical Bioscience) - MSc(MBS)<br>This study investigated the effects of TKIs on the growth and proliferation of MCF-7 breast carcinoma cells in culture. MCF-7 cells were exposed to different concentrations of TKIs alone and in combination with each other. Inhibition of cell growth by TKIs used individually occurred in a dose- and time-dependent manner. When EGFR Inhibitor I, EGFR Inhibitor II/BIBX1382 and the multi-specific EGFR/ErbB-2/ErB-4 Inhibitor were used in combination with each other at equimolar log dose concentrations, the combined effects on cell growth was significantly different to inhibitors used individually as reflected in a decreased EC50 (IC50) during combination treatments. Generally, for the combinations with DOX, CPL and the TKIs, synergistic as well as antagonistic effects were observed at isoeffective concentrations with resultant decreases in dose reduction indices (DRIs) implying greater efficacies with the respective combinations. In this study, conventional PCR was used to detect and illustrate the presence of the EGFR gene in the samples, while RT-qPCR was used to determine the mRNA expression levels of this gene in MCF-7 breast carcinoma cells

Dans cette thèse, la fonctionnalisation redox du MoS₂ a été considérée pour le développement de dispositifs électrocommutables. Des monocouches de MoS₂ exfoliées mécaniquement ont été fonctionnalisées avec du 6-(ferrocényl)hexanethiol et caractérisées de manière approfondie par un large éventail de techniques expérimentales. La combinaison de ces techniques à de la modélisation théorique a permis d’accéder à une meilleure connaissance des propriétés électroniques du MoS₂ fonctionnalisé par du ferrocène. De plus, une étude de microscopie électrochimique (SECM) a permis de mieux comprendre l’alignement des bandes d’énergie à l’interface MoS₂/électrolyte. Enfin, des nanofeuillets de MoS₂ ont été fonctionnalisés par du ferrocène et une autre molécule électroactive donnant lieu à deux processus redox réversibles, le tétrathiafulvalène (TTF). Le succès de la fonctionnalisation a été confirmé par des techniques de caractérisations à plusieurs échelles. Les mesures électriques des dispositifs à base de TMD ont conduit à des résultats prometteurs vers l’objectif de la mise en oeuvre de dispositifs électrocommutables à partir de MoS₂ fonctionnalisé par des molécules électroactives<br>In this thesis, the molecular functionalization of MoS₂ with electroactive molecules was explored for the development of electroswitchable devices. Mechanically exfoliated MoS₂ monolayers were functionalized with 6-(ferrocenyl)hexanethiol and thoroughly characterized by a broad range of techniques. The electronic properties of the ferrocene-functionalized MoS₂ were investigated through the combination of multiple experimental techniques and theoretical modeling. Further insights on the energy band alignment at the MoS₂/ electrolyte interface were gained through scanning electrochemical microscopy (SECM) investigations. Finally, solution-processed MoS₂ nanosheets were functionalized with ferrocene and multiredox tetrathiafulvalene (TTF) derivatives. The successful functionalization was confirmed by multiscale characterization techniques. Electrical measurements of TMD-based devices showed promising results toward the goal of the implementation of electrochemically switchable devices from redox-functionalized MoS₂

L’objectif de cette étude est de proposer des filtres compacts, innovants, qui pourront parfaitement s’intégrer au sein d’équipements de réception hyperfréquences, actuels ou futurs, que ce soient des applications radars ou de guerre électronique. S’appuyant sur les matrices de couplage, les structures physiques déjà existantes ainsi que sur les technologies multicouches, de nouveaux types de couplages ont été investigués et de nouvelles structures de filtrage ont été développées, comme les filtres inter-digités verticaux (VIF), les filtres « serpents » ou les filtres inter-digités repliés (FIF). Une méthode de synthèse a également été élaborée dans le cas des VIF, cette synthèse bénéficie des apports de la RBDM (Rectangular Boundary Division Method), permettant de calculer plus finement les couplages s’établissant au sein des VIF. La validation des filtres proposés, par la fabrication et la mesure de structures de référence, a rendu possible le développement de dispositifs plus aboutis. Cette étude offre ainsi au concepteur tout un panel de filtres compacts innovants<br>The purpose of this study is to propose innovating compact filters, to be integrated within future or current microwave equipments, such as radars or electronic warfare devices. Learning on coupling matrices theory, already existing structures and multilayer technologies, new kinds of coupling have been investigated and new filtering structures have been developed such as the Vertical Inter-digital Filters (VIF), the “snake” filters or the Folded Inter-digital Filters (FIF). A method to synthesize VIF has been elaborated, and benefits from contributions of the Rectangular Boundary Division Method (RBDM) to calculate more precisely couplings in VIF structures. The validation of the proposed filters, by the manufacturing and the measurement of reference structures, made possible the development of more accomplished devices. As a result, this study brings to designers a whole sample group of innovating and compact filters

The work contained within this thesis is concerned with a theoretical investigatiop of both laminar and thermally driven types of cavity flow, together with an analysis of their associated mixing processes which find applications to Industrial mixing and also to the environment. The mixing efficiency has been viewed from two perspectives namely the tracking of a selection of fluid particles, and also the simulation of the dispersive mixing of a coloured fluid element as carried along by the flow. This thesis also incorporates features of both Newtonian and a wide range of non-Newtonian fluids.

The polymerisation of dithienylethylenes (cis and trans 1,2-di(3-thienyl) ethylene, DTE-cis and DTE-trans), thiophene dithiolenes (4,5-dithiophen-3-yl-[1,3] dithiol-2-one, Th-3,3,4,5-di-thiophen-2-yl-[1,3]dithiol-2-one, Th-2,2,4-thiophen-3- yl-5-thiophen-2-yl-[1,3]dithiol-2-one, Th-2,3 and 4,5-di-(5-methyl-thiophen. -3-yl)- [1,3]dithiol-2-one, Th-3,3Me), and nickel dithiolenes (bis[1,2-di(3-thienyl)-1,2-ethenedithiolene] nickel tetrafluoroborate, Ni(Th-3,3)2 and bis[1,2-di(2-thienyl)-1,2-ethenedithiolene] nickel tetrafluoroborate, Ni(Th-2,2)2) was carried out and the resultant polymers were analysed by electrochemical and spectroelectrochemical methods. The voltammetric behaviour of the electropolymerised PDTE-cis and PDTEtrans suggested that polarons and bipolarons were produced upon oxidation. Nevertheless, the formation of a conducting state was not observed in the SNIFTIRS measurements. Upon I2 chemical doping, PDTE-cis and PDTE-trans showed increased stability in the doped state and the SNIFTIRS response of PDTE-trans, revealed similar IR characteristics to those of conductive polymers. The voltammetric and IR behaviour of the dithiolenes, PTh-3,3, PTh-2,2 and PTh-2,3 was similar to that of many conducting polymers. From the UV-Visible spectra of the neutral films it was determined that PTh-3,3, PTh-2,2 and PTh-2,3 have a bandgap of 2.04,2.30 and 2.18 eV, respectively. The electrochemical and SNIFTIRS results for PTh-3,3Me were noticeably different from the other dithiolene polymers revealing a poorly conductive polymer. The solid-state modification of PTh-3,3 to produce a new TTF derivatised polythiophene was carried out. The inclusion of TTF into the PTh-3,3 film was confirmed by voltammetry and IR data that also suggested that only partial modification of the polymer had occurred. The voltammetry of polyNi(Th-3,3)2 and polyNi(Th-2,2)2 showed a redox couple attributed to the electrochemistry of the nickel dithiolene centre, confirming the incorporation of the metal complex into the polymer. Whilst the SNIFTIR spectra of polyNi(Th-3,3)2 showed the characteristic features of metal dithiolenes, the IR behaviour of polyNi(Th-2,2)2 was more comparable to that of conducting polythiophenes. This result indicates that charge delocalisation in polyNi(Th-2,2) occurs through the Ni dithiolene sites disrupting the typical electroactivity of the dithiolene unit. For the polyNi(Th-3,3)2 films, the nickel dithiolene sites seem to be electronically isolated, preserving the aromaticity of the dithiolene system.

Based on research conducted from September 2008 through June 2011, this thesis explores the construction of gender and sexuality identities in Cyprus vis-à-vis the socio-political, legal and cultural context within which it is enabled or inhibited. More specifically, it examines how predominant discourses of nationhood and national identity as well as the processes, norms, institutions and mechanisms of Europeanization, affect local approaches to the relationship between national identity, gender and sexuality. -- Chronologically, the thesis covers the period between the early 1990s - when a Cypriot gay man brought a case before the European Court of Human Rights against the Republic of Cyprus - up to the present. However, it also makes references to the 1974 Turkish invasion and occupation of the island, as well as to the events that preceded and followed it, since these have been determinative of the importance assigned to Cypriot national identity narratives by local actors. -- Part of the data examined includes fifty-five interviews with prelates of the Orthodox Church of Cyprus, Greek-Cypriot political elites, military officials, representatives of women’s groups, as well as Greek-Cypriot and Turkish-Cypriot lesbian, gay, bisexual, trans* and queer individuals and activists. Through the utilization of a research design that draws on Foucaultian analysis, queer theory, law and national identity studies, the thesis argues that the Cypriot discursive landscape both restricts and enables the negotiation and reconfiguration of identity-formation processes. Namely, although nationalistic, androcentric, patriarchical and heterocentric essentialisms continue to permeate the Cypriot socio-political milieu, nationalism is characterized both by inherent contradictions and by the ability to reinvent itself.

Ester liquids including both natural ester and synthetic ester are being considered as potential alternatives to mineral oil, due to their better environmental performance and for some liquids their higher fire point. Although these liquids have been widely used in distribution and traction transformers, it is still a significant step to adopt ester liquids in high-voltage power transformers because the high cost and severe consequence of a factory test failure and the high level of safety and reliability required in service for these units, tend to lead to a cautious approach to any step change in technology. Lightning impulse strength as basic insulation level is of importance for insulation design of power transformers and lightning impulse test is commonly required in the factory routine tests for high-voltage power transformers, so this thesis is aimed to investigate the electrical performances including pre-breakdown and breakdown of natural ester and synthetic ester under impulse voltage. Two types of field geometry were considered in the study, one is sphere-sphere configuration which represents the quasi-uniform fields inside a transformer and another is strongly non-uniform point-plane configuration which represents the situation of a defect or a source of discharge. In quasi-uniform field study, standard breakdown tests were carried out under negative lightning and switching impulse voltages. Influence of various testing methods on the measured lightning breakdown voltage was studied and the 1% lightning withstand voltage was obtained based on Weibull distribution fitting on the cumulative probability plot built up using the approximately 1000 impulse shots. As for strongly non-uniform field study, streamer propagation and breakdown event in ester liquids either with or without pressboard interface were investigated at various gap distances under both positive and negative lightning impulse voltages. A relationship between the results under lightning impulse and previously published results under step voltage was built up to predict the lightning breakdown voltage of ester liquids at very large gaps. The results indicated that impulse strengths of ester liquids for both breakdown and withstand in a quasi-uniform field, are comparable to those of mineral oil. In a strongly non-uniform field, streamers in ester liquids propagate faster and further, than in mineral oil at the same voltage level. Thus breakdown voltages of ester liquids are generally lower than those of mineral oil, which could be as low as 40% at a large gap distance of approximately 1000 mm. Introduction of parallel pressboard interface has no influence on the streamer propagation and thus does not weaken the breakdown voltage, but it tends to reduce the acceleration voltage particularly for mineral oil under positive polarity. Last but not least, a unique phenomenon of secondary reverse streamer (SRS) was observed in ester liquids, which occurs subsequently and well after the extinction of the primary streamer (PS) propagation within a single shot of impulse voltage and has the reverse polarity to the PS. The formation mechanism of SRS is explained mainly due to the reverse electric field induced by the residual space charges left by the PS.

As substâncias farmacêuticas utilizadas na terapêutica humana e animal são classificadas como contaminantes emergentes, juntamente com produtos de higiene pessoal e cosméticos, e atingem o meio aquático por diferentes vias. Os danos associados aos fármacos em compartimentos ambientais se manifestam em baixas concentrações, podendo afetar indivíduos, populações e comunidades. Neste estudo, duas espécies de microcrustáceos de água doce do género Daphnia (D. magna e D. longispina) foram expostas a dois compostos de uso farmacêutico, nomeadamente o antipirético analgésico paracetamol e o antifúngico piritionato de zinco, de forma aguda e crónica. Os objetivos foram registrar a toxicidade desses fármacos considerando parâmetros como mortalidade (cálculo de EC50), reprodução, efeito comportamental (natação), stress oxidativo (catalase e GSTs), e neurotoxicidade (AChE) após exposições a concentrações subletais de ambos os fármacos. Alterações no comportamento natatório e efeitos oxidativos ( evidenciado pela alteração das actividades das enzimas catalase e GSTs) foram observados após exposição ao paracetamol para as duas espécies testadas. A exposição ao piritionato de zinco demonstrou ter efeitos neurotóxicos e oxidativos, com alteração no comportamento natatório. Não houve alteração na reprodução dos indivíduos expostos. Os dados obtidos demonstram que níveis ecologicamente relevantes de fármacos, após formas de exposição realistas, podem alterar parâmetros críticos em duas espécies distintas de microcrustáceos de água doce, embora com padrões de toxicidade nem sempre coincidentes. É possível assim concluir que a avaliação de diferentes níveis de resposta e a integração de dados ecotoxicológicos para espécies de organismos-testes diferentes, é uma estratégia que permite avaliar de forma mais concreta os efeitos subtis e nem sempre coincidentes que os fármacos podem causar no ambiente.<br>Pharmaceutical substances used in human and animal therapy are classified as emerging contaminants, together with personal care products and cosmetics, and reach the aquatic environment through different routes. Damage associated with drugs in environmental compartments is manifested in low concentrations and can affect individuals, populations and communities. In this study, two species of freshwater microcrustaceans of the genus Daphnia (D. magna and D. longispina) were exposed to two compounds of pharmaceutical use, namely the antipyretic analgesic paracetamol and the antifungal zinc pyrithione, acute and chronic. The objectives were to record the toxicity of these drugs considering parameters such as mortality (EC50 calculation) and reproduction; behavioral effect (swimming), oxidative stress (catalase and GSTs), and neurotoxicity (AChE) after exposure to sublethal concentrations of both drugs. Changes in swimming behavior and oxidative effects (evidenced by altered activities of catalase and GSTs enzymes) were observed after exposure to paracetamol for the two species tested. Exposure to zinc pyrithione has been shown to have neurotoxic and oxidative effects, with changes in swimming behavior. There was no change in the reproduction of exposed individuals. The data obtained demonstrate that ecologically relevant levels of drugs, after realistic forms of exposure, can alter critical parameters in two distinct species of freshwater microcrustaceans, although with not coincident patterns of toxicity. It is thus possible to conclude that the evaluation of distinct levels of response and the integration of ecotoxicological data into species of different test organisms is a strategy that allows to evaluate in a more concrete way the subtle and not always coincident effects that the drugs can cause in the environment.<br>Mestrado em Toxicologia e Ecotoxicologia

碩士<br>中山醫學大學<br>公共衛生學系碩士班<br>95<br>In this study changes in the swimming velocity behavior of the freshwater cladoceran Daphnia pulex were used as a indicators of pesticides and surfactant stress. The swimming velocity responses were determined by a real time image analysis, using a digital camera and a Pentium-PC equipped with. For a sequence of 30 images per group, where 10 daphnids were moving simultaneously, the trajectories have been reconstructed in binary image sequences. As biological endpoints, we defined the average swimming velocity. In the pesticides experiments that had been exposed to pesticides for 0 to 30 min, it was observed that the daphnia became significantly increase or decrease positively phototactic swimming velocity after 5 min of exposure to pesticide compared to control animals that had not been exposed to pesticide. In the surfactant experiments that had been exposed to pesticides for 0 to 60 min, it was observed that the daphnia became significantly increase or decrease positively phototactic swimming velocity after 15 min of exposure to surfactant compared to control animals that had not been exposed to surfactant. In flow-through experiments that lasted for 15 and 30 min and during which there were repeated measurements, there was again a significant effect of pesticides and surfactant exposure on the swimming velocity behavior of the daphnia. Irrespective of treatment, time had a significant effect. Results suggest that swimming velocity behavior can be used to detect pesticides within a few min, in short-term as well as in longer-lasting experiments with continuous flow- through and repeated stimulation of the daphnia. In the study changes in the phototactic behavior velocity of D. pulex can be used to detect surfactant pesticides within a few mins, in short term as well as in experiments with repeated light stimuli, similar to the conditions in this study daphnia swimming behavior monitor system.

A implementação de novas legislações com vista a regulamentar a introdução de novos compostos nos produtos para consumo como os desenvolvidos na indústria cosmética, introduziu a necessidade de se desenvolverem testes in vitro que substituíssem os clássicos testes in vivo de modo a ser possível aumentar o número de testes toxicológicos e, ao mesmo tempo, diminuir o uso de animais em laboratório. Pretende-se construir uma base de dados acerca dos compostos usados o mais detalhadamente possível, de modo a maximizar a segurança dos mesmos, abrangendo testes nas mais diferentes áreas da toxicologia. Neste âmbito, surge em 2004 o projecto ReProTect que pretende desenvolver métodos alternativos para o estudo da toxicidade reprodutiva. A complexidade do ciclo reprodutivo torna necessário dividir o seu estudo em partes, desenvolvendo diversos testes alternativos com endpoints diferentes. Nos últimos anos tem-se colocado em questão a segurança imputada aos diferentes filtros UV presentes nos protectores solares. O octocrileno é um filtro UV relativamente recente sendo a informação disponível acerca deste composto, em mamíferos, muito escassa. O presente trabalho tem como principal objectivo testar o potencial tóxico desta substância utilizando para isso um dos testes desenvolvidos durante o projecto ReProTect. O teste de maturação in vitro de oócitos de bovino (bIVM) permite analisar a influência que o octocrileno pode ter durante o processo de maturação dos oócitos nomeadamente na conclusão do processo de meiose. O endpoint deste teste reflecte a concentração de composto a partir da qual 50% dos oócitos são inibidos de concluir a meiose (EC50) sendo os resultados apresentados em percentagem de oócitos em MII. Foram testadas cinco concentrações de octocrileno entre 0,195HM-1,560HM. Os resultados obtidos demonstram uma tendência decrescente de oócitos em MII à medida que se aumenta a concentração. Apesar de se observar valores de percentagem de MII inferiores a 50% (valor mínimo obtido:37%), a determinação do valor de EC50 do octocrileno não pôde ser confirmada devido ao reduzido valor de amostragem. O facto de não existir informações prévias acerca da potencial toxicidade reprodutiva do octocrileno faz com que se considere que este trabalho representa um importante contributo na medida em que se identifica a gama onde provavelmente se situa o valor de EC50 do octocrileno valor que, a confirmar-se, encontra-se abaixo dos 50HM definidos como concentração de EC50, abaixo da qual todos os compostos são considerados como positivos no que se refere ao estudo da toxicidade reprodutiva.<br>The implementation of new legislation to regulate the introduction of new compounds in consumer products such as those developed in the cosmetics industry, has introduced a need to develop in vitro assays that replace the in vivo tests traditionally used in order to be able to increase the number of toxicological test at the same time that reducing the use of laboratory animals. It’s intended to build a database on the compounds used in as much detail as possible in order to maximize data security, comprising testing in different areas of toxicology. In this context, in 2004 was developed the ReProTect project that aims to develop alternative approaches to the study of reproductive toxicity methods. The complexity of the reproductive cycle does not allow the study of their sensitivity to potentially toxic compounds in a global manner, it is necessary divide into parts, and develop several alternative tests with different endpoints. In the last years, several publications had questioned the imputed safety of UF filters present in sunscreens. Octocrylene is a relatively new UV filter witch have very few data reported. There is no information about, among the others, his potential reproductive toxicity. This study aims to test the potential toxicity of this substance using one of the tests developed during the ReProTect project. The in vitro maturation of bovine oocytes test (bIVM) allows to analyze the influence that the octocrylene may have during the process of oocyte maturation particularly at the conclusion of the process of meiosis. The endpoint of the test compound reflects the concentration from which 50% of the oocytes are inhibited from completing meiosis (EC50). The results are presented in % of oocytes that concluded meiosis. Five concentrations of octocrylene between the range of 0,195µM-1,560µM were incorporated during in vitro maturation of bovine oocytes were. The results were observed in visible inverted microscope (400x) and fluorescence (400x). The results showed a downward trend in the % of MII oocytes as the concentration increased. It was possible to observe values under the EC50. A minimum of 37% of MII oocytes was reached. Despite that, the determination of the EC50 value of octocrylene could not be confirmed. It is necessary a larger sample and a greater number of replicates to do it. This study can be an important contribution doing an initial screening of octocrylene concentrations, identifying the possible range of the EC50 value. This takes more importance if we consider that for EC50 under 50µM, the compounds are considered positive to toxicity reproduction. The values tested in this study are far below of 50µM.

碩士<br>國立交通大學<br>環境工程系所<br>94<br>This research is aimed to estimate the low effect concentrations of 108 chemicals on Pseudokirchneriella subcapitata (green algae) assessed by a closed system BOD bottle test. Toxicants including benzenes, alkanes, phenols, anilines, aldehydes, nitrile, alcohols, ketones, pesticides, and PAHs were obtained NOEC, LOEC, EC10 and NEC. Among these parameters, NOEC was found to be generally smaller than EC10 and were apparently lower than LOEC and NEC. For non-polar narcotics , polar narcotics and reactive toxicants, distinct linear relationship were identified between EC50 and low effect concentration. For non-polar narcotic chemicals, the Quantitative structure-activity relationships (QSAR) of four parameters were based on the octanol-water partition coefficient (Log P) was established with R2 and Q2 above 0.85. Further, for polar narcotic chemicals, the discriminating parameters including log P, ELUMO (Energy of the lowest unoccupied molecular orbital)were chosen to predict the toxicity effectively. These equations also show a good prediction. Finally, compared to literature data, results from this study show that the closed system BOD bottle test is greater sensitivity than conventional batch test and a good correlation was found in comparison with the fish (Fathead minnow). Hence, algal toxicity test can be considered as a surrogate test for estimating the toxicity of chemicals to Fathead minnow.

Study of biosensors has become an essential part of research in biotechnology. Biosensors as fast, portable, highly sensitive, and low-cost bioanalytical detection devices have been utilized in many fields of human activity. The first part of the presented work focuses on electrochemical biosensors for rapid environmental screening of herbicides as water pollutants. A sol-gel immobilization method for a photosystem II (PSII) complex is studied in order to enhance the sensitivity and the signal strength and stability of a PSII-based biosensor. Computer simulations of a PSII biosensor are employed with the aim to find out how the immobilization membrane properties influence the biosensor parameters. Newly developed immobilization by a thin-layer membrane based on the results of computer simulations and revised measurement protocols are presented. The second part of the work is devoted to synthesis and electrochemical detection of newly developed metal labels for electrochemical immunosensors. The synthesis of dendrimer-encapsulated silver nanoparticles and biorecognition properties of biotin-nanocomposite conjugates are discussed. For detection of synthesized labels, a microfluidic detector was manufactured and tested and different approaches to packing of a microfluidic chip employing polydimethylsiloxane (PDMS) were investigated. Newly designed microstructures for a microfluidic separator of magnetic beads (MBs) were studied by computer simulations. The separator was made and trapping of MBs for the further employment in MBs-based immunoassays are presented

7 ABSTRACT Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. The role of these domains in forming of ATP-binding pocket and receptor function was investigated by using alanine scanning mutagenesis of the R203- L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor and by examination of the responsiveness to ATP and orthosteric analog agonists 2- (methylthio)adenosine 5'-triphosphate, adenosine 5'-(γ-thio)triphosphate, 2'(3'-O-(4- benzoylbenzoyl)adenosine 5'-triphosphate, and α,β-methyleneadenosine 5'- triphosphate. ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. These experiments, together with homology modeling, indicate that residues of the first group located in the upper part of...

Dissertação de Mestrado em Engenharia Civil, especialidade de Estruturas, apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra.<br>Com o constante desenvolvimento da Engenharia Civil um pouco por todo Mundo, e com o cada vez maior intercâmbio de Engenheiros entre diversos países, torna-se necessário que estes possuam um nível de conhecimento mais vasto com o objectivo de uma melhor adaptação às realidades presentes de cada país. A madeira cada vez mais se torna um material com grande importância, na vida quotidiana e na economia, principalmente em países da América do Norte e um pouco por toda a Europa com maior incidência no Norte. Neste contexto, pretende-se com este estudo, realizar uma análise comparativa do regulamento Europeu - EC5 – “Projecto de Estruturas de Madeira Parte 1-1 – Regras Comuns e Regras para Edifícios” e do regulamento Brasileiro - NBR 7190 – “Projecto de Estruturas de Madeira”. Esta análise terá como objectivo principal apontar as principais diferenças e semelhanças em termos filosofia base e estruturação das respectivas normas. Para além de se analisarem aspectos relacionados com as bases para a análise, isto é, requisitos fundamentais, acções e combinações de acções necessários para a verificação da segurança de estruturas de madeira, analisam-se também aspectos directamente relacionados com as propriedades físicas e mecânicas da madeira e seus derivados. Relativamente à metodologia de cálculo e dimensionamento de estruturas de madeira são analisados os principais critérios de verificação dos estados limites (Estados Limites Últimos e Estados Limites de Utilização), bem como aspectos relacionados com o dimensionamento de ligações. No final dessa análise serão apresentados dois estudos de caso (pavimento e viga de cobertura), dimensionados de acordo com cada uma destas normas, onde serão apontados as principais diferenças entre elas.<br>With the constant changes in Civil Engineering all over the world, and the interchanging of Engineers between countries, it is essential that they possess a vast knowledge with the purpose of a better adjustment to different realities in different countries. Wood is becoming a very important material, not only in our daily lives and in the economy, but as well as a construction material, mainly in countries in North America and some in Europe, especially in the North of Europe. In this context, this study intends to make a comparative analyses of the European regulations - EC5 – “Design of timber structures Part 1-1- General: Comum rules and rules for buildings” and the Brazilian regulations - NBR 7190 – “Design of timber structures”. This analyses’ main purpose will be to point out the differences and similarities in terms of a philosophic basis and the structure of the relating regulations. Furthermore, the analyses will focus on the fundamental requisites, actions and combinations of actions required for the verification of the security of the wood structures, it also analyses aspects directly related with the physical and mechanical properties of the wood and its by-products (derivatives). In relation to (concerning, relatively) the methodology of calculation and the dimensioning of the wood structures, the main criteria of the verification of limited states are analyzed (Ultimate Limit States and Serviceability Limit States), so are the aspects related with the linkage dimensioning. In the final analyses, two studies of this case will be presented (floor element and roof beam), dimensioned according to each of these regulations, where the main differences between both will be pointed out.

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2019<br>Kaposi’s sarcoma-associated herpesvirus (KSHV) is one of the seven recognized human tumor viruses. It is the etiologic agent underlying Kaposi sarcoma, primary effusion lymphoma and multicentric Castleman’s disease. This gammaherpesvirus establishes a lifelong persistence infection in the host and displays a lifecycle with two distinct phases: a short productive lytic phase and a prolonged latent phase. Latency-associated nuclear antigen (LANA) is the major latent gene and is strongly expressed in all forms of KSHV-associated malignancies. LANA mediates episomal replication, segregation and persistence, which is required for long-term maintenance of viral DNA. LANA proteins are also modulators of transcription through E3-ubiquitin ligase activity. Ubiquitination is an essential regulatory mechanism in eukaryotes, controlling a wide range of cellular pathways, including protein degradation. KSHV, like other viruses, has evolved mechanisms to hijack protein degradation pathways in order to establish an environment that favors its propagation. LANA protein has a bipartite SOCS-box motif divided into a Cullin-box and a BC-box located within its C-terminal and N-terminal regions, respectively. The BC-box interacts with an Elongin C and establishes a LANA-Elongin BC complex, which associates with a Cullin/Rbx1 module thus reconstituting a complex protein with E3 ubiquitin-ligase activity. This complex protein assembles an EC5S ubiquitin ligase, which enables the virus to hijack the E3 ligase components of the cell, thus modulating the ubiquitination pathway of its host. In this project, by taking advantage of the MHV68 in vivo model to study KSHV pathogenesis, chimeric viruses with engineered mutations within the BC-box were generated in order to assess the importance of the BC-box motif within the kLANA N-terminal region. Results showed that mutations within kLANA BC-box neither affect protein expression or viral growth in vivo and viral lytic phase in the lungs was not significantly affected. Latency results were not conclusive and therefore the importance and role of kLANA BC-box is still unclear and viral phenotypes need to be reassessed.<br>Os herpesvírus pertencem à grande família de vírus, Herpesviridae e distinguem-se pela sua capacidade de infetar uma enorme variedade de hospedeiros. Até à data, foram identificados oito herpesvírus capazes de infetar humanos e estima-se que quase toda a população adulta esteja infetada por pelo menos um dos vírus pertencentes a esta família. Os os herpesvírus humanos são capazes de se adaptar ao meio intracelular do hospedeiro e escapar ao seu sistema imunitário estabelecendo uma infeção crónica que pode permanecer em estado de latência durante toda a vida do hospedeiro. No entanto, estes vírus normalmente não são causadores de doenças graves, a não ser que o sistema imunitário do hospedeiro esteja extremamente debilitado. A família Herpesviridae pode ser dividida em 3 subfamílias: alfa, beta e gama. E, tendo em conta os vírus capazes de infetar humanos, a família alfa inclui os herpesvirus simplex (HHV-1 e HHV-2) e o vírus varicela-zóster (HHV-3). Os herpesvírus pertencentes à subfamília beta são o citomegalovírus (HHV-5), HHV-6 (variantes A e B) e ainda o HHV-7. Finalmente, os membros da família gama são o vírus Epstein-Barr (HHV-4) e o herpesvírus associado ao sarcoma de Kaposi (HHV-8) e são associados a alguns tipos de doenças cancerígenas. Estes vírus são capazes de induzir a proliferação de células B infetadas através de reações que ocorrem no centro germinativo e expandir a latência viral às células B de memória, que são o maior reservatório de latência dos gamaherpesvírus. Em particular, o herpesvírus associado ao sarcoma de Kaposi (KSHV), um dos sete vírus causadores de cancro até hoje conhecidos, evoluiu no sentido de perturbar os mecanismos celulares normais do hospedeiro como o crescimento celular, a apoptose e até mesmo o sistema imunitário bem como as respostas antivirais. Esta contínua desregulação celular ao longo dos anos pode eventualmente levar ao desenvolvimento de doenças neoplásticas, principalmente em indivíduos imunocomprometidos. Sabe-se que o KSHV é o agente etiológico responsável pelo linfoma primário de efusão primário, pela doença de Castleman multicêntrica e pelo sarcoma de Kaposi, que é o tipo de cancro mais comum em pacientes infetados com o vírus da imunodeficiência humana (HIV). O ciclo de vida do KSHV, tal como os outros herpesvírus, pode ser distinguido em duas fases distintas: uma fase lítica e uma fase de latência. Após a infeção inicial, os genes de ambas as fases são expressos mas após alguns ciclos de replicação viral, a fase lítica decresce e a fase de latência é estabelecida. Para que o genoma viral se mantenha nas células em divisão, o epissoma viral associa-se às histonas e estabelece-se como um cromossoma extra no núcleo 4 das células infetadas com expressão genética muito limitada. Esta reduzida expressão genética ajuda o vírus a escapar ao sistema imunitário do hospedeiro e ao mesmo tempo garante a sua sobrevivência e persistência viral. Uma das principais características deste tipo de vírus é sua capacidade de persistir no hospedeiro através da manutenção da latência viral, o que requer proteínas virais e celulares. Cerca de 90% das células infetadas possui o programa genético necessário à manutenção da latência viral. O programa de latência traduz-se num locus, onde são expressos vários genes que codificam para proteínas essenciais à latência viral, tal como a LANA (Latency-associated Nuclear Antigen). A LANA é considerada a proteína responsável pela persistência viral e, consequentemente pela latência viral. É uma proteína multifuncional, sendo maioritariamente responsável pela segregação, replicação e manutenção do epissoma. No entanto, a LANA pode também interferir com os mecanismos de transcrição e interagir com diversas proteínas celulares e interferir com os mecanismos anti-tumorais do hospedeiro. A LANA, atuando como uma ubiquitina ligase E3, é capaz de regular a transcrição e sinalizar para degradação proteossomal supressores tumorais tais como o von Hippel-Lindau (VHL), o p53 ou ainda o nuclear factor-kappa B (NF-B). A ubiquitinação está presente em todas as células eucarióticas e é um mecanismo regulatório essencial no ciclo celular, na apoptose, na endocitose ou ainda na resposta imunitária. Sendo assim, o KSHV, desenvolveu mecanismos com o objetivo de modular o seu funcionamento a seu favor. A ubiquitinação é um processo que ocorre através de uma cascata enzimática constituída por 3 enzimas: E1 (enzima ativadora da ubiquitina), E2 (enzima conjugadora) e E3 (ubiquitina ligase). Numa primeira etapa há a ligação da ubiquitina à E1 que, depois de ativada, é transferida para uma E2 que, juntamente com uma E3, transferem a ubiquitina para o substrato proteico para que seja degradado via proteossoma. A enzima E3 parece ser a responsável pelo reconhecimento específico do substrato e existem vários tipos de E3, tal como as Ellongin BC-Cullin5-SOCS (EC5S). As EC5S são complexos proteicos constituídos por várias subunidades, incluindo uma Cullin5 que se liga a uma Rbx1, formando assim o módulo Cullin 5-Rbx1, um heterodímero Elongin BC e uma proteína de reconhecimento de substrato SOCS (supressor of cytokine signaling). Todas as proteínas SOCS possuem uma homologia de sequência de 40 aminoácidos conservada – motivo SOCS-box. Este motivo SOCS-box é o responsável pela interação entre a Elongin BC e a Cullin5, estabelecendo a ligação entre o substrato de ubiquitinação e a E2. O complexo Elongin BC é um regulador positivo da RNA polimerase II e possui duas subunidades reguladoras (B e C). Este complexo desempenha um papel importante na regulação da transcrição e ainda ajuda a estabelecer a ligação entre as proteínas SOCS e as Cullin 5. Curiosamente, a proteína viral LANA possui uma SOCS-box 5 bipartida: BC-box + Cullin-box, localizadas na região C-terminal e N-terminal da proteína, respetivamente. Esta sequência proteica BC-box interage com uma Elongin C e estabelece um complexo proteico LANA-Elongin BC, que por sua vez ao ligar-se a um complexo Cullin5/Rbx reconstitui o complexo proteico: Ellongin BC-Cullin5-SOCS. Este complexo assemelha-se ao EC5S, funcionando como uma ubiquitina ligase, modulando os mecanismos de ubiquitinação do hospedeiro a favor do vírus. A infeção causada pelo KSHV é naturalmente limitada a humanos, por isso é muito importante o estabelecimento de um modelo animal de infeção para que o seu mecanismo de patogénese viral possa ser estudado in vivo. Visto que o herpesvirus murídeo 68 (MHV68), um vírus endémico que infeta o rato-do-campo Europeu e é capaz de infetar ratinhos de laboratório (Mus musculus), partilha alguma homologia de sequência genómica com o KSHV e codifica uma proteína homóloga à LANA do KSHV (mLANA), pode ser utilizado para a infeção de ratinhos de laboratório, providenciando um excelente modelo de estudo para o KSHV. A mLANA também possui uma região C-terminal, é expressa nas células B do centro germinativo e está envolvida na persistência do epissoma viral. Por último, a mLANA também é capaz de regular a transcrição genómica através de um complexo proteico EC5S, que é mediado por uma “SOCS-box” viral que partilha alguma homologia com a SOCS-box presente na kLANA. Neste projeto, tirando partido do modelo de infeção já estabelecido no laboratório, foram gerados vírus quimera (v-kLANA Elo-BC 46 e v-KM Elo-BC 34) com mutações na BC-box da kLANA (T212A, L213A, N214A, P215A, I216A, C217A) com o objetivo de avaliar a importância deste motivo na patogénese viral in vivo. O v-kLANA é um vírus quimera em que a mLANA foi substituída pela kLANA completa, incluindo a sua região 5´UTR e o v-KM é um vírus quimera que contém uma proteína de fusão entre a kLANA e a mLANA, ou seja, possui a kLANA mas com a região C-terminal da mLANA. Após mutagénese e reconstituição dos vírus quimera mutantes, 80 ratinhos foram divididos em 4 grupos distintos de infeção e inoculados com o vírus correspondente: 2 grupos com os vírus controlo (v-kLANA e v-KM) e 2 grupos com os vírus mutantes (v-kLANA Elo-BC 46 e v-KM Elo-BC 34). Aos dias 5, 7 e 10 pós infeção os ratinhos foram sacrificados e os pulmões foram devidamente extraídos e aos dias 10, 14 e 21 pós infeção os baços foram cirurgicamente removidos, post mortem. Após o processamento dos pulmões e baços, foi possível calcular os títulos virais na fase lítica e latente, respetivamente, e ainda averiguar o número de células positivas nos baços para DNA viral. Os resultados demonstraram que as mutações inseridas na BC-box da kLANA não afetaram a expressão proteica e, no geral, também não afetaram a fase lítica do vírus nos pulmões. Estes resultados permitem concluir que os vírus mutantes v-kLANA Elo-BC 46 e v-KM 6 Elo-BC 34 são ambos viáveis. No entanto, os resultados in vivo não permitiram tirar conclusões acerca da importância e possível função da BC-box na latência, o que significa que estudos futuros terão de ser feitos no sentido de confirmar ou refutar os fenótipos virais descritos neste projeto.<br>KSHV;LANA;;u;SOCS-box motif;BC-box;Cullin-box;EC5S ubiquitin ligase

Many viruses have developed numerous strategies to recruit and take advantage of cellular protein degradation pathways to evade the cellular viral immune system. One such virus is the Kaposi´s Sarcoma associated herpesvirus (KSHV), first discovered in Kaposi´s Sarcoma lesions found in AIDS patients. Latency-Associated Nuclear Antigen (LANA) is a KSHV multifunctional protein responsible for tethering viral DNA to the chromosome ensuring maintenance and segregation of the viral genome during cell division. Besides its main role of viral maintenance, LANA also physically interacts with several host proteins to modulate cell functions. One such function is to recruit the EC5S ubiquitin-ligase complex by interacting with Elongin BC complex and Cullin 5 protein, which in turn ubiquitinate substrates such as NF-κB and p53 to allow persistent viral infection. Like any other post-translation modifications, ubiquitination is reversible through deubiquitination enzymes (DUBs). LANA also interacts with ubiquitin specific protease 7 (USP7), a deubiquitination enzyme involved in regulation of several proteins including p53. Interaction with USP7 is made through a conserved peptide motif, which is also present in LANA. This work addresses the role of LANA in the recruitment and modulation of the ubiquitination and deubiquitination pathways. Despite the continued efforts in uncovering new LANA interacting partners to form a functional EC5S ubiquitin-ligase complex, only MHV-68 LANA interacted directly with Elongin BC, other interactions were not direct and may require a linker protein. On the other hand, LANA interaction with USP7 was able to be analysed by X-ray structure determination. In addition to a conserved P/AxxS motif, a novel Glutamine (Gln) residue from KSHV LANA was shown to make a specific interaction with USP7. This Gln residue is also present in other herpesvirus protein and hence it might be a conserved motif within herpesviruses.