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Relative Bio-Equivalence of Salbutamol MDIs Without and With the Attached Spacers. Development and validation of novel HPLC methods for the determination of salbutamol (and terbutaline) in urine excreted post-inhalation for bioequivalence and pharmacokinetic studies of Salbutamol MDIsMazhar, Syed H.R. January 2018 (has links)
This research explored in-vitro and in-vivo performance of three salbutamol metered dose inhalers (MDIs): Ventolin Evohaler (Evo), Airomir (Airo) and Salamol. In the in-vitro studies, critical quality attributes of the MDI using an Andersen cascade impactor (ACI) were examined and included measurement of fine particle dose (FPD) and total delivered dose (TDD). Bioequivalence studies were conducted in humans using the urinary pharmacokinetic method. Post-inhalation urinary excretion of salbutamol in the first 0.5 hour (lung deposition, USAL0.5) and over 24 hours (total systemic bioavailability, USAL24) were compared to determine the bioequivalence of the MDIs. The spacers recommended for use with these inhalers were also studied, and charcoal block studies were performed to assess the extent of USAL0.5.
The three MDIs had FPD (μg) of 78, 91 and 89, respectively; the latter pair was equivalent. Their USAL0.5 (6, 7 & 7 μg) was however not bioequivalent. These MDIs delivered equivalent dose (177, 174 & 180 μg) which reflected on their USAL24 (101, 84 & 97 μg). Nevertheless, USAL24 was inequivalent between Evo and Airo.
The FPD of Evo with Volumatic (VOL), AeroChamber Plus (AERO) and Able spacer was 78, 68 and 74 μg, respectively. The AERO treatment method was not equivalent to the MDI while VOL and Able were equivalent between them. Spacer USAL0.5 (16, 15 & 14 μg) was not bioequivalent to the MDI but to each other. The spacer in-vitro TDD (95, 85 & 92 μg) was inequivalent to the MDI treatment method. In contrast, their USAL24 was bioequivalent (97, 85 & 90 μg).
The FPD of Airomir with AERO (95 μg) was in-vitro equivalent while USAL0.5 (15 μg) of this treatment method was bio-inequivalent to the MDI alone. On the contrary, the TDD (110 μg) and USAL24 (84 μg) of AERO were respectively in-vitro inequivalent and bioequivalent to the MDI alone.
The FPD (μg) of Salamol MDI alone and with VOL (84) and AERO (86) as well as between the spacers was equivalent. However, the USAL0.5 of the MDI was not bioequivalent to spacers (20 and 18 μg) despite being equivalent between the spacers. In contrast, the respective TDD (103 and 95 μg) of spacer treatment methods were in-vitro inequivalent to the MDI alone albeit having bioequivalent USAL24 (86 and 87 μg).
The variations in the in-vitro performance of the three MDIs are most likely due to differences in their formulations and designs. As the performance metrics of the MDI influence lung deposition, substituting one MDI with another can have clinical implications.
Although the spacers reduced in-vitro TDD of the MDI to about half, their use increased lung deposition by over two folds, the magnitude of which varied with the MDI and spacer type. Despite significant decrease in dose delivery, the total systemic bioavailability with the spacers was similar to that with the MDI alone. This systemic bioequivalence is more likely due to greater USAL0.5 with the spacers. The results of the charcoal block studies reinforced this outcome.
The present study is unique as it used a clinically relevant salbutamol MDI dose (two puffs), assessed results for equivalence and analysed ACI deposition data further as stage groups. The deposition on adjacent ACI stages were grouped together as coarse, fine and extra-fine particle masses to identify their more likely deposition sites in the human respiratory tract. Moreover, this thesis describes highly sensitive and novel HPLC and SPE methods, developed and validated to quantify salbutamol in urinary and aqueous matrices.
As the clinical effects of MDIs are related to their lung deposition, the current work emphasizes the importance of spacer use. Nevertheless, differences in dose delivery between spacers may have clinical consequences. Hence, only the specific spacer recommended for use with the MDI should be used. / World Federation, Stanmore, London and Sadaat Welfare Foundation, Bradford, West Yorkshire
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Growth and biodegradation by Sporidiobolales yeasts in vanillin-supplemented mediumGonzález Gaarslev, Natalia January 2017 (has links)
Studies of biodegradation in lignins by basidiomycetes yeasts show the conversion of lignin in various degradation products among which vanillin, a valuable substance, suggested to be a strong inhibitor of both fermentation and growth of yeasts, stands. Sporidiobolales yeasts used in these experiments were aimed to be identified by their highly conserved ITS region as well as studied in vanillinsupplemented medium through, vanillin-supplemented plates, TLC and Neubauer’s chamber to find out which, among the several isolates tested, were the most resistant ones, understand how they take up vanillin and how their growth is affected by the presence of the phenolic compound. Two strains were identified as Rhodotorula babjevae. One of them, L4, together with LS22, Rhodosporidium kratochvilovae, could withstand and biodegrade high concentrations of vanillin, producing biodegradation products with Rf values similar to the ones know for vanillic acid and vanillyl alcohol. Better growth in medium supplemented with small doses of vanillin was found, as well as disparity among the same species and their metabolic features, therefore, herbicides resistance was suggested as a reason for strains divergence. Further morphological-species comparison could also describe if there exist a relation between them. / Estudios sobre la biodegradación de ligninas por levaduras basidiomicetes muestran la conversión de lignina en distintos productos de degradación, entre los cuales se encuentra la vainillina, un fuerte inhibidor de la fermentación y el crecimiento de levaduras. Las levaduras Sporidiobolales utilizadas en estos experimentos han intentado ser identificadas a través de la región ETI, muy conservada, además de estudiadas en medios suplementados con vainillina mediante placas suplementadas con vainillina, CCF y cámara de Neubauer para averiguar cuáles son las cepas más resistentes, entender cómo metabolizan la vainillina y cómo su crecimiento se ve afectado por la presencia de dicho compuesto. Dos cepas fueron identificadas como Rhodotorula babjevae. Una de ellas, L4, junto con con la cepa LS22, Rhodosporidium kratochvilovae, pudieron soportar y biodegradar elevadas concentraciones de vainillina, originando productos de biodegradación con valores de Rf similares a los del ácido vanílico y alcohol vanílico previamente conocidos. Se encontró un crecimiento mejor en medios suplementados con pequeñas dosis de vainillina además de una disparidad entre mismas especies y sus características metabólicas, así, herbicidas han sido sugeridos como una posible causa en dicha divergencia. Una futura comparación morfología-especie podrá describir si existe relación entre ambos.
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Taxonomy and Symbiosis in Associations of Physciaceae and Trebouxia / Taxonomie und Symbiose in Assoziationen von Physciaceen und TrebouxiaHelms, Gert 06 November 2003 (has links)
Die Familie der Physciaceen (lichenisierte Ascomyceten) und deren kompatible Photobionten wurden mit Hilfe von nrITS-Sequenzierungen untersucht. Es wurde Frisch- oder Herbarmaterial bearbeitet, das weltweit gesammelt worden war und 23 der 27 Physciaceengattngen repräsentierte. Die Sequenzdaten erlaubten eine differenzierte taxonomische Bearbeitung beider Biontengruppen. Basale Linien der Physciaceenphylogenie waren eng korreliert mit der Verteilung mehrerer phänotypischer Merkmale. Es konnte gezeigt werden, daß die Caliciaceen, eine andere Flechtenfamilie, die Schwestergruppe zu einer der vier Hauptlinien der Physciaceen bilden. Alle Proben der Physciaceen waren mit Algen aus der Gattung Trebouxia assoziiert. Ein Datensatz von über 300 Trebouxia nrITS-Sequenzen wurde zusammengestellt, der eine zuvor ungekannte Diversität innerhalb der Gattung Trebouxia repräsentiert. Die Taxonomie dieser Gattung wurde revidiert und ein System zur Abgrenzung und Zuordnung von nrITS-Varianten vorgeschlagen, das eine Strukturierung der gefundenen Diversität erlaubt. Viele der untersuchten Physciaceenarten erschienen hoch selektiv in Bezug auf ihre kompatiblen Photobionten. Im Gegensatz dazu konnte bei keinem der Photobionten eine Beschränkung auf nur eine Mycobiontenlinie gezeigt werden. Die Beschränkung vieler Mycobionten auf einen bestimmten Photobionten wurde als eine ökologische Abhängigkeit des Mycobionten von seinem kompatiblen Photobionten interpretiert. Daher wurde untersucht, ob Artbildungsereignisse in Trebouxia, Artbildungsereignisse in den assoziierten Physciaceen auslösen können. In einem Vergleich der Trebouxia- mit der Physciaceenphylogenie konnten jedoch keine korrelierten Verzweigungsmuster festgestellt werden. Hauptlinien der Trebouxien waren allerdings mit Umweltparametern, wie z.B. Substrat-pH und Makroklima korreliert. Die Evolution der Physciaceen war von diesen Faktoren offensichtlich deutlich weniger abhängig.Die nrSSU-Gene der Physciaceen enthielten mehr Introns als die aller anderen bekannter Organismengruppen. Der einzigartige Datensatz konnte genutzt werden, um konservierte Regionen innerhalb dieser Introns zu identifizieren. Auf diese konservierten Regionen konnten Primer konstruiert werden, die mit allen Introns einer Insertionsstelle kompatibel waren. Mit Hilfe dieser Primer konnten Introns detektiert werden, die bei der nrSSU-Sequenzierung unerkannt geblieben waren.
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Ekologie hub, asociovaných s tlejícím dřevem v ekosystémech přirozených lesů / Ecology of deadwood-associated fungi in the ecosystems of nature-like forestsZrůstová, Petra January 2014 (has links)
Dead wood plays an important role in forest ecosystems in the context of C dynamics, nutrient cycling, forest regeneration and biodiversity. Decaying wood sustains biodiversity by providing habitats and energy for fungi, bacteria, invertebrates, and many other organisms. Dead wood is resistant to decomposition and its decay is driven mainly by filamentous fungi. Community structure of wood- inhabiting fungi changes during decomposition, but the relationship between substrate quality and decomposer community is still poorly understood. This work studied fungal community composition with respect to tree species, stage of decay, volume and physico-chemical properties (such as pH, carbon and nitrogen content) of dead wood. Fungi were identified using next generation sequencing approaches - 454-pyrosequencing and Illumina MiSeq sequencing. Tree species, volume of dead wood (branches x logs) and stage of decay were the main variables affecting fungal community composition. Higher enzyme activities and content of fungal biomass indicate faster colonization of small branches than tree trunks by fungi. Fungal community composition, wood chemical properties and enzyme activities changed during decomposition. Both content of nitrogen and fungal biomass increased during decomposition. Enzyme activites peaked...
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Populační struktura zlatohlávka tmavého Oxythyrea funesta (Poda, 1761) a fylogeneze rodu Oxythyrea Mulsant, 1842 / Population structure of flower chafer Oxythyrea funesta (Poda, 1761) and phylogeny of the genus Oxythyrea Mulsant, 1842Vondráček, Dominik January 2012 (has links)
Eleven species are distinguished in the genus Oxythyrea Mulsant, 1842 (Coleoptera: Scarabaeidae) nowadays. They are not divided into subspecies. Diversity of the genus is concentrated in the Mediterranean and Oxythyrea funesta (Poda, 1761) inhabit a wide area in the western Palearctic Region. It was observed in last decades, that O. funesta retreated from central Europe to south and then recolonized it back including new areas in northern regions. Master thesis is focused on resolving population structure of O. funesta and partial phylogeny of the genus Oxythyrea using molecular genetic methods. 145 individuals of O. funesta and 15 individuals of five other species of the genus Oxythyrea appear in analysis. We acquired sequences of mitochondrial genes cytochrome oxidase I (807 bp), cytochrome b (381 bp) and nuclear gene internal transcribed spacer 1 (946 bp) from these specimens. The results of phylogenetic analysis confirmed so far the only one existing interpretation of relationships within the genus Oxythyrea based on morphological data. We also confirmed complicated relationships between O. funesta and O. pantherina, which also appear in the historical development of their taxonomy. We detected different genetic lineage in Sicily, southern Italy and Tunisia using phylogenetic trees and haplotype...
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Conception d'espaceurs pour relever les défis de bioconjugaisonMelkoumov, Alexandre 08 1900 (has links)
No description available.
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Estudos moleculares de Anopheles albitarsis e Anopheles triannulatus (Diptera: Culicidae) capturados em criadouro na planície de inundação do Mato Grosso do Sul, Brasil / Molecular studies of Anopheles albitarsis and Anopheles triannulatus (Diptera: Culicidae) captured in flood plain breeding sites in Mato Grosso do Sul, BrazilNeves, Amanda 27 January 2010 (has links)
A malária é uma doença grave, cujos vetores são mosquitos do gênero Anopheles. Esse gênero é composto por cerca de 500 espécies e aproximadamente 45 são responsáveis pela transmissão do parasito em todo o mundo. Alguns destes vetores são componentes de complexo de espécies crípticas como Complexo Albitarsis e Complexo Triannulatus. O Complexo Albitarsis é formado por quatro espécies: An. albitarsis s.s, An. albitarsis B, An. marajoara e An. deaneorum. O Complexo Triannulatus é composto por três espécies: An. triannulatus, An. halophylus e An. triannulatus C. Algumas técnicas moleculares são utilizadas para auxiliar na distinção destas espécies crípticas. Dessa forma, DNA de An. albitarsis s.l e An. triannulatus s.l., capturados na Usina de Porto Primavera, foram amplificados para ITS2 e para o gene ND4 do DNAmt. Todas as amostras foram submetidas à técnica de RFLP. Algumas amostras foram seqüenciadas diretamente ou clonadas para posterior seqüenciamento a fim de se confirmar a espécie. Das amostras do Complexo Albitarsis, 62,85% foram identificadas como An. deaneorum para ITS2-RFLP. As restantes foram amplificadas para ND4-RFLP e foram identificadas como An. albitarsis s.s. Observou-se, no entanto, que as amostras do Complexo Albitarsis não exibiram similaridade total com as depositadas no GeneBank sendo que para ITS2-PCR estas foram identificadas como An. albitarsis s.s. e An. deaneorum, enquanto que para o gene ND4 todas foram identificadas como An. albitarsis B. O ITS2-RFLP para o An. triannulatus s.l demonstrou polimorfismos entre as espécies. No seqüenciamento, estas amostras foram similares àquelas depositadas no GeneBank. O emprego de marcadores moleculares podem, em parte, auxiliar na distinção de espécies crípticas, porém outros marcadores devem ser avaliados a fim de se elucidar a identificação do Complexo Albitarsis. / Malaria is a severe disease whose vectors are mosquitoes belonging to the genus Anopheles. This genus contains 500 species of which approximately 45 are responsible for the worldwide transmission of these parasites. Some of these vectors belong to cryptic species such as those of the Albitarsis and Triannulatus Complex. The Albitarsis Complex is composed of four species: An. albitarsis s.s., An. albitarsis B, An. marajoara and An. deaneorum. The Triannulatus Complex contains three species: An. triannulatus, An. halophylus and An. triannulatus C. We used molecular techniques to differentiate these cryptic species. Thus, DNA of An. albitarsis s.l and An. triannulatus s.l, captured at the Porto Primavera Dam had their ITS2 and their mtDNA, ND4 genes amplified. All samples were analyzed by the RFLP technique. Some samples were directly sequenced while others were cloned for subsequent sequencing for species confirmation. Within the Albitarsis Complex, 62.85% were identified as An. deaneorum by RFLP-ITS2. The remaining were amplified by RFLP-ND4 and identified as An. albitarsis s.s. However the results of sequencing of the samples of the Albitarsis Complex did not overlap entirely with those deposited in the GeneBank since those amplified by RFLP-ITS2 were identified as An. albitarsis s.s. and An. deaneorum while those obtained by RFLP-ND4 were identified as An. albitarsis B. Also RFLP-ITS2 of A. triannulatus s.l. contained polymorphic regions among different species. By sequencing, these samples were similar to those deposited in the GeneBank. The use of molecular markers did, in some instances help to distinguish species within cryptic complexes, however other markers need to be evaluated to elucidate further identification of the Albitarsis Complex.
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Estudo dos fatores interferentes na adaptação marginal de próteses confeccionadas em titânio comercialmente puro e ligas alternativas / Study of determining factors for the marginal fit of prosthesis made of commercially pure titanium and alternative alloysSoriani, Natércia Carreira 08 April 2011 (has links)
O desajuste de copings obtidos por fundição por cera perdida está sujeita à vários fatores tais como: contração de fundição do metal, expansão do revestimento, uso de espaçadores, entre outros. O objetivo do estudo foi avaliar o desajuste marginal pré e pós cimentação de copings obtidos com três ligas de metais básicos - Ni-Cr-Be (VB), Ni-Cr (V2) e Co-Cr (KE) fundidos a partir de dois revestimentos - Termocast (TE) e Microfine (MI) e Titânio comercialmente puro (TR) fundido com dois revestimentos - Rematitan Plus (RP) e Rematitan Ultra (RU), nas condições E0, sem espaçador, E1, com uma camada de espaçador e E2, com duas camadas de espaçador. A partir de uma matriz metálica, foram obtidos 240 copings, sendo 10 para cada combinação metal-revestimento-espaçador. Foi utilizado Microscópio Óptico para determinação do desajuste nas condições pré e pós cimentação, efetuada com cimento de fosfato de zinco. Foi realizado teste de tração pós cimentação para determinação da carga de remoção dos copings da combinação KE-MI. Foram realizados ensaios dilatométricos para a definição dos coeficientes de expansão térmico linear dos metais e dos revestimentos, determinação da expansão de presa dos revestimentos, além de ensaio para determinação da variação dimensional das fundições e análises de microscopia eletrônica de varredura (MEV). Os dados de desajuste marginal foram analisados estatisticamente pelos testes Anova e Student-Newman-Keulsa que revelaram significância estatística (0,05) para os fatores isolados. Para os metais, os valores de desajuste (μm) foram V2 (85) = VB (89) < KE (119) < TR (171) e para os revestimentos, MI (68) < TE (128) < RP (153) < RU (188). Para o espaçador os desajustes foram E2 (76) < E1 (125) < E0 (147). Os menores valores de desajuste pós cimentação foram obtidos pelas combinações: VB-MI-E2 (44), V2-MI-E2 (47), VB-MI-E1 (52), VB-MI-E0 (55), TR-RU-E2 (67), V2-MI-E1(70), V2-TE-E2 (77), V2-MI-E0 (86), VB-TE-E2 (103), todas as demais combinações tiveram valores acima de 120 μm, adotado como referência clinicamente aceitável. Houve diferença significante para as cargas de remoção entre as condições E2 (49 Kgf) < E1 (74 Kgf) < E0 (102 Kgf). Os valores de expansão de presa foram: TE (1,05%), MI (0,88%), RP (0,19%) e RU (0,02%). Para o ensaio dilatométrico, os valores de contração dos metais foram: KE (0,270%), V2 (0,262%), VB (0,256%) e TR (0,160%). Para os revestimentos, houve diferença significante, entre MI (0,74%) e RU (-1,63%). A análise da variação dimensional revelou diferença significante para os metais: V2 (0,08%) ≥ VB (-0,12%) ≥ KE (-0,21%) ≥ R (-0,85%) e para os revestimentos: MI (0,09%) > TE (-0,26%) = RP (-0,50%) > RU (-1,19%). Os revestimentos MI e RU apresentaram micro estrutura mais refinada, contendo cristais mais homogêneos e menos porosidade que os revestimentos RP e TE. Com base nos dados analisados, é válido afirmar que, embora a utilização de revestimentos com boas propriedades dilatométricas facilite a confecção de copings com ajuste marginal adequado, a utilização de espaçadores pode ser indispensável para obtenção de resultados clínicos satisfatórios. / The marginal fit of copings made by lost-wax casting are subject to different variation factors, such as contraction of molten metal, investment thermal expansion, die spacer use and others. This study aimed to evaluate the marginal adaptation of copings before and after cementation, obtained with three basic alloys - Ni-Cr-Be (VB), Ni-Cr (V2) and Co-Cr (KE), melted with two investments - Termocast (TE) and Microfine (MI) - and commercially pure titanium (TR) melted with Rematitan Plus (RP) and Rematitan Ultra (RU), in the conditions: E0 (without die spacer), E1 (one die spacer layer) and E2 (two die spacer layers). From a metal matrix, 240 copings were obtained, 10 for each combination metal-investment-spacer. An optical microscope was used to determine the misfit before and after the cementation with zinc phosphate cement. A tensile strength test was carried out after the cementation in order to determine the remotion load. Dilatometric tests were used to calculate the linear thermal expansion coefficients of metals and investments, to determine the setting expansion of the investments and to determine the dimensional variation as well. After the tests, the data were analyzed statistically. The analysis of variance for the marginal adaptation showed statistical significance (0.05) for all variation factors. For the metals, the misfit, in μm, was: V2 (85) = VB (89) < KE (119) < TR (171); and for investments, MI (68) < TE (128) < RP (153) < RU (188). The misfit for factor die spacer was: E2 (76) < E1 (125) < E0 (147). Having accepted the value of 120 μm as a reference limit of clinically acceptable results, the lowest misfit values for different combinations after cementation were: VB-MI-E2 (44), V2-MI-E2 (47), VB-MI-E1 (52), VB-MI-E0 (55), RU-TR-E2 (67 μm), V2-MI-E1 (70), E2-V2-TE (77), V2-MI-E0 (86), VB-TE-E2 (103). All the other combinations had values above 120 μm. There was a significant difference for tensile strength under the conditions E2 (49 Kgf), E1 (74 Kgf) and E0 (102 kgf). The setting expansion values were: TE (1.05%), MI (0.88%), RP (0.19%) and RU (0.02%). According to the dilatometer test, the values of contraction for the metals were: KE (0.27%), V2 (0.262%), VB (0.256%) and TR (0.16%), and for the investments: MI (0.74%) and RU (-1.63%). The analysis of variance revealed significant differences for metals dimensional variation: V2 (0.08%) ≥ VB (-0.12%) ≥ KE (-0.21%) ≥ TR (-0.85%) and investments: MI (0.09%) > TE (-0.26%) = RP (-0.50%) > RU (-1.19%). In conclusion, the use of investments with good dilatometric properties improves the marginal fit of the copings, but the die spacer use may be essential for obtaining satisfactory clinical results.
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GaAs/AlAs ASPAT diodes for millimetre and sub-millimetre wave applicationsAbdullah, Mohd January 2018 (has links)
The Asymmetric Spacer layer Tunnel (ASPAT) diode is a new diode invented in the early 90s as an alternative to the Schottky barrier diode (SBD) technology for microwave detector applications due to its highly stable temperature characteristics. The ASPAT features a strong non-linear I-V characteristic as a result of tunnelling through a thin barrier, which enables RF detection at zero bias from microwaves up to submillimetre wave frequencies. In this work, two heavily doped GaAs contact layer on top and bottom layers adjacent to lightly doped GaAs intermediate layers, enclose undoped GaAs spacers with different lengths sandwiching an undoped AlAs layer that acts as a tunnel barrier. The ultimate ambition of this work was to develop a MMIC detector as well as a frequency source based on optimized ASPAT diodes for millimetre wave (100GHz) applications. The effect of material parameter and dimensions on the ASPAT source performances was described using an empirical model for the first time. Since this is a new device, keys challenges in this work were to improve DC and RF characteristic as well as to develop a repeatable, reproducible, and ultimately manufacturable fabrication process flow. This was investigated using two approaches namely air-bridge and dielectric-bridge fabrication process flows. Through this work, it was found that the GaAs/AlAs heterostructures ASPAT diode are more amenable to the dielectric-bridge technique as large-scale fabrication of mesa area up to 4Ã4Âμm2 with device yields exceeding 80% routinely produced. The fabrication of the ASPAT using i-line optical lithography which has the capability to reduce emitter area to 4Ã4Âμm2 to lower down the device capacitance for millimetre wave application has been made feasible in this work. The former challenge was extensively studied through materials and structural characterisations by a SILVACO physical modelling and confirmed by comparison with experimental data. The I-V characteristic of the fabricated ASPAT demonstrated outstanding scalability, demonstrating robust processing. A fair comparison has been made between ASPAT and SBD fabricated in-house; indicating ASPAT is extremely stable to the temperature. The RF characterisations were carried out with the aid of Keysight ADS software. The DC characteristic from fabricated GaAs/AlAs ASPAT diodes were absorbed into an ADS simulation tool and utilized to demonstrate the performance of MMIC 100GHz detector as well as 20GHz/40GHz signal generators. Zero bias ASPAT with mesa area of 4Ã4Âμm2 with video resistance of 90KΩ, junction capacitance of 23fF and curvature coefficient of 23V-1 has demonstrated detector voltage sensitivity above 2000V/W, while the signal source conversion loss and conversion efficiency are 28dB and 0.3% respectively. An estimate noise equivalent power (NEP) for this particular device is 18.8pW/Hz1/2.
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Estudos moleculares de Anopheles albitarsis e Anopheles triannulatus (Diptera: Culicidae) capturados em criadouro na planície de inundação do Mato Grosso do Sul, Brasil / Molecular studies of Anopheles albitarsis and Anopheles triannulatus (Diptera: Culicidae) captured in flood plain breeding sites in Mato Grosso do Sul, BrazilAmanda Neves 27 January 2010 (has links)
A malária é uma doença grave, cujos vetores são mosquitos do gênero Anopheles. Esse gênero é composto por cerca de 500 espécies e aproximadamente 45 são responsáveis pela transmissão do parasito em todo o mundo. Alguns destes vetores são componentes de complexo de espécies crípticas como Complexo Albitarsis e Complexo Triannulatus. O Complexo Albitarsis é formado por quatro espécies: An. albitarsis s.s, An. albitarsis B, An. marajoara e An. deaneorum. O Complexo Triannulatus é composto por três espécies: An. triannulatus, An. halophylus e An. triannulatus C. Algumas técnicas moleculares são utilizadas para auxiliar na distinção destas espécies crípticas. Dessa forma, DNA de An. albitarsis s.l e An. triannulatus s.l., capturados na Usina de Porto Primavera, foram amplificados para ITS2 e para o gene ND4 do DNAmt. Todas as amostras foram submetidas à técnica de RFLP. Algumas amostras foram seqüenciadas diretamente ou clonadas para posterior seqüenciamento a fim de se confirmar a espécie. Das amostras do Complexo Albitarsis, 62,85% foram identificadas como An. deaneorum para ITS2-RFLP. As restantes foram amplificadas para ND4-RFLP e foram identificadas como An. albitarsis s.s. Observou-se, no entanto, que as amostras do Complexo Albitarsis não exibiram similaridade total com as depositadas no GeneBank sendo que para ITS2-PCR estas foram identificadas como An. albitarsis s.s. e An. deaneorum, enquanto que para o gene ND4 todas foram identificadas como An. albitarsis B. O ITS2-RFLP para o An. triannulatus s.l demonstrou polimorfismos entre as espécies. No seqüenciamento, estas amostras foram similares àquelas depositadas no GeneBank. O emprego de marcadores moleculares podem, em parte, auxiliar na distinção de espécies crípticas, porém outros marcadores devem ser avaliados a fim de se elucidar a identificação do Complexo Albitarsis. / Malaria is a severe disease whose vectors are mosquitoes belonging to the genus Anopheles. This genus contains 500 species of which approximately 45 are responsible for the worldwide transmission of these parasites. Some of these vectors belong to cryptic species such as those of the Albitarsis and Triannulatus Complex. The Albitarsis Complex is composed of four species: An. albitarsis s.s., An. albitarsis B, An. marajoara and An. deaneorum. The Triannulatus Complex contains three species: An. triannulatus, An. halophylus and An. triannulatus C. We used molecular techniques to differentiate these cryptic species. Thus, DNA of An. albitarsis s.l and An. triannulatus s.l, captured at the Porto Primavera Dam had their ITS2 and their mtDNA, ND4 genes amplified. All samples were analyzed by the RFLP technique. Some samples were directly sequenced while others were cloned for subsequent sequencing for species confirmation. Within the Albitarsis Complex, 62.85% were identified as An. deaneorum by RFLP-ITS2. The remaining were amplified by RFLP-ND4 and identified as An. albitarsis s.s. However the results of sequencing of the samples of the Albitarsis Complex did not overlap entirely with those deposited in the GeneBank since those amplified by RFLP-ITS2 were identified as An. albitarsis s.s. and An. deaneorum while those obtained by RFLP-ND4 were identified as An. albitarsis B. Also RFLP-ITS2 of A. triannulatus s.l. contained polymorphic regions among different species. By sequencing, these samples were similar to those deposited in the GeneBank. The use of molecular markers did, in some instances help to distinguish species within cryptic complexes, however other markers need to be evaluated to elucidate further identification of the Albitarsis Complex.
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