Dissertations / Theses on the topic 'IncF plasmid'

A produção de beta-lactamases de espectro estendido (ESBL) tornou-se um desafio em saúde pública por restringir as opções terapêuticas para o tratamento de infecções causadas por bactérias gram-negativas. O objetivo desse estudo foi avaliar a ocorrência de estirpes produtoras de ESBL nas granjas de suínos brasileiras, bem como caracterizar os plasmídeos carreadores dos genes blaESBL quanto ao grupo de incompatibilidade, tamanho e presença de genes de resistência adicionais. As estirpes foram isoladas em meio MacConkey e identificadas por MALDI-TOF. Posteriormente, os valores de concentração inibitória mínima foram determinados por microdiluição e/ou ágar diluição para aminoglicosídeos, carbapenens, cefalosporinas, fluoroquinolonas, tetraciclinas, sulfas e cefalosporinas associadas a inibidores competitivos. Os genes codificadores de beta-lactamases foram identificados por PCR assim como o grupo de incompatibilidade dos respectivos plasmídeos carreadores e o grupo filogenético das estirpes de E. coli. A análise de clonalidade foi realizada por ERIC-PCR e MLST. Finalmente, o ambiente genético do gene blaCTX-M-15 foi determinado por PCR e/ou sequenciamento, sendo que os plasmídeos carreando genes blaESBL foram transferidos às estirpes receptoras E. coli TOP10 e C600 por transformação e conjugação, respectivamente, e parcialmente sequenciados. As estirpes de Escherichia coli produtoras de CTX-M-2 foram as mais prevalentes, sendo endêmicas no estado de Minas Gerais. Além disso, é relatada a presença da enzima CTX-M-15 em estirpes de E. coli (ST224, ST410, ST1284), Klebsiella pneumoniae, Enterobacter cloacae e Pseudomonas aeruginosa (ST3201). O gene blaCTX-M-15 esteve associado a plasmídeos IncF e foi transferido com sucesso para a estirpe receptora E.coli TOP10, plasmídeos IncF também foram associados a presença do gene blaCTX-M-2. O gene blaCTX-M-8 foi detectado em quatro novos STs de E. coli (ST5845, ST5847, ST5848 e ST5350) e não foi adquirido pelas estirpes receptoras. Estes dados indicam que a vigilância de fenótipos resistentes na produção suína deve de ser considerada uma prioridade, assim como a preferência ao uso de antimicrobianos de espectro estrito a fim de evitar a disseminação desses fenótipos nas granjas e sua possível transmissão para população humana.<br>Extended-spectrum beta-lactamase production (ESBL) became a great challenge regarding public health because limit the therapeutic options to treat infections by gram-negative bacteria. The aim of this study were evaluate the occurrence of ESBL producers in Brazilian swine farms and characterize blaESBL-carrying plasmids by sizing and incompatibility group and presence of additional resistance genes. Strains were isolated in MacConkey agar and identified by Maldi-Tof. Next, the minimal inhibitory concentration values were determined by microdilution and/or agar dilution to aminoglycosides, carbapenems, cephalosporins, fluoroquinolones, tetracyclines, sulfas and cephalosporin/inhibitors association. Betalactamase encoding genes, plasmid incompatibility group and Escherichia coli phylogenetic group were determined by PCR. Clonal relatedness was evaluated by ERIC-PCR and MLST. Finally, the blaCTX-M-15 genetic environment was determined by PCR and/or sequencing and blaESBL-carrying plasmids transferred to E. coli TOP10 and C600 receptor strains by transformation and conjugation, respectively, and partially sequenced. CTX-M-2-producing E. coli were the most prevalent phenotype, which were endemic in Minas Gerais State. Moreover, the CTX-M-15 enzyme emerged among E. coli (ST224, ST410, ST1284), Klebsiella pneumoniae, Enterobacter cloacae e Pseudomonas aeruginosa (ST3201) strains. The blaCTX-M-15 was associated with IncF plasmids, which were successfully transferred to E.coli TOP10, similarly, IncF plasmids were found harboring the blaCTX-M-2. The blaCTX-M-8, detected in four novel E. coli sequence types (ST5845, ST5847, ST5848 e ST5350), was not acquired by receptor strains. Thus, the surveillance of resistant phenotypes in swine production must be established as a priority as well as narrow spectrum antimicrobials prescription antimicrobial instead broad spectrum to prevent the dissemination of these phenotypes in farms and their transmission to human population.

IncC plasmids are large conjugative broad host range plasmids important in the spread of resistance genes conferring resistance to a wide range of clinically important antibiotics. In this study, fundamental aspects of the biology of IncC plasmids and the related IncA plasmids were examined. Traditional incompatibility testing confirmed that IncA and IncC plasmids are stable together for more than 100 generations in the absence of antibiotic selection and are therefore compatible. Entry exclusion experiments revealed that an IncC plasmid in the recipient could completely prevent the conjugative transfer of another IncC or IncA plasmid. IncA plasmids were also able to completely exclude IncC plasmids. This level of exclusion was several orders of magnitude higher than observed in other characterised systems. The gene required in the recipient, eexC in IncC plasmids and eexA in IncA plasmids, was predicted based on the position of exclusion genes in known exclusion systems and found to be sufficient to exclude both IncA and IncC plasmids. IncA and IncC plasmids are able to mobilise the integrative mobilisable element (IME) Salmonella genomic island 1 (SGI1) and its variants. These element types interact in several specific ways. The presence of an SGI1 variant in the donor allowed an IncC plasmid to avoid exclusion exerted by EexC in the recipient but not when the complete plasmid when present, indicating there are further interactions preventing entry of IncC plasmids by conjugation. Further exclusion experiments showed that SGI1 in the recipient reduced conjugative transfer of IncC plasmids by 15-240 fold. However, IncC plasmids did not exclude SGI1. IncC plasmids have gained increasing attention with >350 sequences now available in GenBank. Examination of the sequences of IncC plasmids used in this study and sequences available in GenBank led to the identification of several important lineages of type 2 IncC plasmids based on the positions of resistance islands. Some lineages are found in a wide number of species and are globally disseminated. Extensive evolution was observed in the resistance islands of these important lineages. In addition, two novel trimethoprim resistance genes were found on IncC plasmids or an IncC plasmid segment and were characterised.

Thesis (DSc)--Stellenbosch University, 2014.<br>ENGLISH ABSTRACT: Plasmids belonging to the IncQ family have an exceptionally broad host-range and are highly mobilizable in the presence of the self-transmissible IncP plasmids. All IncQ plasmids identified to date have certain features in common. The feature that distinguishes them most from all other plasmids is that they have a unique mechanism of replication. Their replicons consist of repA, repB and repC genes encoding a replicase, primase and DNA-binding proteins respectively. All IncQ plasmids contain at least three 22-bp iterons (or 20-bp iterons with 2-bp spacers) that are identical in sequence and to which the RepC DNA-binding protein binds. They replicate by means of a unique strand-displacement mechanism that is considered to place a limit on their size. Replication proceeds by a partially single-stranded intermediate that is believed to result in an increased likelihood of structural instability with an increase in plasmid size. The most compact backbone of IncQ plasmids is approximately 5.9-kb and the largest natural IncQ plasmid reported is 14.2-kb. Although the mobilization regions of IncQ plasmids are not as unique as the replicons, they are all characterized by the primase of the replicon being fused to the relaxase of the mobilization genes. The remainder of the mobilization genes may vary substantially in number and sequence between plasmids and have been subdivided into at least four distinct lineages. This dissertation consists of twenty one manuscripts published during the period 1984 to 2012. The focus is almost entirely on the IncQ plasmid subfamily known as IncQ2. Most of the earlier work was on determining the nature and extent of the replicons, mobilization genes and the toxin-antitoxin plasmid stability system. A strong theme in the latter work focussed on using the natural variation among the IncQ2 plasmids as a means to understand IncQ plasmid evolution. The collection of articles comprises a combination of original research and reviews.<br>AFRIKAANSE OPSOMMING: Plasmiede wat aan die IncQ familie behoort kom ‘n uitsonderlike wye gasheerselreeks voor en is hoogs mobiliseerbaar deur middel van die selfoordraagbaar IncP plasmiede. Alle IncQ plasmiedes wat tot datum identifiseer is het sekere gemeenskaplike eienskappe. Die eienskap wat hulle van alle ander plasmiedes onderskei is hul unieke dupliseringsmeganisme. Hul dupliseringsmeganisme bestaan uit repA, repB en repC gene wat onderskeidlik ‘n helikase, ‘n ‘primase’ en ‘n DNSbindingsproteïen enkodeer. Die IncQ plasmiede het ten minste drie 22-bp iterone (of 20-bp iterone met 2-bp skeidingsnukleotiede) met ‘n identiese nukleotiedvolgorde en waaraan die RepCbindingsproteïen bind. Hulle dupliseer deur middel van ‘n unieke DNA-string-vervangingsmeganisme wat ‘n beperking op hul grootte plaas. Tydens replikasie word ‘n intermediêre struktuur gevorm wat gedeeltelik enkelstring is en dit is blykbaar die rede vir ‘n verhoging in strukturële onstabilitiet as die plasmied groter word. Die kleinste ruggraat onder die IncQ plasmiede is min of meer 5.9-kb en die grootste natuurlike IncQ plasmied wat gerapporteer is, is 14.2-kb. Alhoewel die mobiliseringsgebied van die IncQ plasmiede nie so duidelik uitkenbaar as die replikons nie, hierdie gebied is gekenmerk deur ‘n ‘primase’ wat aan ‘n ‘relaxase’ in die mobiliseringsgene gekoppel is. Die oorblywende mobiliseringsgene verskil in beide getal en nukleotiedvolgorde tussen plasmiede en is gebruik om die plasmiede in vier duidelike oorsponggroepe in te deel. Hierdie proefskrif bestaan uit een-en-twintig artikels wat tussen 1984 en 2012 gepubliseer is. Die fokus is hoofsaaklik op die IncQ plasmiedsubfamilie wat as IncQ2 bekend is. Baie van die vroeër werk het oor die aard en omvang van die duplisering en mobiliseringsgene asook die toksienteentoksien plasmiedstabiliseringsmeganisme hanteer. ‘n Sterk tema in die latere werk was om die natuurlike variasie onder die IncQ2 plasmiede te bestudeer ten einde IncQ plasmiedevolusie te verstaan. Die publikasie versameling bestaan uit ‘n kombinasie van oorspronklike navorsing en oorsigartikels.

L'îlot génomique Salmonella Genomic Island 1 (SGI1) est un élément intégratif et mobilisable, support de nombreux gènes de résistance aux antibiotiques, et identifié chez de nombreux genres bactériens. Le transfert de SGI1 requiert spécifiquement la présence d'un plasmide conjugatif du groupe d'incompatibilité IncA/C. Les régulateurs globaux AcaCD des plasmides IncA/C activent l’excision de SGI1 qui, une fois sous forme d’un intermédiaire extrachromosomique circulaire, va pouvoir être transféré en utilisant la machinerie de conjugaison encodée par les plasmides IncA/C (mobilisation conjugative en trans). Depuis la description de SGI1, plusieurs études ont relaté une apparente stabilité de SGI1 au cours des générations bactériennes. Cependant, des observations préliminaires indiquaient des difficultés de cohabitation entre SGI1 et les plasmides IncA/C. L’objectif de ce travail était d’étudier la stabilité de SGI1 et sa compatibilité avec les plasmides conjugatifs IncA/C dont dépend sa mobilité. L’opéron putatif S026- S025 de SGI1 a été identifié comme constituant un système Toxine-Antitoxine (TA) qui a été appelé sgiAT. Le rôle de ce système TA dans la stabilité de SGI1 a été mis en évidence en présence d'un plasmide IncA/C. De plus, l’incompatibilité entre SGI1 et les plasmides IncA/C a été démontrée expérimentalement pour la première fois. La stabilité de SGI1 est liée à son intégration chromosomique. Cependant, lorsque SGI1 est excisé du chromosome et donc vulnérable (il peut être perdu), c’est-à-dire en présence d’un plasmide IncA/C, le système TA sgiAT joue un rôle important dans le maintien de SGI1 dans les populations bactériennes<br>The multidrug resistance Salmonella Genomic Island 1 (SGI1) is an integrative mobilizable element identified in several enterobacterial pathogens. This chromosomal island requires specifically the presence of a conjugative IncA/C plasmid to be excised and transfered by conjugation (mobilization in trans). Preliminary observations suggest stable maintenance of SGI1 in the bacterial host but paradoxically also incompatibility between SGI1 and IncA/C plasmids. Here, using a Salmonella enterica serovar Agona clonal bacterial population as model, we demonstrate that a Toxin-Antitoxin (TA) system encoded by SGI1 plays a critical role in its stable host maintenance when an IncA/C plasmid is concomitantly present. This system, designated sgiAT for Salmonella genomic island 1 Antitoxin and Toxin respectively, thus seems to play a stabilizing role in a situation where SGI1 is susceptible to be lost through plasmid IncA/C-mediated excision. Moreover and for the first time, the incompatibility between SGI1 and IncA/C plasmids was experimentally confirmed

KPC (Klebsiella pneumoniae carbapenemases) são -lactamases da classe A de Ambler globalmente disseminadas, com 10 variantes, sendo predominates KPC-2 e KPC-3. O objetivo deste trabalho foi estudar a genética e epidemiologia molecular de enterobactérias resistentes aos carbapenêmicos isoladas no Brasil. Sessenta e quatro enterobactérias resistentes aos carbapenêmicos foram analisadas: 57 Klebsiella pneumoniae (Kp), 5 Enterobacter cloacae (Ecl), 1 Serratia marcescens (Sm) e 1 Citrobacter freundii (Cf), de diferentes pacientes, em seis hospitais e em duas distintas regiões do Brasil. Identificação e testes de sensibilidade antimicrobiana foram realizados por sistemas semi-automáticos e métodos padronizados. A relação clonal foi estabelecido por Pulsed-field gel electrophoresis (PFGE) e também por tipagem por sequenciamento de multilocus no caso de K. pneumoniae. A presença de genes que codificam carbapenemases e -lactamases de espectro estendido foi pesquisada. A caracterização de blaKPC-2, do ambiente genético e de plasmídeos incluiu PCR e sequenciamento, análises de RFLP, S1-PFGE e hibridação. Os isolados Kp corresponderam a 5 pulsotipos, por PFGE, ligados a 6 tipos de sequência (ST): KPA-ST258 (n = 51 com 6 subtipos), KpA6-ST11 (n = 1), KPB-ST327 (n = 1), KPC-ST44 (n = 2), KPD-ST437 (n = 1) e KPE-ST48 (n = 1). Ecl foram agrupados em clones e e, Sm e Cf representam um clone cada. Todos os isolados foram resistentes aos -lactâmicos, sensíveis à colistina e tigeciclina e mostraram fenótipos variáveis contra aminoglicosídeos, quinolonas, nitrofurantoína e sulfametoxazol-trimetoprim. Heterorresistência a carbapenêmicos foi observada para isolados de Kp e Cf, conforme relatado anteriormente com produtores de KPC-2 e VIM. Esse estudo relata a disseminação do gene blaKPC-2 nos estados de São Paulo e Rio de Janeiro facilitada por clones de K. pneumoniae pertencentes ao globalmente disseminado Complexo Clonal CC258 (ST258, ST437 e ST11) e uma diversidade de plasmídeos (IncFII-KpA, IncN-Kp e Ecl, IncL/M-Sm e Cf e, dois plasmídeos não-tipáveis carreando Tn4401a ou Tn4401b) disseminados com sucesso entre as enterobactérias. Constitui também a primeira descrição do ST258 no Brasil associada a um surto em um hospital universitário da cidade de Ribeirao Preto. Este trabalho apontou a alta diversidade de elementos genéticos disponíveis abrigando blaKPC-2. Isso poderia ampliar enormemente a disseminação desse gene no Brasil como também no continente.<br>KPC (Klebsiella pneumoniae carbapenemases) are globally spread -lactamases of the Ambler class A comprising 10 variants, KPC-2 and KPC-3 being predominant. The objective of this work was study the genetic and molecular epidemiology of carbapenem resistant-enterobacterial isolates in Brazil. Sixty-four carbapenem resistant isolates were analyzed: 57 Klebsiella pneumoniae (Kp), 5 Enterobacter cloacae (Ecl), 1 Serratia marcescens (Sm) and 1 Citrobacter freundii (Cf) from different patients at six hospitals in two different Brazilian regions. Identification and antimicrobial susceptibility testing were accomplished by using semiautomatic systems and standard methods. Clonal relatedness was established by Pulsed-Field Gel Electrophoresis (PFGE) and also by multilocus sequence typing in the case K. pneumoniae isolates. The presence of genes encoding carbapenemases and extended spectrum -lactamases was searched. Characterization of blaKPC-2, genetic environment and plasmids included PCR and further sequencing, RFLP analyses, S1-PFGE and hybridization. The Kp isolates corresponded to 5 PFGE types linked to 6 sequence types (ST): KpA-ST258 (n=51 comprising 6 subtypes), KpA6-ST11 (n=1), KpB-ST327 (n=1), KpC-ST44 (n=2), KpD-ST437 (n=1) and KpE-ST48 (n=1). Ecl isolates were grouped in and clones and, Sm and Cf represent one clone each. All isolates were resistant to -lactams, susceptible to colistin and tigecycline and showed variable phenotype against aminoglycosides, quinolones, nitrofurantoin and trimethoprim-sulfamethoxazole. Heteroresistance to carbapenems was observed for Kp and Cf isolates, as previously reported to KPC-2 and VIM producers. This study reports the spread of blaKPC-2 in Sao Paulo and Rio de Janeiro states facilitated by globally spread CC258-K. pneumoniae clones (ST258, ST11, ST437) and a diversity of plasmids (IncFII-KpA, IncN-Kp and Ecl, IncL/M-Sm and Cf and, two untypeable plasmids carrying Tn4401a or Tn4401b) successfully disseminated among Enterobacteriaceae species. It also constitutes the first description of ST258 in Brazil which was associated with a hospital outbreak in Ribeirao Preto city. This work pointed out the high diversity of available genetic elements harboring blaKPC-2. This might greatly amplify the dissemination of KPC genetic in Brazil and within of the South America continent.

Plasmid ColIb-P9 is a 93.2 kb self-transmissible plasmid, belonging to the I1 incompatibility group. Whilst much data had been gained concerning the molecular biology of conjugation mediated by this plasmid, a lack of information exsisted concerning the genetic organisation of the transfer genes. A physical map of the plasmid was constructed by detailed restriction analysis of DNA fragments sub-cloned from ColIb-P9. These fragments were also used to locate the positions of the transfer gene sog and the origin of transfer. Transposons Tn5 and Tnl723 were used to construct insertion mutants at defined points in ColIb-P9 and the effect of these on the expression of various transfer-related functions was studied. Using this technique, the probable location of the genes encoding the thick and thin sex pili were identified and also the site of the plasmid-encoded nuclease gene. The exact location of the entry exclusion gene was also determined. Complementation studies using the sub-cloned fragments of ColIb-P9 and a set of cosmid-clones generated from ColIbdrd-1 indicated that a positive regulator of the expression of the transfer genes exsisted and that this was composed of two genetically distinct elements. Studies involving wild type ColIb-P9 (drd+) indicated that this positive regulatory system is subject to negative control in cells containing the drd+ plasmid. The information gained from these studies was combined into a model of the organisation of the transfer genes of ColIb-P9. This defines at least three separate Tra regions, covering some 50 kb of the plasmid, with the origin of transfer located at one end of the transfer region.

Plasmid DNA partitioning is a crucial process for the transfer of at least a single copy of plasmid to the daughter cells during bacterial cell division. Partitioning for various low-copy number plasmids involves a DNA-binding protein (ParB), a centromere-like DNA site ( parS) and a ParA-family protein. Interestingly, the RK2 plasmid encodes two ParA proteins of different lengths. The longer protein is IncC1 (364 a.a), while IncC2 lacks a N- terminal domain of 105 amino acids (IncC NTD). The secondary structure of IncC NTD by NMR spectroscopy and other biophysical methods has been determined as random coil. It appears to bind DNA weakly and non-specifically. The expression and purification of IncC1 and IncC2 proteins was optimized. The two proteins and IncC NTD were characterized using various biophysical methods including Circular Dichroism, Analytical Ultracentrifugation, Small Angle X-ray Scattering, Size Exclusion Chromatography-Multi Angle Light Scattering, and EMSAs. Bacterial two hybrid assays and chemical crosslinking showed the two IncC proteins form homo- and hetero-dimers and interact with KorB protein. IncC1 and IncC2 proteins bind to DNA, non-specifically. IncC1 binds DNA weakly in the absence of nucleotides but IncC2 protein was found to bind DNA only in the presence of nucleotides (ADP, ATP).

Thesis (PhD (Microbiology))--University of Stellenbosch 2010.<br>ENGLISH ABSTRACT: Two isogenic plasmids, pRAS3.1 (11,851-bp) and pRAS3.2 (11,823-bp), were identified as tetracycline resistance plasmids occurring within Aeromonas salmonicida subsp. salmonicida and atypical A. salmonicida subsp. salmonicida strains that were isolated from salmon aquaculture farms in Norway (L'Abee-Lund and Sorum, 2002). Although sequence analysis showed that, except for the repC gene, the replication and mobilization genes of the two pRAS3 plasmids are similar to that of the two IncQ-2 plasmids pTF-FC2 and pTC-F14, incompatibility testing during the course of this study revealed that the replicons of the two pRAS3 plasmids were compatible with the replicons of the IncQ-1α, ß] and y plasmids RSF1010, pIE1107 and pIE1130, as well as with the IncQ-2α and ß plasmids, pTF-FC2 and pTC-F14, respectively. Through sequence analysis it was suggested that the repC gene of the ancestral pRAS3 plasmid was probably acquired during a gene exchange event with a yet to be identified plasmid. The difference in the RepC of the pRAS3 plasmids compared to that of the other IncQ-like plasmids against which the pRAS3 plasmids were tested for incompatibility was thus suggested to be a likely reason for the compatibility of the two pRAS3 plasmid replicons with these IncQ-1 and IncQ-2 plasmids. Two previously unidentified genes, encoding two small 108 and 74-aa proteins distantly related to the PemIK (Bravo et al., 1987; Tsuchimoto et al., 1988) and MazEF (Masuda et al., 1993) TA systems, were found to be present between repB and repA genes of the two pRAS3 plasmids. Cloning of these two genes onto an unstable pOU82-test vector increased the stability of the vector from 35 to 98% after ~72 generations, thus suggesting that like the PasABC and PasAB systems of pTF-FC2 and pTC-F14, these two genes encode proteins which function as a toxin-antitoxin (TA) system. Although located in a similar position on the plasmids, the TA system of the two pRAS3 plasmids and the Pas systems of pTF-FC2 and pTC-F14 are unrelated, suggesting that these two types of TA systems were acquired independently from each other. Based on the sequence similarity and genetic organization of pRAS3 compared to the IncQ-2α and ß] plasmids pTF-FC2 and pTC-F14, respectively, but given that the pRAS3 plasmids were compatible with both pTF-FC2 and pTC-F14, as well as other IncQ-like plasmids, it was suggested that the two pRAS3 plasmids be classified into a new IncQ-2y subgroup. A comparison of the sequences of the two pRAS3 plasmids to each other by L'Abee-Lund and Sorum (2002) revealed that, apart from a number of point mutations within the tetAR tetracycline resistance genes of the two plasmids, the only other differences between them are that pRAS3.1 has 4 tandem copies of 22-bp iteron repeats within its origin of vegetative replication (oriV), and 5 tandem copies of CCCCCG 6-bp repeats near the origin of transfer (oriT), while pRAS3.2 has only three and four copies of each of the two repeated sequences, respectively. As the two pRAS3 plasmids are likely to have arisen from the same ancestor, this raised the question of how the copy numbers of these two different types of repeat sequences affected the ability of pRAS3.1 and pRAS3.2 plasmids to compete within a host cell as well as within a population of host cells, and therefore, why both of these isogenic plasmids have managed to persist in the environment. The plasmid copy numbers (PCN) of pRAS3.1 and pRAS3.2 were estimated to be 45 ± 13 and 30 ± 5 plasmids per chromosome, respectively. By creating a series of pRAS3.1 derivative plasmids with 3 to 7 copies of the 22-bp iterons and 4 or 5 copies of the 6-bp repeats, it was shown that an increase in the number of iterons brought about a decrease in PCN, probably due to an increased ability to bind RepC, while an increase in the number of 6-bp repeats from 4 to 5 brought about an increase in repB transcription, and the higher levels of RepB resulted in an increase in PCN. Thus the reason for pRAS3.1 having a ~1.5-fold higher PCN than pRAS3.2, even though it has 4 x 22-bp iterons compared to the 3 x 22-bp iterons of pRAS3.2, was that it had a higher level of repB transcription due to having 5 x 6-bp repeats in its mobB-mobA/repB promoter region compared to the 4 x 6-bp repeats of pRAS3.2. The differences in the number of iterons and 6-bp repeats, and hence PCN, did not have an effect on the stability of the two wild type (WT) plasmids or their derivatives even when the TA system was neutralized by having a copy of the TA genes present on a vector in trans and it was argued that the relatively high PCN of the two pRAS3 plasmids was sufficient to ensure plasmid stability through random distribution. As the two pRAS3 plasmids were mobilized at similar frequencies difference in PCN and mobB-mobA/repB transcription did not seem to affect their mobilization frequency. When pRAS3.1 and pRAS3.2 were competed intracellularly as coresident plasmids, pRAS3.1 was able to displace pRAS3.2 from 98% of the host cells within ~20 generations. The displacement of pRAS3.2 by pRAS3.1 was found to be as a result of pRAS3.1 having 4 x 22-bp iterons, which enabled pRAS3.1 to titrate of the communal pool of available RepC initiator proteins. Plasmids with 5 or 7 x 22-bp iterons, were however less effective at displacing a plasmid with 3 iterons, and it was speculated that plasmids with more than 4 x 22-bp iterons within their oriV were less successful at initiating replication than was a plasmid with 3 iterons within its oriV. A direct correlation was found between the PCN of a pRAS3 plasmid and the metabolic burden it imposed on its host. Thus pRAS3.1, as a result of its ~1.5-fold higher PCN than pRAS3.2 placed a small but significantly higher (~2.8%) metabolic load on its host compared to pRAS3.2. It was concluded that pRAS3.1 had a competitive advantage over pRAS3.2 when these plasmids were coresident within a single host (as would have been when the two plasmids first diverged from each other) as it was able to displace pRAS3.2. However, as a result of pRAS3.2 having a lower PCN, it placed a smaller metabolic burden on an isogenic host and this resulted in pRAS3.2 having an advantage over pRAS3.1 at the population level. Sequence remnants of pRAS3.2 from horizontal gene transfer suggested that pRAS3.2 was the original pRAS3 plasmid and thus that pRAS3.1 evolved from pRAS3.2. As the pRAS3.1 derivative plasmids that were constructed during the course of this study are likely to have been intermediates in the evolution of pRAS3.1 from pRAS3.2, I was able to speculate on the stepwise evolution of pRAS3.1 from pRAS3.2 based on the characteristics of these plasmids, and thus, how both macro- and microevolutionary events have contributed to the evolution of these two plasmids.<br>AFRIKAANSE OPSOMMING: Die twee isogeniese plasmiede, pRAS3.1 en pRAS3.2, was geidentifiseer as tetrasiklien weerstandbiedende plasmiede wat in Aeromonas salmonicida subsp. salmonicida en nie-tipiese A. salmonicida voorkom (L'Abee-Lund and Sorum, 2002). DNS volgorde analise deur L'Abee-Lund en Sorum (2002) het gewys dat die gene verantwoordelik vir replisering (uitsluitend die repC) en mobililisering naverwant is aan die van twee IncQ-2 plasmiede, pTF-FC2 en pTC-F14. Eksperimente tydens hierdie studie het egter gewys dat die repliserende sisteme van die twee pRAS3 plasmiede versoenbaar is met die repliserende sisteme van die IncQ-1α, ß and y plasmiede RSF1010, pIE1107 en pIE1130, sowel as die IncQ-2α en ß plasmiede, pTF-FC2 and pTC-F14, onderskeidelik. Analise van die aminosuur volgorde van die pRAS3 RepC proteien het gedui daarop dat die proteien taamlik verskil van die RepC proteiene van die naverwante plasmiede pTF-FC2 en pTC-F14, sowel as die van die IncQ-1 tipe plasmiede, en daar was voorgestel dat die voorsaat pRAS3 plasmied moontlik die repC geen bekom het vanaf 'n ander, nog onbekende, plasmied deur middel van horisontale geen uitruiling. Die verskil in die RepC van die pRAS3 plasmiede teenoor die van die ander IncQ plasmiede waarteen hulle getoets was vir onversoenbaarheid, was waarskynlik die rede waarom die pRAS3 plasmiede versoenbaar was met die IncQ-1 en IncQ-2 plasmiede. DNS volgorde analise tydens hierdie studie het die teenwoordigheid van twee, vantevore ongeidentifiseerde, klein 108 en 74 aminosuur proteiene onthul wat ver langs verwant is aan die PemIK (Bravo et al., 1987; Tsuchimoto et al., 1988) en MazEF (Masuda et al., 1993) toksien-antitoksien sisteme. Die gene wat kodeer vir hierdie toksien-antitoksien proteine kom tussen die repB en die repA gene van die twee pRAS3 plasmiede voor. Klonering van die toksien-antitoksien gene van die pRAS3 plasmiede op 'n ander onstabiele plasmied het die stabiliteit van hierdie plasmied verhoog van 35 tot en met 98% na ~72 generasies. Hierdie experiment het dus bevestig dat, soos die PasABC en PasAB sisteme van pTF-FC2 en pTC-F14 onderskeidelik, die twee gene 'n toksien-antitoksien sisteem kodeer wat die stabiliteit van 'n plasmied binne 'n bakteriese populasie kan verbeter. Alhoewel die toksien-antitoksien gene van pRAS3 op 'n soortgelyke posisie op die pRAS3 plasmiede voorkom as wat die pasABC en pasAB gene op hulle onderskeidelike pTF-FC2 en pTC-F14 plasmiede voorkom, is hulle nie verwant nie en dus was dit voorgestel dat die twee tipe toksien-antitoksien sisteme onafhanklik van mekaar verkry is. Aangesien die DNS volgorde en genetiese rangskikking van pRAS3 teenoor die IncQ-2α en ß plasmiede pTF-FC2 en pTC-F14, onderskeidelik, soortgelyk is, asook die feit dat die pRAS3 plasmiede versoenbaar was met pTF-FC2 en pTC-F14, sowel as ander IncQ tipe plasmiede, word dit voorgestel dat die twee pRAS3 plasmiede in 'n nuwe IncQ-2y subgroep ingedeel word. 'n Vergelyking van die DNS volgorde van die twee pRAS3 plasmiede deur L'Abee-Lund and Sorum (2002) het gewys dat, behalwe vir 'n paar puntmutasies binne die tetAR tetrasiklien weerstandsgene, verskil die twee net in die opsig dat pRAS3.1 het 4 agtereenvolgende kopiee van 22-bp 'iteron' herhalings wat gelee is binne sy replikasie oorsprong en 5 kopiee van 'n CCCCCG 6-bp herhaling wat naby sy oorsprong van oordrag gelee is, terwyl pRAS3.2 net 3 en 4 kopiee het van elk van die onderskeie volgorde herhalings. Dus die bestaan van twee plasmiede met verskillende kopiegetalle van die twee verskillende tipe DNA volgorde herhalings, maar wat vermoedelik afkomstig is vanaf dieselfde stam plasmied, bring die volgende oorhoofse vrae aangaande die plasmiede na vore: hoe beinvloed die DNS volgorde herhalings die vermoe van die twee plasmiede om binne 'n enkele gasheersel te kompeteer vir die beskikbare plasmied repliserings masjinerie, en hoe beinvloed dit die plasmied-gasheersel verhouding en dus hulle vermoe om te kompeteer op die populasie vlak, en laastens, hoekom het beide weergawes van die plasmied bly voortbestaan in die omgewing? Die plasmied kopiegetalle van pRAS3.1 en pRAS3.2 was eksperimenteel beraam by ongeveer 45 ± 13 en 30 ± 5 plasmiede per chromosoom in E. coli, onderskeidelik. Deur 'n reeks van pRAS3.1 derivate te skep met 3 tot 7 'iteron' herhalings en 4 of 5 kopiee van die 6-bp herhalings was dit bewys dat 'n toename in die hoeveelheid 'iterons' 'n afname in die plasmied kopiegetal veroorsaak, vermoedelik deur 'n verbeterde vermoe om RepC te bind, terwyl 'n verhoging van 4 tot 5 kopiee van die 6-bp herhaling 'n afname in die kopiegetal te weeg gebring het. Die repB geen van 'n plasmied met 5 x 6-bp herhalings was ~2-voud hoer uitgedruk as die van 'n plasmied met 4 x 6-bp herhalings, en dit was verder bewys dat 'n verhoogde vlak van repB transkripsie vanaf 'n L-arabinose induseerbare promoter in trans van 'n pRAS3 plasmied met 4 x 6-bp herhalings het 'n ~2-voud verhoging in plasmied kopiegetal teweeg gebring. Die rede dat pRAS3.1 'n ~1.5-voud hoer plasmied kopiegetal gehad het as pRAS3.2, was as gevolg van 'n hoer vlak van repB uitdrukking weens die feit dat pRAS3.1 5 x 6-bp herhalings in die mobB-mobA/repB promoter area het terwyl pRAS3.2 net 4 van die 6-bp herhalings in dieslefde posisie het. Sou pRAS3.1 4 x 22-bp 'iterons' gehad het, maar saam met 4 x 6-bp herhalings soos pRAS3.2, dan sou die plasmied kopiegetal 23 ± 2 plasmiede per chromosoom gewees het. Die verskil in die hoeveelheid 'iterons' en 6-bp herhalings, en dus die plasmied kopiegetal, het nie 'n effek op die stabiliteit van die wilde tipe plasmiede of hulle derivate gehad nie, selfs al was die toksien-antitoksien sisteem geneutraliseer deurdat daar 'n kopie van die toksien-antitoksien sisteem op 'n ander plasmied in trans van die pRAS3 plasmiede en hul derivate geplaas was. Die relatiewe hoe plasmied kopiegetal van die pRAS3 plasmiede, wat moontlik hoog genoeg was om plasmied stabiliteit deur middel van toevallige uitdeling te verseker, was voorgestel as die rede vir die hoe mate van plasmied stabiliteit. Soortgelyke frekwensies van mobilisasie vir pRAS3.1 en pRAS3.2 (0.032 ± 0.014 en 0.021 ± 0.013 transkonjugate per donateur, onderskeidelik) was waargeneem. Dus het dit geblyk dat die verskil in uitdrukking van die mobB-mobA/repB operon, sowel as die plasmied kopiegetal van die twee pRAS3 plasmiede, nie die mobiliserings frekwensie beinvloed het nie. Intrasellulere kompetisie tussen pRAS3.1 en pRAS3.2 het gewys dat pRAS3.1 die vermoe gehad om binne ~20 generasies pRAS3.2 vanuit 98% van die gasheerselle te skop. Daar was gewys dat die teenwoordigheid van 4 x 22-bp 'iterons' in die oorsprong van replikasie van pRAS3.1 die rede was vir die vermoe van hierdie plasmied om pRAS3.2 uit te kompeteer binne die gasheersel, moontlik deurdat die 4 x 22-bp 'iterons' beter in staat was om die RepC protein te bind. Die vermoe van plasmiede met 5 of 7 x 22-bp 'iterons' om te kompeteer met 'n plasmied met net 3 x 22-bp 'iterons' was toenemend swakker in vergelyking met die van 'n plasmied met 4 x 22-bp 'iterons', en hierdie waarneming het gelei tot die voorstel dat plasmiede met meer as 4 x 22-bp 'iterons' nie so suksesvol was om replikasie te inisieer soos ¡¥n plasmied met 3 x 22-bp 'iterons' nie. 'n Direkte korrelasie was gevind tussen die plasmied kopiegetal van 'n pRAS3 plasmied en die metaboliese lading wat die plasmied op die gasheersel geplaas het. Dus het pRAS3.1, met 'n plasmied kopiegetal van ~1.5-voud hoer as die van pRAS3.2, 'n effens hoer (~2.8%) metababoliese lading op die gasheersel as pRAS3.2 geplaas. In gevolge van die inter- en intrasellulere kompetiesie eksperimente, was dit ge-argumenteer dat pRAS3.1 'n mededingende voordeel bo-oor pRAS3.2 binne 'n gasheersel (soos wat dit sou gewees het kort nadat die twee plasmiede van mekaar uiteengevloei het) gehad het omdat dit in staat was om pRAS3.2 vanuit die gasheersel te skop. Aan die ander kant het pRAS3.2 'n laer plasmied kopiegetal en dus 'n laer metaboliese lading op die isogeniese gasheersel geplaas het, en daardeur het pRAS3.2 weer op die populasievlak die kompeterende voordeel bo-oor pRAS3.1 gehad. Die eienskappe van pRAS3.2 was meer soortgelyk aan die van ander IncQ-tipe plasmiede as wat die eienskappe van pRAS3.1 was, en dus word dit voorgestel dat pRAS3.1 vanaf pRAS3.2 afkomstig was. Omdat die derivaat plasmiede wat geskep was vanaf pRAS3.1 tydens hierdie studie moontlike tussengangers in die ontwikkeling van pRAS3.1 vanaf pRAS3.2 was, kan gespekuleer word, gebaseer op die eienskappe van hierdie plasmiede, oor die “stapsgewyse manier” waarmee pRAS3.1 vanaf pRAS3.2 ge-evolueer het, en dus hoe beide makro- en mikro-evolusionere gebeurlikhede bygedra het tot die evolusie van genoemde plasmiede.

Thesis (MSc)--University of Stellenbosch, 2003.<br>ENGLISH ABSTRACT: The 14.2 kb plasmid pTC-FI4 was isolated from the moderately thermophilic (45°- 50°C), highly acidophilic (pH 1.5 to 2.5), chemolithotrophic bacterium Acidithiobacil/us caldus and has a replicon that is closely related to the promiscuous, broad host-range, IncQ-family of plasmids. The region containing the mobilization genes was sequenced and encoded five Mob proteins and an origin of transfer, which are related to the DNA processing (Tral) region of IncPI plasmids, rather than to the three Mob protein systems of the IncQ-l-group plasmids (e.g. plasmids RSFIOIO or R1162). Plasmid pTC-F14 is the third example of an IncQ family plasmid that has five mob genes, with the others being pTF-FC2 and pRAS3.1. The minimal region that was essential for mobilization included the mobA, mobB and the mobC genes as well as the oriT. The mobD and mobE genes were non-essential, but together enhanced the mobilization frequency by approximately 300-fold. The repB gene increased the mobilization frequency but was not essential for mobilization. Mobilization of pTC-F14 between Escherichia coli strains by a chromosomally integrated RP4 plasmid was more than 3500-fold less efficient than the mobilization ofpTF-FC2. When both plasmids were co-resident in the same E. coli host, pTC-FI4 was mobilized at almost the same frequency as pTF-FC2. This enhanced pTC-FI4 mobilization frequency was due to the presence of a combination of the pTF-FC2 mobD and mobE gene products, the functions of which are still unknown. pTF-FC2 could mobilize the oriT of pTC-FI4 whereas pTC-F14 could only mobilize the pTFFC2 oriT if provided with some of the mobilization genes from the pTC-FC2 mobilization region. Unexpectedly either the mobEDC genes or the mobAB genes would allow the mobilization of the pTF-FC2 oriT by pTC-F14 even though there was no common gene between the two subsets. No evidence for any negative effect on the transfer of one plasmid by the related, potentially competitive plasmid was obtained.<br>AFRIKAANSE OPSOMMING: Die 14.2 kb plasmied, pTC-F14, is uit die matig termofiliese (45°C tot 50°C), hoogs asidofiliese (pH 1.5 tot 2.5), chemolitooutotrofiese bakterium Acidithiobaci/lus caldus geisoleer en beskik oor 'n replikon wat verwant is aan die vanaf die IncQ-familie van plasmiede. Hierdie plasmiede is alom bekend vir hulle promiskuïteit tydens konjugasie asook hul vermoë om in 'n groot aantal verskillende gasheer organismes te kan repliseer. DNA volgorde analise van die mobiliserings area het 'n oordrags oorsprong asook vyf oop leesrame onthul wat nader verwant is aan die DNA prosseserings gene van die Tral area op die IncP 1 plasmiede, as die van die mobiliserings stelsel van die IncQ-l-groep plasmiede. Plasmied pTC-Fl4 is die derde voorbeeld, saam met pTF-FC2 en pRAS3.1, van 'n IncQ-tipe plasmied met 'n vyfgeen mobiliserings sisteem. Die kleinste area op die plasmied nodig vir mobilisering van pTC-Fl4 is bepaal, en het die mobA, mobB en mobC gene sowel as die oordrags oorsprong ingesluit. Saam, was die mobD en mobE gene verantwoordelik vir 'n 300- voud toename in die mobilisasie frekwensie van pTC-Fl4 alhowel die gene nie absoluut nodig was vir mobilisering van die plasmied nie. Die repB geen het ook bygedra tot die frekwensie waarteen die volledige plasmied gemobiliseer was, maar hierdie geen was ook nie nodig vir mobilisering van die pTC-F14 plasmied nie. Die frekwensie waarteen pTC-Fl4 tussen Escherichia coli rasse beweeg het tydens konjugasie, terwyl gebruik gemaak is van 'n chromosomaal geintegreerde RP4 plasmied, was ongeveer 3500-voud laer as die van pTF-FC2. Indien beide pTC-Fl4 en pTF-FC2 in dieselfde E. coli gasheer aangetref word, word beide plasmiede teen ongeveer dieselfde frekwensie gemobiliseer. Die verhoogde frekensie vir pTC-Fl4 was as gevolg van die teenwoordigheid van beide die mobD en mobE gene van die pTF-FC2 plasmied, waarvan die funksies nog onbekend is. Plasmied pTF-FC2 kon die oordrags oorsprong van pTC-Fl4 mobiliseer waarteenoor plasmied pTC-FI4 die oordrags oorsprong vanafpTF-FC2 slegs kon mobiliseer indien een van twee dele van die pTF-FC2 mobiliserings gene voorsien word (al was daar geen oorvleuling tussen die twee nie). Alhoewel die plasmiede moontlik kon kompeteer op die vlak van plasmied oordrag is geen negatiewe kompetesie waargeneem tussen dié twee verwante plasmiede nie.

This thesis describes the development of new types of fiber optic sensors for the measurement of mechanical quantities such as displacement, vibration and acceleration. Also, it describes the realization of specific acquisition systems designed to interrogate the developed sensors. Since optical fibers have been historically associated with high speed telecommunication links because of their very large bandwidth and low attenuation, there is a great interest for their employment in sensor applications. Fiber sensors represent a promising solution in many fields since fibers can be used for the measurement of several quantities, not only mechanical as those investigated in this work, but also chemical with the possibility to detect specific chemical or bio-chemical molecules. Among the physical quantities to be detected, the displacement measurement is required in some applications, especially in structural civil and mechanical fields, where it is possible to evaluate the cracks evolution, providing information about the safety of the structure under monitoring in order to detect eventually risky situations. All the developed sensors are able to measure the displacement along one or two axis, that can be employed also during vibration tests especially at high frequencies, and also acceleration sensors to monitor acceleration at low frequencies. The developed sensors are based on plastic optical fibers instead of the traditional glass fibers, which are traditionally employed in optical communications. This change is related to the aim of realizing sensors maintaining the excellent typical characteristics of the fibers, such as electromagnetic immunity, intrinsically fire safety and flexibility of applications, but with costs comparable to those of commercial electromechanical sensors. Indeed, nowadays, the commercial fiber optic sensors are based on glass fibers because they have very good performance. However, they find limited applications due to the high costs of their complex interrogation systems and also for the procedure required to splice the fibers. On the other hand, plastic optical fibers represent a promising alternative because of their geometrical and optical properties that allow employing low-cost non coherent sources such as LED and also simplifying the procedure for the sensor connection and installation. Therefore, the design of the proposed plastic optical fiber displacement sensors is described with the sensor practical arrangement and the realized prototypes. An acquisition system has been designed and realized to characterize the sensors and the characterization results are also provided. Moreover, the development and the characterization of a plastic optical fiber sensor able to measure the displacement in two directions have been described. The main drawback of the developed sensors are stability issues and for this reason laboratory and in situ-tests have been carried out in order to verify the sensor performance over the time. I The results obtained with the stability tests have highlighted the necessity to develop displacement sensors with increased stability. To this aim, a compensation technique based on two different wavelengths has been developed. The same working principle of the developed displacement sensor has been exploited to realize a fiber vibrometer to be employed during the vibration monitoring for measuring without contact the vibrations of the device under test. The sensor development, a suitable calibration procedure developed to overcome the problem of real targets with a non uniform reflectivity, and the experimental tests have been described. Furthermore, the preliminary results concerning the feasibility study of a plastic optical fiber accelerometer are reported.

Les éléments génétiques mobiles (EGM) sont des structures génétiques fréquemment associées à la dissémination de gènes de résistance aux antibiotiques (GRA). Dans ce travail, nous avons utilisé deux EGM comme « proxies », les intégrons de classe 1 et les plasmides IncP-1, afin de mieux comprendre (i) le devenir possible des GRA une fois relargués dans un écosystème fluvial (l’Orne, France), ainsi que (ii) l’effet des pressions anthropiques sur leur persistance. À partir d'analyses de l'eau des rivières, nous avons pu montrer que les deux EGM ne se comportaient pas de la même manière. L'entrée des intégrons de classe 1 dans le système fluvial semblait être diffuse plutôt que ponctuelle, tandis que l'abondance du plasmide IncP-1 est relativement stable le long de la section de la rivière étudiée (23 km), indiquant ainsi une origine plutôt indigène. Les intrants anthropiques tels que les stations d’épuration des eaux usées ne semblent pas affecter l’abondance des EGM en raison d’un niveau trop élevé de dilution des effluents. Par ailleurs, il est intéressant de noter que les bactéries porteuses d’EGM semblaient être enrichies sur les matières en suspension, susceptibles de servir de véhicule pour amener des communautés de bactéries plus riches en EGM vers les sédiments. L'analyse de deux carottes de sédiment indique clairement que seules les couches supérieures présentent un niveau élevé de bactéries porteuses d’EGM. Ces abondances diminuent dans les couches plus profondes où seules des zones ponctuelles présentent des microréservoirs avec des abondances d’EGM plus élevées. Pour une carotte sédimentaire au moins, nous avons pu montrer que l'abondance relative d’EGM corrèle négativement la présence de polluants tel que le plomb ou certains HAP<br>Mobile genetic elements (MGEs) are genetic structures frequently associated to the dissemination of antibiotic resistance genes (ARGs). In this work, we used two of them as proxies, class 1 integrons and IncP-1 plasmids, to better understand (i) the possible fate of ARGs once released in a river ecosystem (Orne, France), as well as (ii) the effect of anthropogenic pressures on their persistence. From river water analyses, we could show that the two MGEs do not behave the same way. The entry of class 1 integrons in the river system appeared to be diffuse rather than punctual, while the abundance of IncP-1 plasmid is relatively stable along the river section studied (23 km) thus indicating a rather indigenous origin. Anthropic inputs such as wastewater treatment plant did not seem to affect the abundance of MGEs because a too high level of effluent dilution. Interestingly, MGE-bearing bacteria appeared to be enriched on suspended material, which is likely to serve as a vehicle to drive MGE-richer communities of bacteria toward the sediments. The analysis of two sediment cores clearly indicates that only the top layers displayed an elevated level of MGE-bearing bacteria. These abundances decrease in deeper layers where only localized zones display micro-reservoirs of elevated MGE abundances. For one sediment core at least, we could show that the relative abundance of MGE negatively correlates with pollutants such as lead or certain PAHs

Le plasmide RP4 et ses dérivés du même groupe d'incompatibilité (IncP-1) sont transférables par conjugaison chez Myxococcus xanthus, où ils se maintiennent intégrés dans le chromosome. Le mode d'intégration de ces plasmides s'avère tout à fait différent de ce que l'on trouve chez d'autres bactéries à gram négatif. Ces plasmides intégrés peuvent subir des remaniements dans des proportions variables selon les cas et sont susceptibles d'être stabilisés. De nouveaux vecteurs sont préparés. Ces vecteurs ont une très grande efficacité de transfert chez plusieurs souches de Myxococcus xanthus. L'insertion dans un de ces vecteurs in vitro du gène appA codant pour la synthèse de la phosphatase hyperacide d'E. Coli, montre que ce gène est exprimé chez Myxococcus xanthus<br>IncP-1 plasmids (RP4 and its derivatives) are self-transmissible to Myxococcus xanthus, and maintained integrated into the host chromosome. The mode of integration is different from the one described for other gram negative bacteria. These plasmids are liable to structural instability, but it is possible to isolate stabilized insertions. New vectors are prepared. They have a high efficiency of transfer into many strains of Myxococcus Xanthus. The gene appA is cloned in vitro in one of these new vectors. Its codes for the synthesis og hyperacid phosphatise of E. Coli. This gene is expressed by Myxococcus Xanthus

Nowadays bandwidth requirements are increasing vertiginously. As new ways and concepts of how to share information emerge, new ways of how to access the web enter the market. Computers and mobile devices are only the beginning, the spectrum of web products and services such as IPTV, VoIP, on-line gaming, etc has been augmented by the possibility to share, store data, interact and work on the Cloud. The rush for bandwidth has led researchers from all over the world to enquire themselves on how to achieve higher data rates, and it is thanks to their efforts, that both long-haul and short-range communications systems have experienced a huge development during the last few years. However, as the demand for higher information throughput increases traditional short-range solutions reach their lim- its. As a result, optical solutions are now migrating from long-haul to short-range communication systems. As part of this trend, plastic optical fiber (POF) systems have arisen as promising candidates for applications where traditional glass optical fibers (GOF) are unsuitable. POF systems feature a series of characteristics that make them very suitable for the market requirements. More in detail, these systems are low cost, robust, easy to handle and to install, flexible and yet tolerant to bendings. Nonetheless, these features come at the expense of a considerable higher bandwidth limitation when compared to GOF systems. This thesis is aimed to the investigate the use of digital signal processing (DSP) algorithms to overcome the bandwidth limitation in short-range optical communications system based on POF. In particular, this dissertation presents the design and development of DSP algorithms on field programmable gate arrays (FPGAs) with the ultimate purpose of implementing a fully engineered 1Gbit/s Ethernet Media Converter capable of establishing data links over 50+ meters of PMMA-SI POF using an RC-LED as transmitter.

The thesis investigates the realization of all-optical sensors for the detection of chemicals and bio-chemicals by exploiting the properties of surface plasma waves (or plasmons) excited at a dielectric-metal interface, the so called phenomenon of surface plasmon resonance (SPR). SPR occurs for light with suitable wavelength, incidence and polarization and manifests itself as a strong attenuation of the light reflection coefficient at the metal-dielectric interface. Supposing to use a broadband light source for the interrogation, this turns out in a deep and narrow notch in the reflected spectrum, the position of which is strongly dependent on the refractive index of the substance (the analyte) in contact with the metal layer. These sensors are characterized by very high sensitivity and, after proper functionalization, are therefore used in chemical and biochemical analysis to detect ppb (part-per-billion) concentrations of specific substances, such as pollutants in water. A further reason of interest is the possibility to conduct label-free analyses, without the use of other chemicals to make the analyte detectable. The construction parameters (e.g. metal used, layer thickness, and incidence angle, just to name a few) play a key role in determining the performance of these sensors. Therefore, in the first part of the thesis a mathematical model is introduced to allow running parametric simulations to optimize the layout according to the different configurations and applications. Then, based on the indications obtained from the simulations, some prototypes both in bulk optic (i.e. using prisms-based setups) and in optical fiber configurations have been realized and fully characterized to validate the implemented models. Optical fiber based sensors are particularly interesting for their small size that makes them well suited in all those applications where it is required the use of a compact, lightweight and minimally invasive system. For this reason, the fiber optic SPR sensor solution is often taken as the reference sensing system throughout the thesis. In a second part of the thesis, in view of long-term monitoring applications, the most critical parameters affecting the sensor resolution have been identified and analyzed in detail. These are: i) the stability of the optical source used to interrogate the sensor; ii) the influence of misalignments and other mechanical instabilities; iii) the effect of temperature fluctuations. As for the optical source stability, the most common source types used in practical setups (halogen lamp, super-continuum laser and SLED) have been measured, and a method to compare their long-term fluctuations and subsequent influence on the sensor performance has been devised. Similar approach based on the analysis of repeated experimental results has been applied also to evaluate the impact of the main mechanical parameters, such as the light beam angle of incidence, both intentionally and unintentionally variable, respectively in the bulk optic setup or in the fiber-based setup. The latter, in particular, is very relevant in the development of fiber sensing probes that to be disposable need to be low cost, and thus they make use of cheap connectors with poor repeatability. Another key issue is the effect of temperature. Indeed, since the refractive index is also function of the temperature and a SPR sensor basically detects changes in the refractive index, if the whole measuring system is not kept at a constant temperature, its drift induces a shift in the position of the plasmon resonance; and this could be erroneously interpreted as a variation in the concentration of the analyte. The solution widely used to reduce this drawback is to stabilize the temperature (typically using Peltier modules) and insert the whole sensing part in robust chassis; this, however, makes the instrument bulky and suitable for lab use only, and definitely not for portable monitoring systems. To overcome these limitations, a new internally compensated sensor configuration has been studied and its behavior assessed through some experiments. In the final part of the thesis all the analyzed aspects and developed solutions are applied to a feasibility study of SPR fiber sensor probes for Continuous Glucose Monitoring application (CGM), a topic of high relevance considering that diabetes constitutes one of the most common diseases, affecting more than 350 million people worldwide, corresponding to more than 8% of the adult population.

The core of my research activity during the Ph.D. period has been the study and the development of Surface Plasmon Resonance (SPR) based sensors for the detection of molecules of biological and medical interest. In particular, between the different configurations allowing plasmon excitation, I have focused my research on the study of nanostructured gratings, which allow to achieve a higher sensitivity than the prism coupled sensors and to miniaturize the measurement system. First my activity focused on the development of an opto-electronic bench able to detect plasmonic signal and to transduce it into an electric one. The test bench must allow varying independently some parameters that are fundamental for plasmonic excitation, such as the incident angle of laser beam, the azimuthal angle between the scattering plane and the grating vector, and the incident light polarization. The light modulated by the grating is transduced into electrical current through a photodiodes array and then acquired by a parameters analyzer. I have realized a versatile bench in order to perform measurements of both reflectance, analyzing the light reflected from the grating, and transmittance. The use of a motorized rotation stage has automated the measurement and it is controlled by a custom National Instruments LabVIEW software: in this way only few initial steps must be manually performed. I have analyzed three types of gratings: - Gold sinusoidal grating, optimized for reflectance measurements in incident light polarization modulation, exploiting the sensitivity increase due to a non-zero azimuthal angle. This grating has been provided us by LaNN laboratory (Laboratory of research for Nanofabrication and Nanodevices) of National Council of Research (CNR) of Padova. The grating has been manufactured through lithography (by Laser Interference Lithography-LIL) of a photoresist deposited over a glass (or silicon wafer), nanostructure replica and thermal evaporation of the gold plasmonic layer. First I have analyzed the bare grating, and then I have measured bulk with different refractive indexes in order to estimate sensor sensitivity. Then I have measured if the sensor is able to detect biological molecules, first through tests of avidin detection, exploiting avidin-biotin binding, and then through tests of DNA detection, via complementary Peptide Nucleic Acid (PNA) immobilization. - Gold digital grating, that exploits light extraordinary transmission. This grating has been fabricated by LaNN laboratory of CNR of Padova through Electron Beam Lithography (EBL) technique, and it has been designed in order to realize a simple and compact detection system, since the only sensing parameter considered is incident light polarization. Grating ability to detect surface changes of refractive index has been evaluated by a functionalization process with dodecanethiol, that is a molecule composed of a chain of twelve carbon atoms that forms a layer of well- known thickness and refractive index. - Silver trapezoidal grating, developed thanks to the collaboration with the Spin-Off Next Step Engineering, that has involved me in the last months of my Ph.D.. In fact, I have participated in grating fabrication exploiting the industrial facilities of the Spin-Off, which allow producing high quantity of low-cost devices, suitable to be a disposable sensor. The manufacturing process consists of the development of a stamper obtained through interferential lithography, the replica molding of polymeric substrate and the metal layer deposition through sputtering. These gratings have been optimized for transmittance measurements and their response as a function of incident light and azimuthal angles has been analyzed. Measurements of bulk with different refractive indexes, in order to estimate sensor sensitivity, and then of grating functionalized with different lengths alkanethiols have been performed. All experimental data have been compared with simulations results. In fact the behavior of the gratings has been studied through different simulation methods. In particular the digital gold grating has been studied through Finite Element Method (FEM) implemented in COMSOL Multiphysics; the vector model has been applied for both sinusoidal gold gratings and trapezoidal silver ones. The latter grating has been also analyzed through Rigorous Coupled Wave Analysis (RCWA). As already mentioned, during the last period of my Ph.D., I have collaborated with Next Step Engineering to develop an innovative industrial process that allows creating both grating for plasmonic events detection and electronic/microfluidic hybrid devices within a single, well-established, production line. With this process I have manufactured all the custom devices I used for my experimental activity. Moreover, this industrial process is the object of an Italian patent that is now pending and I am one of the inventors. During my PhD I have also developed microfluidic devices through a particular technique of polymer etching, able to create clear-cut profiles without deforming the planar structure, and also through suitable changes of production process adopted by Next Step Engineering, previously mentioned. The former devices have been used with silver gratings for the measurements of bulk with different refractive indexes.<br>Il tema principale dell’attività di ricerca che ho svolto durante il mio periodo di Dottorato in Scienza e Tecnologia dell’Informazione è stato lo studio e lo sviluppo di sensori basati sull’effetto di risonanza plasmonica per la rilevazione di molecole di interesse medico e biologico. In particolare, tra le varie configurazioni che permettono l’eccitazione plasmonica, mi sono focalizzata sullo studio dei reticoli nanostrutturati, i quali permettono di raggiungere elevate sensibilità, se paragonati ai dispositivi accoppiati con prisma, e di miniaturizzare e integrare il sistema di misura come obiettivo nel lungo periodo. Inizialmente la mia attività si è concentrata sullo sviluppo di un banco opto-elettronico che permettesse di rilevare il segnale plasmonico e trasdurlo in un segnale elettrico. Il banco doveva essere in grado di variare indipendentemente alcuni parametri determinanti per l’eccitazione plasmonica, ossia l’angolo di incidenza del fascio laser, l’angolo azimutale tra il piano di scattering e il vettore del reticolo, e la polarizzazione della luce incidente. La luce modulata dal reticolo viene poi trasformata in corrente elettrica attraverso un array di fotodiodi, e quindi acquisita attraverso un analizzatore di parametri. Ho mirato a realizzare un banco molto versatile in modo da poter effettuare misure sia di riflettanza, andando ad analizzare la luce riflessa dal reticolo, sia di trasmittanza, analizzando la luce trasmessa dal campione. L’introduzione di uno stadio motorizzato ha permesso di rendere la misura più automatizzata e gestibile via software, attraverso un programma custom sviluppato in LabVIEW, e lasciando manuali solo pochi passaggi iniziali. Ho analizzato tre tipologie diverse di reticoli: - Reticolo d’oro con superficie sinusoidale, ottimizzato per effettuare misure in riflessione con modulazione della polarizzazione della luce incidente, sfruttando l’aumento di sensibilità derivante dall’angolo azimutale non nullo. Tale reticolo è stato fornito dal laboratorio LaNN (Laboratorio di ricerca per la Nanofabbricazione e i Nanodispositivi) del Consiglio Nazionale delle Ricerche (CNR) di Padova. Il reticolo è stato realizzato attraverso litografia interferenziale di uno strato di fotoresist deposto su un vetrino (o silicio), da cui è stato ricavato uno stampo che permette la replica della nano struttura; infine, attraverso un’evaporazione termica, è stato depositato uno strato d’oro. Inizialmente ho analizzato il reticolo in condizione “fresh”; successivamente ho effettuato misure di “bulk” con indici di rifrazione diversi, per poter stimare la sensibilità del sensore. Ho poi misurato la capacità del dispositivo nel rilevare molecole di interesse biologico, dapprima attraverso prove di rilevazione di avidina presente in una soluzione, sfruttando il legame avidina-biotina, poi con prove di rilevazione di singole catene di DNA, attraverso l’immobilizzazione sulla superficie della nanostruttra di acido peptidonucleico (PNA) complementare. - Reticolo d’oro digitale, ideato per sfruttare il fenomeno di trasmissione straordinaria della luce. Tale reticolo è stato realizzato dal laboratorio LaNN del CNR di Padova attraverso la tecnica di litografia a fascio di elettroni (Electron Beam Lithography-EBL) e nasce con l’obiettivo di creare un sistema di rilevazione estremamente semplice, poiché l’unico parametro di sensing, e quindi variabile, è la polarizzazione della luce incidente. La capacità del sistema di discriminare variazioni superficiali di indice di rifrazione è stata valutata funzionalizzando il reticolo con dodecanethiol, ossia una molecola composta da una catena di dodici atomi di carbonio in grado di formare uno strato di dimensioni e indice di rifrazione noti. - Reticolo trapezoidale in argento, nato dalla collaborazione con lo Spin-Off Next Step Engineering, che mi ha coinvolta nell’ultimo periodo di dottorato. Infatti, ho partecipato in prima persona alla realizzazione del sensore, sfruttando le facilities industriali a cui l’azienda ha accesso, permettendo di produrre dispositivi a basso costo e in elevate quantità, quindi adatti ad un utilizzo di tipo “usa e getta”. Il processo di fabbricazione prevede la realizzazione di uno stampo attraverso litografia interferenziale, una fase di replica a stampo su substrato polimerico e la deposizione di uno strato metallico per polverizzazione catodica. Tali sensori sono stati ottimizzati per la misura della luce trasmessa e si è analizzato il comportamento al variare dell’angolo di incidenza e dell’angolo azimutale. Si è quindi misurato il comportamento del sensore in presenza di bulk ad indici di rifrazione diversi per la stima della sensibilità, e successivamente si sono effettuate misure funzionalizzando il campione con alcantioli di diversa lunghezza. I risultati sperimentali sono stati confrontati con quelli ottenuti dalle simulazioni. Infatti si è studiato il comportamento di ogni reticolo attraverso metodi di simulazione diversi. In particolare il reticolo digitale in oro è stato studiato attraverso il metodo degli elementi finiti (FEM) implementato in COMSOL Multiphysics, il modello vettoriale è stato applicato sia per lo studio del reticolo sinusoidale in oro che del reticolo trapezoidale in argento. Quest’ultimo reticolo è stato analizzato anche attraverso il metodo RCWA (Rigorous Coupled Wave Analysis). Come già accennato, durante l’ultimo periodo di dottorato ho contribuito a sviluppare, in collaborazione con lo Spin-Off dell’università di Padova Next Step Engineering, un innovativo processo di produzione industriale che consente di creare non solo reticoli per la rilevazione di segnali plasmonici, ma anche dispositivi ibridi elettronici/microfluidici per applicazioni biologiche e mediche, all’interno di una singola linea produttiva automatizzata. Con questo processo ho prodotto i reticoli in argento, che ho utilizzato per la mia attività sperimentale. Il processo di produzione è oggetto di un brevetto italiano attualmente in fase di deposito, di cui sono uno degli inventori. Durante il dottorato ho approfondito anche lo sviluppo di dispositivi microfluidici sia attraverso tecniche di incisione polimerica, in grado di creare profili di taglio netti senza deformarne la struttura planare, sia apportando le appropriate modifiche al processo produttivo utilizzato da Next Step Engineering, precedentemente citato. I dispositivi realizzati sono stati utilizzati per le misure di bulk a diversi indici di rifrazione utilizzando i reticoli in argento.

Face plastic surgery (PS) plays a major role in today medicine. Both for reconstructive and cosmetic surgery, achieving harmony of facial features is an important, if not the major goal. Several systems have been proposed for presenting to patient and surgeon possible outcomes of the surgical procedure. In this work, we present a new 3D system able to automatically suggest, for selected facial features as nose, chin, etc., shapes that aesthetically match the patient’s face. The basic idea is suggesting shape changes aimed to approach similar but more harmonious faces. To this goal, our system compares the 3D scan of the patient with a database of scans of harmonious faces, excluding the feature to be corrected. Then, the corresponding features of the k most similar harmonious faces, as well as their average, are suitably pasted onto the patient’s face, producing k+1 aesthetically effective surgery simulations. The system has been fully implemented and tested. To demonstrate the system, a 3D database of harmonious faces has been collected and a number of PS treatments have been simulated. The ratings of the outcomes of the simulations, provided by panels of human judges, show that the system and the underlying idea are effective.

Plasma magnetic control is one of the core engineering issues to be tackled in a fusion device. Over the last years, model based approaches have been proposed to face this issue, proving their effectiveness and allowing to reduce the time span needed for control testing and validation. The first part of this work is intended to give an overview of the subject, from the historical milestones to the underlying physics; the most common techniques for tokamak plasmas electromagnetic modeling and control are also introduced and discussed. After this introduction, a general architecture for plasma magnetic control in tokamaks is proposed. Finally, the proposed solution is applied to the Experimental Advanced Superconducting Tokamak (EAST) tokamak, where a new plasma magnetic control architecture was developed and implemented during the 2016-2018 experimental campaigns, and to the Japan Torus-60 Super Advanced (JT-60SA) device, which is currently under construction in Japan.

La grande variabilità del sistema Al-Si è causa di diversi problemi all’interno di linee di produzione. La stabilità, la prevedibilità, e il controllo di processi e fenomeni è una condizione cardine all’interno delle produzioni di massa che ne prevedono l’impiego. La creazione di un quadro teorico complessivo entro cui inserire i comportamenti del sistema in esame è vitale per poter raggiungere l’obiettivo di avere elevate e consistenti rese produttive.

Part 1. Space vehicles re-entering earth's atmosphere produce a shock wave which in turns results in a bow of plasma around the vehicle body. This plasma signicantly affects all radio links between the vehicle and ground, since the electron plasma frequency reaches beyond several GHz. In this work, a model of the propagation in plasma issue is developed. The radiofrequency propagation from/to antennae installed aboard the vehicle to the ground stations (or Data Relay Satellites) can be predicted, and the position of this antennae improved before a mission launch. Part 2. The Surface Integral Equation is one of the most used methods in the simulation of electromagnetic problems. The method used a discretized description of the surface on which a number of basis functions are needed. In the case of multi-scale structures, the test-object has regions with high details that require a fine mesh, together with flat surfaces where the current can be properly described with a coarser mesh. The goal of this work is to develop an automatic tool that identies the regions to be refined in a initial coarse mesh (dened only by geometry) using electromagnetic characteristics of the problem. It avoid the use of more unknowns that the actually needed (computational cost) and permits use a geometric mesh as base for different problems, adapting to each electromagnetic incidence automatically.

L'objet de ce mémoire de thèse est d’étudier une méthode de simulation numérique d'un modèle dit “Full wave” de la propagation dans un tokamak d'une onde électromagnétique injectée par un ensemble d'antennes installées sur le bord de l'enceinte de confinement. Une méthode de simulation par éléments finis de Lagrange est déployée. Dans le premier chapitre est introduit le modèle physique considéré pour décrire la propagation d'une onde électrostatique d'une fréquence proche de la résonance hybride dans un plasma dit froid et confine par un champ magnétique à l'intérieur d'un tokamak. La propagation des ondes électromagnétiques est modélisée par les équations de Maxwell. Une approximation de la solution harmonique en temps est considérée. Dans le chapitre deux sont rappelées les formulations varationnelles mixtes et mixtes augmentées déjà étudiées précédemment. Ces formulations nous permettent de chercher des solutions dans (H¹(Ω))³ et donc une approximation en éléments finis conformes dans cet espace. Le chapitre trois est dédié à la présentation de la discrétisation des équations du modèle en trois dimensions d’espace. Dans le chapitre quatre il est démontré le caractère bien posé du système d'équations discret lorsqu'on considère une approximation de type Taylor-Hood ℙ₂-ℙ₁. Un résultat d’existence et unicité de la solution dans le cas d'un "tore" polyédrique est présenté. Le chapitre 5 est dédié aux simulations numériques. En premier on explicite les termes du tenseur diélectrique K ainsi que ses dérivées, qui sont nécessaires au montage de la matrice de raideur du système. Les premières simulations concernent le cas où la densité des électrons et des ions est constante. On présente ensuite des résultats dans le cas où les densités ont un profil parabolique. Le cas où le vecteur d'onde est une fonction de la distance au centre du tokamak est également considéré<br>The object of this thesis is to study a numerical simulation method of a so-called “Full-wave” model of the propagation in a tokamak of an electromagnetic wave injected by a set of antennas installed in the boundary of the containment chamber. A Lagrange finite element approximation method is deployed. In the first chapter, we introduced the physical model considered to describe the propagation of an electrostatic wave of a frequency close to hybrid resonance in a so-called cold plasma, plasma confined by a magnetic field inside a tokamak. The propagation of electromagnetic waves is modeled by Maxwell's equations. This work contemplates an approximation of the time-harmonic solution. In the second chapter, we recall the mixed and mixed variational formulations already studied in the past. These formulations allow us to find solutions in (H¹(Ω))³ and therefore a conformal finite element approximation in this space. Chapter three is dedicated to the presentation of the discretization of the model's equations in three dimensions of space. In chapter four it is demonstrated the well-posed character of the discrete system of equations when one considers a Taylor-Hood type approximation ℙ₂-ℙ₁. In this section, we proved a result about the existence and the uniqueness of the solution in the case of a polyhedral “torus”. Chapter 5 is dedicated to numerical simulations. First are introduced the terms of the dielectric tensor K and its derivatives, needed to mount the stiffness matrix of the system. The first simulations concern the case where the density of electrons and ions is constant. Then results are presented in the case where densities have a parabolic profile. It is also considered the case where the wave vector is a function of the distance at the center of the tokamak

In this thesis the study of different innovative antenna systems is presented. The antenna designs that have been analyzed and that are described in this thesis can be divided in three main groups based on their application: phased-array antennas working on surfaces that change shape in time, millimeter-wave antennas for skin cancer diagnosis, and gaseous plasma antennas for satellite communications. As far as the first topic is concerned, the research activity presented in this thesis focuses on the study of the strengths and limitations of a specific pattern recovery technique: the projection method. This technique has been adopted to retrieve the radiation properties of linear and planar arrays placed on surfaces whose shape changes in time according to different geometrical deformation; moreover, its effectiveness was assessed for both broadside and beam steering arrays, leading to a novel and simpler formulation of this pattern recovery technique for arrays whose beam is tilted towards different directions. The results have been obtained both through full-wave numerical simulations in CST Microwave Studio and through measurements performed in collaboration with the North Dakota State University (NDSU), Fargo, North Dakota, USA. Regarding skin cancer diagnosis, a novel substrate integrated waveguide probe for early-stage skin cancer detection has been designed as well: this probe is cheap and easy to fabricate and can achieve high accuracy in detecting small early-stage skin cancer, thus providing a tool with the potential of being adopted as a real aid for skin cancer diagnosis. The probe has been tested both through full-wave numerical simulations and through measurements on a skin phantom realized at The University of Queensland with the support of the Microwave Team. The study about gaseous plasma antennas for satellite communications has been mainly numerical. Different designs have been conceived with the aim of exploiting the advantages of using plasma elements while at the same time avoiding the limitations related to this novel technology. A practical implementation of these designs is now the subject of a collaboration with CISAS B. Colombo, an aerospace research center of the University of Padova.

Deformation phenomena in shape memory alloys involve stress-, temperature-induced phase transformations and crystallographic variant conversion or reorientation, equivalent to a twinning operation. In near equiatomic NiTi, Ti rich compositions can exist near room temperature as a monoclinic B19' martensitic phase, which when deformed undergoes twinning resulting in strains as large as 8%. Upon heating, the martensite transforms to a cubic B2 austenitic phase, thereby recovering the strain and exhibiting the shape memory effect. Ni rich compositions on the other hand can exist near room temperature in the austenitic phase and undergo a reversible martensitic transformation on application of stress. Associated with this reversible martensitic transformation are macroscopic strains, again as large as 8%, which are also recovered and resulting in superelasticity. This work primarily focuses on neutron diffraction measurements during loading at the Los Alamos Neutron Science Center at Los Alamos National Laboratory. Three phenomena were investigated: First, the phenomena of hysteresis reduction and increase in linearity with increasing plastic deformation in superelastic NiTi. There is usually a hysteresis associated with the forward and reverse transformations in superelastic NiTi which translates to a hysteresis in the stress-strain curve during loading and unloading. This hysteresis is reduced in cold-worked NiTi and the macroscopic stress-strain response is more linear. This work reports on measurements during loading and unloading in plastically deformed (up to 11%) and cycled NiTi. Second, the tension-compression stress-strain asymmetry in martensitic NiTi. This work reports on measurements during tensile and compressive loading of polycrystalline shape-memory martensitic NiTi with no starting texture. Third, a heterogeneous stress-induced phase transformation in superelastic NiTi. Measurements were performed on a NiTi disc specimen loaded laterally in compression and associated with a macroscopically heterogeneous stress state. For the case of superelastic NiTi, the experiments related the macroscopic stress-strain behavior (from an extensometer or an analytical approach) with the texture, phase volume fraction and strain evolution (from neutron diffraction spectra). For the case of shape memory NiTi, the macroscopic connection was made with the texture and strain evolution due to twinning and elastic deformation in martensitic NiTi. In all cases, this work provided for the first time insight into atomic-scale phenomena such as mismatch accommodation and martensite variant selection. The aforementioned technique of neutron diffraction for mechanical characterization was also extended to engineering components and focused mainly on the determination of residual strains. Two samples were investigated and presented in this work; first, a welded INCONEL 718 NASA space shuttle flow liner was studied at 135 K and second, Ti-6Al-4V turbine blade components were investigated for Siemens Westinghouse Power Corporation. Lastly, also reported in this dissertation is a refinement of the methodology established in the author's masters thesis at UCF that used synchrotron x-ray diffraction during loading to study superelastic NiTi. The Los Alamos Neutron Science Center is a national user facility funded by the United States Department of Energy, Office of Basic Energy Sciences, under Contract No. W-7405-ENG-36. The work reported here was made possible by grants to UCF from NASA (NAG3-2751), NSF CAREER (DMR-0239512), Siemens Westinghouse Power Corporation and the Space Research Initiative.<br>Ph.D.<br>Department of Mechanical, Materials and Aerospace Engineering;<br>Engineering and Computer Science<br>Materials Science and Engineering

Antibiotic resistance is increasing worldwide closely linked with antibiotic misuse and overuse. Antibiotic gene resistance is commonly disseminated through MGEs (mobile genetic elements), especially plasmids. IncP-1 plasmids are BHR presumably found in several bacterial families and have been associated with antibiotic resistance and tolerance to metals. The identification of plasmid groups has been frequently done using the PBRT (PCR-based replicon typing) approach which assigns plasmids to Inc groups, including the IncP-1 plasmid group. The efforts to further characterize these structures, especially when associated with antibiotic resistant bacteria, has been lacking, and consequently, information is scarce and dispersed. Therefore, a systematic analysis was carried out to understand the occurrence, distribution, and genetic traits of IncP-1 plasmids associated with antibiotic resistant bacteria. To do so, a bibliographic search strategy was followed, where the Scopus platform was used to look for studies that used the PBRT method developed by Carattoli et al. (2005) for plasmid identification and that actually detected IncP-1 plasmids structures. Article collection for a period of 5 years resulted in 96 eligible articles, which were used to retrieve relevant information about IncP-1 plasmids occurrence and features. The articles combined reported the identification of 629 IncP-1 replicons. Altogether, the bacterial hosts of IncP-1 plasmids were found in 32 countries and were collected from a variety of environmental sources. Bacterial hosts belonged to 28 species distributed in 10 genera, of Enterobacteriaceae and Pseudomonadaceae families and were resistant to 10 different antibiotic classes, harboring 32 different resistance genes. IncP-1 plasmid-positive bacteria usually harbored at least 2 different Inc groups. Of all the IncP-1 plasmids identified, 229 (~36%) were further described, their sizes ranging from 35 to 320 kb and have been associated with medically important resistance genes and additional genetic elements that could potentiate gene dissemination. Furthermore, we studied the molecular diversity of previously reported IncP-1 plasmids occurring in 9 Escherichia coli isolates from an UWTP in Portugal. This was accomplished via PCR amplification of the 281 bp trfA gene fragment sequences and transfer to new well-known bacterial hosts for further characterizations. Amplicon sequencing showed 100% identity in all trfA fragments suggesting genetic structure conservation association to similar bacteria and environments. Similarity searching of the trfA fragment sequence was used to select closely related fully sequenced IncP-1β1 plasmids for comparisons. As a result, 63 closely related replicons were selected for comparative analysis and found to be usually large genetic structures, isolated mainly from wastewater and soil, harboring genetic determinants associated with resistance to aminoglycosides, sulphonamides, and β-lactams, and with tolerance to Hg. Attempts to transfer the 9 IncP-1 plasmids to a recipient strain were unsuccessful probably due to the chosen selective markers. The high prevalence of mer operon genes in these IncP-1β1 plasmids, suggesting mercury tolerance, could be used in a future work as a selective marker to transfer these 9 IncP-1 plasmids another bacterial host allowing for proper genomic characterization of these promiscuous structures.<br>A resistência aos antibióticos está a aumentar em todo o mundo, intimamente associada ao uso incorreto e excessivo de antibióticos. Os genes de resistência a antibióticos são comummente disseminados por meio de EGMs (elementos genéticos móveis), especialmente plasmídeos. Os plasmídeos IncP-1 têm amplo espetro de hospedeiros, são presumivelmente encontrados em várias famílias de bactérias, e têm sido associados à resistência a antibióticos e tolerância a metais. A identificação de grupos de plasmídeos tem sido frequentemente realizada utilizando a abordagem PBRT (PCR-based replicon typing), que atribui plasmídeos a grupos de incompatibilidade, incluindo o grupo IncP-1. Os esforços para caracterizar melhor essas estruturas, principalmente quando associadas a bactérias resistentes a antibióticos, têm falhado e, consequentemente, as informações são escassas e dispersas. Portanto, uma análise sistemática foi realizada para compreender a ocorrência, distribuição e características genéticas de plasmídeos IncP-1 associados a bactérias resistentes a antibióticos. Para tal, a plataforma Scopus foi utilizada para realizar uma pesquisa bibliográfica, para selecionar estudos que identificaram plasmídeos IncP-1 através do método PBRT desenvolvido por Carattoli et al. (2005). A pesquisa abrangeu um período de 5 anos e resultou em 96 artigos elegíveis, os quais foram usados para a obtenção de informações relevantes sobre a ocorrência e características dos plasmídeos IncP-1. Os artigos combinados relataram a identificação de 629 replicões IncP-1. Ao todo, os hospedeiros bacterianos dos plasmídeos IncP-1 foram encontrados em 32 países e foram coletados de uma variedade de fontes ambientais. Os estudos identificaram hospedeiros bacterianos pertencentes a 28 espécies, distribuídas em 10 géneros das famílias Enterobacteriaceae e Pseudomonadaceae, foram identificados como resistentes a 10 classes de antibióticos, possuindo 32 genes de resistência diferentes, e pelo menos apresentando replicões de 2 grupos Inc diferentes. De todos os plasmídeos IncP-1 identificados, 229 (~ 36%) foram descritos posteriormente, e seus tamanhos variam de 35 a 320 kb, foram associados a genes de resistência clinicamente importantes e a elementos genéticos adicionais que poderiam potenciar a disseminação de genes. Além disso, foi estudada a diversidade molecular de plasmídeos IncP-1 identificados anteriormente em 9 isolados de Escherichia coli de uma UWTP em Portugal. Isso foi realizado por meio da amplificação por PCR de um fragmento do gene trfA de 281 bp, e tentativa de transferência para novos hospedeiros bacterianos bem conhecidos para posterior caracterização. A sequenciação dos amplicões mostrou 100% identidade entre fragmentos trfA sugerindo conservação das suas estruturas genéticas e associação com bactérias e ambientes semelhantes. A pesquisa por similaridade da sequência do fragmento trfA foi usada para selecionar e comparar plasmídeos IncP-1β1 totalmente sequenciados. Isso resultou na análise de 63 replicões, geralmente grandes estruturas genéticas, isolados de águas residuais e solos, possuindo determinantes genéticos associados à resistência a aminoglicosídeos, sulfonamidas e βlactâmicos, e à tolerância ao Hg. As tentativas de transferir os 9 plasmídeos IncP-1 para uma estirpe de laboratório não tiveram sucesso, provavelmente devido aos marcadores seletivos escolhidos. A ampla prevalência de genes do operão mer nestes plasmídeos IncP-1β1, sugerindo tolerância ao mercúrio, poderia ser usada em um trabalho futuro como um marcador seletivo para a transferência dos 9 plasmídeos IncP-1 de água residual para outro hospedeiro, permitindo a caracterização genética dessas estruturas.<br>Mestrado em Microbiologia

碩士<br>國立臺灣大學<br>電機工程學研究所<br>90<br>Plasma Display Panel (PDP) is a potential product. It has advantages over other display panels by wide viewing angle, large size, and thinness. In this thesis, the digital logic system of 50-inch PDP is analyzed in detail. There are two main functions in the system. One is to display image data processed by the system on the panel. The other is to control high-voltage switches with waveform compiler system. Because of the high resolution, PDP has to process large image data. The objective of the system is to reduce the processed time of image data as much as possible. It can enlarge time percentage of displaying image to enhance brightness of PDP. In this thesis, input and output data format of the digital logic system and specifications of components are introduced first. Then designs of the digital logic system and the waveform compiler system are illustrated. At last, the simulation results are shown to verify the design.

碩士<br>逢甲大學<br>電子工程所<br>94<br>This is investigates the optimization conditions for using the microwave plasma with operation frequency of 2.45 GHz to clean the DRAM leadframe before performing the wire-bonding process. First, using the Auger Electron Spectroscopy (AES) and the X-Ray Photoelectron Spectroscopy (XPS) to analyze the contamination conditions on the bond pads and to obtain the clean criteria of determining the nature and charge-in quality and to provide references for the plasma gas selection. According to the assembly factory experiences, the wire-bonding strength and the resin viscosity on the leadframe can be improved when the contact angles are around 15~35 degrees by applying the plasma cleaning technique to the wafer surface. This experiment is established upon a requirement of 25 degrees to obtain the parameter optimization after plasma cleaning. Then, using both the wire-pull and the ball-shear experimental results to verify the bonding quality after applying the plasma cleaning. Since the pollution distribution on the bond pad is random and may cause difference to the bonding strength, an assisted statistic method has been used the to confirm the improvement results. Finally, the reliability testing has been performed on the products to verify if they may match the requirements.

碩士<br>國立成功大學<br>高階管理碩士在職專班(EMBA)<br>102<br>The research is meant to understand the environment of global semiconductor equipment suppliers and simultaneously discuss the competitive situation among Taiwanese suppliers in this very environment. Through S-C-P (Structure-Conduct-Performance) analysis model, competitor analysis, and interfirm rivalry, our research subjects are the semiconductor equipment suppliers which are two companies in Taiwan, HMI (Hermes Microvision Inc.) and GPT (Grand Plastic Technology Corp.). We conclude that this given research method can help to realize the adversity the industry encounters and to come up with systematic solutions, providing the suppliers a chance to take advantage of Taiwan's leading technique in wafer manufacturing. Furthermore, the contributions of our study for the semiconductor equipment suppliers are to facilitate the acquisition of technique, integration of the supply chain, promoting the independence of companies, and achieving the goal of localization of the semiconductor industry. Given that semiconductor equipment market provides a harsh competitive environment, only those who remain the advanced position in technology can set foot in the field. In HMI’s case, the company has more international strategies and advantages in technology than GPT. Moreover, HMI is active in expanding their external supply chain and optimizing the internal organization. Namely, HMI isolates the core department and transforms it to a division as the preparation for future industry.

A recently proposed route to magnetized target fusion (MTF) has been developed [4] which utilizes an array of high velocity pulsed plasma accelerators, fired in unison at a target plasma. The plasma accelerators are required to be capable of reproducible results of 0.2mg of hydrogen plasma at velocities in excess of 200km/s and be possible of operating at 10Hz. No previously developed pulse plasma accelerator is capable of these results. The purpose of this thesis was to develop a plasma accelerator for application to the proposed fusion scheme. The previously unexplored possibility of using a piezoelectric valve in these devices was investigated. The piezoelectric valve that was developed is capable of generating reproducible, short, well defined hydrogen pulses with longitudinal temperatures below 2K. Presently the valve can deliver a maximum output of 0.022mg of hydrogen gas. A unique coaxial plasma accelerator, the Lica was developed that has three main features that set it apart: 1) it uses a piezoelectric valve to deliver short well defined hydrogen pulses into the accelerator, 2) the gas is linearly injected into the device and 3) it uses a novel preionization method. Currently the Lica is unable to offer the performance required for the proposed fusion scheme, the bulk of the plasma generated in this device appears to be travelling the region of 40-50km/s at a temperature of 3000K to 5000K. There are a few anomalies in the operation of this device: 1) it appears to accelerate a series of plasma sheaths in the regions of 40km/s, 50km/s and 60-80km/s, 2) the final plasma velocity appears to be independent of the acceleration distance and in some instances high velocity plasma in the region of 200km/s was observed. A numerical finite element model (FEM) electromagnetic model called MATAC was developed to try and simulate the operation of the Lica, because it was shown that simple analytical models are inadequate. The preliminary modelling efforts predicted the final velocity of the bulk of the plasma to be 82.6km/s. A spin off from the development of the numerical model was the extension of the upwinding scheme of [157] to quadratic and cubic FEM elements.

Marine debris is a major persistent pollutant of oceans worldwide, with far reaching effects on the inhabitants and users of the marine environment. Marine plastic pollution is recognised as a key threatening process in Australia because of the harm it can cause wildlife through ingestion and entanglement (Chapter 1). More than 56 % of seabird species globally have been impacted by marine debris, but little is known about marine debris interactions in East Australian seabirds. The southern Great Barrier Reef (GBR) supports a high percentage of breeding and nesting seabirds, however no research existed on the interactions of marine debris with wildlife in this ecologically important area. This project was the first to examine the prevalence of marine debris on southern GBR beaches, and the first to examine marine debris interactions with two seabird species, the wedge‐tailed shearwater (Ardenna pacifica) and the brown booby (Sula leucogaster). A risk matrix for marine debris interactions was applied to help inform decision makers about this pollution threat. The utility of the created risk matrix is that it can be expanded to other organisms or regions within Australia. The prevalence of marine debris within offshore sites in the southern GBR (Capricorn‐Bunker Group of islands) was examined, and compared to levels of marine debris on near‐shore beaches (Sunshine Coast, Queensland, and Coffs Coast, New South Wales) (Chapter 2). Near‐shore sites had double the amount of debris items (0.08 m‐2) than offshore (0.04 m‐2) sites, with significantly higher rates of accumulation near‐shore than offshore (U = 0.000, p = 0.050). Levels of debris increased between the first and second sampling periods at near‐shore beaches only, indicating the possible influence of environmental conditions on the deposition and exhumation of debris items. More sampling is needed to confirm this trend. A Marine Debris Pollution Index was developed to aid in monitoring debris pollution at surveyed sites based on mean amounts and size of collected debris items. Off/white‐clear coloured debris items dominated survey collections (39 % nearshore; 31% offshore). Hard plastic items were the most prevalent debris type at both near‐shore and offshore sites (56 % and 42 %, respectively). Some differences in debris type existed between near and offshore beaches, for instance more fibrous plastic and sheet plastic were recovered at near‐shore sites, and these differences were attributed to source influences. For example, near‐shore sites were more heavily influenced by land‐based sources of pollution (F[2,60] = 546.811, p = 0.021). Plastic ingestion was studied in wedge‐tailed shearwaters (Chapter 3) at offshore GBR sites and near‐shore locations in southern QLD and northern NSW that were also surveyed for beach marine debris (Chapter 2). Overall on average ~13 % of late‐stage wedge‐tailed shearwater chicks had been fed marine debris plastic over two survey seasons with these birds most often ingesting off/white‐clear coloured plastics (~40 %). Interestingly, significant differences existed between debris colour and material type on surveyed beaches compared to that fed to chicks nesting offshore suggesting a selective feeding pattern on these plastics. This trend was not apparent in late‐stage chicks nesting at near‐shore sites. Ingestion appeared to be more frequent in near‐shore birds (~17 %), although this was not a significant pattern (F[1, 11] = 4.792, p = 0.065). While, the number of ingested plastic pieces per bird was significantly higher offshore (U = 40.000, p = 0.032). Marine debris ingestion did not appear to have a negative interaction on the health of surveyed late‐stage chicks as indicated by their body condition (U = 1091.00, p = 0.204). Marine debris from oceanic sources was common in the nests of brown boobies with over one‐third containing this anthropogenic material (Chapter 4). Using a novel photographic technique developed for surveying the nest material of this species, differences in levels of nest marine debris items were determined. The outcomes of this technique detected that the Swain Reefs nests were considerably more contaminated (58 %) than Capricorn‐Bunker nests (11 %). Hard plastic debris dominated Swain Reefs anthropogenic nest material (82 %) and beach debris (77 %) at all surveyed locations. There was a higher prevalence in nests for blue‐purple coloured plastic items in Swains and Fairfax nests (28 % and 29 %, respectively). Specific debris items were chosen by brown boobies for use within nests, limiting the use of nests as a substitute for beach surveys as an indicator of environmental debris levels. The use of marine debris within the nest material of brown boobies did not appear to negatively affect the brown boobies during the sampled periods, with no birds found entangled. A new risk matrix developed by modifying the IMO matrix (IMO, 2014) was applied to determine the level of risk marine debris posed to a region (Chapter 5). The marine debris beach survey data and seabird interaction data gathered from this research was applied to the risk matrix as a proof of concept demonstration. A traffic light colouring was used with the matrix based on marine debris interactions and amounts in in the environment. The results of the applied risk matrix from this study were ‘green’ and ‘yellow’ for seabirds in the southern GBR and nesting locations on the East Australian coastline. The yellow designation given to birds at Heron Island and those nesting in the Swain Reefs is at a tolerable risk level, but calls for actions to be taken to reduce marine debris levels when possible. The green designation given to birds at Northwest Island and Muttonbird Island, represent a more broadly acceptable risk level that requires monitoring and should be reduced further when practical. Further data is needed (i.e. more birds sampled) to provide a more accurate understanding of the risk to seabirds in the surveyed areas however. Recommendations from this research include the need for continued monitoring of marine debris levels (and interactions) on Australian beaches to address this issue; to ascertain the effectiveness of any preventative measures; and to gauge the continued performance of current legislation aimed at mitigating or ameliorating the marine debris problem. Management strategies targeted at the marine debris issue in south‐east Australia could be strengthened through enforcement of current legislation. This is especially important as increased ship traffic is anticipated throughout the region and shipping debris has been shown to be one of the common sources of marine debris in the surveyed areas. This thesis aimed to provide a baseline on the current presence and interactions of marine debris with two seabird species common to this ecologically important area. A number of new and novel tools were developed to undertake this monitoring and can be used by others to help best inform upon this pollution threat. The data collected and presented herein will provide new insights that will aid both the management of seabirds in this region and to improve the management of marine debris.

This study investigated the suitability of using waste plastics as an additive in biodiesel through assessing the performance, emissions and combustion characterises of a diesel engine. Firstly, the waste plastics were dissolved into different biodiesels. Then, the dissolved solution was mixed with standard diesel to make diesel-biodiesel-plastic blends. These plastic blends were then tested in a 4-cylinder, 4-stroke, diesel engine. These analyses indicated that waste plastics can potentially be used as fuel additives along with the diesel-biodiesel blends in diesel engines. There exists little research in this field, so the comprehensive study reported in this thesis is important and significant. The novelty of this work lies in identifying the biodegradable solvents that can properly dissolve waste plastics and demonstrate the beneficial effects of plastics in reducing harmful gas emissions and improving engine performance.

In recent years, microwave, millimeter-wave, and THz applications such as medical and security imaging, wireless power transfer, and near-field focusing, just to mention but a few, have gained much attention in the area of ICT due to their potentially high social impact. On one hand, the need of highly-directive THz sensors with tunable radiating features in the far-field region has recently boosted the research activity in the design of flexible, low-cost and low-profile devices. On the other hand, it is of paramount importance to focus energy in the near-field region, and thus the generation of limited-diffraction waves in the microwave and millimeter-wave regime is a topic of recent increasing interest. In this context, leaky-wave theory is an elegant and extremely useful formalism which allows for describing in a common fashion guiding and radiating phenomena in both the near field and the far field, spanning frequencies from microwaves to optics passing through THz. In this PhD thesis we aim to exploit the intrinsic versatility of the leakywave approach to design advanced radiating systems for controlling the far-field radiating features at THz frequencies and for focusing electromagnetic radiation in the near field at millimeter waves. Specifically, the use of relatively new materials such as graphene and liquid crystals has been considered for the design of leaky-wave based radiators, achieving very promising results in terms of reconfigurability, efficiency, and radiating capabilities. In this context, an original theoretical analysis has provided new general formulas for the evaluation of the radiating features (e.g., half-power beamwidth, sidelobe level, etc.) of leaky-wave antennas. Indeed, the current formulations are based on several simplifying hypotheses which do not allow for an accurate evaluation of the beamwidth in different situations. In addition to the intriguing reconfigurable capabilities offered by leaky waves in far-field applications, interesting focusing capabilities can be obtained in the near field. In particular, it is shown that leaky waves can profitably be used to generate limited-diffraction Bessel beams by means of narrow-band radiators in the microwave range. Also, the use of higher-order leaky-wave modes allows for achieving almost the same performance in the millimeter-wave range, where previous designs were subjected to severe fabrication issues. Even more interestingly, the limited-diffractive character of Bessel beams can also be used to generate limited-diffraction pulses as superpositions of monochromatic Bessel beams over a considerable fractional bandwidth. In this context, a novel theoretical framework has been developed to understand the practical limitations to efficiently generate limited-diffraction, limited-dispersion pulses, such as X-waves, in the microwave/millimeter-wave range. As a result of this investigation, a class of wideband radiators has been thoroughly analyzed, showing promising capabilities for the generation of both zeroth-order and higher-order Xwaves. The latter may pave the way for the first localized transmission of orbital angular momentum in the microwave range.