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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
121

A Phage Display System to Profile the DNA-binding Specificities of C2H2 Zinc Fingers

Lam, Kathy 07 January 2011 (has links)
Knowing the sequence specificities of transcription factors allows us to surmise their functions and establish their regulatory roles in genomes. The most common DNA-binding domain among eukaryotic transcription factors is the Cys2His2 zinc finger domain; however, despite their prevalence, the specificities of the majority of Cys2His2 zinc finger proteins remain unknown due to the difficulty in assaying them. My objective was to develop a new phage displayed-based assay, in which individual Cys2His2 domains are displayed on phage in an otherwise constant three-finger protein scaffold. In Chapter 2, I discuss evidence for the modularity of the Cys2His2 domain, since my assay requires that zinc fingers be modular. In Chapter 3, I describe my results on the development of this phage display-based assay. This work provides support for a new strategy to determine the specificities of individual zinc fingers, which can be used to infer specificities for multi-finger Cys2His2 proteins.
122

Molecular typing and evolution of Salmonella enterica serovar Typhimurium

Hu, Honghua January 2005 (has links)
Salmonella enterica serovar Typhimurium is a common cause of salmonellosis among humans and animals worldwide. In Australia, Typhimurium is responsible for over half of the salmonellosis cases. The Anderson phage-typing scheme is the primary means of long-term surveillance of Typhimurium outbreak isolates, and has played an important role in epidemiology. However, there exist quite a number of strains of Typhimurium that cannot be defined by the phage-typing scheme. Furthermore, the knowledge of evolutionary relationships among isolates of different phage types is still very limited and the genetic basis of phage type variation remains largely unknown. To address these issues, this study focused on molecular typing and evolution of Typhimurium. Fluorescent amplified-fragment length polymorphism (AFLP) was applied to 46 Typhimurium isolates comprising nine phage types in Australia using the restriction enzymes MseI and EcoRI and MseI +1 / EcoRI +1 primer pair combinations. The selected phage types, DT9, DT135, DT64, DT44, DT126, DT12a, DT1, DT141 and DT108, have been dominant or frequent phage types in animal and human infections in Australia in recent years. AFLP in the present study showed a very good discrimination power with Simpson index of diversity of 0.98, 35 different AFLP patterns were observed in the 46 isolates studied. The tree based on AFLP patterns showed good correlation with phage type, grouped most Typhimurium isolates by phage type, and differentiated all nine phage types. Furthermore, 84 phage-type specific polymorphic AFLP fragments, for which presence or absence correlated with phage type (including 25 with one exception to phage-type specificity) were observed in the 46 strains studied. Eighteen phage-type specific AFLP fragments were cloned and sequenced. Sixteen are of known genes or have a homologue in the databases. It was found a predominance of phage and plasmid genes rather than mutational changes in the AFLP fragments studied. Of the 18 cloned and sequenced AFLP fragments, only four relate to mutational changes in the S. enterica chromosome, the other 14 comprise DNA of mobile elements: nine are phage related, three are plasmid related and two are gain of DNA from unknown origin. Twelve of the 18 sequenced phage-type specific AFLP markers are polymorphic because the DNA is present or absent as indicated by Southern hybridization. Two of these markers were successfully used in preliminary PCR-based typing of 30 DT9 and 29 DT135 isolates from worldwide collections. 27 of the 30 DT9 isolates and all DT135 isolates tested were correctly categorized. The results implied a good potential to use the sequence of these fragments as the basis for a multiplex PCR or a microarray based molecular �phage� typing method for Typhimurium. This thesis also studied the molecular evolutionary relationships among the same set of 46 Typhimurium isolates using mutational changes detected by AFLP, or analysis of intergenic regions and their flanking genes in genome sequences. The complete genome sequence of Typhimurium LT2 was analysed by computer modelled AFLP. The polymorphic AFLP fragments, which matched with the modelled LT2 AFLP fragments, were amplified and sequenced by LT2 genome based primers to determine the changes. Forty-nine intergenic regions with higher pairwise differences between LT2 and Typhi CT18 were amplified and sequenced using LT2 genome based primers for one isolate of each phage type. 51 polymorphic sites were detected consisting of 18 in AFLP fragments and 33 in intergenic regions or their flanking genes. PCR-RFLP (restriction fragment length polymorphism) and SNaPshot were used to further investigate the distribution of the single nucleotide polymorphisms (SNPs) detected in intergenic regions in all isolates studied. Of the 18 mutational changes detected in AFLP fragments, eight were indels (insertions / deletions) and ten single base substitutions. Of the eight indels, four were in genes, three in intergenic regions, and one covered adjacent intergenic and coding regions. The four indels in genes all caused frameshift mutations, including three single base indels and one 19 bp deletion. Of the ten substitutions, one was in an intergenic region and nine in genes comprising three synonymous and six non-synonymous substitutions. Of the 33 polymorphic sites detected from sequences of 23 intergenic regions and their flanking genes, one was IS200 insertion and 32 single nucleotide polymorphisms (SNPs), of which 30 were single base substitutions and two were single base indels. Nine of the 33 variations were found in the flanking genes, which were all single base substitutions comprising four synonymous, four non-synonymous substitutions and one non-sense mutation. More non-synonymous than synonymous substitutions were found for those in coding regions within Typhimurium, indicating that slightly deleterious intraspecies mutations can be fixed within clones, such as various lineages of Typhimurium. The 51 polymorphic sites, which were inferred from sequences of both mutation related AFLP fragments, and intergenic regions and their flanking genes, gave a single phylogenetic tree of the 46 Typhimurium isolates studied. All sequences involved were compared with the homologous sequences in the available S. enterica genome sequences for serovars Typhi, Paratyphi A, Gallinarum, Enteritidis and Pullorum and this enabled the determination of the direction of the mutational changes in the isolates studied and the root of the phylogenetic tree. There were only two events inferred to have occurred twice, the remaining 49 polymorphisms can be explained by a single event. The data indicated that Typhimurium has a very strong clonal structure with a very low level of recombination over the time for diversification of Typhimurium as majority of clonal variations are from point mutations rather than recombination. The phylogenetic tree based on mutational changes showed that most Typhimurium isolates of a given phage type are in the same evolutionary group, but that some phage types appear to have arisen more than once. Comparison of the phylogenetic tree with AFLP data gave examples of unrelated isolates of a given phage type having common AFLP fragments comprising plasmid or phage genes, supporting the view that phage type can be determined by presence of specific phages or plasmids. The mutation-based tree showed that six of the nine phage types studied appeared to have a single origin, at least for the isolates studied. It also found that DT1 and DT44 had two independent origins even for the limited set of strains used. The distribution of DT12a isolates into two groups could be explained that the group of three DT12a isolates were derived from the other group of four DT12a isolates, where the root of the tree might be. The data also confirmed that DT64 arose from DT9. The phylogenetic tree that was generated based on essentially mutational changes provides clear relationships of the closely related Typhimurium isolates with high level of consistency and reasonable confidence. This study provided one of the few analyses of relationships of isolates within a clone. Matching actual AFLP with computer modeled AFLP and sequencing intergenic regions provide very good new strategies to identify mutational polymorphisms and to study the molecular evolutionary relationships in the closely related isolates.
123

Seleção e caracterização de peptídeos recombinantes do Mycobacterium leprae ligantes à IgG por meio da tecnologia de phage display / Seleção e caracterização de peptídeos recombinantes do Mycobacterium leprae ligantes à IgG por meio da tecnologia de phage display

Lima, Mayara Ingrid Sousa January 2011 (has links)
Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-30T21:26:19Z No. of bitstreams: 1 Mayara Ingrid Sousa Lima Seleção e caracterização de peptideos....pdf: 1915651 bytes, checksum: 4954d0969cc99ed5643d45ee27772173 (MD5) / Made available in DSpace on 2012-07-30T21:26:19Z (GMT). No. of bitstreams: 1 Mayara Ingrid Sousa Lima Seleção e caracterização de peptideos....pdf: 1915651 bytes, checksum: 4954d0969cc99ed5643d45ee27772173 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia,Brasil / A hanseníase é uma doença infecciosa crônica, causada pelo Mycobacterium leprae, que apresenta manifestações clínicas variadas. Essas variações refletem em diferenças que vão de uma forte resposta imune celular com controle do crescimento do bacilo, no pólo tuberculóide, a uma anergia em resposta celular, no pólo virchoviano. A caracterização do perfil antigênico do M. leprae frente a esse quadro de múltiplos aspectos clínicos representa uma ferramenta fundamental para o desenvolvimento de novas plataformas para um diagnóstico diferencial mais sensível e/ou desenvolvimento de unidades vacinais. Dessa forma, o objetivo desse trabalho foi selecionar e caracterizar peptídeos miméticos de antígenos do M. leprae reativos contra IgGs totais purificadas de pacientes com hanseníase. Para a seleção foi utilizada a tecnologia de phage display, usando bibliotecas randômicas de peptídeos expressos em fagos filamentosos. Foi realizada uma seleção com IgGs de pacientes Tuberculóides e outra com IgGs de pacientes Virchovianos. A validação dos peptídeos foi realizada utilizando o imunoensaio ELISA, o teste de redução de colônias e análise de bioinformática. Após a pré-validação e sequenciamento foram encontradas 17 mimotopos para o pólo Vichorviano e 12 no pólo Tuberculóide. Foram validados 4 peptídeos, sendo 2 do pólo Tuberculóide (T03, T04) e 2 do pólo Virchoviano (V06 e V13). Os peptídeos TALFPWL (T03) e YSTTLSY (T04) foram imunorreativos em soros de pacientes paucibacilares, bem como em pacientes Virchovianos, além de terem alinhado com proteínas de membrana do M. leprae com potencial antigênico. O peptídeo V06 apresentou especificidade de 100% e sensibilidade de 94,74%, o que se complementa com os dados do teste de redução da pIII, o qual obteve uma taxa de redução de 82% em soros Virchovianos. O peptídeo V13 também foi reativo e apresentou similaridades com chaperonas e proteínas de membrana. Este estudo aponta perspectivas para a identificação de novos antígenos, propiciando a descoberta de novos alvos biológicos com potencial diagnóstico e/ou terapêutico. / Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which has varied clinical manifestations. These variations reflect differences that spans from a strong cellular mediated immunity and bacili growth control the tuberculoid pole to a poor T cell immunity at the lepromatous pole. The antigenic profile characterization in both clinical forms represents a fundamental tool for the development of new platforms for a differential diagnosis more sensitive and/or development of vaccine units. Thus, the objective was to select and characterize mimetics peptides antigens of M. leprae reactive against total IgG purified from leprosy patients. The phage display technology was used for selection using random peptides libraries expressed on filamentous phages. A selection was performed with IgGs from tuberculoid patients and other IgGs of lepromatous patients. Peptides validation was performed using the ELISA immunoassay, the plaque reduction test and bioinformatics analysis. After the pre-validation and sequencing were found 17 valid sequences for the lepromatous pole and 12 tuberculoid pole. Four peptides were validated, two of tuberculoid pole (T03, T04) and two lepromatous pole (V06 and V13). The peptides TALFPWL (T03) and YSTTLSY (T04) were imunoreatives in sera from paucibacillary patients and in lepromatous patients. They had alignment with membrane proteins of M. leprae antigenic potential. The V06 peptide showed 100% specificity and 94.74% sensitivity, which is supplemented with the plaque reduction test, who obtained a reduction rate of 82% in lepromatous sera. The V13 peptide was also reactive and showed similarities with chaperones and membrane proteins. This study presents insights for new antigens identification, leading to discovery of new biological targets with potential diagnostic or therapeutic
124

Seleção e caracterização de peptídeos recombinantes miméticos de antígenos do vírus da dengue por "PHAGE DISPLAY"

Santos, Paula de Souza 28 February 2006 (has links)
Dengue fever is caused by an arbovirus of the Flaviridae family, transmitted from person to another through an intermediate fly vector, Aedes aegypti. It is a tropical and subtropical infectious disease characterized by fever and strong pain in joints, which could also lead to bleeding in its hemorrhagic form. In this investigation, Phage Display technology was used to identify recombinant peptides with affinity to polyclonal antibodies (IgY) raised in immunized chickens with total proteins of the DENV-3 culture. Animal sera was purified in a HiTrap column and concentrated through dialysis. The IgY s were immunoreactive against DENV cultures, but were not type specific. Phage clones were selected from a random peptide library (PhD-7) in four cycles of biopanning against IgY. Selected phages were amplified in deepwell microtiter plates and submitted to ELISA tests. Immunoreactive clones against IgY were sequenced, translated and analysed through bioinformatics. Fourteen distinct clones were selected and aligned against viral proteome sequences and among themselves. Three consensus sequences among phage clones were detected: VLRN, APP and LPP. The peptide search in BLASTp showed similarity to the following viral proteins: polyprotein, envelop, and the nonstructural proteins NS1, NS2a, NS3 and NS5. All of them have matched with DENV-1, -2, -3, and/or -4 sequences, corroborating with the lack of type specificity of the raised IgY. Considering the VLRN motif, the analyses of antigenicity indexes of similar peptides demonstrated that its antigenicity is highly influenced by neighboring residues. Three-dimensional analysis of the DENV-2 capsid protein, with the alignment of the VLRN motif, have identified two target sequences, NRVSTVQQL and EIGRMLNILNRR, that are present in the polyprotein of all four viral types, which may contain the two possible domains VxRN and LRN, respectively. Six selected phages have presented known protein domains, and five of them presented specific phosphorylation and glycosylation sites, similar to known eukaryotic viruses; however, they may not be physiologically active sites in the dengue virus. Finally, the peptides were used to detect human IgG and IgM. ELISA tests were performed in two patients with isolated infections of DENV-1 or DENV-3. The reactivity of the 14 clones was superior to total antigens obtained from cultures, but they were not type specific. / RESUMO - GERAL A dengue é uma doença infecciosa febril aguda, transmitida de uma pessoa doente a uma pessoa sadia pela picada da fêmea contaminada de um mosquito Aedes aegypti. A doença é causada por um arbovírus, membro da família Flaviviridae e do gênero flavivirus. Existem quatro tipos de vírus da dengue: Dengue 1, Dengue 2, Dengue 3 e Dengue 4, e há um grande espectro de manifestações clínicas da doença, sendo as duas principais, a dengue clássica e a dengue hemorrágica. O diagnóstico geralmente é baseado em sintomas, e laboratorialmente é tardio, no entanto vários testes específicos para cada sorotipo. A tecnologia do Phage Display (exposição de biomoléculas em fagos) tem sido amplamente utilizada no mapeamento de epítopos de diversos antígenos, constituintes de vários agentes causadores de doenças. Com o objetivo de selecionar peptídeos recombinantes expressos no capsídeo de bacteriófagos produziu-se soro policlonal em galinhas previamente sensibilizadas com proteínas totais do vírus da dengue tipo 3. Após 4 ciclos de seleção de uma biblioteca de fagos com peptídeos recombinantes lineares de 7 resíduos contra a IgY policlonal, 14 fagos imunorreativos foram selecionados e suas seqüências de ácidos nucléicos foram posteriormente analisadas por bioinformática. Foi possível identificar domínios protéicos comuns entre os peptídeos, sendo que o principal domínio mapeado foi VLRN. Testes subseqüentes se fazem necessários para a melhor caracterização destes peptídeos, incluindo possíveis aplicações diagnósticas e vacinais dos peptídeos selecionados. RESUMO - CAPÍTULO I A dengue é causada por um arbovírus da família Flaviridae, transmitido de uma pessoa à outra através de um hospedeiro intermediário, o mosquito Aedes aegypti. É uma doença infecciosa tropical e subtropical caracterizada por febre e dor intensa nas articulações, além de sangramentos intensos quando na sua forma hemorrágica. Neste trabalho utilizou-se da tecnologia de exposição de peptídeos randômicos em fagos, phage display, no intuito de identificar peptídeos recombinantes com afinidade a anticorpos policlonais (IgY) gerados em galinhas imunizadas com proteínas totais do vírus da dengue tipo 3. As IgYs dos animais foram purificadas em coluna HiTrap e concentradas por diálise. As IgYs foram imunorreativas contra todas as culturas de DENV, mas não foram tipo específico. Os clones foram selecionados de uma biblioteca de fagos randômicos (PhD-7) por quatro ciclos de seleção contra as IgYs. Os fagos selecionados foram amplificados em placas deepwell e submetidos a testes ELISA. Clones imunorreativos contra IgY foram seqüenciados, traduzidos e analisados por bioinformática. Quatorze clones distintos foram selecionados e alinhados contra a seqüência de dados do proteoma viral e entre todos eles. Três seqüências consenso entre os clones foram detectadas: VLRN, APP e LPP. A procura por prováveis seqüências protéicas do vírus da dengue no BLAST mostrou similaridade a diversos sítios protéicos virais: poliproteína, envelope e as proteínas não-estruturais NS1, NS2a, NS3 e NS5. Todas elas se alinharam com as seqüências dos tipos DENV-1, -2, -3, e/ou -4, corroborando com a falta de especificidade das IgYs. Considerando o motivo VLRN, as análises dos índices de antigenicidade dos peptídeos similares demonstraram que estes índices são altamente influenciados pelos resíduos vizinhos. Análise 3-D da proteína do capsídeo viral do tipo DENV-2, com superposição do motivo VLRN, identificou duas seqüências alvos, NRVSTVQQL e EIGRMLNILNRR, que estavam presentes na poliproteína dos quatro tipos virais, os quais podem conter dois prováveis domínios, VxRN e LRN, respectivamente. Domínios de fosforilação e glicosilação, encontrados em vírus eucarióticos, foram similares às seqüências presentes em cinco fagos recombinantes selecionados, o que não implica que estes motivos sejam fisiologicamente ativos no vírus da dengue. Finalmente, os peptídeos foram usados para detectar IgG e IgM humana. Testes ELISA foram realizados em dois pacientes com infecções isoladas de DENV-1 ou DENV-3. A reatividade dos 14 clones foi superior àquela observada para antígenos totais obtidos das culturas, embora não tenha sido específica. / Mestre em Genética e Bioquímica
125

Targeting Astrogliosis: Isolation and Characterization of Astrocyte Specific Single Chain Antibody Fragments

January 2013 (has links)
abstract: Specificity and affinity towards a given ligand/epitope limit target-specific delivery. Companies can spend between $500 million to $2 billion attempting to discover a new drug or therapy; a significant portion of this expense funds high-throughput screening to find the most successful target-specific compound available. A more recent addition to discovering highly specific targets is the application of phage display utilizing single chain variable fragment antibodies (scFv). The aim of this research was to employ phage display to identify pathologies related to traumatic brain injury (TBI), particularly astrogliosis. A unique biopanning method against viable astrocyte cultures activated with TGF-β achieved this aim. Four scFv clones of interest showed varying relative affinities toward astrocytes. One of those four showed the ability to identify reactive astroctyes over basal astrocytes through max signal readings, while another showed a statistical significance in max signal reading toward basal astrocytes. Future studies will include further affinity characterization assays. This work contributes to the development of targeting therapeutics and diagnostics for TBI. / Dissertation/Thesis / M.S. Bioengineering 2013
126

Estratégias terapêuticas para inibir o crescimento de biofilme produzido por cepas multirresistentes de Pseudomonas aeruginosa representativas de clones e/ou genótipos de resistência endêmicos no Brasil. / Therapeutic strategies to inhibit the growth of biofilm produced by strains of multiresistant Pseudomonas aeruginosa representative of clones and/or exhibiting resistance genotypes endemic in Brazil.

Rodrigo Cantamessa Gonçalves 10 February 2015 (has links)
Pseudomonas aeruginosa é um patógeno multirresistente capaz de produzir um biofilme protetor contra antibacterianos (ATB). O presente estudo avaliou estratégias terapêuticas contra biofilmes de cepas multirresistentes de P. aeruginosa representativas de clones e/ou genótipos de resistência endêmicos no Brasil. Os biofilmes foram formados in vitro utilizando um modelo adaptado do MBEC Assay e as estratégias terapêuticas utilizaram bacteriófagos líticos, combinação de ATB e/ou uso de força iônica alta (meio FIA). A aplicação de bacteriófagos líticos (φSPM-1) e a combinação de Aztreonam (ATM) e Piperacilina/Tazobactam (PPT), não foram capazes de eliminar o biofilme. Biofilme formado em meio FIA possui CIM similar ao modelo planctônico, tanto para ATM (4 mg/mL) quanto para PPT (16 mg/mL). Ambos os ATB apresentaram CIM reduzida (inferior a 2 mg/mL) quando aplicados em conjunto com meio FIA. Dependendo da concentração de NaCl, a aplicação de meio FIA possui efeito bactericida sobre bactérias planctônicas e efeito bacteriostático sobre biofilmes já formados. / Multidrug-resistant Pseudomonas aeruginosa is a pathogen capable of producing a protective biofilm against antibiotics (ATB). The present study evaluated therapeutic strategies against biofilms of multidrug-resistant strains of P. aeruginosa representative of clones and/or exhibiting resistance genotypes endemic in Brazil. Biofilms were formed in vitro using an adapted model of MBEC Assay and the therapeutic strategies used lytic bacteriophages, combination of ATB and/or use of high ionic strength (HIS medium). The application of lytic bacteriophages (φSPM-1) and the combination of Aztreonam (ATM) and Piperacillin / Tazobactam (PPT) were unable to remove the biofilm. The application of HIS during biofilm formation restored the bacteriostatic effect of both ATM (4 mg/mL) and PPT (16 mg/ml). Both ATB showed reduced MIC values (less than 2 mg/mL) when applied in conjunction with HIS medium. It was shown that HIS has a bacteriostatic or bactericidal effect on planktonic growth, which depend on the NaCl concentration, and bacteriostatic activity against mature biofilm.
127

Development of a novel high throughput method for identifying phage-host pairs in an extreme environment

Olonade, Israel Temiloluwa January 2017 (has links)
Philosophiae Doctor - PhD / There are approximately 10³¹ bacteriophages in the biosphere, outnumbering bacteria 10:1, hence, the dynamic and specific nature of phage-host interactions exerts significant influence on microbial communities. Bacteriophages also represent the reservoir of the highest known genetic diversity making them a potential source of novel biotechnological products. However, the isolation of novel bacteriophages is limited by the observation that less than 1% of bacterial hosts have been cultured. This study aimed to bypass this problem by developing novel culture independent approaches to improve our ability to isolate novel phage-host pairs. Samples were collected from an abandoned copper prospecting site near the Gobabeb Desert Research and Training Station and a Salt lake located in the Swakopmund region of the Namibian desert. Two approaches were explored in this study namely viral tagging and reverse metaviromics. For viral tagging, fluorescently labelling the environmental phage fraction before challenging the environmental bacterial fraction with tagged phages proved difficult. This was most likely due to the complex interaction of the labelling agent with phages and requires further studies. For the reverse metaviromics approach, total DNA from the environmental phage fractions was extracted, sequenced and analyzed for novel phages. Analysis of the phage diversity showed that the copper site was dominated by tailed viruses as has been shown for other extreme arid environments. However, the saline site was atypical of marine environments, with tailed viruses being the most abundant, suggesting that the diversity present is not only driven by salinity. Using the metaviromic sequence data to guide the selection of potential bacterial hosts, two strategies were employed. In the first, putative hosts were predicted based on similarity of phage sequences to those identified in databases. Media targeting these specific genera were employed, 8 bacterial species were isolated and based on 16S rRNA similarity to the closest known species were identified as Halomonas caseinilytica, Halomonas eurihalina, Halomonas sinaiensis, Idiomarina loihiensis, Marinobacter xestospongiae, Virgibacillus salarius and two Salinivibrio species. The 16S rRNA analysis also suggested that H. sinaiensis, V. salarius and both Salinivibrio species are novel. All 8 isolates were challenged with the environmental phage fraction. A novel phage, SMHB1, was isolated on one of the Salinivibrio spp. and is only the second characterized phage ever described for this genus. SMHB1 is a 32 kb myovirus, with a head diameter of 56 nm, and a tail length of 106 nm. The second approach involved the design of fluorescently labelled probes targeting phages identified from the metaviromic sequence data. In a control E. coli system to detect cloned phage DNA fragments, 87% of the interrogated cells showed significant hybridization of the phage specific probe to the target. The optimized method was applied to a simulated environmental bacterial fraction and a detection limit of 1:100 was observed for the bacteria containing the phage DNA fragment of interest. This study demonstrates the possibility of improving the specificity of isolating phage-host pairs in a culture-independent manner by incorporating sequence data in the experimental design; and contributes to our knowledge of the phage diversity of an understudied extreme environment.
128

Comparative Gene Expression Analyses of Campylobacter jejuni Strains Isolated from Clinical, Environmental and Animal Sources

Azzi, Ghiwa January 2013 (has links)
Campylobacter species are the primary cause of bacterial food-borne diarrhoea worldwide. Comparative genomic analyses of Campylobacter strains reveal genome plasticity providing insight into the evolution of virulence traits. The goal of this study was to identify genes important for infectivity and for naturally occurring variability in phenotypic traits in C. jejuni and C. coli strains. Transcriptome and phenotype analyses were conducted to determine if genetic and phenotypic characteristics could be attributed to the source of the strains. Isolates from water sources had higher biofilm formation than animal strains. Clinical strains had decreased sensitivity to hydrogen peroxide as well as increased adherence and invasion when compared to animal strains. A number of genetic differences were observed; however, without further analysis it is difficult to determine which of these impact virulence in Campylobacter. Ultimately, this project will lead to the identification of markers associated with strains of Campylobacter causing illness.
129

Domain Antibody Fragment Phage Display as a Biomarker Discovery Tool for Traumatic Brain Injury

January 2020 (has links)
abstract: Traumatic brain injury (TBI) affects an estimated 1.7 million people in the United States each year and is a leading cause of death and disability for children and young adults in industrialized countries. Unfortunately, the molecular and cellular mechanisms of injury progression have yet to be fully elucidated. Consequently, this complexity impacts the development of accurate diagnosis and treatment options. Biomarkers, objective signatures of injury, can inform and facilitate development of sensitive and specific theranostic devices. Discovery techniques that take advantage of mining the temporal complexity of TBI are critical for the identification of high specificity biomarkers. Domain antibody fragment (dAb) phage display, a powerful screening technique to uncover protein-protein interactions, has been applied to biomarker discovery in various cancers and more recently, neurological conditions such as Alzheimer’s Disease and stroke. The small size of dAbs (12-15 kDa) and ability to screen against brain vasculature make them ideal for interacting with the neural milieu in vivo. Despite these characteristics, implementation of dAb phage display to elucidate temporal mechanisms of TBI has yet to reach its full potential. My dissertation employs a unique target identification pipeline that entails in vivo dAb phage display and next generation sequencing (NGS) analysis to screen for temporal biomarkers of TBI. Using a mouse model of controlled cortical impact (CCI) injury, targeting motifs were designed based on the heavy complementarity determining region (HCDR3) structure of dAbs with preferential binding to acute (1 day) and subacute (7 days) post-injury timepoints. Bioreactivity for these two constructs was validated via immunohistochemistry. Further, immunoprecipitation-mass spectrometry analysis identified temporally distinct candidate biological targets in brain tissue lysate. The pipeline of phage display followed by NGS analysis demonstrated a unique approach to discover motifs that are sensitive to the heterogeneous and diverse pathology caused by neural injury. This strategy successfully achieves 1) target motif identification for TBI at distinct timepoints and 2) characterization of their spatiotemporal specificity. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2020
130

Phage-Displayed Random Peptide Libraries in Mice: Toxicity After Serial Panning

Krag, David N., Fuller, Susan P., Oligino, Lyn, Pero, Stephanie C., Weaver, Donald L., Soden, Amy L., Hebert, Christopher, Mills, Sadie, Liu, Chen, Peterson, Daniel 16 October 2002 (has links)
Purpose: In vivo screening of phage-displayed random peptide libraries (RPLs) has been used to identify peptide ligands to targets found on endothelial cells of blood vessels supplying specific tissues such as brain, kidney, and tumor tissue. Peptides that bind specifically to blood vessels supplying tumor tissue have been conjugated to cytotoxic agents and used to successfully eradicate tumors in a mouse model. With the ultimate goal of developing similar methods for treating human cancer, we describe an in vivo RPL screening process that, unlike previous in vivo experiments, does not harm the animal being screened. Methods: RPLs were administered to FVB, BalbC, and tumor-bearing MRL/MpJ-fasLPR mice in a variety of dosing formats. Tumor nodules were excised 10 min following infusion and phage were amplified from the specimens. Phage were reinjected into the same animal within 48 h. This process was repeated twice for a total of three in vivo screens of mouse tumor tissue within the same animal. Mice were observed for systemic side effects, histopathologic damage, and presence of phage in organs. Peptide sequences were determined from several third-pan phage clones. Results: Overall there was minimal toxicity from administration of single or repeat doses of RPLs. Amino acid consensus sequences were identified and some of the sequences were similar to those of peptide ligands that bind matrix metalloproteinases. Conclusions: Serial administration of an RPL is well tolerated and serial panning in individual mice leading to consensus sequence motifs is possible. Based on these preclinical data the Food and Drug Administration has approved the implementation of human clinical trials with this technique.

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