Dissertations / Theses on the topic 'Autotransporters'
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1
Orner, Sherko A. "Functional characterisation of Neisseria meningitidis autotransporters MSPA and APP." Thesis, University of Nottingham, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.490049.
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2
Sims, Peter Vincent. "Biogenesis of BapF : a novel acylated Bordetella autotransporter." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/41811.
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Autotransporters are a superfamily of Gram-negative secreted proteins, composed of a Cterminal
domain which forms a β-barrel in the outer membrane and plays a significant role in
translocation of the N-terminal passenger domain to the cell surface. A subfamily of
autotransporters is N-terminally acylated during their biogenesis, a post-translational
modification demonstrated to be essential for their function. Considering the autotransporter
passenger domain is secreted beyond the outer membrane, how translocation can occur in the
presence of N-terminal acyl groups is undetermined. The work in this thesis describes the
biogenesis of the Bordetella autotransporter F (BapF) from B. bronchiseptica RB50, which is Nterminally
acylated during the initial stages of its secretion to the cell surface. The acyl groups
attached to BapF at the Cys28 residue were shown to be subsequently removed by signal
peptidase 1 cleavage between residues Ala34 and Ala35, indicating that BapF forms an acylated
intermediate in its secretion pathway. Studying the secretion of BapF mutants in which Nterminal
acylation was ablated revealed that the passenger domain was still capable of reaching
the cell surface; the surface-expressed passenger domain mediated phenotypes of cellular
aggregation and an increased rate of culture sedimentation, similar to that observed in E. coli
cultures expressing the wild-type BapF protein. However, cells expressing these mutants
appear to have damaged outer membranes, potentially due to the observed increase in fulllength
protein accumulating in the periplasm. Alternatively, E. coli cells expressing a BapF
mutant in which signal peptidase 1 cleavage is blocked do not exhibit obvious aggregation and
sedimentation phenotypes. Yet, an independent passenger domain is clearly produced. Based
on the results presented in this thesis, it can be hypothesized that sequential processing of the
BapF signal peptide, producing an acylated intermediate in the secretion pathway, helps to
regulate the passage of BapF through the periplasm ultimately permitting surface expression. In
addition, bioinformatic and molecular analysis strongly suggest the BapF passenger domain
folds into a β-propeller structure, and if proven to do so, BapF will be the first autotransporter
reported with this conformation
3
Bokhari, Syed Habib. "Characterisation and secretion mechanism of Bordetella pertussis autotransporter proteins." Thesis, University of Glasgow, 2002. http://theses.gla.ac.uk/1507/.
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The identification and characterisation of new virulence determinants of 5. pertussis is providing important information for understanding the colonisation and survival strategies of the microorganism. B. pertussis deploys a range of surface-associated components to enable its successful colonisation of the host. Bap-5 has been identified as a new member of the B. pertussis autotransporter family of proteins that includes PRN, BrkA, TCF and Vag-8, largely due to its homology at the C-terminus and some other similar regions such as the RGD (integrin-binding) and SGXG (glycosaminoglycan-binding) motifs. The bap-5 gene also exists in B. bronchiseptica and B. parapertussis. Characteristic upstream regulatory sequences such as a ribosome-binding site were not seen in bap-5, but a potential heptameric BvgA-binding motif was identified. The expression of Bap-5 was confirmed by RT-PCR and Western blotting and was shown to be bvg dependent. Although Bap-5 does not possess a typical signal sequence like pertactin (PRN), its surface localisation was confirmed by agglutination and immunofluorescence assays. A potential role for Bap-5 in infection was studied by generating Bap-5 deficient mutants in two strains of B. pertussis. An allelic exchange procedure with the suicide vector pSS1129 carrying the bap-5 gene disrupted with a kanamycin-resistance cassette was used. PCR and Southern blotting confirmed the replacement of the wild-type bap-5 gene with the mutated version. Moreover, SDS-PAGE and Western blotting of outer-membrane preparations of B. pertussis Taberman wild-type and its Bap-5-deficient mutant showed a clear difference in their outer-membrane profile at ~79.9kDa presumably representing the unprocessed form and bands at ~65 kDa and ~16 kDa may represent the processed forms of the protein. The Bap-5 characterisation studies showed that the Taberman Bap-5-deficient strain was less able than the parent strain to colonise the lower respiratory tract of mice and adhesion studies (in vitro) showed that the Taberman parent was better in adhering to certain cell types than the Bap-5-deficient mutant.
4
Contreras, Morales Elis Rosella. "La planeación inadecuada del transporte público de Toluca. Caso de estudio la empresa Autotransportes Suburbanos de la ciudad de Toluca y Zona Industrial , S.A de C.V. ( ATSUZI )." Tesis de Licenciatura, Universidad Autónoma del Estado de México, 2013. http://hdl.handle.net/20.500.11799/49262.
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La Zona Metropolitana de Toluca durante las últimas décadas ha tenido un
crecimiento
demográfico y una expansión urbana importante, lo cual ha generado cambios
en las estructura de la ciudad, así como en la prestación de servicios públicos; en este
sentido, la oferta del servicio de transporte, se ha dado en forma deficiente, la capacidad de
dotar infraestructura y de circulación ha sido desordenada, parcial, segmentada y la
movilidad de las personas se ha visto obstruida, paralizando el tránsito en sectores de la
ciudad en horas pico prolongadas.
Esta investigación, surge ante la falta de
referentes analíticos en la planeación de
rutas del transporte público en la zona metropolitana
y el
rezago de documentación
significativ
a.El objetivo de la presente investigación es analizar el transporte público urbano de la Zona Metrop oli tana de Toluca, en particular la empresa “Autotransportes Suburbanos de la Ciudad de Toluca y Zona Industrial, S.A. de C.V. (ATSUZI) en un contexto de movilidad urbana. La investigación se realizó con apoyo de información estadística, analizando la problemática del transporte público urbano existente en la ciudad y con ello realizar sugerencias de reordenamiento y de mov ilidad de la población de esta zona metropolitana.
5
Dufailu, Osman Adamu. "Role of meningococcal Neisserial autotransporter lipoprotein (NalP) in host pathogenesis." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/41127/.
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Neisseria meningitidis (Nm) can cause life-threatening bacterial meningitis and septicaemia. The high mortality of meningitis is associated with meningococcal invasion of the central nervous system (CNS) by breaching of the blood-brain barrier (BBB). Nm elaborates several cell-surface and secreted virulence factors that promote host invasion and colonisation. One important class are the autotransporter proteins. Adhesion and penetration protein (App), meningococcal serine protease A (MspA), Immunoglobulin A1 protease (IgA1p) and Neisserial autotransporter lipoprotein (NalP) are serine protease autotransporters of Nm; all have previously been demonstrated to have roles in meningococcal virulence. For App and MspA, uptake by host cells, nuclear localisation, histone clipping and induction of apoptosis have been described in our laboratory. However, a time course for the efficient uptake and nuclear localisation of these proteins into human brain microvascular endothelial cells (HBMECs) and details regarding the mechanism for histone clipping remained unknown. To address these points, both App and MspA and proteolytically-inactive mutant derivatives were expressed using the pColdTF vector system and purified under native conditions. Using confocal scanning microscopy optimal uptake and nuclear localisation of recombinant fusion proteins containing the functional passenger domains of both App and MspA in HBMECs was shown to occur 8 h-post exposure. Furthermore, the requirement for the active site serine residue in both autotransporters for H3.1 clipping was demonstrated, and human coagulation factor V (FV) was shown to be an additional substrate for both proteins. NalP is a cell-surface maturation protease which processes App, MspA and other meningococcal surface proteins such as IgA1P, LbpB and NhbA, and thus modulates the cell surface and secretome of the organism. Previous studies aimed at functional characterisation of NalP have typically relied on the phenotypic comparison of wild-type and nalP-mutant derivatives. Here an active recombinant NalP was expressed and purified, and used to investigate the interaction of NalP with host cells in order to more comprehensively elucidate the role of NalP during meningococcal interaction with the host. A recombinant NalP (rNalP) passenger domain was purified under non-denaturing conditions using immobilized nickel chromatography. Although rNalP had apparent molecular weight 8-10 kDa less than that of NalP secreted by wild-type meningococci, it was functional as determined by its ability to process human complement 3 (C3). rNalP was shown to cleave human coagulation factor V (FV), a proteolytic event which is likely to contribute to bacterial pathogenesis. Binding and uptake of rNalP into human cells was demonstrated by flow cytometry and confocal microscopy. Interestingly, rNalP was differentially localised to different cellular compartments in different cell types. Treatment of HBMECs with rNalP resulted in increased levels of IL-6 and IL-8, and decreased levels of TNF-α in culture supernatants. rNalP was shown to clip histone 2B but not other histone proteins. Using a re-tagging approach a number of rNalP-interacting host proteins were identified. These included proteins of the candidate membrane, cytoplasm, cytoskeleton endoplasmic reticulum, mitochondrion, and nucleus. Some of these proteins are likely to be involved in trafficking of NalP and the cellular response to this protein. Overall, the findings of this study expand our knowledge on the contribution of three autotransporters to meningococci pathogenesis and provide a platform to further explore the host response to NalP uptake and the epigenetic changes associated with autotransporter host interactions, which may guide the development of future therapeutic interventions.
6
Gagnon, Elizabeth. "The requirement of putative autochaperone motifs for autotransporter passenger domain folding." Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/43502.
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7
Darch, Owen Matthew. "A study on the function of the Pseudomonas aeruainosa autotransporter PA0328." Thesis, University of Nottingham, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.523036.
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8
Noofeli, Mojtaba. "Genetic analysis and characterisation of the BapC autotransporter of bordetella pertussis." Thesis, University of Glasgow, 2008. http://theses.gla.ac.uk/105/.
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The autotransporters are a family of extracellular proteins, found in various Gram-negative bacteria, that have many different functions but appear to have a similar mechanism of export. In B. pertussis, the virulence-regulated proteins Pertactin, BrkA, Tcf, and Vag8 have structural homology at their C-termini (30-kDa) and the N-terminal of the mature proteins share structural characteristics such as RGD and SGXG motifs. Recently, another member of the B. pertussis autotransporter family, Bap-5 (Blackburn, 2000) (GenBank accession number AF081494) or BapC (GenBank accession number AJ277634.1) was identified. The present work has suggested that BapC, like BrkA, is a serum-resistance factor. B. pertussis brkA, bapC double and bapC single mutants were created, and showed greater sensitivity to killing by normal human serum than their wild-type strains but they were not as sensitive as a bvg mutant strain. Competition assays also showed an important role for BapC, like BrkA, in virulence of B. pertussis strains after intranasal infection in the mouse. Moreover, the brkA, bapC double and bapC single mutants were found to be more sensitive to the antimicrobial peptide, cecropin P1, than the parent strain. Nucleotide and amino acid analyses of the bapC region spanning the poly(C) and poly(G) tracts of a number of B. pertussis strains showed minor nucleotide and amino acid polymorphisms in some strains but it appeared that all had an ORF that would be able to produce some form of BapC.
9
Ait-Tahar, Kamel. "Identification and characterisation of AutA : an autotransporter protein of Neisseria meningitidis." Thesis, University of Nottingham, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.394868.
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10
Yoo, Christopher Charles. "Investigating the Role of Trimeric Autotransporter Adhesins in Fusobacterium nucleatum Pathogenesis." Thesis, Virginia Tech, 2019. http://hdl.handle.net/10919/101683.
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Fusobacterium nucleatum is a Gram-negative bacterium that serves as a bridging organism in polymicrobial biofilms within the oral cavity. Although the bacterium is abundant in healthy gingival tissue, recent studies have found that F. nucleatum is associated with a wide-spectrum of human diseases which include periodontal disease, preterm birth, endocarditis, colorectal cancer, and pancreatic cancer. Previous studies of F. nucleatum virulence have uncovered two surface adhesins, Fap2 and FadA, that interact with the surface of human cells; however, the study of new virulence factors was previously limited as there was no gene deletion system available to functionally analyze F. nucleatum proteins.
Interestingly, F. nucleatum has a diverse landscape of structurally unique surface adhesins called Type 5c secreted trimeric autotransporter adhesins (TAAs), which are a family of proteins that are historically known for their contributions to bacterial pathogenesis. This dissertation encompasses the use of recombinant protein expression systems and newly developed gene deletion technology to provide a foundational understanding of the contribution of Type 5c secreted proteins in F. nucleatum pathogenesis. Our results show that the presence of TAAs on the surface of F. nucleatum contribute to the bacterium's ability to bind and invade human cells, establishing the need to characterize other F. nucleatum surface proteins.
Additionally, our studies analyzed the proinflammatory landscape induced by F. nucleatum through the identification of specific cytokines that are being secreted during in vitro infections of human cells. Cytokine signaling is a critical aspect of the host cell immune response as it promotes the recruitment of immune cells to the site of infection for efficient clearance of bacterial pathogens. While it has been well established that F. nucleatum modulates the secretion of IL-8, our studies identified that the bacterium also promotes the secretion of CXCL1, which is an important signaling protein that promotes tumor metastases. Overall, the work provided in this dissertation has delivered the initial characterization of TAAs in F. nucleatum virulence, a framework for future studies of Type 5c secreted proteins in Fusobacterium pathogenesis, and the role of Fap2 and FadA in promoting pro-inflammatory and pro-metastatic signaling from colorectal cancer cells.Master of Science in Life Sciences
11
Mehat, Jai. "Characterisation of CapC, a novel strain-specific autotransporter in Campylobacter species." Thesis, University of Surrey, 2017. http://epubs.surrey.ac.uk/841594/.
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Campylobacter jejuni and Campylobacter coli are recognised as the principal causative agents of bacterial gastroenteritis in the developed world. However, despite the identification of factors integral to infection, characterisation of the virulence strategies employed by Campylobacter remains a significant challenge. Bacterial autotransporter proteins comprise the largest and most diverse class of secretory proteins in Gram-negative bacteria; notably almost all previously characterised autotransporter proteins contribute to bacterial virulence to some extent. The aim of this study was to characterise CapC, a newly identified, strain-specific gene predicted to encode an autotransporter protein, and to examine the contribution of this factor to the virulence of Campylobacter jejuni. The capC gene was initially confirmed as being encoded by approximately 60% of C. jejuni and C. coli human clinical and poultry associated isolates. Moreover, CapC was confirmed as a member of the autotransporter family through the use of bioinformatic prediction tools and the localisation site of this protein was determined as the outer membrane of C. jejuni. Targeted mutagenesis of the capC gene in C. jejuni 81116 and C. jejuni M1 and subsequent comparison of wild-type and isogenic mutant strains demonstrated that CapC contributes directly to virulence in the Galleria mellonella invertebrate model (p=0.00017; p=0.002323). Furthermore, tissue culture assays using non-polarised, partially differentiated Caco-2 and T84 intestinal epithelial cells indicate that deletion of CapC resulted in significantly decreased adhesion and invasion efficiency. Additional analyses indicated that CapC primarily contributes to adhesion to intestinal epithelial cells. Additional assays showed that deletion of the capC gene has no significant phenotypic effect on cytotoxicity in a Caco-2 cell model. A secondary aim of this study was to examine the distribution of capC amongst campylobacters and to establish any potential genetic associations of this virulence determinant. Using publically vi available genome sequences, capC was established to be highly prevalent in C. jejuni (67.9%) and C. coli (84%). Campylobacter autotransporter proteins were also shown to be present in truncated and full length forms. Interestingly, full length CapC was shown to be primarily associated with the ST-45, ST-283 and ST-573 clonal complexes in C. jejuni and the ST-828 clonal complex in C. coli. Furthermore, this study detailed the identification of a novel autotransporter in Campylobacter species, tentatively designated as CapD. This autotransporter was found to be genetically distinct from CapC and is the most prevalent autotransporter identified in Campylobacter species. The studies presented in this thesis indicate that CapC is a strain-specific virulence determinant in Campylobacter species that is associated with select lineages of C. jejuni and C. coli. CapC contributes to the integral infection process of adhesion however further studies are required to fully elucidate the exact nature of this interaction. Additionally, it can be concluded that possession of Campylobacter autotransporter proteins is dependent on genetic background.
12
Ackermann, Nikolaus. "Das Yersinia-Adhäsin YadA, ein oligomerer Autotransporter als Prototyp der Oca-Familie." Diss., lmu, 2005. http://nbn-resolving.de/urn:nbn:de:bvb:19-32890.
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13
Jarmander, Johan. "Improved detection and performance of surface expression from the AIDA-I autotransporter." Licentiate thesis, KTH, Bioprocessteknik (stängd 20130101), 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-121561.
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Surface expression of recombinant proteins has attracted a lot of attention due to its potential in applications such as enzyme production, vaccine delivery and bioremediation. Autotransporters have been used for surface expression of a variety of proteins, but the expression systems reported in literature have typically been inflexible and incapable of detecting proteolysis, thereby limiting surface expression yield. In this thesis, a modular surface expression system, utilizing dual tag detection, was therefore created. It was based on the adhesin involved in diffuse adherence (AIDA-I) autotransporter, and was here used to express the model proteins SefA and H:gm on the cell surface of Escherichia coli. Due to the dual tag detection system, proteolysed H:gm could be successfully verified on the cell surface. By optimizing cultivation conditions, surface expression yield of SefA was increased by 300 %, and proteolysis reduced by 33 %. While proteolysis could not be eliminated completely, the work presented in this thesis is a major step towards a general system for surface expression of a wide range of proteins in varied applications.QC 20130506
14
Hassan, Hoda Abdel-Hadi. "Identification and characterisation of app : an immunogenic autotransporter protein of Neisseria meningitidis." Thesis, University of Nottingham, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368253.
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15
Rossiter, Amanda Eve. "Deciphering the autotransporter pathway of Gram-negative bacteria : from regulation to secretion." Thesis, University of Birmingham, 2011. http://etheses.bham.ac.uk//id/eprint/3046/.
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Autotransporters represent a diverse family of virulence effectors that are secreted from Gram-negative bacteria by the Type V Secretion System. Their initial description coined the term ‘Autotransporter’ to embody the notion that their three-part architecture governs their navigation through the bacterial cell envelope. The Pet cytotoxic autotransporter is secreted by the diarrhoeal pathogen, Enteroaggregative Escherichia coli (EAEC) and was used as a model to study autotransporter biogenesis. Following a global transposon mutagenesis of EAEC, novel accessory factors were identified that are required for Pet biogenesis, including the transcription factors CRP and Fis, periplasmic chaperones and components of the β-barrel assembly machinery (BAM) complex. Using both in vivo and in vitro techniques, we show that the pet promoter is co-dependent on CRP and Fis. We present a novel co-activation mechanism whereby CRP is placed at a non-optimal position for transcription initiation, creating dependence on Fis for full activation and show that this co-activation mechanism extends to functionally similar autotransporters. Furthermore, we highlight novel components of the BAM complex required for AT secretion. This work builds on previous studies that, in recent years, have challenged the ‘auto’ nature of this secretion process causing a paradigm shift towards a much more complex mechanism of AT secretion than initially suggested.
16
Mandyoli, Lungelo. "Structural characterization of EtpA an adhesin from enterotoxigenic Escherichia coli (ETEC)." Diss., University of Pretoria, 2016. http://hdl.handle.net/2263/60835.
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Enterotoxigenic Escherichia coli (ETEC) encompass a group of diverse bacterial pathogens that collectively cause hundreds of millions of diarrheal cases annually, mostly in developing countries. As part of its infection strategy, ETEC invades and colonizes small intestinal epithelial cells where it secretes heat-labile and/or heat-stable enterotoxins, inducing diarrhoea. The ability of ETEC to invade human epithelial cells is a hallmark of its pathogenicity and is controlled by a set of plasmid and chromosome encoded virulence factors. They include EtpA, a 170 kDa plasmid encoded autotransporter. During infection, EtpA functions as an adhesin linking flagellin at the tip of ETEC flagella to the host cell surface and allowing ETEC to deposit its toxins. Antibodies targeting either EtpA or the conserved regions of flagellin impair delivery of the heat-labile toxin in vitro, and prevent intestinal colonization of mice following gastrointestinal challenge with ETEC. EtpA is thus critical to the pathogenicity of ETEC. In this study, a truncated version of EtpA (35 kDa) termed N-terminal EtpA69-445 or N-EtpA69-445 was cloned and produced as an N-terminal GST-tagged cytoplasmic fusion protein in E. coli BL21 cells. The protein was purified by affinity chromatography on glutathione agarose beads. However, the yield of the pure protein was poor due to its limited solubility.
As an alternative, a 57 kDa truncated version of EtpA (N-EtpA69-607) was produced as a secreted C-terminal His6-tagged fusion protein in E. coli TOP10 cells. The protein was purified to homogeneity by metal affinity chromatography (MAC) using Ni-NTA and ion exchange chromatography (IEC) on a Mono S 10/100 GL column. Biophysical characterization of N-EtpA69-607 using circular dichroism (CD) spectroscopy revealed the typical spectrum of a β-helical protein. The in silico modelled structure of the protein confirmed N-EtpA69-607 to be a β-helical protein. CD spectra recorded at increasing temperatures indicated N-EtpA69-607 to be thermally highly stable retaining its fold up to 95°C. Dynamic light scattering (DLS) experiments showed that N-EtpA69-607 is polydisperse in solution forming higher oligomers. Lead crystallization conditions of N- EtpA69-607 were determined but the crystals were too small for X-ray data collection. This study thus represents a significant step towards the characterization of the three dimensional structure of EtpA and understanding its structure-function relationship.Dissertation (MSc)--University of Pretoria, 2016.
Biochemistry
MSc
Unrestricted
17
Gustavsson, Martin. "Surface expression using the AIDA autotransporter : Towards live vaccines and whole-cell biocatalysis." Licentiate thesis, KTH, Bioprocessteknik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-48575.
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The area of surface expression has gathered a lot of interest from research groups all over the world and much work is performed in the area. Autotransporters have been used for surface expression in Gram-negative bacteria. One of the more commonly used autotransporters is the Adhesin Involved in Diffuse Adherence (AIDA) of pathogenic Escherichia coli. The surface expression of enzymes and vaccine epitopes offer several advantages. Surface expressed enzymes gain similar properties to immobilised enzymes, mainly simplified handling and separation using centrifugation. Surface expressed vaccine epitopes can have longer half-lives inside the animal that is to be immunized and surface groups on the host cell can act as adjuvants, increasing the immune response and leading to a better immunisation. However, while much basic research is directed towards mechanisms of surface expression using autotransporters there are few reports regarding production of surface expressed protein. Thus the aim of this work was the optimisation of the yield and productivity of surface expressed protein. Protein Z, an IgG-binding domain of Staphylococcal protein A, was used as a model protein for the investigation of which cultivation parameters influenced surface expression. The choice of cultivation medium gave the largest impact on expression, which was attributed to effects based on the induction of the native promoter of AIDA. The AIDA system was then used for the expression of two Salmonella surface proteins, SefA and H:gm, with potential for use as vaccine epitopes. SefA was verified located on the cell surface, and H:gm was found in the outer membrane of the host cell, though only in proteolytically truncated forms lacking the His6-tag used for detection. This proteolysis persisted in E. coli strains deficient for the outer membrane protease OmpT and was concluded to be dependent on other proteases. The removal of proteolysis and further optimisation of the yield of surface-expressed protein are important goals of further work.QC 20111123
Vinnova: BIO-AMINES
SIDA Vietnam: Production of viral proteins for vaccine development
18
Beriotto, Irene. "Optimising the autotransporter system for secretion and display of heterologous proteins on GMMA." Thesis, University of Birmingham, 2017. http://etheses.bham.ac.uk//id/eprint/7652/.
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The Pet Autotransporter protein was engineered and recently proposed as recombinant protein production (RPP) system. This system allows targeting the protein of interest in the culture supernatant fraction. The reduction of diversity and quantity of process impurities and size and number of downstream steps required, increase the overall process robustness and speed-up the process development time for RPP. In the context of this study the platform was investigated for the production of a “difficult” E.coli protein with commercial relevance, C1275. Pet autotransporter was suitable for the production and one step-purification of a protein with comparable purity, thermal stability and immunogenicity to that produced using conventional technology. Additionally, the autotransporter platform was tested for the first time in scaled-up production conditions. The system was compatible with fermentation and the scaled-up conditions resulted in high yield of antigen production even though a further system optimization would be required for a one step-purification process.
19
Latatujeva, Oksana. "Strateginių pokyčių valdymas krovininio transporto versle." Master's thesis, Lithuanian Academic Libraries Network (LABT), 2014. http://vddb.library.lt/obj/LT-eLABa-0001:E.02~2009~D_20140626_182827-47733.
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Temos aktualumas. Dar visai neseniai krovinius gabenančios bendrovės lengva ranka pirko naujus vilkikus, plėtė veiklos apimtis taip uždirbdamos nemažus pelnus. Dabartiniu metu situacija yra kardinaliai pasikeitusi. Ekonomikos nuosmukis sukėlė didžiulius krovinių gabenimo verslo nuostolius: užsakymų mažėja, sąnaudos didėja, įmonės bankrutuoja. Daugumai vežėjų kyla klausimai: kokiomis priemonėmis išvengti krovinių gabenimo verslo nuostolių, kaip juos sumažinti, ką daryti, kad verslas būtų pelningas, kaip didinti veiklos efektyvumą, kokių verslo pertvarkymo programų imtis ir pan. Tai yra probleminiai klausimai, kuriuos kiekvieną dieną kelia krovinius gabenančios bendrovės. Reaguodami į ekonominę krizę, vadovai gali susigundyti atidėti į šalį ilgalaikius strateginius planus ir pasirinki skubotas sprendimus. Tačiau, tikėtina, kad iš dabartinės situacijos išeis tos bendrovės, kurios sutelks dėmesį į strateginius pokyčius, jų valdymą organizacijoje. Tai reiškia, kad vadovai turi peržiūrėti esamas strategijas, strateginius tikslus, juos keisti arba koreguoti, atsižvelgiant į rinkos diktuojamas sąlygas. Tai yra sudėtingas procesas, reikalaujantis daug žinių, įgūdžių, išteklių bei pasiruošimo. Šis procesas reiškia nusistovėjusios tvarkos pasikeitimą, naujų darbo metodų įvedimą, naujų strateginių tikslų ir uždavinių iškėlimą, kuriems gali būti pritariama arba priešinamasi, todėl įmonėje parinkta strateginių pokyčių valdymo sistema turi būti itin gerai apgalvota ir įvertinta. Tai aktualu... [toliau žr. visą tekstą]SUMMARY The relevance of the theme. The recession of the economy have caused enormous loss for freight transport businesss. Many transporters are asking such questions: what means can help to avoid the loss of the freight transport businesss, how to increase the efficiency of the business, what business reorganisation programs should be undertaken, etc. These are problematic questions, which are held by transport companies every day. It is believable, that from this current situation will leave that companies, which will be concentrated into strategic changes and their management in the organization. This process means the change of the routine, the introduction of the new work methods, the raising of new strategic aims and tasks, which can be approved or opposed. Therefore, the selected management of the strategic changes, should be especially well considered and valued. It is relevant for every organization, which seeks the competitive advantage in the market. Aim of research – find out the potential of improving strategic change management in freight transport business. Tasks of research: 1. To reveal the part of strategic changes and to explore it’s management. 2. To accomplish the analysis of freight transport business. 3. To review the methodology of strategic changes management in freight transport business. 4. To research strategic changes management in freight transport business. 5. To find out the possibilities of improving strategic changes in freight transport... [to full text]
20
Scaglione, Patricia. "THE STRUCTURAL AND FOLDING CHARACTERISTICS OF THE PLASMID-ENCODED TOXIN FROM ENTEROAGGREGATIVE ESCHERICHIA COLI." Master's thesis, University of Central Florida, 2008. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/3880.
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Plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a member of the autotransporter subfamily termed SPATE (serine protease autotransporters of Enterobacteriaceae). Autotransporters, which are the most common Gram-negative secreted virulence factors, contain three functional domains: an amino terminal leader sequence, a mature protein or passenger domain, and a carboxy-terminal β domain. The leader sequence targets the protein to the periplasmic space and the β domain then forms a β-barrel pore in the outer membrane of the bacterium which allows the passenger domain to enter the external milieu. In some cases the passenger domain is cleaved from the β-barrel at the extracellular surface to release a soluble toxin. This is thought to be a self-contained process that does not require chaperones or ATP for folding and export of the passenger domain. Pet produces cytotoxic effects through cleavage of its target, the actin-binding protein α- fodrin. Pet is secreted into the extracellular environment, but its target lies within the cytosol. To reach its target, Pet moves from the cell surface to the ER where it triggers ER-associated degradation (ERAD) to enter the cytosol. ERAD is a normal cellular process in which improperly folded proteins are exported from the ER to the cytosol for degradation. Other toxins that utilize this pathway are AB toxins such as cholera toxin (CT) and ricin. The A subunits of these toxins are thermally unstable, and this facilitates their ERAD-dependent translocation into the cytosol. Pet, however, is not an AB toxin. We predict that thermal unfolding is not the mechanism Pet employs to exploit ERAD. It was necessary to purify the toxin first in order to study the structural properties and ER export of Pet. Surprisingly, purified Pet eluted as two close peaks by size exclusion chromatography. Both peaks were Pet as demonstrated through immunoblotting. The folding efficiency of autotransporters has not been extensively elucidated, and based on our purification results, we hypothesized that there is inefficiency in the folding of autotransporters, specifically Pet. A toxicity assay showed that Pet peak one did not display cytopathic activity while Pet peak two did. CD and fluorescence spectroscopy measurements also detected structural differences between the two variants of Pet and demonstrated that Pet peak one was an unfolded variant of Pet peak two. Native gel electrophoresis and biophysical measurements indicated that Pet peak one did not exist as a dimer or aggregate. Our results indicate there are two forms of Pet, and thus the folding process of autotransporters appears to be inherently inefficient. Active Pet (peak two) was used for further biophysical measurements and biochemical assays. Circular dichroism and fluorescence spectroscopy showed that the secondary and tertiary structures of Pet are maintained at physiological temperature, 37°C. Thermal unfolding of Pet occurred at temperatures above 50°C. Fluorescence quenching of Pet was also performed and demonstrated that, at 37°C, there are solvent-exposed aromatic amino acids. The slight structural alterations to Pet at physiological temperature as well as the exposed hydrophobic residues could trigger ERAD. In addition, a modeled structure of Pet revealed a hydrophobic loop which is surface-exposed and a likely target for toxin-ERAD interactions. The data suggests that translocation of Pet mediated by ERAD can occur by a mechanism different from certain AB toxins. An open, hydrophobic conformation likely triggers ERAD, but may also contribute to poor folding.M.S.
Department of Biomolecular Science
Burnett College of Biomedical Sciences
Molecular and Microbiology MS
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CERECEDO, ESCALANTE ABDEEL, and DE SANTIAGO JAVIER VALENCIA. "BARRERAS AL CRECIMIENTO DE UNA MICRO, PEQUEÑA Y MEDIANA EMPRESA DE AUTOTRANSPORTE FEDERAL DE CARGA: EL CASO DE TRANSPORTES VALENCIA." Tesis de Licenciatura, UNIVERSIDAD AUTÓNOMA DEL ESTADO DE MÉXICO, 2018. http://hdl.handle.net/20.500.11799/79810.
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La presente tesis aborda el tema del crecimiento de las micro, pequeñas y medianas empresas de Autotransporte de carga (MiPymes). Se sabe que las tasas de crecimiento en estas empresas son muy bajas, y que a su vez las tasas de muerte en las mismas son muy altas. Luego, nos surge la interrogante de indagar ¿cuáles son las barreras que limitan el crecimiento de este tipo de empresas?
Para realizar la presente investigación se utilizó el método de estudio de casos intrínseco que nos permite indagar a profundidad las causas a una pregunta definida. Se comenzó con un estudio de la literatura sobre cuáles son las razones que se saben a la fecha son limitantes del crecimiento de estas empresas. Posteriormente se diseñó un cuestionario que fuera capaz de indagar nuevas razones aún no explicadas. Se aplicó el instrumento de investigación en una empresa que será el estudio de caso, de donde se derivaron conclusiones.
Se encontró que además de las barreras identificadas en el estudio de la literatura, las cuales son: 1) Inseguridad en el autotransporte federal de carga, 2) Preparación empresarial, 3) Capacitación de operadores, 4) Financiamiento, 5) Competitividad en el sector, existen otras reveladas por nuestro estudio, las cuales son: 6) Influencia familiar en el manejo de la empresa, 7) Prácticas deficientes de administración.
Las dos últimas se explican en tanto que las empresas de esta categoría normalmente son empresas familiares, donde diversos miembros de una familia forman parte de los actores principales en la toma de decisiones. Esto hace que las decisiones lejos de ser racional, sean con una carga afectiva o revelando los vínculos de amistad y esto arroja decisiones no precisamente enfocadas a los mejores resultados operativos y financieros de la empresa. Por otro lado, la misma estructura familiar hace que exista poca profesionalización en la administración de las funciones empresariales y con ello el ejercicio de malas prácticas perjudicando el desempeño empresarial, de nueva cuenta en la empresa.
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Grin, Iwan [Verfasser], and Dirk [Akademischer Betreuer] Linke. "Assisted Secretion of a Trimeric Autotransporter Adhesin from Salmonella / Iwan Grin ; Betreuer: Dirk Linke." Tübingen : Universitätsbibliothek Tübingen, 2015. http://d-nb.info/1197058044/34.
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Coutte, Loïc. "Caractérisation et rôle biologique de la protéase SphB1 impliquée dans la maturation de la FHA de Bordetella pertussis au cours de sa sécrétion." Paris 6, 2004. http://www.theses.fr/2004PA066071.
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Ashgar, Sami S. "Identification and characterisation of two novel autotransporter proteins, designated CapA and CapB, in Campylobacter jejuni." Thesis, University of Nottingham, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408625.
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Doebel-Hickok, Monte. "Characterizing the secretion and function of TcfA : a unique autotransporter and virulence factor in Bordetella pertussis." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/64235.
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Autotransporters (ATs) are an important family of proteins that are essential for the virulence of a variety of Gram-negative bacteria. The vast majority of ATs possess a classical right handed β-helical structure which facilitates the vectorial secretion of the protein. However, not all ATs possess this classical structure. Tracheal colonization factor (TcfA) of B. pertussis is one of only a few ATs that are predicted to be relatively unstructured or to possess a coil structure. It is not known what factors are important for the secretion of non- β-helical ATs. This study sought to characterize the secretion of TcfA which could also reveal more broadly applicable requirements for the secretion of ATs and other surface exposed proteins. This thesis characterized the secretion of TcfA both in E. coli as well as in B. pertussis. The study determined that TcfA has special secretion requirements that are not met when the protein is expressed in E. coli. The unique B. pertussis chaperone Par27 was identified as an important factor for secretion via both its disruption in B. pertussis as well as via its insertion in E. coli. However, it was also determined that there are additional factors that E. coli is lacking that are important for the secretion of TcfA. The study also sought to characterize potential virulence functions of TcfA. It is known that TcfA contributes to the pathogenesis of B. pertussis, but its specific role remains to be elucidated. This study used modeling to provide support for the theory that TcfA binds Factor H in B. pertussis. However, a factor H surface binding assay determined that TcfA is not the only factor that binds the complement regulatory protein Factor H in B. pertussis. Another uniquely structured AT, BapB, was hypothesized as the potential additional factor that binds Factor H. However, additional studies are required to determine the importance of BapB. Furthermore, the study determined that TcfA does not play a large role in the serum survival of B. pertussis. In summary, this thesis characterized the secretion and some potential virulence functions of TcfA, but it also raised many additional questions.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Gustavsson, Martin. "Influence of recombinant passenger properties and process conditions on surface expression using the AIDA-I autotransporter." Doctoral thesis, KTH, Bioprocessteknik (stängd 20130101), 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-122230.
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Surface expression has attracted much recent interest, and it has been suggested for a variety of applications. Two such applications are whole-cell biocatalysis and the creation of live vaccines. For successful implementation of these applications there is a need for flexible surface expression systems that can yield a high level of expression with a variety of recombinant fusion proteins. The aim of this work was thus to create a surface expression system that would fulfil these requirements. A novel surface expression system based on the AIDA-I autotransporter was created with the key qualities being are good, protein-independent detection of the expression through the presence of two epitope tags flanking the recombinant protein, and full modularity of the different components of the expression cassette. To evaluate the flexibility of this construct, 8 different model proteins with potential use as live-vaccines or biocatalysts were expressed and their surface expression levels were analysed. Positive signals were detected for all of the studied proteins using antibody labelling followed by flow cytometric analysis, showing the functionality of the expression system. The ratio of the signal from the two epitope tags indicated that several of the studied proteins were present mainly in proteolytically degraded forms, which was confirmed by Western blot analysis of the outer membrane protein fraction. This proteolysis was suggested to be due to protein-dependent stalling of translocation intermediates in the periplasm, with indications that larger size and higher cysteine content had a negative impact on expression levels. Process design with reduced cultivation pH and temperature was used to increase total surface expression yield of one of the model proteins by 400 %, with a simultaneous reduction of proteolysis by a third. While not sufficient to completely remove proteolysis, this shows that process design can be used to greatly increase surface expression. Thus, it is recommended that future work combine this with engineering of the bacterial strain or the expression system in order to overcome the observed proteolysis and maximise the yield of surface expressed protein.QC 20130516
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Aragão, Annelize Zambon Barbosa 1984. "Vat (vacuolating autotransporter toxin) produzida por APEC (avian pathogenic Escherichia coli) = efeitos intracelulares e distribuição filogenetica." [s.n.], 2010. http://repositorio.unicamp.br/jspui/handle/REPOSIP/317304.
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Orientador: Tomomasa YanoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-15T17:06:12Z (GMT). No. of bitstreams: 1 Aragao_AnnelizeZambonBarbosa1984-_M.pdf: 1442209 bytes, checksum: 20b02ae5cd0351830e8e623b5e9c17a4 (MD5) Previous issue date: 2010
Resumo: Escherichia coli isoladas de lesões de celulite aviária em frangos de corte produzem uma citotoxina que causa intensa vacuolização citoplasmática em células de origem aviária, mas não em células de mamíferos. Esta citotoxina foi denominada Vat (vacuolating autotransporter toxin) e apresenta massa molecular de 56 kDa e ponto isoelétrico de 6,37. Estudos preliminares comprovaram que a Vat induz, em frangos de corte, respostas inflamatórias liberando as citocinas TNF-a e IL-10, indicando o seu papel relevante no desenvolvimento da celulite aviária. A dose citotóxica para 50% das células foi determinada em 40 µg.mL-1. A Vat aplicada sobre cultura de células FEG (fibroblasto de embrião de galinha) induz perda da viabilidade em 50% células após 18 horas de ensaio. Ensaios com a toxina indicam que, após 24 horas, os lisossomos mostram-se bastante comprometidos (37% viáveis), a viabilidade mitocondrial decresce e se mantém durante as 24 horas do ensaio em cerca de 55%, havendo uma redução acentuada das mitocôndrias viáveis após 36 horas da aplicação da Vat. A membrana plasmática das células FEG também sofre alterações de integridade, liberando 66,7 unidades de LDH (lactato desidrogenase)/mL, após 12 horas de ensaio (pico máximo de liberação, que corresponde a 51,7% do conteúdo total de LDH em células FEG). A Vat induz alterações no citoesqueleto das células FEG e os ensaios com o marcador fluorescente Acridine Orange indicam que há um aumento de RNA no citoplasma das células. Neste trabalho o gene vat foi detectado em amostras de APEC (filogrupos A e B1), UPEC e SEPEC (ambas em filogrupos B2 e D). Todas as amostras positivas para vat foram examinadas em células, sendo que 54% (27/50) apresentaram atividade vacuolizante em células FEG. Nossos resultados indicam que a Vat é uma toxina que induz diversas ações intracelulares, podendo ser chamada de toxina multifuncional
Abstract: Escherichia coli isolated from avian cellulitis lesions in broilers produce a cytotoxin that causes intense vacuolation in avian cells, but not in mammals cells. This cytotoxin, called Vat (vacuolating autotransporter toxin), has a 56 kDa protein and has a isoeletric point of 6.37. Preliminary studies have shown the Vat induce inflammatory response in broilers, by releasing cytokines TNF-a and IL-10, what indicates it can act in the avian cellulitis development. The cytotoxic dose for 50% of the cells was fixed at 40 µg.mL-1. The tests using CEF (chicken embryo fibroblasts) cells indicate Vat leads to cell viability loss in 50% of the cells after 18 hours. The tests with the toxin indicate that after 24 hours the lysosomes are 37% viable, the mitochondrial viability decreases after 24 hours (about 55%), with a remarkable reduction of viable mitochondria after 36 hours of Vat application. The CEF cells' plasma membrane of also loses integrity, releasing 66.7 units LDH (lactato dehydrogenase)/mL, after 12 hours of test (which is 51.7% of the total LDH content in CEF cells). The CEF cells' cytoskeleton also changed when exposed to Vat. Essays involving the fluorescent dye Acridine Orange indicate a RNA increase in the cytoplasm of cells. In this work the vat gene was detected in samples of APEC (A and B1 philogroups), UPEC and SEPEC (B2 and D philogroups). All vat positive samples have been tested for vat in cells, and 54% (27/50) of them have shown vacuolizing activity in CEF cells. The results indicate Vat is a toxin which induces various intracellular actions, therefore it can be called a multifunctional toxin
Mestrado
Microbiologia
Mestre em Genética e Biologia Molecular
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Ngwamidiba, Maxime. "Etude moléculaire des gènes SCA1 et SCA2 codant des protéines autotransporteurs chez les membres du genre " rickettsia"." Aix-Marseille 2, 2006. http://www.theses.fr/2006AIX20660.
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Des analyses d'ADN sur les restes des soldats de la Grande Armée de Napoléon (1812) ont révélé la présence entre autre de Rickettsia prowazekii. Pourtant la rickettsiologie ne commençera qu'avec les travaux de Ricketts et von Prowazek en 1910, et ne cessera de s'alimenter d'espèces et de pathologies nouvelles. En tant que premières bactéries intracellulaires strictes décrites, la taxonomie des rickettsies rassemblait initialement sur la base de ce critère, un grand nombre de genres bactériens ultérieurement reclassés avec l'avènement du séquençage et la découverte d'horloges moléculaires telle que la sous-unité 16S de ARN ribosomique ou le cytochrome C. Pour l'identification des espèces de Rickettsia, de nombreux critères phénotypiques dont la morphologie, les tests de fixation du complément, de neutralisation de toxines, de sérotypage et les profils protéiques ont longtemps été utilisés. Mais c'est la comparaison des séquences de gènes, dont ompA, ompB et sca4, qui ont permis d'identifier très précisément les espèces du genre Rickettsia et de proposer une classification phylogénique fiable. Cependant, la position phylogénique d'espèces telles que Rickettsia helvetica, Rickettsia canadensis et Rickettsia bellii n'a pu être déterminée avec certitude. Aussi, l'analyse basée sur la concaténation de plusieurs gènes, associée aux caractères phénotypiques peut constituer une meilleure alternativeThe history of rickettsioses is probably as ancient as human civilisation. The first documented cases of rickettsioses dates back to 1812. In early part of the last century (1910) Ricketts and von Prowazek laid the foundation of modern rickettsiology. Their pioneering works eventually led to the recognition of new species and Rickettsiales infections. As soon as Rickettsia are the first strictly intracellular bacteria described, its taxonomy gathered on the basis of this criterion, and a great number of kinds of bacteria which will be identified only with the advent of the sequencing and the discovery of molecular clocks such as ribosomal 16S RNA and cytochrome C. Many phenotypic criterion such as morphology, tests of complement, neutralization of toxins, mousse serotyping and SDS-page proved reliable. However, gene comparison (ompA, ompB and sca4) will make it possible to very precisely determine the species containing of the genus Rickettsia and to suggest a classification supported by high bootstrap values as well as antibiotics tests. Nevertheless, the phylogenetic position of species such Rickettsia helvetica, Rickettsia canadensis and Rickettsia bellii could not be given with precision, and the polyphasic analysis of the classification of the Rickettsia species based on genes concatenation associated with phenotypic characters available might be alternatives for Rickettsia phylogeny
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Shahid, Shakeel Ahmad [Verfasser]. "Structure and function of the autotransporter protein YadA : a solid-state MAS NMR study / Shakeel Ahmad Shahid." Berlin : Freie Universität Berlin, 2012. http://d-nb.info/1030383162/34.
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Ashawesh, Mahmoud. "Investigation of the spiral secretion pattern of the serine protease autotransporter, EspC, using innovative fluorescent labelling approaches." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/38029/.
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Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen belonging to the Enterobacteriaceae and considered to be a leading cause of acute infantile diarrhoea in developing nations. During onset of infection, EPEC inject a variety of effector proteins into the host epithelial cells using the type III secretion system (T3SS). These effectors are encoded within the pathogenicity island (PAI) called the locus of enterocyte effacement (LEE). EPEC also secretes the non-LEE encoded serine protease EspC (EPEC secreted protein C) into the extracellular milieu. This autotransporter (AT) exits EPEC through a type V secretion system (T5SS) and is subsequently delivered into host cells by the T3SS. The precise steps by which EspC is secreted are still unknown. By using both traditional and advanced optical microscopes, it is shown here that EspC tagged with two different fluorescent labels locates to a structure that resembles the spiral bacterial cytoskeleton when it has its β-barrel domain attached. It was discounted that the spiral formation was an artefact of aggregation. Moreover, production of another AT (AaaA, derived from Pseudomonas aeruginosa) also generated spiral structures that resemble the ones observed with EspC. Perturbing the structure of the bacterial cytoskeleton actin homologue MreB with the S-(3,4-dichlorobenzyl) isothiourea compound (A22) disrupted the localization of EspC-mCherry. Furthermore, producing EspC-mCherry in an E. coli SecA mutant generated altered localization patterns. Collectively, these results indicate that the spiral localization of EspC is dependent upon its C-terminal β-barrel domain, the Sec translocon and the actin homologue MreB and this spiral secretion pathway seems to be conserved among ATs. Further analysis is required to reveal the molecular mechanism underlying spiral formation to unravel the mystery of AT secretion, thereby enabling the development of new therapeutic targets and treatments.
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Sakamoto, Chizuko. "Study of the regulatory network of the surface autotransported adhesin antigen 43 in escherichia coli." Paris 7, 2013. http://www.theses.fr/2013PA077205.
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Ag43 est une protéine de la membrane externe permettant à E. Coli d'auto-agréger. Elle promeut la formation de biofilms et est impliquée dans la persistance de souches d' E. Coli uropathogènes dans le tractus urinaire. Cette adhésine est régulée par un mécanisme de variation de phase très bien décrit : l'expression d'agn43 résulte de la compétition entre la méthylase Dam (activateur) et du régulateur transcriptionnel OxyR (represseur). C'est pourquoi des cellules clonales peuvent soit exprimer agn43 (phase ON) ou non (phase OFF). A l'heure de l'écriture de cette thèse, ce mécanisme reste le seul connu pour la régulation d'agn43, malgré que le rôle de la variation de phase in vivo soit toujours à l'étude. Bien que le 'switch' de phases d'agn43 conduit à une population hétérogène de bactéries ON et OFF, les études menées sur Ag43 considèrent rarement les biais potentiels associés à la variation de phase. Nous avons donc suivi le status ON / OFF d'agn43 génétiquement et phénotypiquement, et nous montrons que l'utilisation de populations ayant aléatoirement un status ON ou OFF pour agn43 peut entraîner des conclusions erronées sur la fonction ou de la régulation d'Ag43. En particulier, nous démontrons que Lrp et MqsR , précédemment identifiés comme des régulateurs d'agn43, ne régulent pas l'expression ou fréquence de commutation ON / OFF d'agn43. Nous montrons également que la formation de biofilms en conditions de flux dynamique n'influence pas la commutation ON / OFF, mais sélectionne physiquement les cellules agrégeant grâce à Ag43. Cela indique qu'une mauvaise interprétation est possible lorsque l'on étudie l'expression de tels gènes au sein de biofilms. De plus, nous apportons la preuve qu'ignorer l'état agn43 ON / OFF initial d'une population d'E. Con donnée risque de biaiser l'analyse des phénotypes associés à d'autres adhésines d'E. Coli, soulignant ainsi l'importance du suivi de la variation de phase d'Ag43. D'autre part, nous montrons que le système à deux composants CpxAR (ou Cpx), impliqué dans la réponse aux stress périplasmiques, peut moduler la fonction d'auto'-agrégation médiée par Ag43. Nous avons déterminé que le système Cpx ne régule pas directement l'expression d'Ag43 , ni sa stabilité, ni sa translocation à la surface mais plutôt indirectement par un mécanisme de « masquage / démasquage » via l'expression de firnbriae de type 1 (opéronfim), une autre adhésine majeure d'E. Coli également régulée par un mécanisme de variation de phase. De plus, nous avons montré que l'expression defim induit le système Cpx via la lipoprotéine N1pE, un inducteur du système Cpx, qui est impliquée dans la détection des surfaces, fournissant la première preuve d' une rétroaction négative des fimbriae de type 1 sur leur propre expression. En outre, nos résultats tendent à montrer qu'OxyR est aussi un répresseur de l'expression defim faisant de lui un régulateur clé des adhésines 'phase-variablez' d'E. Coli (Ag43 et Fim). Cette étude fournit donc un nouvel aperçu de la régulation d'Ag43 en particulier en ce qui concerne ses interactions avec d'autres adhésinesAg43 is an outer-membrane protein enabling E. Coli to auto-aggregate. It promotes the formation of biofilms and is implicated in the persistence of uropathogenic E. Coli strains in the urinary tract. This adhesin is regulated by a mechanism of phase variation which is very well-documented : the expression of agn43 results of the competition between the Dam methylase (activator) and the transcriptional regulator OxyR (repressor). Fiente, sister-cells can either express agn43 (phase ON) or not (phase OFF). Up-to-date, this is the only known mechanism of regulation of agn43, although the in vivo role of phase variation is still poorly understood. Although the agn43 regulatory switch leads to a heterogeneous population of ON and OFF bacteria, studies of Ag43 seldom consider potential biases associated with phase variation. We monitored agn43 ON/OFF phase-variation status genetically and phenotypically and we show that the use of populations with random agn43 ON or OFF status could result in misleading conclusions about Ag43 function or regulation. In particular, we demonstrate that Lrp and MqsR, previously identified as agn43 regulators, do not regulate agn43 expression or ON/OFF switch frequency. We also show that biofilm formation in dynamic flow conditions does not influence agn43 ON/OFF switching but physically selects aggregating agn43 ON cells. This indicates that misinterpretation is possible when studying gene expression within biofilms. Moreover, we provide evidence that ignoring the initial agn43 ON/OFF status of the E. Coli populations studied is likely to bias analyses of phenotypes associated with other E. Coli adhesins thereby emphasizing the importance of monitoring Ag43 phase-variation. On another hand, we show that the two-component system CpxAR (or Cpx), implicated in periplasmic stress response, can modulate the function of Ag43- mediated auto-aggregation. We have determined that the Cpx system is not directly regulating Ag43 expression, stability nor its translocation to the surface but rather indirectly by a `masking/unmasking' mechanism via the expression of type 1 fimbriae (fim operon), another major phase-variable adhesin of E. Coli. Moreover, we have shown that the expression offim induces the Cpx system via the lipoprotein N1pE, inducer of the Cpx system, which is involved in surface sensing, providing the first evidence of a negative feedback of type 1 fimbriae on their own expression. Furthermore, our results tend to show that OxyR is also a repressor offim expression making it a master regulator of the phase-variable adhesins of E. Coli (Ag43 and Fim). This study therefore provides a new insight into Ag43 regulation in particular regarding its interactions with other adhesins
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Casasanta, Michael Anthony. "Laying the Genetic and Molecular Foundation for the Study of Fusobacterium Nucleatum in Relation to Human Health and Disease." Diss., Virginia Tech, 2019. http://hdl.handle.net/10919/88484.
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Fusobacterium nucleatum is a Gram-negative, anaerobic bacterium that is a member of the human oral microbiota. Although it is a normal resident of the mouth, it is associated with a number of human diseases including: sepsis, inflammatory bowel disease (IBD), and colorectal cancer (CRC). Despite the important association of F. nucleatum with human health and disease, remarkably little is known about the molecular mechanisms underlying these infections. This knowledge gap can, in part, be attributed to a lack of molecular tools and experimental workflows. Creating the genetic tools to fill this knowledge gap is an imperative undertaking for the future development of treatments for diseases involving F. nucleatum. Previous work in the field has assigned functions to just a handful of Fusobacterium proteins (Fap2, FadA), and only two of those proteins have a well-defined role in the host-pathogen relationship. This dissertation contains work that lays the molecular and genetic foundation for future studies involving F. nucleatum by creating a unique gene deletion system while simultaneously establishing broadly applicable experimental workflows and molecular tools to study initial bacterial attachment and invasion processes crucial to Fusobacterium virulence. Marker-less gene deletions confirm the importance of Fap2 in host-cell attachment and invasion and suggest a lesser role in invasion for FadA, representing a significant revision to the Fusobacterium-host relationship. Also, our system allows for the overexpression and purification of virulence factors directly from Fusobacterium for the first time. This permits us to study aspects of Fusobacterium protein biology that were previously impossible and will provide further insights into the nature of Fusobacterium virulence. A custom suite of molecular tools was also developed to facilitate recombinant expression of these proteins in general laboratory settings using simple E. coli protein expression systems. We have used these new technologies to express and purify a number of potential Fusobacterium virulence factors as detailed in this dissertation.
Also contained in this dissertation is the application of these breakthroughs to probe the function of a novel F. nucleatum outer membrane phospholipase, FplA. Phospholipases are important virulence factors in a number of well-studied human pathogens including Pseudomonas aeruginosa and Legionella pneumophila, where they interfere with host cellular signaling processes to increase intracellular bacterial survival. Our data show that FplA is a Class A1 phospholipase (PLA1) with robust catalytic activity capable of binding to and cleaving a number of lipid types. Additionally, we show that it has the ability to bind to important host signaling lipids including phosphatidylinositol 3, 5-bisphosphate and phosphatidylinositol 3, 4, 5-triphosphate. These data suggest FplA may play a role in manipulating the intracellular processes of host cells. Taken together, work in this dissertation provides tools and experimental frameworks for the future study of F. nucleatum pathogenesis while identifying and initially characterizing a new, potentially significant, virulence factor in FplA.Doctor of Philosophy
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Hernández, Casas Luis Arturo. "ANÁLISIS COMPARATIVO DE LA REGULACIÓN DEL AUTOTRANSPORTE DE MERCANCÍAS ENTRE MÉXICO Y ESTADOS UNIDOS, 1980-2016." Tesis de Licenciatura, Universidad Autónoma del Estado de México, 2018. http://hdl.handle.net/20.500.11799/95307.
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En el presente trabajo, se analizará y comparará el marco regulatorio del envío de mercancías vía terrestres en México y Estados Unidos, 1980- 2016, con la finalidad de probar la hipótesis la cual versa sobre lo siguiente: una adecuada implementación de la regulación existentes en el envío de mercancías permitiría abrir nuevos mercados, articular regiones y desarrollar el comercio nacional e internacional, en México y Estados Unidos
2 Outsourcing: también conocidos como subcontratación de servicios para realizar operaciones de la empresa, consiguiendo un número de clientes mayor
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Para ello el trabajo se ha dividido en cuatro capítulos en los cuales se aborda lo siguiente. Como primer capítulo se desarrollan los conceptos y tipologías de logística, con el propósito de dar a conocer la diferencia entre cada una de ellas. En un segundo capítulo se habla del transporte de mercancías a nivel internacional, mostrando los diferentes tipos de transporte y sus respectivos organismos reguladores, resaltando como se ha ido transformando. El tercer capítulo analiza de las regulaciones vigentes en el autotransporte de carga tanto en Estados Unidos como en México, así mismo se detecta lo que falta hacer y conocer. En un cuarto capítulo se desarrolla un análisis comparativo de las regulaciones imperantes, así como una discusión de los que se detecta falta hacer. Lo anterior permite sustentar la necesidad de crear un plan no sólo de ampliación de la infraestructura sino también de conservación a largo plazo de la ya existente para sacar el máximo provecho a este medio de transporte, pero sobre todo dar a conocer las diferentes regulaciones que existen y como se puede generar un mayor conocimiento de leyes y reglamentos del transporte de mercancías.
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Medina, Estrada Juan Antonio. "“TIPIFICAR EN EL CÓDIGO PENAL FEDERAL LA PRESTACIÓN DEL SERVICIO PÚBLICO DE AUTOTRANSPORTE DE PASAJEROS SIN AUTORIZACIÓN”." Tesis de Licenciatura, Universidad Autónoma del Estado de México, 2013. http://hdl.handle.net/20.500.11799/67176.
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El Autotransporte Federal de Pasajeros, es el medio de transporte que traslada a los usuarios de un lugar a otro, a través de carreteras federales, el cual ha sido y es, durante todavía bastante tiempo, el medio de transporte más utilizado y por ende el más importante para el movimiento de personas.
El sector del Autotransporte, es de gran importancia en la economía del país, al ser el medio por el cual la mayor parte de la población se traslada hacia sus lugares de trabajo, a lugares de esparcimiento entre otros destinos, esta movilidad de masas en relación a los ingresos económicos que representa, ha dado lugar a la aparición y a la existencia de autotransportistas que compiten deslealmente con aquellos que lo prestan de manera legal, ya que los primeros realizan una actividad sin tener el permiso otorgado por la Secretaría de Comunicaciones y Transportes, lo cual provoca que la sociedad quede expuesta y en un estado de indefensión en contra de las conductas que se generan al realizar esta ilícita actividad.
Por ello, una persona que preste servicios de Autotransporte Federal de Pasajeros en un vehículo que circula sin cumplir con los ordenamientos aplicables, y que además no tiene permiso correspondiente, debe traducirse no sólo en una violación a la ley, sino que además, vulnera la intención de seguridad y prevención que ha establecido el legislador a lo largo de la historia, cuyo efecto es salvaguardar y conservar el orden en el Sociedad y del Estado de Derecho, respecto de este servicio.
Seguir permitiendo este tipo de conductas sin algún tipo de sanción eficaz, provoca que otros las observen y las realicen, ya que las posible multas que se imponen, puedan ser pagadas o impugnadas, en este sentido, lo anterior no interrumpe de manera definitiva la prestación del servicio en esas condiciones, ya que éste se puede seguir prestando aun cuando no se page o cuando aún todavía no se resuelva la situación jurídica de la impugnación de la sanción.
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Turner, David Patrick James. "Identification and characterisation of autotransported serine protease A (AspA) in Neisseria meningitidis : a novel subtilisin-like serine protease." Thesis, University of Nottingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272370.
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Fleetwood, Filippa. "Bacterial display systems for engineering of affinity proteins." Doctoral thesis, KTH, Proteinteknologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-156420.
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Directed evolution is a powerful method for engineering of specific affinity proteins such as antibodies and alternative scaffold proteins. For selections from combinatorial protein libraries, robust and high-throughput selection platforms are needed. An attractive technology for this purpose is cell surface display, offering many advantages, such as the quantitative isolation of high-affinity library members using flow-cytometric cell sorting. This thesis describes the development, evaluation and use of bacterial display technologies for the engineering of affinity proteins. Affinity proteins used in therapeutic and diagnostic applications commonly aim to specifically bind to disease-related drug targets. Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is a critical process in various types of cancer and vascular eye disorders. Vascular Growth Factor Receptor 2 (VEGFR2) is one of the main regulators of angiogenesis. The first two studies presented in this thesis describe the engineering of a biparatopic Affibody molecule targeting VEGFR2, intended for therapeutic and in vivo imaging applications. Monomeric VEGFR2-specific Affibody molecules were generated by combining phage and staphylococcal display technologies, and the engineering of two Affibody molecules, targeting distinct epitopes on VEGFR2 into a biparatopic construct, resulted in a dramatic increase in affinity. The biparatopic construct was able to block the ligand VEGF-A from binding to VEGFR2-expressing cells, resulting in an efficient inhibition of VEGFR2 phosphorylation and angiogenesis-like tube formation in vitro. In the third study, the staphylococcal display system was evaluated for the selection from a single-domain antibody library. This was the first demonstration of successful selection from an antibody-based library on Gram-positive bacteria. A direct comparison to the selection from the same library displayed on phage resulted in different sets of binders, and higher affinities among the clones selected by staphylococcal display. These results highlight the importance of choosing a display system that is suitable for the intended application. The last study describes the development and evaluation of an autotransporter-based display system intended for display of Affibody libraries on E. coli. A dual-purpose expression vector was designed, allowing efficient display of Affibody molecules, as well as small-scale protein production and purification of selected candidates without the need for sub-cloning. The use of E. coli would allow the display of large Affibody libraries due to a high transformation frequency. In combination with the facilitated means for protein production, this system has potential to improve the throughput of the engineering process of Affibody molecules. In summary, this thesis describes the development, evaluation and use of bacterial display systems for engineering of affinity proteins. The results demonstrate great potential of these display systems and the generated affinity proteins for future biotechnological and therapeutic use.QC 20141203
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Gawarzewski, Iris [Verfasser], and Joachim [Akademischer Betreuer] Jose. "Determination of the AIDA-I ß-barrel crystal structure: getting the clue to the autotransporter secretion pathway / Iris Gawarzewski. Gutachter: Joachim Jose." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2014. http://d-nb.info/1046404458/34.
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Beckett, Brian. "Structural aspects of adhesin involved in diffuse adherence (AIDA-I) and autotransporter adhesin heptosyltransferase (Aah) and their roles in «Escherichia coli» aggregation." Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=121300.
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Aggregated Escherichia coli (E. coli) expressing the adhesin involved in diffuse adherence (AIDA-I) cause severe cases of fatal neonatal diarrhea. AIDA-I is a glycoprotein, located in the outer membrane of Gram negative E. coli. It causes bacterial aggregation. Autotransporter adhesin heptosyltransferase (Aah) is an E. coli glycosyltransferase that glycosylates AIDA-I. In the first part of this M.Sc. thesis, we observed both non-aggregated and aggregated AIDA-I molecules by single particle transmission electron microscopy and electron tomography in order to provide insights into the poorly understood mechanism of AIDA-I-mediated attachment. Our analysis showed that non-aggregated AIDA-I were elongated molecules ~320 Å long and ~50 Å wide composed of a single AIDA-I polypeptide. While the length and thickness of the molecules were constant, we observed individual molecules with variable curvatures indicating the molecule is flexible. Our results suggest the presence of different structural domains in AIDA-I, resembling 5 "beads on a string" in 2-D averages. At low concentration of β-octylglucoside, AIDA-I molecules aggregated into "spider"-shaped oligomers with elongated "arms" looking similar to monomeric AIDA-I. Interactions between the arms of adjacent oligomers were indicative of the cis-trans interaction of AIDA molecules.In the second part of this M.Sc. thesis, we observed the Aah molecules. Imaging of Aah showed that it assembles into a six fold symmetric ring-shaped complex of ~200 Å in diameter with a ~160 Å pore. Given the 660 kDa measured molecular weight by gel filtration, it is likely that is ring-shaped complexes observed are dodecamers. Aah is the only bacterial glycosyltransferase to have been reported so far to assemble into an oligomer.Taken together, our study provides valuable information on the molecular arrangement of AIDA-I and Aah, and provides the building stones for further studies on the interaction AIDA-I between cells as well as on the glycosylation of AIDA by Aah.E. coli exprimant l'adhésine impliquée dans l'adhérence diffuse (AIDA-I) causent de graves cas de diarrhée néonatale fatale. AIDA-I est une glycoprotéine, située dans la membrane externe de Gram négatif E. coli. Elle provoque l'agrégation bactérienne. L'autotransporteur adhésine heptosyltransférase (Aah) est une glycosyltransférase chez E. coli qui glycosyle AIDA-I.Dans la première partie de ce mémoire de maîtrise, nous avons observé les molécules d'AIDA sous leurs formes non agrégées et agrégées par microscopie électronique, particules isolées et tomographie électronique afin de donner un aperçu sur le mécanisme d'attachement AIDA. Notre analyse a montré que les molécules AIDA-I sous leurs formes non agrégées étaient des molécules allongées mesurant environ 320 Å en longueur et 50 Å en largeur, composées d'un seul polypeptide AIDA-I. Alors que la longueur et l'épaisseur des molécules sont constantes, nous avons observé des molécules individuelles avec des courbures variables indiquant que la molécule est flexible. Nos résultats suggèrent la présence de différents domaines structuraux dans Aida-I, ressemblant à 5 « perles sur une chaîne » dans nos moyennes deux-dimensionnels. À faible concentration de β-octoglucoside, les AIDA-I molécules s'organisent oligomères ressemblant des « araignées » avec « bras » allongés. Ceux-ci sont d'apparence similaire aux monomères d' AIDA -I. Des interactions entre les bras d'oligomères adjacents ont été observées. Elles pourraient correspondre à l'interaction trans-cis de molécules AIDA-I.Dans la deuxième partie de ce mémoire de maîtrise, nous avons observé les molécules de l'Aah. La visualisation de l'Aah a montré que Aah assemble en un complexe six fois symétrique en forme d'anneau de ~ 200 Å de diamètre avec un trou de ~ 160 Å. Étant donné le poids moléculaire mesuré par filtration sur gel de était de 660 kDa, il est probable que les complexes en forme d'anneau observés sont des dodecamères. Aah est la première glycosyltrsanferase bactérienne jusqu'à lors rapportée à assembler en un oligomère. Pris ensemble, notre étude fournit des renseignements précieux sur l'arrangement moléculaire de AIDA-I et Aah et fournit les pierres de construction pour poursuivre des études sur l'interaction AIDA-I entrecellules, ainsi que sur la glycosylation d'AIDA-I par Aah.
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Bräutigam, Cornelia [Verfasser], and I. B. [Akademischer Betreuer] Autenrieth. "BamB, BamC und BamE und ihre Bedeutung für die Biogenese der Autotransporter-Adhäsine YadA, Invasin und Intimin / Cornelia Bräutigam ; Betreuer: I. B. Autenrieth." Tübingen : Universitätsbibliothek Tübingen, 2017. http://d-nb.info/1199463523/34.
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Gailiūnaitė, Ernesta. "Autotransporto triukšmo tyrimai Kėdaniuose." Bachelor's thesis, Lithuanian Academic Libraries Network (LABT), 2010. http://vddb.laba.lt/obj/LT-eLABa-0001:E.02~2010~D_20100903_081757-16993.
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Autotransportas yra pagrindinis miesto triukšmo šaltinis. Didėjant automobilizacijai, didėja ir triukšmo lygis. Leistinieji triukšmo lygiai viršijami ne tik miesto centre, bet ir miesto viešosiose tyliosiose zonose, mokyklų, darželių – lopšelių, gyvenamųjų namų teritorijose. Dėl šių priežasčių yra trikdomas poilsis ir kenkiama sveikatai. Atliekant autotransporto triukšmo tyrimus Kėdainių mieste pasirinkta 16 vietų: mokyklų, darželių – lopšelių, viešųjų tyliųjų miesto zonų, parkų, ligoninės, gyvenamųjų namų teritorijose. Lyginant su higienos norma gauti rezultatai, leidžiantys parašyti išvadas ir pateikti pasiūlymus triukšmo mažinimui. Mažinant autotransporto triukšmo sklaidą Kėdainių mieste būtina taikyti prevencines ir triukšmo mažinimo priemones. Mažinti automobilių kiekį mieste, įrengti dviračių ir pėsčiųjų trasas, dažniau naudotis viešuoju transportu.Transport is a major source of urban noise. Automobilization increases, and increases the noise level. The permissible noise levels are exceeded, not only downtown, but quiet in public and urban areas, schools and nurseries - Nursery school and residential areas. For those reasons, interfere with recreation and detrimental to health. The road noise in Kedainiai selected 16 places: schools, nursery - Nursery school, public dormant urban areas, parks, hospitals, residential areas. Compared with the hygiene rule, the results obtained allow to write the findings and suggestions for noise reduction. Reducing motor noise in the dissemination of Kedainiai need for preventive measures and noise reduction. Reduce the amount of parking in the city, equipped with bicycle and pedestrian trails, a greater use of public transport.
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Kmitas, Evaldas. "Autotransporto keliama oro cheminė tarša Kelmėje." Master's thesis, Lithuanian Academic Libraries Network (LABT), 2011. http://vddb.laba.lt/obj/LT-eLABa-0001:E.02~2011~D_20110602_120656-97255.
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Kiekvienasis metais vis didėjant autotransporto kiekiui keliuose, didėja ir jų į aplinką išmetamų teršalų kiekiai. Todėl galime numanyti, jog Lietuvoje autotransportui išaugus 2,5 karto per paskutinį dešimtmetį tiek kartų išaugo ir į aplinką išmetamų kenksmingų deginių kiekiai, kurie labiausiai jaučiami didžiuosiuose miestuose ar miesteliuose. Greta Kelmės miesto yra keli dideli cheminės oro taršos šaltiniai: kelias Ryga-Kaliningradas, Kelmė – Raseiniai ir pagrindinė miesto sankryža, kur susidaro didžiausi teršalų kiekiai. Šiais keliais nuolat juda intensyvūs autotransporto srautai, kurių dalis patenka į Kelmės miestą. Todėl buvo atliktas autotransporto srautų įtakos Kelmės miesto aplinkos orui tyrimas: ištirti autotransporto srautai Kelmėje; išmatuota anglies monoksido kiekiai ore; teoriškai apskaičiuoti autotransporto amžiaus ir jo eksploatacinių parametrų įtaka deginių kiekiui ir jų sudėčiai; eksperimentiškai ištirta autotransporto cheminė tarša.The volume of vehicle traffic, growing every year, increases the emissions of toxic substances and pollutes the environment. Consequently, the road traffic in Lithuania has increased by 2.5 times over the last decade. Big cities and small towns have noticed the effect of harmful exhaust emissions on the environment and people health most. A large number of chemical sources of air pollution are located near Kelme city: road Riga – Kaliningrad, Kelme – Raseiniai and the main city crossroads, which produces the most significant contaminants. These roads are constantly moving the intense flows of motor vehicles, the part of which falls within the town of Kelme. Therefore the aim of our research was to study the impact of the road traffic on the air quality in Kelme city, to investigate the traffic flows, to measure the concentration of carbon monoxide in the air, theoretically calculate the impact of vehicle age and its operating parameters on the exhaust emissions and its composition. The experimental research revealed the chemical pollution of the vehicles .
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Andersson, Ken G. "Combinatorial Protein Engineering Of Affibody Molecules Using E. Coli Display And Rational Design Of Affibody-Based Tracers For Medical Imaging." Doctoral thesis, KTH, Proteinteknologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-213451.
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Directed evolution is today an established strategy for generation of new affinity proteins. This thesis describes the development of a cell-display method using Escherichia coli for directed evolution of Affibody molecules. Further, the thesis describes rational design of Affibody-based tracers, intended for future patient stratification using medical imaging. Fusing recombinant proteins to various autotransporters is a promising approach for efficient surface display on the surface of E. coli, as well as for construction of high-complexity libraries. In paper I, we successfully engineered an expression vector for display of Affibody molecules using the autotransporter AIDA-I. In paper II, a large Affibody library of 2.3x109 variants was constructed and screening using FACS resulted in new specific binders in the nanomolar range. In paper III, we demonstrated Sortase-mediated secretion and conjugation of binders directly from the E. coli surface. The three following studies describe rational design of Affibody-based tracers against two cancer-associated targets for molecular imaging. First, anti-HER3 Affibody molecules were labelled with 111In, and SPECT imaging showed that the conjugates specifically targeted HER3-expressing xenografts. Furthermore, labeling with 68Ga for PET imaging showed that tumor uptake correlated with HER3 expression, suggesting that the tracers have potential for patient stratification. The last study describes the development and investigation of anti-EGFR Affibody-based imaging agents. Labeled with 89Zr, the Affibody tracer demonstrated higher tumor uptake at 3 h post injection than the anti-EGFR antibody cetuximab at 48 h post injection. In conclusion, this thesis describes new tools and knowledge that will hopefully contribute to the development of affinity proteins for biotechnology, therapy and medical imaging in the future.QC 20170904
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Bäcklund, Emma. "Impact of glucose uptake rate on recombinant protein production in Escherichia coli." Doctoral thesis, KTH, Bioprocessteknik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-34019.
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Escherichia coli (E. coli) is an attractive host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve high product quantities. In this thesis, the impact of the glucose uptake rate on the production of recombinant proteins was studied, aiming at improving and optimising production of recombinant proteins in E. coli. E. coli can be cultivated to high cell densities in bioreactors by applying the fed-batch technique, which offers a means to control the glucose uptake rate. One objective of this study was to find a method for control of the glucose uptake rate in small-scale cultivation, such as microtitre plates and shake flasks. Strains with mutations in the phosphotransferase system (PTS) where used for this purpose. The mutants had lower uptake rates of glucose, resulting in lower growth rates and lower accumulation of acetic acid in comparison to the wild type. By using the mutants in batch cultivations, the formation of acetic acid to levels detrimental to cell growth could be avoided, and ten times higher cell density was reached. Thus, the use of the mutant strains represent a novel, simple alternative to fed-batch cultures. The PTS mutants were applied for production of integral membrane proteins in order to investigate if the reduced glucose uptake rate of the mutants was beneficial for their production. The mutants were able to produce three out of five integral membrane proteins that were not possible to produce by the wild-type strain. The expression level of one selected membrane protein was increased when using the mutants and the expression level appeared to be a function of strain, glucose uptake rate and acetic acid accumulation. For production purposes, it is not uncommon that the recombinant proteins are secreted to the E. coli periplasm. However, one drawback with secretion is the undesired leakage of periplasmic products to the medium. The leakage of the product to the medium was studied as a function of the feed rate of glucose in fed-batch cultivations and they were found to correlate. It was also shown that the amount of outer membrane proteins was affected by the feed rate of glucose and by secretion of a recombinant product to the periplasm. The cell surface is another compartment where recombinant proteins can be expressed. Surface display of proteins is a potentially attractive production strategy since it offers a simple purification scheme and possibilities for on-cell protein characterisation, and may in some cases also be the only viable option. The AIDA-autotransporter was applied for surface display of the Z domain of staphylococcal protein A under control of the aidA promoter. Z was expressed in an active form and was accessible to the medium. Expression was favoured by growth in minimal medium and it seemed likely that expression was higher at higher feed rates of glucose during fed-batch cultivation. A repetitive batch process was developed, where relatively high cell densities were achieved whilst maintaining a high expression level of Z.QC 20110608
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Kochuparampil, Jumey. "Probing Individual Complexes of Self-Associating Autotransporters by Single Molecule Force Spectroscopy." Thesis, 2013. http://spectrum.library.concordia.ca/977272/16/MSc__Jumey_Kochuparampil.pdf.
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Self-associating autotransporters (SAAT) are proteins that play a major role in bacterial auto-aggregation. Members of this family include the adhesin involved in diffuse adherence (AIDA-I) and the enterotoxigenic invasion locus b (TibA) proteins, which represent powerful model systems to study how bacteria promote tight association leading to auto-aggregation. Herein, the homologous interactions of SAATs were systematically determined by using single molecule force spectroscopy (SMFS), which measures the force required to pull apart two interacting molecular entities at the piconewton scale. In this study, a detailed force curve evaluation and in depth histogram analyses were developed. The unbinding force of the AIDA-I/AIDA-I interaction was measured at different loading rates (ranging from 780 to 14000 pN/s). The rate of complex dissociation, koff (0.3 s-1), and the activation energy barrier distance, xb (0.4 nm) were calculated based on the rupture forces. This first single molecule study of the AIDA-I/AIDA-I interaction confirms a very strong self-association that is in line with the important role that SAATs play in the pathogenicity of bacteria. The TibA protein self-association showed a highly selective interaction similar to that of AIDA-I. These single molecule experiments are consistent with previously performed bulk measurements and indicate that self-association between the different SAAT proteins are more specific among the homo-protein pairs (AIDA-I/AIDA-I and TibA/TibA having an unbinding force of 52 ± 13 pN and 64 ± 14 pN, respectively, at a loading rate of 5020 pN/s) than in hetero-protein pairs (AIDA-I/TibA).
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Spahich, Nicole Ann. "A Tale of Two Proteins: Insights into the Haemophilus influenzae Hap and Hia Autotransporters." Diss., 2011. http://hdl.handle.net/10161/5002.
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Nontypeable Haemophilus influenzae (NTHi) is a common commensal in the human nasopharynx that can cause localized respiratory tract diseases such as otitis media, bronchitis, and pneumonia. NTHi adheres to respiratory epithelial cells, a critical step in the process of colonization enabled by bacterial surface adhesive structures called adhesins. One group of NTHi adhesins are autotransporters, proteins that have an N-terminal signal sequence, a C-terminal β-barrel domain, and an internal passenger domain with effector function. The goal of this work was to increase our understanding of two NTHi autotransporters, Hap and Hia.
Hap is a monomeric autotransporter that mediates adherence to epithelial cells and extracellular matrix (ECM) proteins. Hap also self-associates with protein on neighboring bacteria, resulting in bacterial aggregation and microcolony formation. The Hap passenger domain contains the regions responsible for adhesive activity. To define the molecular mechanism of Hap adhesive activity, we crystallized the Hap passenger domain. Characterization of the crystal structure revealed an N-terminal globular domain and a more ordered, prism-like C-terminal domain. Interestingly, Hap crystallized as a multimer, suggesting that Hap-Hap interactions occurred in the passenger domain. Progressive deletions of the β-loops that comprise the C-terminal region disrupted Hap-Hap interactions and led to a defect in bacterial settling. To further support that the C-terminal domain was responsible for Hap-Hap interactions,
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we purified the wild type and truncated passenger domains and conjugated the proteins to latex beads. By light microscopy we visualized bead aggregation when the wild type passenger domain was conjugated to the beads, but not when the truncated passenger domain was conjugated. These results show that the C-terminal portion of the Hap passenger domain is responsible for Hap-Hap interactions leading to multimerization. Hap multimerization could be important in microcolony formation that leads to biofilm formation in vivo.
The ECM binding domain in located in the final 511 amino acids of the Hap passenger domain. To pin-point the region of the ECM protein fibronectin that is recognized by Hap, we spotted small fragments of fibronectin onto nitrocellulose membranes and incubated the membrane with purified Hap passenger domain. Far Western analysis using Hap antibody revealed that the smallest fibronectin region necessary for binding was comprised of the first two type III repeats, FNIII(1-2). To define the regions of Hap responsible for interaction with fibronectin, we mutated motifs in the Hap passenger domain that are important for fibronectin binding in other bacterial proteins. Based on assessment by ELISA, many of the mutations located between amino acids 525-725 caused reduced bacterial binding to fibronectin. However, no mutation totally ablated binding, suggesting that a larger Hap region is involved in fibronectin binding.
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In an additional study, we identified a relationship between Hap levels in the outer membrane and the expression of lipopolysaccharide (LPS) biosynthesis enzymes. Through Western and qPCR analysis, we found that mutation of the rfaF, pgmB, lgtC, kfiC, orfE, rfbP, lsgB and lsgD genes involved in the synthesis of LPS oligosaccharide core in H. influenzae strain Rd/HapS243A resulted in loss of Hap in the bacterial outer membrane and a decrease in hap transcript. In contrast, the same mutations had no effect on outer membrane localization of H. influenzae P5 and IgA1 protease or levels of the p5 or iga1 transcripts, suggesting a Hap-specific effect. Elimination of the HtrA periplasmic protease resulted in a return of Hap to the outer membrane and restoration of wild type levels of hap transcript. We speculate that the lack of certain LPS biosynthesis enzymes causes Hap to mislocalize and accumulate in the periplasm, where it is degraded by HtrA. This degradation then leads to a decrease in hap transcript. lgtC is one of several phase variable LPS biosynthesis genes. Using an antibody against the epitope formed in part by the lgtC gene product, we identified lgtC phase-off bacteria by Western analysis of colony blots. Consistent with our previous observations, in lgtC phase off bacteria Hap was absent from the outer membrane and hap transcript was reduced. By analyzing a lgtC/lic2A double mutant, we found that Hap localization in the outer membrane and hap transcript levels were not related to LPS size but instead to the functions of the LPS synthesis enzymes themselves. This relationship could be beneficial to bacteria in vivo as a way to regulate Hap expression.
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Early models suggested that autotransporters do not require accessory factors for folding and OM insertion. However, mounting recent evidence has suggested that the Bam complex is required for OM localization of most β-barrel proteins, including autotransporters. We studied the role of the Bam complex in OM localization of the trimeric autotransporter Hia. We expressed Hia in E. coli strains with mutations in the Bam complex and found that BamA and BamD were needed for Hia localization, while BamB, BamC, and BamE were not necessary. In further studies, we mutated the C-terminus of Hia and found that the final and third-to-last amino acids were the most important for outer membrane localization.
In summary, this work provides insights into the regulation and adhesive activity of Hap and the outer membrane localization of Hia. We have learned important details about these factors that shed light on aspects of H. influenzae disease and could lead to new antimicrobial therapies.
Dissertation
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Chávez, Solís Dulce Yazmín, and Juárez Elí Angélica Silva. "Sistema de Información Contable-Fiscal para una Persona Física dedicada al Autotransporte de Carga en General, Estudio de caso: “Autotransportes Lozada”." Tesis de Licenciatura, 2014. http://hdl.handle.net/20.500.11799/30807.
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En la presente investigación se pretende aplicar un sistema de información contable – fiscal, el cuál consistirá en diseñar a Autotransportes Lozada una forma para ir adquiriendo una disciplina fiscal, esto es, ayudarle a manejar correctamente sus ingresos, costos y gastos, apegándose a las disposiciones fiscales vigentes.
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Charbonneau, Marie-Ève. "Étude de la biogenèse de l'autotransporteur AIDA-I d'Escherichia coli." Thèse, 2012. http://hdl.handle.net/1866/8561.
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Les autotransporteurs monomériques, appartenant au système de sécrétion de type V, correspondent à une famille importante de facteurs de virulence bactériens. Plusieurs fonctions, souvent essentielles pour le développement d’une infection ou pour le maintien et la survie des bactéries dans l’organisme hôte, ont été décrites pour cette famille de protéines. Malgré l’importance de ces protéines, notre connaissance de leur biogenèse et de leur mécanisme d’action demeure relativement limitée.
L’autotransporteur AIDA-I, retrouvé chez diverses souches d’Escherichia coli, est un autotransporter multifonctionnel typique impliqué dans l’adhésion et l’invasion cellulaire ainsi que dans la formation de biofilm et d’agrégats bactériens. Les domaines extracellulaires d’autotransporteurs monomériques sont responsables de la fonctionnalité et possèdent pratiquement tous une structure caractéristique d’hélice β. Nous avons mené une étude de mutagenèse aléatoire avec AIDA-I afin de comprendre la base de la multifonctionnalité de cette protéine. Par cette approche, nous avons démontré que les domaines passagers de certains autotransporteurs possèdent une organisation modulaire, ce qui signifie qu’ils sont construits sous la forme de modules fonctionnels.
Les domaines passagers d’autotransporteurs peuvent être clivés et relâchés dans le milieu extracellulaire. Toutefois, malgré la diversité des mécanismes de clivage existants, plusieurs protéines, telles qu’AIDA-I, sont clivées par un mécanisme qui demeure inconnu. En effectuant une renaturation in vitro d’AIDA-I, couplée avec une approche de mutagenèse dirigée, nous avons démontré que cette protéine se clive par un mécanisme autocatalytique qui implique deux acides aminés possédant un groupement carboxyle. Ces résultats ont permis la description d’un nouveau mécanisme de clivage pour la famille des autotransporteurs monomériques.
Une des particularités d’AIDA-I est sa glycosylation par une heptosyltransférase spécifique nommée Aah. La glycosylation est un concept plutôt récent chez les bactéries et pour l’instant, très peu de protéines ont été décrites comme glycosylées chez E. coli. Nous avons démontré que Aah est le prototype pour une nouvelle famille de glycosyltransférases bactériennes retrouvées chez diverses espèces de protéobactéries. La glycosylation d’AIDA-I est une modification cytoplasmique et post-traductionnelle. De plus, Aah ne reconnaît pas une séquence primaire, mais plutôt un motif structural. Ces observations sont uniques chez les bactéries et permettent d’élargir nos connaissances sur la glycosylation chez les procaryotes. La glycosylation par Aah est essentielle pour la conformation d’AIDA-I et par conséquent pour sa capacité de permettre l’adhésion. Puisque plusieurs homologues d’Aah sont retrouvés à proximité d’autotransporteurs monomériques putatifs, cette famille de glycosyltranférases pourrait être importante, sinon essentielle, pour la biogenèse et/ou la fonction de nombreux autotransporteurs.
En conclusion, les résultats présentés dans cette thèse apportent de nouvelles informations et permettent une meilleure compréhension de la biogenèse d’une des plus importantes familles de protéines sécrétées chez les bactéries Gram négatif.Monomeric autotransporters, a family of proteins that use the type V secretion pathway, are important mediators of virulence for many bacterial pathogens. Many functions important for host colonization and survival have been described for these proteins. Despite the recognized importance of this family of proteins, the mechanisms that are required for the biogenesis and functionality of monomeric autotransporters still remain poorly understood. The Escherichia coli adhesin involved in diffuse adherence (AIDA-I) is a classical multifunctional autotransporter protein that mediates bacterial aggregation and biofilm formation, as well as adhesion and invasion of cultured epithelial cells. Extracellular domains of autotransporters are responsible for the protein function and fold into a characteristic β-helical structure. We performed a random mutagenesis of the AIDA-I passenger domain in order to identify regions involved in the various phenotypes associated with the expression of this protein. Our study suggests that the passenger domain of AIDA-I possesses a modular organization, which means that AIDA-I is built with individual functional modules. Autotransporter passenger domains can be cleaved from the β-domain and released into the extracellular milieu. However, despite the fact that diverse cleavage mechanisms have been previously described, many autotransporters, like AIDA-I, are cleaved by an unknown mechanism. By monitoring the in vitro refolding and cleavage following by site-directed mutagenesis, we showed that AIDA-I processing is an autocatalytic event that involves two acidic residues. Our results unveil a new mechanism of auto-processing in the autotransporter family. AIDA-I is one of the few glycosylated proteins found in Escherichia coli. Glycosylation is mediated by a specific heptosyltransferase encoded by the aah gene, but little is known about the role of this modification and the mechanism involved. Our findings suggest that Aah represents the prototype of a new large family of bacterial protein O-glycosyltransferases that modify various substrates recognized through a structural motif. Furthermore, we showed that glycosylation occurs in the cytoplasm by a cotranslational mechanism. These observations are unique in bacteria and represent a significant advance in our comprehension of prokaryotic glycosylation. We also showed that glycosylation is required to ensure a normal conformation of AIDA-I and, as a consequence, is necessary for its cell-binding function. The finding that other autotransporters or large adhesin-encoding genes are linked to Aah homologue-encoding genes suggests that glycosylation may be important, if not essential, for the function of these proteins, as for AIDA-I. In conclusion, the results presented in this thesis bring new information about the autotransporter family and also give new insight into the mechanisms that are important for different aspects of the biogenesis of monomeric autotransporters.
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Oliver, David C. "Structure/function studies of the Bordetella pertussis autotransporter protein BrkA :." Thesis, 2005. http://hdl.handle.net/2429/16936.
Full textAbstract:
The autotransporter secretion system represents a fundamental strategy that Gramnegative
bacteria have evolved to deliver an array of functionally diverse proteins to the
cell surface. As the name implies, the autotransporter secretion system does not employ
specific accessory factors; all of the information necessary for movement of a substrate
polypeptide across the two membranes of the cell envelope is encoded within a single
gene product. Autotransporters are modular multidomain proteins consisting of an N -
terminal signal peptide, a passenger domain that contains the effector function(s) to be
delivered to the cell surface, and a C-terminal domain termed the translocation unit. The
signal peptide directs translocation across the inner membrane and the translocation unit
facilitates export across the outer membrane. How this seemingly simple protein
secretion strategy functions is largely unknown. This study addresses the secretion
mechanism of the BrkA protein, a known virulence factor of Bordetella pertussis (the
causative agent of whooping cough) and a putative autotransporter protein. Using a
combination of genetic, biochemical, bioinformatic and cell biological approaches, BrkA
was shown here to be a bona fide autotransporter protein and was established as a model
system for studying autotransporter secretion. Structural and functional dissection of
BrkA revealed a multidomain architecture consisting of a signal peptide, a passenger
domain that contains the BrkA effector functions (serum resistance and adherence), and a
C-terminal translocation unit. Significantly, a region termed the junction that is located
at the C-terminus of the BrkA passenger domain was identified to be required for
passenger folding during secretion. The conservation of this domain in a functionally
diverse group of autotransporter proteins suggested that it plays an important role in
secretion. The demonstration that the junction region mediated BrkA passenger folding
when supplied in trans as a separate polypeptide suggests that it can function as an
intramolecular chaperone. Further dissection of the BrkA junction revealed a sub-region
that is not required for passenger folding but is required for secretion of a "folding
competent" native BrkA passenger. These findings have been integrated with our current
knowledge of autotransporter secretion to generate a working model of BrkA secretion
that may be applicable to other autotransporter proteins.Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Doyle, Matthew Thomas. "Polarity and secretion of Shigella flexneri IcsA: a classical autotransporter." Thesis, 2015. http://hdl.handle.net/2440/98693.
Full textAbstract:
The classical autotransporter IcsA is an essential virulence factor for the enteropathogen Shigella flexneri as it provides adherence properties and allows intra- and intercellular spreading in the colonic mucosa. IcsA is an outer membrane surface protein that specifically hijacks host-cell actin recruiting and polymerising complexes allowing actin polymerisation as a form of actin-based motility (ABM). Importantly, since IcsA is localised specifically at one end of the bacterium (the pole), the resulting ABM is unidirectional which is a requirement for efficient S. flexneri dissemination. However, the molecular mechanisms that generate IcsA polarity remain poorly understood. Furthermore, IcsA is a member of the autotransporter family of secreted virulence factors (Type Va). Although many steps in the autotransporter pathway have been elucidated, it is still poorly understood how these diverse proteins are efficiently translocated to the bacterial cell surface. As such, this thesis investigates the two arms of IcsA biogenesis: (1) polar targeting and (2) autotransport. Regarding IcsA polarity, it was identified here that the IcsA-specific outer membrane protease IcsP localises to the septa of dividing S. flexneri and to the opposing pole relative IcsA. The concentration of IcsP was higher at the septum than the pole showing a life cycle dependent distribution of IcsP. This provides the basis of a model where IcsP is important during division of S. flexneri for setting up (and the continued maintenance of) IcsA polarity by the proteolysis of misdirected IcsA. Further, multiple previous reports have suggested that the S. flexneri lipopolysaccharide O-antigen surface structure can influence IcsA polarity by augmenting membrane fluidity or by asymmetric masking of IcsA surface exposure. These notions were tested here resulting in data that clearly refutes these models and agues simply that IcsA exposure is masked symmetrically over the bacterial cell surface. Finally, IcsA itself contains polar targeting (PT) regions that direct it to the pole by an, as yet, unclear mechanism. Examination of these regions revealed that the central PT (cPT) is most important in polarity augmentation and contains critical polarity targeting function residues. Regarding IcsA autotransport, a highly conserved but uncharacterised autotransporter motif was scrutinized for potential biogenesis functions and was designated in this work as the passenger-associated transport repeat (PATR). It was found that the PATR plays a critical role in the efficient secretion of IcsA to the cell surface. Strikingly, bioinformatics analyses revealed that the PATR delineates an important separate autotransporter sub-type with unique functions, composition, and architecture.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Biological Sciences, 2015.
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Côté, Jean-Philippe. "Les autotransporteurs auto-associatifs d’Escherichia coli : de facteurs de virulence à déterminants sociaux." Thèse, 2013. http://hdl.handle.net/1866/10508.
Full textAbstract:
Les autotransporteurs monomériques représentent le système de sécrétion le plus simple et le plus utilisé chez les bactéries à Gram négatif. Les autotransporteurs monomériques sont des protéines modulaires qui contiennent toute l’information pour leur sécrétion dans leur séquence. Les phénotypes associés à l’expression d’un autotransporteur peuvent être très variés et, souvent, les autotransporteurs sont des protéines multifonctionnelles. C’est le cas notamment des autotransporteurs AIDA-I, TibA et Ag43 d’Escherichia coli qui promouvoient l’adhésion et l’invasion de cellules épithéliales, l’auto-agrégation des bactéries et la formation de biofilm. Ces trois autotransporteurs ont d’ailleurs été regroupés dans une même famille, appelée les autotransporteurs auto-associatifs (SAATs). À cause de leur fonctionnalité, les SAATs sont considérés comme étant d’importants facteurs de virulence d’Escherichia coli. Toutefois, il existe plusieurs différences entre les SAATs qui ne sont pas bien comprises, si bien que leur rôle pour les bactéries n’est toujours pas bien compris.
Nous avons donc d’abord caractérisé TibA, le membre des SAATs le moins bien étudié à l’aide d’une étude structure-fonction. Nous avons observé que TibA était une protéine modulaire et que son domaine fonctionnel était composé de deux modules : un module d’auto-agrégation en N-terminal et un module d’adhésion en C-terminal. En comparant nos résultats avec ceux obtenus pour les autres SAATs, nous avons réalisé que l’organisation des trois SAATs était très variée, c’est-à-dire que les trois SAATs sont composés de modules différents. Nous avons par ailleurs observé cet arrangement en modules lorsque nous avons analysé plusieurs séquences d’aidA, suggérant qu’un mécanisme d’échange et d’acquisition de modules était à la base de l’évolution des SAATs. Sans surprise, nous avons aussi observé que la famille des SAATs ne se limitait pas à AIDA-I, TibA et Ag43 et ne se limitait pas à Escherichia coli.
La comparaison a aussi révélé l’importance du phénotype d’auto-agrégation dans la fonctionnalité des SAATs. Nous avons donc entrepris une étude du mécanisme d’auto-agrégation. Nos résultats on montré que l’auto-agrégation était le résultat d’une interaction directe SAAT/SAAT et ont mis en évidence un mécanisme similaire à celui utilisé par les cadhérines eucaryotes. De plus, nous avons observé que, comme les cadhérines, les SAATs étaient impliqués dans des interactions homophiliques; un SAAT interagit donc spécifiquement avec lui-même et non avec un différent SAAT.
Finalement, les SAATs font parties des quelques protéines qui sont glycosylées chez Escherichia coli. Nous avons déterminé que le rôle de la glycosylation de TibA était de stabiliser la protéine et de lui donner la flexibilité nécessaire pour moduler sa conformation et, ainsi, être pleinement fonctionnelle.
Globalement, nos résultats suggèrent que les SAATs sont des molécules « cadhérines-like » qui permettent la reconnaissance de soi chez les bactéries. Une telle habilité à discriminer entre le soi et le non-soi pourrait donc être utilisée par les bactéries pour organiser les communautés bactériennes.Autotransporters are versatile virulence factors of Gram-negative bacteria and use one of the simplest and most widespread secretion system in bacteria. The name autotransporter originate from the observation that all the information needed for the secretion of the protein is encoded in its own sequence, meaning that autotransporters do not need a specialized secretion apparatus. Many autotransporters are multifunctional proteins and can perform a large variety of functions. The self-associating autotransporters (SAATs), represented by AIDA-I, TibA and Ag43, are such multifunctional proteins and can mediate the adhesion and invasion of epithelial cells, the auto-aggregation of bacteria and the formation of biofilm. Because of these functionalities, SAATs are considered important virulence factors of Escherichia coli. However, there are many differences between the SAATs and we still do not know their exact role for the bacteria. Therefore, we have realized a structure-function study of TibA, the least studied SAAT. Our study showed that TibA is a modular protein and that the functional domain of TibA is composed of two modules: an N-terminal module responsible for auto-aggregation and a C-terminal module responsible for adhesion. Our results showed that the organization of AIDA-I, TibA and Ag43 is different and that the SAATs represent different assemblies of modules. We also observed the modular organization when we analyzed various sequence of aidA, suggesting that the SAATs have evolved by a mechanism of domain shuffling. Not surprisingly, we have found new SAATs in Escherichia coli and in other proteobacteria. Our results also highlighted the importance of auto-aggregation in the functionality of the SAATs. We therefore assessed the mechanism of SAAT-mediated auto-aggregation of bacteria. Our results showed that SAATs mediate auto-aggregation of bacteria through direct SAAT/SAAT interactions and that these interactions were reminiscent of the interactions made by cadherin molecules in eukaryotes. We further observed that the SAATs were involved in homophilic interactions, as it is the case with cadherin molecules. SAATs are part of the few proteins that are glycosylated in Escherichia coli. We therefore characterized the glycosylation of TibA and found that glycosylation of TibA stabilized the protein and allowed the protein to modulate its conformation, resulting in a fully functional protein. Taken together, our results suggest that the SAATs may be cadherin-like molecules by bacteria in order to discriminate between self and non-self. Such an ability to discriminate self from non-self is rarely evoked in bacteria, but could play a role in the organization of multicellular communities.