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Technical University of Lisbon

1. Santos, Ana Rita Rodrigues dos. Utilização de mutantes para identificação de genes envolvidos na produção de biofilmes de Listeria monocytogenes.

Degree: 2015, Technical University of Lisbon

Mestrado em Engenharia Alimentar - Instituto Superior de Agronomia

Listeria monocytogenes is a bacteria considered difficult to eradicate in food industry duo to its ability to form biofilms. The ability of biofilm formation was evaluated in five L. monocytogenes strains (the wild type EGDe and four mutants of this strain, at gene fliF, fliI, tatAC and lmo0364). The Tat system is responsible for secretion of proteins in their final phase of conformation. The lmo0364 gene encodes for a transcrptional regulator that has been poorly studied. The flagella are proteins involved in the mobility of the bacteria and play a crucial role in the initial and subsequent steps of biofilm formation and cell attachment. The crystal violet, the ruthenium red and the enumeration of cells in stainless steel assays were used. The biofilm production was evaluated at 25 ° C and 37 ° C in TSB and MWB medium. The results indicate a reduced ability of flagellar mutants to produce biofilm whencompared to its wild type, showing the importance of flagella to produce biofilm in L. monocytogenes

Advisors/Committee Members: Brito, Maria Luísa de Castro, Lourenço, António Abreu Afonso.

Subjects/Keywords: Listeria monocytogenes; biofilm; mutant; exoproteins; flagella; secretion systems

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APA (6th Edition):

Santos, A. R. R. d. (2015). Utilização de mutantes para identificação de genes envolvidos na produção de biofilmes de Listeria monocytogenes. (Thesis). Technical University of Lisbon. Retrieved from https://www.rcaap.pt/detail.jsp?id=oai:www.repository.utl.pt:10400.5/8513

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

Santos, Ana Rita Rodrigues dos. “Utilização de mutantes para identificação de genes envolvidos na produção de biofilmes de Listeria monocytogenes.” 2015. Thesis, Technical University of Lisbon. Accessed November 15, 2019. https://www.rcaap.pt/detail.jsp?id=oai:www.repository.utl.pt:10400.5/8513.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

Santos, Ana Rita Rodrigues dos. “Utilização de mutantes para identificação de genes envolvidos na produção de biofilmes de Listeria monocytogenes.” 2015. Web. 15 Nov 2019.

Vancouver:

Santos ARRd. Utilização de mutantes para identificação de genes envolvidos na produção de biofilmes de Listeria monocytogenes. [Internet] [Thesis]. Technical University of Lisbon; 2015. [cited 2019 Nov 15]. Available from: https://www.rcaap.pt/detail.jsp?id=oai:www.repository.utl.pt:10400.5/8513.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Santos ARRd. Utilização de mutantes para identificação de genes envolvidos na produção de biofilmes de Listeria monocytogenes. [Thesis]. Technical University of Lisbon; 2015. Available from: https://www.rcaap.pt/detail.jsp?id=oai:www.repository.utl.pt:10400.5/8513

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation


University of Helsinki

2. Ahmed, Mohamed Adel. The impact of carbohydrate source on protein and antigen secretion in Lactobacillus rhamnosus GG.

Degree: Department of Food and Environmental Sciences; Helsingfors universitet, Agrikultur- och forstvetenskapliga fakulteten, Institutionen för livsmedels- och miljövetenskaper, 2015, University of Helsinki

Lactobacillus rhamnosus GG (GG) is one of the most studied probiotics worldwide and it has proved to confer health benefits to human. However, the exact mechanism of its probiotic action is still not fully understood. Some proteins secreted by probiotics, such as p75 (Msp1) and p40 (Msp2) in GG, are reported to play an important role in gut epithelial homeostasis. The aim of this work was to develop a static biofilm plate model for analyzing exoproteomes (all secreted proteins) produced by L. rhamnosus GG biofilms. First, different culture volumes (7 mL and 10 mL), incubation times and protein precipitation methods were tested to optimize conditions in order to maximize the protein yield. Next, the impact of different sugars on the exoproteome composition of the GG cells during planktonic and biofilm mode of growth was explored. Finally, the planktonic and biofilm exoproteins were subjected to antigen profiling and protein identification. The 6-well Polystyrene Microtiter plate was used as the static biofilm model for inducing the biofilm formation of GG. Biofilms were formed for 24 h and 48 h and the protein secretion from each time point was assessed by precipitating the supernatant proteins using 10% trichloroacetic acid (TCA)/acetone or 2-D Clean Up kit (GE Healthcare). The purified proteins were subjected to one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1-DE) and the proteins were visualized using Coomassie blue staining. 1-DE immunoblotting using antibodies raised against whole GG cells was used to analyze antigens produced by the GG biofilms under the optimized biofilm formation conditions. The antigens produced by the GG biofilm cells were compared to those produced by the GG cells during planktonic growth (from over- night cultures) and the cross-reacting proteins were visualized using an IR800-conjugated secondary antibody. The immunoblots were scanned using an Odyssey Infrared Imaging System (Licor). In addition, the impact of two different carbohydrate sources on the antigen profiles was also explored. Finally, in-liquid tryptic digestion coupled with Liquid Chromatography- tandem Mass Spectrometry analysis (LC-MS/MS) was used to identify and compare exoproteomes produced by the biofilm cells cultured in the presence of the tested carbohydrates. The results showed that the commercial 2-D Clean Up kit is better than 10% TCA protein precipitation/acetone washing, because it produced clear protein patterns with less background. However, the TCA/acetone–protocol resulted in detection of higher number of proteins in 1-DE gels. Comparative immunoblot analyses of the planktonic and biofilm exoproteins at 24 h and 48 h time points revealed a clear difference in antigen profiles between the two modes of growth. In addition, the utilized carbon source was found to have a great impact on the antigen abundances and/or export. Using LC-MS/MS, 36 exoproteins were identified from the GG biofilm cultures (identification score ≥ 40, p < 0.05). Most of the identified proteins were…

Subjects/Keywords: Lactobacillus; probiotics; biofilm; planktonic; exoproteins; exoproteome; TCA; SDS PAGE; immnoblotting; LC-MS/MS; Livsmedelsteknologi; Food Technology; Elintarviketeknologia

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APA (6th Edition):

Ahmed, M. A. (2015). The impact of carbohydrate source on protein and antigen secretion in Lactobacillus rhamnosus GG. (Masters Thesis). University of Helsinki. Retrieved from http://hdl.handle.net/10138/157472

Chicago Manual of Style (16th Edition):

Ahmed, Mohamed Adel. “The impact of carbohydrate source on protein and antigen secretion in Lactobacillus rhamnosus GG.” 2015. Masters Thesis, University of Helsinki. Accessed November 15, 2019. http://hdl.handle.net/10138/157472.

MLA Handbook (7th Edition):

Ahmed, Mohamed Adel. “The impact of carbohydrate source on protein and antigen secretion in Lactobacillus rhamnosus GG.” 2015. Web. 15 Nov 2019.

Vancouver:

Ahmed MA. The impact of carbohydrate source on protein and antigen secretion in Lactobacillus rhamnosus GG. [Internet] [Masters thesis]. University of Helsinki; 2015. [cited 2019 Nov 15]. Available from: http://hdl.handle.net/10138/157472.

Council of Science Editors:

Ahmed MA. The impact of carbohydrate source on protein and antigen secretion in Lactobacillus rhamnosus GG. [Masters Thesis]. University of Helsinki; 2015. Available from: http://hdl.handle.net/10138/157472


Texas Medical Center

3. Stinemetz, Emily K; and#60;pand#62;0000-0001-6477-4687and#60;/pand#62. Evaluating the impact of post-translational modifications by the secreted zinc metalloprotease, GelE, on the major autolysin of E. faecalis, AtlA, and a stress-induced protein, SalB.

Degree: PhD, 2017, Texas Medical Center

AtlA is the major peptidoglycan hydrolase of E. faecalis involved in cell separation of dividing cells. SalB is a secreted stress-induced protein regulated by the CroRS system. In addition, these two proteins also appear to be affected by the virulence factor, gelatinase (GelE). GelE is a secreted zinc metalloprotease known to impact various cellular functions by post- translational modification of protein substrates. The overall objective of this work was to understand how GelE cleavage of secreted proteins, specifically AtlA and SalB, changes their function. Herein, I discovered that GelE modifies both AtlA and SalB. As visualized by Western blot analysis and flow cytometry, when GelE is expressed, AtlA exists in a N-terminally truncated form. Furthermore, N-terminal-sequencing analysis identified the GelE-cleavage site within AtlA to occur near the catalytic region, Domain II. Thus, cleavage removes the majority of the N-terminal T/E rich region, Domain I. Truncation of AtlA at this site caused no significant difference in the peptidoglycan hydrolysis activity compared to the full-length protein. Nevertheless, the modification of AtlA was shown to be required for cell separation and the completion of cell division. Additionally, GelE-modified AtlA was shown to localize to the cell septum. Taken together, these results demonstrate that post-translational modification of AtlA by GelE regulates AtlA septum localization and successful cell separation. Similarly, in the presence of GelE, SalB was found in multiple fragments. Western blot and flow cytometry analysis demonstrated that SalB was found in the media supernatant, but not associated with the cell surface. Overall, this dissertation demonstrates that GelE post-translationally modifies these two secreted proteins, AtlA and SalB, impacting the function of AtlA in cell division. Future experiments will strengthen our knowledge of how these modifications impact E. faecalis virulence. Advisors/Committee Members: Barrett Harvey, Ph.D., Jeffrey Actor, Ph.D., Danielle Garsin, Ph.D..

Subjects/Keywords: GelE; gelatinase; AtlA; autolysin; SalB; septum localization; peptidoglycan hydrolase; Enterococcus faecalis; exoproteins; Bacteriology; Laboratory and Basic Science Research; Medicine and Health Sciences; Pathogenic Microbiology

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Stinemetz, E. K. a. (2017). Evaluating the impact of post-translational modifications by the secreted zinc metalloprotease, GelE, on the major autolysin of E. faecalis, AtlA, and a stress-induced protein, SalB. (Doctoral Dissertation). Texas Medical Center. Retrieved from http://digitalcommons.library.tmc.edu/utgsbs_dissertations/743

Chicago Manual of Style (16th Edition):

Stinemetz, Emily K; and#60;pand#62;0000-0001-6477-4687and#60;/pand#62. “Evaluating the impact of post-translational modifications by the secreted zinc metalloprotease, GelE, on the major autolysin of E. faecalis, AtlA, and a stress-induced protein, SalB.” 2017. Doctoral Dissertation, Texas Medical Center. Accessed November 15, 2019. http://digitalcommons.library.tmc.edu/utgsbs_dissertations/743.

MLA Handbook (7th Edition):

Stinemetz, Emily K; and#60;pand#62;0000-0001-6477-4687and#60;/pand#62. “Evaluating the impact of post-translational modifications by the secreted zinc metalloprotease, GelE, on the major autolysin of E. faecalis, AtlA, and a stress-induced protein, SalB.” 2017. Web. 15 Nov 2019.

Vancouver:

Stinemetz EKa. Evaluating the impact of post-translational modifications by the secreted zinc metalloprotease, GelE, on the major autolysin of E. faecalis, AtlA, and a stress-induced protein, SalB. [Internet] [Doctoral dissertation]. Texas Medical Center; 2017. [cited 2019 Nov 15]. Available from: http://digitalcommons.library.tmc.edu/utgsbs_dissertations/743.

Council of Science Editors:

Stinemetz EKa. Evaluating the impact of post-translational modifications by the secreted zinc metalloprotease, GelE, on the major autolysin of E. faecalis, AtlA, and a stress-induced protein, SalB. [Doctoral Dissertation]. Texas Medical Center; 2017. Available from: http://digitalcommons.library.tmc.edu/utgsbs_dissertations/743

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