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Goey, Cher Hui. "Cascading effects in bioprocessing : the impact of cell culture environment on CHO cell behaviour and host cell protein species." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/52703.
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One of the reasons for the rejection of new drugs during clinical trials is the presence of host cell proteins (HCPs) in the drug formulation. HCPs are immunogenic impurities that can compromise patient safety. Moreover, proteolytic and binding HCPs compromise the integrity, and, hence, the stability and efficacy of a recombinant protein. Therefore, HCPs should be removed from the bioprocess train as soon as possible. Current downstream purification platforms are challenged by HCP-mAb co-elution. A Quality by Design strategy to overcome this problem is to reduce the number of HCPs in the downstream feedstock by tracing their source back to upstream culture and eliminating it. Previous studies have shown that upstream cell culture parameters, e.g. harvest time and culture temperature, significantly affect HCP profiles. However, little is known about how host cells coordinate and regulate their molecular machinery under different cell culture environment that results in different HCP profiles. This study presents experimental results linking cell culture temperature and key process indicators of CHO cell cultures and post-Protein A purification (mAb titre, HCP level and HCP species) by considering the cellular behaviour in terms of cell growth, cell cycle distribution and cell health). This study involved the application of single-use fed-batch bioreactors to culture IgG4 producing GS-CHO cell line, cell health and cell cycle analysis with NucleoCounter, Protein A purification and proteomic analysis with HCP ELISA kits and LC-MS/MS. Cells were more robust under mild hypothermia: over 90% of cells were maintained in a healthy state until the decline phase, and the onset of apoptosis was less evident compared to the results for physiological temperature. IgG4 titre and HCP level at harvest were comparable between the two cases. However, mild hypothermia reduced the HCP variety in HCCF by 36%, including 44% and 27% lower proteases and chaperones, respectively. The differences in HCP variety at harvest resulted in a significantly different HCP profile post-Protein A purification between the two cases. Half of the critically immunogenic HCPs species as determined by the CHOPPI tool were different between the two cases. This study shows that cell culture conditions significantly affect the HCP profile at harvest and that of purified samples.
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Carr, Sharon. "Adenovirus and its interaction with host cell proteins /." St Andrews, 2007. http://hdl.handle.net/10023/219.
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Qashqari, Fadi Saleh I. "Regulation of host cell proteins by adenovirus oncoproteins." Thesis, University of Birmingham, 2017. http://etheses.bham.ac.uk//id/eprint/8020/.
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Adenovirus early region proteins, ElA, E1B-55K, E4orf3 and E4orf6 regulate host cell processes to facilitate viral replication. E4orf3 suppress host cell anti-viral activities through association with host cell proteins in E4orf3 nuclear-track structures, whilst E1B-55K, E4orf3 and E4orf6 are all recruited to viral replication centres during infection to promote viral DNA replication and inhibit host cell antiviral activities. Immunoprecipitation coupled to mass spectrometry identified Toplla as an Ad12 E4orf3-binding protein that localized with E4orf3 in adenovirus-infected cells. It was determined that Toplla expression was induced during infection, and that Toplla was required for the adenovirusdependent stabilization of p53. It was also established that, despite their ability to cooperate functionally, Toplla and p53 do not associate physically during infection. Immunoprecipitation coupled to mass spectrometry was also used to identify host cell proteins recruited to viral replication centres during adenovirus infection. The RP A-1 binding protein, Smarcall, and the FACT complex histone chaperone protein, SSRPl were identified as host cell proteins recruited to viral replication centres during infection. Following recruitment to viral replication centres Smarcall was found to be degraded in an E1B-55K and E4orf6 dependent manner, whilst SSRPl was found to be stable during infection and was not targeted for degradation.
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Bjertsjö, Rennermalm Anna. "Staphylococcal cell wall associated proteins : characteristics and host interactions /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-542-9/.
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Huang, Edwin P. C. Biotechnology & Biomolecular Sciences Faculty of Science UNSW. "Recombinant protein production utilising a metallothionein expression system and a Super-CHO cell line." Awarded by:University of New South Wales. School of Biotechnology and Biomolecular Sciences, 2006. http://handle.unsw.edu.au/1959.4/24940.
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A novel metal-inducible and amplifiable metallothionein (MT) expression system, pNK, was firstly optimised and characterised for the production of a reporter protein, human growth hormone (hGH) in a suspension CHO cell line grown in a serum-free media. The pNK-based hGH production was demonstrated in cadmium-free condition under various fermentation modes (batch, fed-batch and perfusion) and scales (flask to bench-top bioreactor). Improvement of specific productivity of recombinant protein from pNK was shown to be possible by addition of butyrate or substrate substitution of glutamine by glutamate. Combination of fed-batch and butyrate addition strategies resulted in more than one gram per litre of hGH being obtained from the pNK expression system in a bioreactor. In the second part of the project, based on a statistical approach suggested by Plackett-Burman (P-B), a chemically-defined and protein-free medium, named Super-CHO protein-free (SPF), was developed to support a Super-CHO cell line, C2.8-325, to grow as a single-cell suspension culture with comparable growth rate and viable cell number as observed in a commercial medium containing undefined additives. Using Dulbecco's Modified Eagle's Medium/Ham's F12 1:1 mixture (DMEM/F12) as the basal medium, a P-B design matrix screened 10 nutritional components. Components shown potentially beneficial for cell growth rate and viable cell number were supplemented to DMEM/F12 to formulate the SPF medium. Finally, the pNK expression system and the Super-CHO cell line were applied simultaneously in an attempt to express a humanised anti-CD48 monoclonal antibody (MAb), IgG1-N2A (N2A-MAb). This aimed to test C2.8-SPF grown in newly developed SPF medium for transfection, clone development and recombinant protein production. A stable and N2A-MAb expressing C2.8-SPF cell line was successfully constructed, and N2A MAb expression was subsequently amplified and demonstrated in various cultivation scales (flask and bioreactor). This project demonstrated that the novel metal-inducible and - amplifiable mammalian expression system, pNK, and the novel mammalian host cell-line, Super-CHO C2.8-SPF, capable of growing as a single-cell suspension culture in a chemically-defined protein-free medium, SPF, could be utilised in combination to provide a new, low-cost, and regulatory-compliant recombinant protein expression platform, suitable for the biopharmaceutical industry to use in the manufacture of therapeutic recombinant proteins.
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Mohamed, Ahmed Attia Ali. "Interaction of hepatitis C virus polymerase with host cell proteins." Thesis, Durham University, 2009. http://etheses.dur.ac.uk/2107/.
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Hepatitis C virus (HCV) interacts with host cell proteins to modify cellular pathways creating a favourable environment that facilitates its replication and persistence. The purpose of the work presented in this thesis was to identify cellular proteins that can interact with NS5B, the virus's RNA-dependent RNA polymerase, that may contribute to the virus's biology. A number of cellular proteins were found to interact with NS5B using the yeast two-hybrid system. These proteins were involved in cellular pathways such as interferon signalling, lipid transport and metabolism, protein trafficking, cell proliferation and apoptosis. Of these, phospholipid scramblase 1 (PLSCR1) and zinc finger protein 143 (ZNF143) were selected for further investigation. The interactions were confirmed in vitro, and, for PLSCR1, the region that interacted with NS5B was determined to be within the amino-terminal region of the protein (61-137 a.a.). NS5B interacted with PLSCR1 and ZNF143 via a single interacting region localized in its N-terminus (1-153 a.a.).Expression of PLSCR1 or ZNF143 enhanced the ability of interferon to stimulate transcription from an interferon-stimulated response element (ISRE) reporter construct. Co-expression with NS5B was found to down-regulate this activity. Expression of a number of interferon-stimulated genes was investigated in the presence of NS5B, PLSCR1 or ZNF143 but no significant effect was observed. Overexpression of PLSCR1 had no effect on HCV sub-genomic replicon replication, while reduction of its expression by short hairpin RNA (shRNA) enhanced replication. Overexpression of ZNF143 was found to have a suppressive effect on replication but downregulating its expression did not enhance replication. In addition to using the yeast two-hybrid system to identify NS5B- interacting proteins, an in vitro pulldown assay coupled with mass spectrometry identified α- and β -tubulin associated with NS5B in vitro and in vivo. Subsequently this association was demonstrated to be an indirect interaction but the intermediatory partner was not identified. The domain that mediated the association with α- and β-tubulin was determined to be within the N-terminus of NS5B (1-153 a.a.). Nocodazole, an inhibitor of tubulin polymerization, had a marked effect on the association of α -tubulin with NS5B displacing it from the complex but had no effect on β -tubulin's association. Utilizing an HCV sub- genomic replicon, nocodazole was shown to have a significant inhibitory effect on replication. Taken together the data presented in this thesis showed that NS5B had a multitude of potential interactions with a variety of cellular proteins. The biological significance of some of these interactions on the cellular response to IFN and replicon replication was investigated. This work has generated a number of novel observations on the interaction between the virus and the cell that warrant future investigation
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Goldman, Merlin Hesper. "The effect of process variables on the glycosylation of gamma-interferon produced in CHO cells." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244743.
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Patel, Amit. "Interaction of enteropathogenic 'Escherichia coli' (EPEC) tir with host cell proteins." Thesis, University of Nottingham, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431869.
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Cowlishaw, Deborah Anne. "Identification of host proteins required for bacteriophage infection of Streptomyces sp." Thesis, University of Nottingham, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367589.
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Rytkönen, Anne. "Molecular studies of Neisseria - host cell interactions /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7140-018-4/.
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Porter, Alison J. "Analysis of the efficiency of selecting GS-CHO cell lines for cGMP manufacture of recombinant proteins." Thesis, University of Manchester, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.692544.
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The market size for biotherapeutics is growing rapidly and the hunt is always on for the next blockbuster drug. A wide range of cell phenotypes are obtained during the process of generating mammalian cell lines suitable for the production of a biotherapeutic such as a monoclonal antibody. A strategy is therefore required for the selection of a cell line with ’desirable’ characteristics, from a population of closely related cell lines, for the successful manufacture of the biotherapeutic. As this process is included in the sequence of activities which determines the minimum time to start cGMP manufacturing of the biotherapeutic, companies are actively seeking to improve its efficiency. Stringent examination of a typical selection strategy revealed that it was capable of identifying high producing cell lines. However, it only isolated a fraction of the highest producing cell lines and ranking positions between assessment stages that form the selection strategy were not consistent. These results suggested that there was potential to improve selection strategies. Models of alternative selection strategies, built by in silico analysis of data typically collected during cell line construction, were not able to identify ’better’ cell lines. Alternative markers for cell lines capable of achieving high product concentrations could be used to improve selection strategies. In this study, parameters that showed a relationship with recombinant product concentration were identified, suggesting the possibility for development of more predictive assessment criteria. However, results also showed that it was unlikely that a single predictive parameter would be identified. The study also highlighted the affect the environment, which may change between assessment stages, had on cell lines. Closer matching of the cellular environment was therefore also suggested as a means to improve selection strategies. This study has highlighted the importance of designing a selection strategy that is highly predictive of the final production process. In the future, cell line selection strategies are likely to require the incorporation of closer matching of the cellular environment and the use of multiple predictive markers which form profiles of cell lines capable of producing the highest product concentrations.
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Dickey, Laura Leigh. "Respiratory synctial virus interactions with host-cell RNA-processing structures and proteins." Thesis, Boston University, 2013. https://hdl.handle.net/2144/10980.
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Thesis (Ph.D.)--Boston University<br>Respiratory syncytial virus (RSV) is a negative-strand RNA virus that causes significant pneumonia-related morbidity and mortality worldwide. There are currently neither vaccines nor effective therapies for RSV. As with other viruses, RSV mRNAs are translated using host-cell machinery, rendering the virus subject to cellular factors that regulate mRNA homeostasis. Stress granules (SGs) and processing bodies (p-bodies) are inter-dependent, stress-response cytoplasmic structures involved in mRNA triage and degradation, respectively. We hypothesized that RSV has evolved to manipulate cellular stress responses in order to facilitate optimal virus propagation. While wild-type (wt) RSV induced SGs in approximately 1% of infected cells, a mutant version of RSV whose Tr region was replaced with an inverted LeC sequence (LeC virus) induced SG formation in approximately 50% to 70% of infected cells. A 12U to A substitution relative to the 5' end of the LeC virus abrogated SG induction. Mixed-infection studies showed that wt RSV was able to prevent LeC-mediated SG induction. Unlike Sendai virus, RSV-mediated prevention of SG formation was independent of SG-associated t-cell intracellular antigen related (TIAR) protein. RSV infection altered neither the number nor distribution of p-bodies; however, p-body-associated decapping protein 1 (dcp1) was phosphorylated throughout RSV infection via the extracellular signal-regulated kinase (ERK) 1/2 pathway. RSV-mediated dcp1 phosphorylation was limited to serine 315, serine 319, and threonine 321. Dcp1 phosphorylation occurred in response to some, but not all, environmental stresses, and dcp1 was not phosphorylated during infection with HIV-1, measles, mumps, or canine distemper virus. Overexpression of dcp1 significantly attenuated RSV cytopathic effects, and preliminary data suggested that dcp1 phosphorylation regulated RSV-induced interleukin-8 production. Finally, an antibody toward cellular SG- and p-body-associated, RNA-binding protein p54 was able to recognize a subset of RSV nucleoprotein (N). p54 and RSV N contain a similar amino acid sequence motif, suggesting that they may have similar or competing activities that are important during RSV replication. Taken together, our results demonstrate that RSV can manipulate cellular RNA-processing structures and proteins to facilitate viral propagation.
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Riaz, Muhammad Suleman. "Investigating the effects of host factors (proteins and non-proteins) on mycobacteria." Thesis, Brunel University, 2018. http://bura.brunel.ac.uk/handle/2438/16060.
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Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis, is one of the leading causes of death due to a single infectious agent and results in more than 1 million human deaths every year. M.tb infection of the host initiates a local inflammatory response, resulting in the migration of a number of host plasma protein and non-protein factors to the site of infection. In addition, some of these factors are also produced locally at the site of infection. It is envisaged that these host factors are likely to come in direct contact with M.tb and immune cells and may modulate the outcome of the infection. In this study, a number of host factors including transferrin, lactoferrin, fibrinogen, C-reactive protein, alpha-2-macroglobulin (α2M), vitronectin, plasminogen, low-density lipoprotein (LDL), high-density lipoprotein (HDL), serotonin, L-alpha dipalmitoyl phosphatidylcholine (DPPC) and platelet activating factor C-16 (PAF C-16) were screened in vitro for their direct effect on the growth of mycobacteria using M.smegmatis as a model. As a result of this screening, PAF C-16, a phospholipid compound was identified that directly inhibited the growth of M.smegmatis and M.bovis BCG in a dose and time-dependent manner. Use of a range of PAF C-16 structural analogues, including Lyso-PAF, PAF C-18, Hexanolamino PAF, 2-O-methyl PAF & Pyrrolidino PAF, revealed that small modifications in structure did not alter the direct growth inhibition property of PAF C-16 and similar levels of M.smegmatis and M.bovis BCG growth inhibition were observed as compared to PAF C-16. Structural dissection of PAF C-16 suggested that the attachment of carbon tail to the glycerol backbone via ether bond at sn-1 position was important for its direct growth inhibition activity against mycobacteria. Microscopy and flow cytometry with PAF C-16 treated M.smegmatis and M.bovis BCG showed damage to the bacterial cell membrane. The addition of membrane-stabilizing agents, α-tocopherol, tween-80 and tween-20, partially mitigated the growth inhibitory effect of PAF C-16. These results suggested that the growth inhibition activity of PAF C-16 against mycobacteria is most likely due to its detergent-like effect, resulting in damage to the bacterial cell membrane. PAF C-16 and its structural analogues were also investigated for their effect on the growth of intracellular M.smegmatis inside THP1 cells. In vitro, PAF C-16, PAF C-18 and Hexanolamino PAF inhibited the growth of intracellular M.smegmatis, whereas, analogues such as Lyso-PAF and 2-O-methyl PAF failed to show any growth inhibitory effect, suggesting that the presence of acetyl group at sn-2 position was important for growth inhibition of intracellular M.smegmatis. Use of PAF receptor antagonists partially mitigated the inhibitory effect of PAF C-16 on the growth of intracellular M.smegmatis, suggesting this inhibition was through receptor-mediated signalling pathways. Blocking of PAF C-16 signalling pathway components such as phospholipase C and phospholipase A2, resulted in the increased survival of intracellular M.smegmatis. Arachidonic acid, a product of PAF C-16 signalling pathway directly inhibited the growth of M.smegmatis. Furthermore, inhibition of iNOS enzyme and antibody-mediated neutralization of TNF-α partially mitigated the inhibitory effect of PAF C-16 on intracellular M.smegmatis growth, suggesting that the production of NO and TNF-α were also involved in PAF C-16 induced intracellular growth inhibition. Overall, this study has identified PAF C-16, its structural analogues such as Lyso-PAF, PAF C-18, Hexanolamino PAF and other compounds including 1-O-hexadecyl-sn-glycerol, miltefosine and hexadecyl lactate with novel anti-mycobacterial activity. Further investigations are needed to demonstrate their effectiveness against M.tb both in vitro and in animal models to assess their therapeutic potential as anti-TB drugs.
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Ratanji, Kirsty. "Investigating the immunogenicity of therapeutic proteins : protein aggregation and host cell protein impurities." Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/investigating-the-immunogenicity-of-therapeutic-proteins-protein-aggregation-and-host-cell-protein-impurities(fda43dd8-2c1e-492b-abd9-e010774d2219).html.
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The development of anti-drug antibodies (ADA) against therapeutic proteins can impact upon drug safety and efficacy. This is a major challenge in the development of biotherapeutics. Various factors have the potential to contribute to protein immunogenicity and the production of ADA. Protein aggregation is one of these factors, though the mechanisms underlying aggregate immunogenicity are poorly understood. In this thesis the effect of protein aggregation on immunogenicity has been investigated. The thermal and/or mechanical stresses required in order to achieve subvisible aggregates of three test proteins were determined. Stressed preparations of proteins were characterised using a suite of biophysical techniques, including dynamic light scattering and circular dichroism. The immunogenic potential of subvisible aggregates of a humanised single chain variable fragment (scFv) and ovalbumin (OVA) was studied following intraperitoneal exposure in BALB/c strain mice. Monomeric proteins induced a T helper (Th) 2 dominant immune response, but when aggregated, the responses gained a Th1 phenotype, with a significant increase in the antigen-specific IgG2a antibody response. Cytokine profiles in supernatants taken from splenocyte-dendritic cell co-cultures were also consistent with aggregated preparations of OVA inducing a Th1-type response. Host cell protein (HCP) impurities can also contribute to immunogenicity. Mass spectrometry analysis of an scFv preparation identified the presence of the Escherichia coli (E.coli) heat shock protein DnaK, amongst other HCP, as an impurity. Protein preparations free from DnaK were spiked with recombinant E.coli DnaK to mimic the HCP impurity. The effect of DnaK on the immunogenicity of aggregated and monomeric scFv preparations was then investigated. BALB/c mice were immunised with monomeric and aggregated preparations, with and without E.coli DnaK at 0.1% by mass. Aggregation alone resulted in an enhanced IgG2a antibody response, and the presence of DnaK increased this further. Comparable investigations were also conducted using mouse albumin; here an increase in immunogenicity was observed with protein aggregation, and the presence of DnaK was found to increase the IgG2a response. Collectively, the evidence presented in this thesis shows that aggregation can impact upon the magnitude and character of induced immune responses, and that subvisible aggregation promotes a Th1 immune skewing. Additionally, E.coli HCP DnaK enhances protein aggregate immunogenicity, which indicates that heat shock proteins, as a class of HCP, could have an adjuvant-like effect on biotherapeutic aggregates.
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Ohmer, Michaela [Verfasser], and Georg [Akademischer Betreuer] Häcker. "Regulation of cell survival by host and viral anti-apoptotic Bcl-2 proteins." Freiburg : Universität, 2016. http://d-nb.info/1152210300/34.
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Schoggins, John Wesley. "Adenovirus host-cell interactions : the role of capsid proteins in transduction, immune activation, and gene targeting /." Access full-text from WCMC:, 2007. http://proquest.umi.com/pqdweb?did=1432771281&sid=1&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Khairalla, Ahmed Samir Mohammed. "Identification of host immune cell receptors mediating the binding of meningococcal App/MspA proteins." Thesis, University of Nottingham, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.575478.
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Neisseria meningitidis, a major cause of bacterial meningitis and septicemia, secretes multiple virulence factors to the extracellular environment, including the adhesion and penetration protein (App) and the meningococcal serine protease A (MspA). Both proteins belong to the serine protease auto transporter family and have the capacity to adhere to host epithelial and endothelial cells. Besides, both proteins have been previously shown to be internalized and translocated to the nuclei of human brain microvascular endothelial cells and dendritic cells (DCs), where they modulate gene expression. However, host cell receptors, endocytic pathways, nuclear translocation mechanisms, and gene expression modulation associated with App or MspA are poorly defined. In an effort to address this knowledge gap, recombinant forms of the passenger domains of both proteins were purified under native conditions using cold shock expression technology. The purified proteins, designated rpdApp and rpdMspA, were used to raise polyclonal, affinity-purified guinea pig antisera. These antibodies facilitated the investigation of interactions between both bacterial proteins and host cell molecules. One of these interactions highlighted in the present study is the entry of rpdApp and rpdMspA into human monocyte-derived immature DCs via receptor-mediated endocytosis. In vitro internalization and blocking assays showed that mannose receptor (MR) and transferrin receptor 1 are involved in the uptake of both proteins into DCs. Besides, two different approaches revealed that the binding of rpdApp and rpdMspA to MR is mediated specifically by the C-type lectin-like carbohydrate recognition domains 4':'7 (CTLD4-7). Additionally, the recognition of both bacterial proteins by these domains was shown to be calcium dependent, in addition to being inhibitable by D-mannose or L- fucose, but not D-galactose. Using gel overlay (Far-Western) analysis, rpdApp and rpdMspA were shown to interact with two nucleocytoplasmic shuttling proteins, namely heat shock protein 70 (Hsp70) and galectin-3 (Gal-3), with some data suggesting that interactions with Gal-3 may be mediated by its carbohydrate-recognition domain. Hsp70, which also interacts with the bacterial proteins, is characterized by possessing lectinic activity towards O-linked ~-N-acetylglucosamine (O-G1cNAc). Given the binding of App and MspA to different lectins, this study sought to examine the glycosylation status of both proteins, with a focus on O-GlcNAc modifications and their possible interplay with phosphorylation. Using in silico prediction methods, multiple sites within each protein were identified as potential sites for phosphorylation, O-GlcNAc modification, and alternative phosphorylation/O-GlcNAc modification. These predictions were supported by a series of Western blot analyses, which reproducibly showed that several meningococcal secreted proteins, including those of the expected size of App or MspA (115-120 kDa), are modified by both O-GlcNAc and tyrosine phosphorylation. The identity of the 115- to 120-kDa proteins was confirmed by stripping and reprobing the blots with antibodies against rpdApp and rpdMspA. However, attempts to detect these post-translational modifications in a sample of meningococcal secreted proteins tagged with TAMRA-alkyne by tandem mass spectrometry were unsuccessful. This may be attributed to the suppressive effect of the attached tag on trypsin cleavage and/or ionization efficiency. Finally, the results of Far-Western and ELISA assays clearly demonstrated the interactions of rpdApp or rpdMspA with various histone subunits. Additionally, both proteins were shown to have trypsin-like serine protease activity and to be capable of cleaving recombinant histone H3.1 in vitro. The proteolytic activity of both proteins on H3.l was shown to be specifically abolished by pre-incubation with serine protease inhibitors. Collectively, these results extend our understanding of the virulence potential of App and MspA beyond the adhesive or invasive functions, and reveal their capacity to participate in multiple interactions with host molecules. Besides, the current findings provide the basis for future studies to explore the epigenetic alterations induced by both proteins. Hopefully, the information gained will provide new insights into meningococcal pathogenesis and will help in the development of improved therapeutic approaches against the disease.
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Sokolova, Nadiia Verfasser], and Theresia [Akademischer Betreuer] [Stradal. "Identification and characterization of interactions between bacterial WxxxE-virulence proteins and host cell proteins / Nadiia Sokolova ; Betreuer: Theresia Stradal." Braunschweig : Technische Universität Braunschweig, 2017. http://d-nb.info/1175817961/34.
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He, Yupeng. "Modulation of the host cell signaling pathways and protein synthesis by hepatitis C virus nonstructural 5A protein /." Thesis, Connect to this title online; UW restricted, 2002. http://hdl.handle.net/1773/11491.
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Curra, Chiara. "Protein trafficking and host cell remodeling in malaria parasite infection." Thesis, Montpellier 2, 2010. http://www.theses.fr/2010MON20219.
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Pour assurer ses besoins de croissance, multiplication, et survie, Plasmodium modifie sa cellule hôte, l'érythrocyte, après l'invasion. Le parasite met en place ainsi un système d'échanges (import/export) avec sa cellule hôte et le milieu extérieur. Nous avons identifié dans la base de données de Plasmodium berghei, le parasite de rongeurs, une famille de gènes, sep, correspondant à la famille etramp chez Plasmodium falciparum. Cette famille de gènes code pour des petites protéines exportées, et conservées dans tout le genre Plasmodium. Les protéines SEP (13?16 kDa) contiennent en N-terminal un peptide signal prédit, un domaine hydrophobe interne, et elles diffèrent au niveau des régions C-terminal et 3' UTR. Toutefois, les protéines SEP sont exprimées à différents moments du cycle de Plasmodium. Durant le cycle érythrocytaire, PbSEP1 et PbSEP3 sont exprimées à partir du stade trophozoïte, et la même quantité de protéine est détectée au stade schizonte et gamétocyte, pendant que PbSEP3 est hautement détectée dans les trophozoïtes mûrs et les gamétocytes. Chez le moustique, PbSEP1 et PbSEP3 sont détectées seulement chez les ookinètes, alors que PbSEP2 est très abondante dans les ookinètes, oocystes, et sporozoïtes des glandes salivaires. Les protéines SEP ont également des localisations différentes. Dans l'érythrocyte, PbSEP1 est localisée dans la membrane de la vacuole parasitophore, alors que PbSEP2 et PbSEP3 sont exportées au-delà de cette vacuole, et sont ainsi localisées dans la cellule hôte, en association avec des structures vésiculaires. Dans cette étude, nous avons identifié les signaux d'adressage des protéines SEP dans la vacuole parasitophore et dans la cellule hôte, chez Plasmodium berghei. L'autre partie du travail, effectuée à l'Université de Montpellier II, a consisté à étudier la localisation de deux protéines du squelette sous- membranaire de l'érythrocyte, la dématine, et l'adducine, durant le développement intra-érythrocytaire de Plasmodium falciparum. Le but de cette étude étant d'identifier un mécanisme potentiel d'internalisation des composants du squelette sous-membranaire de l'érythrocyte dans le parasite. Des études d'immuno-localisation ont montré que la dématine et l'adducine sont internalisées à partir du stade trophozoïte, et sont localisées probablement à la vacuole parasitophore (membrane et/ou lumière). Cette internalisation a été confirmée par des études de fractionnement cellulaire et d'accessibilité à la protéinase K, montrant que la dématine est totalement internalisée, alors l'adducine ne l'est que partiellement, suggérant une localisation de la protéine à la périphérie du parasite<br>Plasmodium endurance depends on the ability of the parasite to reorganize the cytosol of the erythrocyte, a terminally differentiated cell, and remodel its skeleton membrane immediately after invasion. In this way the parasite can organize the import/export of the molecules necessary to its survival. The comprehension of cellular trafficking mechanisms which occur during Plasmodium infection is a very important step and fundamental contribute to understand the biology of the malaria parasite.We identified in database of the rodent malaria parasite Plasmodium berghei the gene family sep, corresponding to etramp in P. falciparum, encoding small exported proteins conserved in the genus Plasmodium. SEP proteins (13?16 kDa) contain a predicted signal peptide at the NH2-terminus, an internal hydrophobic region while they differ in their C-terminal region; the genes share the upstream regulative region while differ in the 3' UTR. Despite this, we showed that SEPs have a different timing of expression and a different localization: in the erythrocytic cycle PbSEP1 and PbSEP3 start to be expressed at trophozoite and the same amount of protein is detected also in schizonts and gametocytes, while PbSEP2 is highly detected in mature trophozoites and even more in gametocytes. In mosquitoes stages PbSEP1 and PbSEP3 are expressed only in ookinetes, while PbSEP2 is very abundant in ookinetes, oocysts and in sporozoites of the salivary glands. SEPs also have a different localization in the iRBC: PbSEP1 is targeted to the membrane of the parasitophorous vacuole, while PbSEP2 and 3 are exported beyond the parasite membrane and translocated to the host cell compartment in association with vesicle-like structures. In this study we identified the specific signals necessary for the correct timing of expression and to direct SEP proteins to the vacuolar membrane and to the host cell compartments.The second part of the work was carried out in Montpellier II University and aims to identify the localization of two RBC membrane skeleton components, dematin and adducin, during Plasmodium falciparum infection. Our purpose is to recognize a possible mechanism of internalization of host cytoskeleton components to the parasite compartments. In fact, IFA experiments carried on iRBCs showed that dematin and adducin start to be internalized at trophozoite stage and localize at the periphery of the parasite, most probably at the parasitophoruos vacuole (PV) membrane/lumen. Dematin and adducin internalization during Plasmodium infection is also demonstrated by subcellular fractionation and proteinase K assay: while dematin is fully internalized, adducin is partially protected and suggesting a localization of the protein at the periphery of the parasite where it can be exposed to PK degradation
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Courtney, Sean C. "Functional Analysis of Host Cell Proteins and Stress Responses that Inhibit West Nile Virus Infection." Digital Archive @ GSU, 2011. http://digitalarchive.gsu.edu/biology_diss/101.
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Resistance to flavivirus-induced disease is conferred by a single gene that encodes oligoadenylate synthetase (Oas) 1b (Oas1b). Oas1b is not a functional synthetase suggesting its anti-flavivirus mechanism is RNase L-independent and that it may be mediated by interactions with other host cell protein(s). A yeast two-hybrid screen was used to identify host cell binding partners of Oas1b. Candidate partners were confirmed by yeast co-transformation and co-immunoprecipitation analyses. Oxysterol binding protein-related 1L (ORP1L) and ATP binding cassette subfamily F 3 (ABCF3) were found to interact with Oas1b. RNAi knockdown studies suggested that ORP1L and ABCF3 form a tripartite complex with Oas1b that is critical for the flavivirus-induced disease resistance mechanism.
Stresses including oxidation, nutrient starvation, and viral infections often induce the formation of stress granules (SGs) in eukaryotic cells. In response to stress, eIF2α kinases phosphorylate eIF2α leading to stalled 48S pre-initiation complexes and SG formation. West Nile virus (WNV) Eg101 infections were previously shown not to induce the formation of SGs. Infections with viruses of other natural WNV strains, as well as a WNV lineage 1/2-based infectious clone (W956IC) were analyzed and only W956IC infections were found to induce SGs. eIF2α kinase knockout MEFs were used to show that the W956IC-induced SGs were PKR-dependent. WNV chimeras were made by inserting Eg101 genes into the W956IC backbone. Chimeras replacing NS5 or NS1 and NS5 or NS1 and NS3 and NS4a reduced SG formation as well as early viral RNA synthesis similar to Eg101 infections. W956IC infections but not Eg101 infections were shown to produce exposed viral dsRNA at early times after infection. The data suggest that natural WNV infections evade the cell SG response by suppressing the amplification of viral RNA until cytoplasmic membranes have been remodeled to protect replication complexes from detection.
It was previously reported that WNV Eg101 infections inhibited the formation of arsenite-induced SGs. The ability of other natural WNV strain infections to inhibit SG formation by arsenite (HRI), DTT (PERK), W956IC co-infection (PKR), and heat shock treatments was assessed. WNV infections only inhibited arsenite-induced SG formation suggesting that WNV infections specifically suppress the response to oxidative intermediates.
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Westlund, Alexander. "Increased expression of therapeutic proteins by identification of 3'-UTRs from high expressing genes in CHO cells." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-157593.
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Therapeutic proteins, a.k.a. biopharmaceuticals, are most commonly produced in expression systems derived from Chinese Hamstery Ovary (CHO) cells, thanks to great capacity of post-translational modifications like secretation, folding and glycosylation. The engineering of cells for regulation of protein expression has many options including knock-in and knock-out of genes, epigenetic studies or improvement of the expression casette of the protein of interest by e.g. promotor variants or modifications of the 5’ and 3’ untranslated region (UTR). The 3’-UTR is therefore a good optimization candidate for attempting to achieve increased expression of therapeutic proteins. The final aim of this study was to identify and design 3’-UTRs for improved expression of therapeutic proteins in HyClone™ CHO cells from GE Healthcare Bio-Sciences AB (GEHC). The impact goal is to increase the efficiency and lower the costs for pharmaceutical companies when producing biopharmaceuticals in the HyClone™ CHO cell line, leading to increased accessibility of monoclonal antibodies (mAbs) on the pharmaceutical market. The study was initiated with bioinformatic analysis of the CHO cell transcriptome from a set of RNA-seq data of HyClone™ CHO to find high expressing, context independent genes. The 3’-UTRs from the best candidate genes were used for construction of plasmids for expression of a Fc-eGFP fusion protein. Nine selected 3’-UTRs were designed, synthesized and cloned into a parent plasmid (pGE0520) creating nine plasmid variants (pGE0523-531). The constructed plasmids were used for evaluation with site directed integration (SDI) into the HyClone™ CHO cell line and expression analysis were performed by flow cytometry and antibody titer measurements from cells with successfully integrated plasmid sorted by fluorescence-activated cell sorting (FACS). Result show a significant effect on protein expression when using different variants of 3’-UTRs. Two variants, pGE0524 and pGE0526, competing with the parent plasmid in expression levels and integration efficiency from SDI, making them candidates for further investigations against the parent plasmid. Results also show good correlation between flow cytometry data from pre- and post-sorting, which can make research for further 3’-UTRs more efficient by evaluations and prediction of expression levels before cell sorting.
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Garcia, Lobato Tavares Raquel. "Host cell responses to Helicobacter pylori secreted factors." Doctoral thesis, Stockholms universitet, Institutionen för molekylär biovetenskap, Wenner-Grens institut, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-148427.
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The infection of the human gastric mucosa by the bacterium Helicobacter pylori can lead to the development of gastritis, gastroduodenal ulcers, and cancer. The factors that determine disease development in a small percentage of infected individuals are still not fully understood. In this thesis, we aimed to identify and functionally characterize novel virulence factors of H. pylori and to understand their effect on host cell responses. In Paper I, we found that JHP0290, an uncharacterized secreted protein of H. pylori, induced macrophage apoptosis concomitant to the release of pro-inflammatory cytokine TNF via the regulation of the Src family of kinases and ERK MAPK pathways. In paper II, we demonstrated that JHP0290 exhibits both proliferative and anti-apoptotic activity, together with a faster progression of the cell cycle in gastric epithelial cells. During these responses, ERK MAPK and NF-κB pathways were activated. Paper III revealed a pro-apoptotic effect of another H. pylori-secreted protein HP1286 in macrophages via the TNF-independent and ERK-dependent pathways. No apoptosis was observed in HP1286-treated T cells or HL60 neutrophil-like cells, suggesting cell-type specific effect of HP1286. In Paper IV, we observed the pro-inflammatory activity of H. pylori secreted protein HP1173 in macrophages. The protein was found to induce TNF, IL-1β, and IL-8 in macrophages through MAPKs, NF-κB, and AP-1 signaling pathways. Furthermore, differential expression and release of JHP0290, HP1286, and HP1173 homologues was observed among H. pylori strains (papers II, III, IV). Due to their ability to regulate multiple host cell responses, proteins JHP0290, HP1286, and HP1173 could play an important role in bacterial pathogenesis.
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Suppiah, Suganthi. "Investigation of Interactions of the Rubella Virus P150 Replicase Protein with Host Cell Proteins in Infected Cells." Digital Archive @ GSU, 2009. http://digitalarchive.gsu.edu/biology_diss/86.
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Due to their simplicity, viruses require the assistance of host factors for various aspects of their replication cycle. This study investigated the interaction of one of the two non-structural replicase proteins of rubella virus (RUBV), P150, with cell proteins. RUBV forms replication complexes for replicating its RNA in association with membranes of endosomes and lysosomes; the thusly modified endosomes/lysosomes are termed cytopathic vacuoles or CPVs. In the first study, a RUBV expressing a FLAG epitope-tagged P150 was used to co-immunoprecipitate putative interacting cell proteins from an infected cell lysate fraction enriched for CPVs using differential centrifugation. However, the only interacting protein identified was the companion RUBV replicase protein P90. Thus, cell proteins do not bind with either sufficient affinity or in stoichiometric amounts to be detected by this method and may not be a component of the virus holoenzyme. In the second study, a proline-rich region within P150 with three PxxPxR consensus SH3 domain-binding motifs was investigated for its ability to bind cell proteins. Substitution mutations (to alanine) of the two prolines were made in each of these motifs with the finding that mutations in the first two motifs led to lower viral titers and a small plaque phenotype with reversion to the wt sequence within one passage. Mutations in the third motif had a wt phenotype and did not revert. However, these mutations did not affect viral RNA synthesis, suggesting that the importance of these motifs is in a later stage of viral life cycle, e.g. virion assembly and release. To extend these findings, the proline hinge region with either the wt or mutant sequence was expressed as a GST-fusion in human cells. Pulldown experiments revealed specific binding with human p32 protein (gC1qR), which was previously shown to interact with the RUBV capsid protein. Binding of p32 with P150 was confirmed. The function of p32 in the RUBV replication cycle is unclear, but could involve virion assembly and release or induction of apoptosis.
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Fischer, Joshua Richard. "Mechanisms of Host Cell Attachment by the Lyme Disease Spirochete: A Dissertation." eScholarship@UMMS, 2005. http://escholarship.umassmed.edu/gsbs_diss/194.
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Host cell binding is an essential step in colonization by many bacterial pathogens, and the Lyme disease agent, Borrelia burgdorferi, which colonizes multiple tissues, is capable of attachment to diverse cell types. Glycosaminoglycans (GAGs) are ubiquitously expressed on mammalian cells and are recognized by multiple B. burgdorferi surface proteins. We previously showed that B. burgdorferi strains differ in the particular spectrum of GAGs that they recognize, leading to differences in the cultured mammalian cell types that they efficiently bind. The molecular basis of these binding specificities remains undefined, due to the difficulty of analyzing multiple, potentially redundant cell attachment pathways and to the paucity of genetic tools for this pathogen. Complementation of a high-passage non-adherent B. burgdorferi strain reveals that the expression of DbpA, DbpB, or BBK32, is sufficient to confer efficient spirochete attachment to 293 epithelial cells. Epithelial cell attachment by DbpA and B was mediated by dermatan sulfate, while BBK32 recognized dermatan and heparan sulfate. The GAG binding properties of bacteria expressing DbpB or DbpA were distinguishable in that DbpB, but not DbpA, promoted spirochetal attachment to C6 glial cells. Furthermore, DbpA alleles from diverse Lyme disease spirochetes exhibit allelic variation with respect to binding decorin, dermatan sulfate, and epithelial cells. Targeted disruption of bbk32 resulted in decreased spirochete binding to fibronectin, GAGs, and mammalian cells. Thus, DbpA, DbpB, and BBK32 may play central but distinct roles in cell type-specific binding by Lyme disease spirochetes. This study illustrates that transformation of high-passage B. burgdorferi strains and targeted gene disruption provide a comprehensive genetic approach to analyze virulence-associated phenotypes conferred by multiple bacterial factors.
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Vatter, Heather. "Analysis of Simian Hemorragic Fever Virus Proteins and the Host Cell Responses of Disease Resistant and Susceptible Primates." Digital Archive @ GSU, 2013. http://digitalarchive.gsu.edu/biology_diss/128.
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African monkey species are natural hosts of simian hemorrhagic fever virus (SHFV) and develop persistent, asymptomatic infections. SHFV was previously shown to also cause a rapid onset fatal hemorrhagic fever disease in macaques. Infection of macaques with a new isolate of SHFV from persistently infected baboon sera, that showed high nucleotide identity with the lab strain LVR, resulted in viremia, pro-inflammatory cytokine and tissue factor production, and symptoms of coagulation defects. Primary macrophages and myeloid dendritic cell cultures from disease-susceptible macaques efficiently replicated SHFV and produced pro-inflammatory cytokines, including IL-6 and TNF-α, as well as tissue factor. Cells from disease resistant baboons produced low virus yields and the immunomodulatory cytokine IL-10. IL-10 treatment of macaque cells decreased IL-6 levels but had no effect on TNF-α levels, tissue factor or virus production suggesting that IL-10 plays a role in modulating immunopathology in disease-resistant baboons but not in regulating the efficiency of virus replication.
SHFV is a member of the family Arteriviridae. The SHFV genome encodes 8 minor structural proteins. Other arteriviruses encode 4 minor structural proteins. Amino acid sequence comparisons suggest that the four additional SHFV minor structural proteins resulted from gene duplication. A full-length infectious clone of SHFV was constructed and produced virus with replication kinetics comparable to the parental virus. Mutant infectious clones, each with the start codon of one of the minor structural proteins substituted, were analyzed. All eight SHFV proteins were required for infectious virus production.
The SHFV nonstructural polyprotein is processed into the mature replicase proteins by several viral proteases including papain-like cysteine proteases (PLPs). Only one or two PLP domains are present in other arteriviruses but SHFV has three PLP domains. Analysis of in vitro proteolytic processing of C- and N-terminally tagged polyproteins indicated that the PLP in each of the three SHFV nsp1 proteins is active. However, the nsp1α protease is more similar to a cysteine protease than a PLP. Analysis of the subcellular localization of the three SHFV nsp1 proteins indicated they have divergent functions.
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Jassim, Amir. "Involvement of the matrix proteins SPARC and osteopontin in the dynamic interaction between tumour and host cells." Thesis, Brunel University, 2016. http://bura.brunel.ac.uk/handle/2438/13399.
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Osteoblasts are highly active cells that are responsible for secreting bone forming components such as collagen type I and matricellular proteins that mediate collagen deposition and mineralisation. SPARC and osteopontin are matricellular proteins that are involved in bone regulation and cell-matrix interactions and are also upregulated in metastatic disease. Secretion of these proteins results in changes to the stromal environment that includes cell migration, angiogenesis, matrix degradation, matrix deposition, bone mineralisation and bone resorption. Signalling pathways not only lead to the expression of target proteins, but also have immediate early effects, for example, on cell adhesion. We asked if the ERK 1 and 2 module of the MAPK pathway was involved in the intracellular trafficking of SPARC and Osteopontin. Membrane trafficking is an essential process that ensures newly synthesised proteins pass from their site of synthesis to the extracellular environment. Using an inhibitor of ERK 1 and 2 activation (U0126), as well as siRNA directed against ERK 1 or 2 individually, a change in intracellular localisation of SPARC and osteopontin was observed in cells treated with U0126 and siRNA against ERK 2 alone, likely in or around the Golgi apparatus. Consistent with the observation above, analysis of protein secretion showed that there was a reduction of total protein secreted (30% reduction) when ERK 1 and 2 activation was prevented together or knock down of ERK 2 alone. A mechanism is proposed where ERK 2 is likely activating a substrate that is allowing SPARC and osteopontin to continue along the secretory pathway. This directly implicates ERK 2 as an important regulator of matricellular protein secretion in osteoblasts. In cancer, Ras mutations can lead to permanent activation of the MAPK pathway leading to cancer cell proliferation and survival, however, we propose another mechanism important in metastasis whereby ERK 2 activation is manipulated to facilitate secretion of matricellular proteins which can then mediate changes to the stromal environment that allow the tumour to metastasise successfully.
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Chang, Po-Hsun. "Characterization of the Outer Membrane of Treponema Pallidum Subsp. Pallidum by Binding Studies Using Antibodies, Complement, and Host Serum Proteins." Thesis, University of North Texas, 1989. https://digital.library.unt.edu/ark:/67531/metadc798468/.
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The major goal of this study was to achieve sustained cultivation of virulent T. pallidum in vitro. The putatuive binding of host proteins to the outer membrane (OM) of intact, virulent T. pallidum subsp. pallidum has been investigated. A major breakthrough was the development of a filtration assay, usinglow protein-binding membrane filters, for the measurement of substances bound to or incorporated into th eOM of T. pallidum. This avoided the conventional manipulations which can damage the fragile OM of T. pallidum. Using this filtration assay, studies on the binding of host serum proteins demonstrated that intact treponemes did not bind host proteins as previously reported. It also indicated that previous studies were probably performed with damaged by this research. The studies on the binding of polyclonal and monoclonal antibodies to intact and detergent treated treponemes provided evidence of the low level binding of antibody to intact treponemes which was greatly enhanced but the removal of the outer membrane with 0.1% Triton X. This research research corroborated that of others which suggests that the outer membrane of T. pallidum contains very little protein or surface exposed antigen.
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Rhein, Bethany Ann. "Ebola virus: entry, pathogenesis and identification of host antiviral activities." Diss., University of Iowa, 2015. https://ir.uiowa.edu/etd/6629.
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Ebola virus (EBOV) is a member of the Filoviridae family of highly pathogenic viruses that cause severe hemorrhagic fever and is the causative agent of the 2014 West Africa outbreak. Currently, there are no approved filovirus vaccines or treatments to combat these sporadic and deadly epidemics. One target for EBOV antiviral therapy is to block viral entry into host cells. Recently, phosphatidylserine (PtdSer) receptors, primarily known for their involvement in the clearance of dying cells, were shown to mediate entry of enveloped viruses including filoviruses. The PtdSer receptors, T-cell immunoglobulin mucin domain-1 (TIM-1) and family member TIM-4, serve as filovirus receptors, significantly enhancing EBOV entry. TIM-dependent virus uptake occurs via apoptotic mimicry by binding to PtdSer on the surface of virions through a conserved PtdSer binding pocket within the amino terminal IgV domain. TIM-4 is expressed on antigen presenting cells (APCs), including macrophages and dendritic cells (DCs), which are critical in early EBOV infection. My studies are the first to define the molecular details of virion/TIM-4 interactions and establish the importance of TIM-4 for EBOV infection of murine resident peritoneal macrophages. In addition, previous work has utilized only in vitro models to establish the importance of the TIM proteins in EBOV entry. My studies are the first to demonstrate the importance of TIM-1 and TIM-4 for in vivo EBOV pathogenesis and to confirm them as relevant targets of future filovirus therapeutics.
Macrophage phenotypes can vary greatly depending upon chemokine and cytokine signals from their microenvironment. Historically, macrophages have been classified into two major subgroups: classically activated macrophages (M1) and alternatively activated macrophages (M2). Macrophages are a critical early target of EBOV infection and my work primarily focused on interferon gamma-stimulated (M1) macrophages since this treatment profoundly inhibited EBOV infection of human and murine macrophages. Interferon gamma treatment blocked EBOV replication in macrophages, reducing viral RNA levels in a manner similar to that observed when cultures were treated with the protein synthesis inhibitor, cycloheximide. Microarray studies with interferon gamma-treated human macrophages identified more than 160 interferon-stimulated genes. Ectopic expression of a select group of these genes inhibited EBOV infection. These studies provide new potential avenues for antiviral targeting as these genes that have not previously appreciated to inhibit infection of negative strand RNA viruses including EBOV. In addition and most exciting, using MA-EBOV, we found that murine interferon gamma, when administered either 24 hours before or after infection, protects lethally challenged mice and significantly reduces morbidity. Our findings suggest that interferon gamma, an FDA-approved drug, may serve as a novel and effective prophylactic or treatment option.
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Arfi, Zulfaquar Ahmad Verfasser], Rainer [Akademischer Betreuer] [Fischer, and Stefan [Akademischer Betreuer] Schillberg. "Development of an immunoassay to detect tobacco host cell proteins during biopharmaceutical development / Zulfaquar Ahmad Arfi ; Rainer Fischer, Stefan Schillberg." Aachen : Universitätsbibliothek der RWTH Aachen, 2015. http://d-nb.info/1129176789/34.
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Arfi, Zulfaquar Ahmad [Verfasser], Rainer [Akademischer Betreuer] Fischer, and Stefan [Akademischer Betreuer] Schillberg. "Development of an immunoassay to detect tobacco host cell proteins during biopharmaceutical development / Zulfaquar Ahmad Arfi ; Rainer Fischer, Stefan Schillberg." Aachen : Universitätsbibliothek der RWTH Aachen, 2015. http://d-nb.info/1129176789/34.
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Thay, Bernard. "Vesicle-mediated and free soluble delivery of bacterial effector proteins by oral and systemic pathogens." Doctoral thesis, Umeå universitet, Institutionen för odontologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-82782.
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Periodontitis, the primary cause of tooth-loss worldwide, is a bacterially induced chronic inflammatory disease of the periodontium. It is associated with systemic conditions such as cardiovascular disease (CVD). However, pathogenic mechanisms of periodontitis-associated bacteria that may contribute to the CVD association are unclear. The aim of this doctoral thesis project was to characterize bacterial mechanisms that can originate from the periodontal pocket and expose the host to multiple effector proteins, thereby potentially contributing to periodontal tissue degradation and systemic stimulation. As our main model, we have used Aggregatibacter actinomycetemcomitans, a Gram-negative species associated with aggressive forms of periodontitis, and with non-oral infections, such as endocarditis. Since Gram-positive species might be more common in periodontitis than previously believed, we have also investigated mechanisms of the multipotent bacterium, Staphylococcus aureus. Using an ex vivo insert model we showed that free-soluble surface material, released during growth by A. actinomycetemcomitans independently of outer membrane vesicles (OMVs), enhanced the expression of several proinflammatory cytokines in human whole blood. A clear LPS-independent effect suggested the involvement of effector proteins in this cytokine stimulation. This was supported by MALDI-TOF-MS and immunoblotting, which confirmed the release of GroEL and peptidoglycan-associated lipoprotein (PAL), in free-soluble form. We next demonstrated that A. actinomycetemcomitans OMVs could deliver multiple proteins including biologically active cytolethal distending toxin (CDT), a major virulence factor, into human gingival fibroblasts and HeLa cells. Using confocal microscopy, the active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. By using a fluorescent probe, B-R18, it was shown that the OMVs fused with lipid rafts in the plasma membrane. These findings suggest that OMVs can deliver biologically active virulence factors such as CDT into susceptible cells of the periodontium. Using A. actinomycetemcomitans vesicles labeled with the lipophilic dye, PKH26, it was shown that the OMVs can be internalized into the perinuclear region of human cells in a cholesterol-dependent manner. Co-localization analysis supported that the internalized OMVs carried A. actinomycetemcomitans antigens. Inhibition assays suggested that although OMV internalization appeared to have a major role in effector protein delivery, additional interactions such as vesicle membrane fusion may also contribute. The OMVs strongly induced activation of the cytosolic pathogen recognition receptors NOD1 and NOD2 in HEK293T-cells, consistent with a role in triggering innate immunity by carrying PAMPs such as peptidoglycan into host cells. Membrane vesicles (MVs) from S. aureus were found to carry biologically active alpha-toxin, a key virulence factor, which was delivered to host cells and required for full cytotoxicity of the vesicles. Confocal microscopy analysis revealed that these MVs, similar to A. actinomycetemcomitans OMVs, interacted with HeLa cells via membrane fusion. Thus, as S. aureus is frequently found in individuals with aggressive periodontitis, MV production could have potential to contribute to the severity of tissue destruction.
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Forsman, Alma. "The Epstein-Barr virus nuclear antigens 1 & 5 : study of virus-host cellular protein interactions /." Göteborg : Institute of Biomedicine, Department of Clinical Chemistry and Transfusion Medicine, 2009. http://hdl.handle.net/2077/21357.
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Lövkvist, Lena. "Receptor interactions between pathogenic bacteria and host cells /." Uppsala : Acta Universitatis Upsaliensis : Uppsala universitetsbibliotek [distributör], 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7782.
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Kanatani, Sachie. "Host-parasite interactions in the dissemination of Toxoplasma gondii." Doctoral thesis, Stockholms universitet, Institutionen för molekylär biovetenskap, Wenner-Grens institut, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-148573.
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Toxoplasma gondii is an obligate intracellular parasite that infects virtually all warm-blooded organisms. Systemic dissemination of T. gondii in the organism can cause life-threatening infection that manifests as Toxoplasma encephalitis in immune-compromised patients. In addition, mounting evidence from epidemiological studies indicates a link between chronic Toxoplasma infection and mental disorders. To better understand the pathogenesis of toxoplasmosis, basic knowledge on the host-parasite interactions and the dissemination mechanisms are essential. Previous findings have established that, upon infection with T. gondii, dendritic cells (DCs) and microglia exhibit enhanced migration, which was termed the hypermigratory phenotype. As a result of this enhanced migration, DCs and microglia are used as vehicle cells for dissemination (‘Trojan horse’) which potentiates dissemination of T. gondii in mice. However, the precise mechanisms behind the hypermigratory phenotype remained unknown. In this thesis, we characterized host-parasite interactions upon infection with T. gondii and investigated the basic mechanisms behind the hypermigratory phenotype of T. gondii-infected DCs and microglia. In paper I, we observed that upon infection with T. gondii, DCs underwent rapid morphological changes such as loss of adhesiveness and podosomes, with integrin redistribution. These rapid morphological changes were linked to hypermotility and were induced by active invasion of T. gondii within minutes. T. gondii-infected DCs exhibited up-regulation of the C-C chemokine receptor CCR7 and chemotaxis towards the CCR7 chemotactic cue, CCL19. In paper II, we developed a 3-dimensional migration assay in a collagen matrix, which allowed us to characterize the hypermigratory phenotype in a more in vivo-like environment. The migration of T. gondii-infected DCs exhibited features consistent with integrin-independent amoeboid type of migration. T. gondii-induced hypermigration of DCs was further potentiated in the presence of CCL19 in a 3D migration assay. In paper III, we identified a parasite effector molecule, a Tg14-3-3 protein derived from parasite secretory organelles. Tg14-3-3 was sufficient to induce the hypermigratory phenotype. Transfection with Tg14-3-3-containing fractions or recombinant Tg14-3-3 protein induced the hypermigratory phenotype in primary DCs and in a microglial cell line. In addition, Tg14-3-3 localized in the parasitophorous vacuolar space and host 14-3-3 proteins were rapidly recruited around the parasitophorous vacuole. In paper IV, we found that mouse DCs dominantly express the L-type voltage-dependent calcium channel, Cav1.3. Cav1.3 was linked to the GABAergic signaling-induced hypermigratory phenotype. Pharmacological inhibition of Cav1.3 and knockdown of Cav1.3 abolished the hypermigratory phenotype in T. gondii infected DCs. Blockade of voltage-dependent calcium channels reduced the dissemination of T. gondii in a mouse model. In paper V, we showed that microglia, resident immune cells in the brain, also exhibited rapid morphological changes and hypermotility upon infection with T. gondii. However, an alternative GABA synthesis pathway was shown to be involved in the hypermigratory phenotype in microglia. In summary, this thesis describes novel host-parasite interactions, including host cell migratory responses and key molecular mechanisms that mediate the hypermigratory phenotype. The findings define a novel motility-related signaling axis in DCs. Thus, T. gondii employs GABAergic non-canonical pathways to hijack host cell migration and facilitate dissemination. We believe that these findings represent a significant step forward towards a better understanding of the pathogenesis of T. gondii infection.<br><p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.</p>
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Miller, Cathy Lea. "Investigation of the role of minute virus of mice (MVM) small non-structural protein NS2 interactions with host cell proteins during MVM infection." free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3025638.
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Huang, Bernice. "Anaplasma phagocytophilum remodels its host cell-derived vacuole into a protective niche by redecorating the vacuolar membrane with select Rab GTPases and bacterial proteins." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/280.
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Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to cause the emerging tick-transmitted disease, human granulocytic anaplasmosis (HGA). Following entry, the pathogen replicates within a host cell-derived vacuole that fails to mature along the endocytic pathway, does not acidify, and does not fuse with lysosomes. Selective fusogenicity is prototypical of many vacuole-adapted pathogens and has been attributed, at least in part, to pathogen modification of the vacuolar inclusion membrane and/or to selective recruitment or exclusion of host trafficking regulators. As a result, the A. phagocytophilum-occupied vacuolar membrane (AVM) provides a unique interface to study the host-pathogen interactions critical to A. phagocytophilum intracellular survival. Diverse vacuole-adapted pathogens; including Chlamydia, Legionella, and Salmonella; selectively recruit host Rab GTPases to their vacuolar membranes to establish replicative permissive niches within their host cells. Rab GTPases coordinate many aspects of endocytic and exocytic cargo delivery. We determined that the A. phagocytophilum-occupied vacuole (ApV) selectively recruits a subset of fluorescently-tagged Rabs that are predominantly associated with recycling endosomes. Another emerging theme among vacuole-adapted pathogens is the ability to hijack ubiquitin machinery to modulate host cellular processes. Mono- and polyubiquitination differentially dictate the subcellular localization, activity, and fate of protein substrates. Monoubiquitination directs membrane traffic from the plasma membrane to the endosome and has been shown to promote autophagy. We show that monoubiquitinated proteins decorate the AVM during infection of promyelocytic HL-60 cells, endothelial RF/6A cells, and to a lesser extent, embryonic tick ISE6 cells. Importantly, tetracycline treatment concomitantly promotes loss of the recycling endosome-associated GFP-Rabs and ubiquitinated proteins and acquisition of the late endosomal marker, Rab7, and lysosomal marker, LAMP-1, implicating bacterial-derived proteins in the ApV's altered fusogenicity. Therefore, we rationalized that A. phagocytophilum-encoded proteins that associate with the AVM may establish interactions with the host cell that are important for intracellular survival. By focusing on A. phagocytophilum proteins that are induced during host infection, we identified the first two bacterial-encoded proteins -- APH_1387 and APH_0032 -- that modify the AVM. Although functional studies are hindered by the lack of a system to genetically manipulate Anaplasma, the pathobiological roles of APH_1387 and APH_0032 are likely unique, as both proteins exhibit very little or no homology with any previously described protein. APH_1387 and APH_0032 are present at the cytoplasmic face of the AVM, therefore they likely interact with host proteins. We demonstrate that ectopic expression of APH_1387 and APH_0032 inhibits the ApV development in A. phagocytophilum infected cells. The results presented in this dissertation contribute to our understanding of how A. phagocytophilum modifies the vacuolar membrane in which it resides to establish a safe haven and evade lysosomal degradation.
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Eccleston, Ruth Charlotte. "A mechanistic model predicting cell surface presentation of peptides by MHC class I proteins, considering peptide competition, viral intracellular kinetics and host genotype factors." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/10038760/.
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Major histocompatability complex class I (MHC-I) proteins present short fragments of pathogenic or cancerous proteins (peptides) on the surface of infected cells for recognition by T lymphocytes which are stimulated upon recognition of foreign peptides. Due to the diversity of peptide sequences and the sequence-specificity of MHC-I alleles, being able to determine which peptides will be presented by which MHC-I alleles and in what proportion could be important for the development of vaccines and treatments based on the presented peptiodome. Machine learning tools, trained on experimental data, are widely used to predict immunogenic peptides. However they are unable to account for the impact the intracellular kinetics of the pathogenic or cancerous protein which will greatly influence the resultant peptidome. Here we describe a mechanistic model of peptide presentation, validated against experimental data, which accounts for intracellular peptide concentration, and can predict the relative cell surface presentation of competing peptides with varying affinities for MHC-I proteins. We demonstrate how combining this mechanistic model with the intracellular kinetics of HIV proteins can provide insight in to the experimentally reported immunogenicity of the viral protein Gag, and show how such a model can be used to predict the most abundant viral peptides presented on the cell surface. Similarly, we predict the HeLa cell peptidome and demonstrate how a simple metric can be used to approximate the abundance of a peptide based solely on protein synthesis and degradation, peptide-MHC affinity and proteasomal cleavage.
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Husson, Gauthier. "Development of host cell protein impurities quantification methods by mass spectrometry to control the quality of biopharmaceuticals." Thesis, Strasbourg, 2017. http://www.theses.fr/2017STRAF066/document.
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Les récents progrès instrumentaux en spectrométrie de masse, notamment en terme de- rapidité de balayage et de résolution, ont permis l'émergence de l'approche « data independent acquisition» (DIA). Cette approche promet de combiner les points forts des approches « shotgun » et ciblées,mais aujourd'hui l'analyse des données DIA reste compliquée. L'objectif de cette thèse a été de développer des méthodes innovantes de spectrométrie de masse, et en particulier d'améliorer l'analyse des données DIA. De plus, nous avons développé une approche originale Top 3-ID-DIA, permettant à la fois un profilage complet des protéines de la cellule hôte (HCP) ainsi qu'une quantification absolue d'HCP clés dans les échantillons d'anticorps monoclonaux (mAb), au sein d'une même analyse.Cette méthode est prête à être implémentée en industrie, et pourrait fournir un support en temps réel aux développements du procédé de production de mAb, ainsi que pour évaluer la pureté des biomédicaments<br>Recent instrumental developments in mass spectrometry, notably in terms of scan speed and resolution, allowed the emergence of “data independent acquisition” (DIA) approach. This approach promises to combine the strengths of both shotgun and targeted proteomics, but today DIA data analysis remains challenging. The objective of my PhD was to develop innovative mass spectrometry approaches, and in particular to improve DIA data analysis. Moreover, we developed an original Top 3-ID-DIA approach, allowing both a global profiling of host cell proteins (HCP) and an absolute quantification of key HCP in monoclonal antibodies samples, within a single analysis. This method is ready to be transferred to industry, and could provide a real time support for mAb manufacturing process development, as well as for product purity assessment
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Ebrahimi, Majid. "Studies of p63 and p63 related proteins in patients diagnosed with oral lichen planus." Doctoral thesis, Umeå : Univ, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1437.
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Ahlqvist, Jenny. "Differences in tropism and viral assembly pathways of human herpesvirus 6A and 6B (HHV-6A and 6B) and association of host cell proteins in HHV-6A virions /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-214-9/.
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Thorsén, Jenny. "Purification of His-tagged Proteins Using WorkBeads 40 TREN as a Pre-Treatment Step Prior Loading Sample onto IMAC Resins with the Purpose to Enhance Performance." Thesis, Uppsala universitet, Biokemi, 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-439642.
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This work is the result of evaluating a novel strategy for the purification of recombinant His-tagged proteins. Proteins purified in this study were the E. coli translational proteins IF-3, RF-1, and RFF. The study aimed to analyse the potential of using Bio-Works WorkBeads™40 TREN, a multimodal anion ion exchange chromatography resin, as a pretreatment step upstream an immobilized metal ion chromatography (IMAC) resin to enhance performance efficiency of His-tagged protein purification. The method demonstrated here shows potential for anyone seeking to increase the purity of His-tagged protein purification or to introduce an effective purification procedure by replacing a polishing step downstream IMAC with WorkBeads 40 TREN upstream IMAC. The latter contributing to guard the IMAC column from heavy bioburden. This study showed that running WorkBeads 40 TREN prior IMAC captures impurities and removes 97-98 % more dsDNA compared to direct IMAC. WorkBeads 40 TREN is therefore highly advantageous to include early in a purification process to remove protein binding DNA fragments. Moreover, WorkBeads 40 TREN increases purity in the final product by capturing more host cell proteins than when running direct IMAC. This concept is general and WorkBeads 40 TREN could be used upstream a variety of resins such as Protein A and RPC.
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Bevington, Joyce M. "Cellular Response to Adenovirus and Adeno- Associated Virus Coinfection." Connect to full text in OhioLINK ETD Center, 2009. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=mco1242921394.
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Dissertation (Ph.D.)--University of Toledo, 2009.<br>"In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 70-80, p. 28-158.
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Jonscher, Ernst Georg Wolfgang [Verfasser]. "Identification of proteins involved in host cell cytosol uptake in the human Malaria parasite Plasmodium falciparum : Identifizierung von Proteinen der Wirtszellzytosolaufnahme des humanen Malaria-Erregers Plasmodium falciparum / Ernst Georg Wolfgang Jonscher." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2018. http://d-nb.info/1223620964/34.
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Danielsson, Niemi Liza. "Host ligands and oral bacterial adhesion studies on phosphorylated polypeptides and gp-340 in saliva and milk /." Doctoral thesis, Umeå : Umeå university, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-32894.
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Lassalle, Damien. "Les pore forming toxin chez les Lophotrochozoaires : exemple des organismes Biomphalaria glabrata/ Schistosoma mansoni A New Assessment of Thioester-Containing Proteins Diversity of the Freshwater Snail Biomphalaria glabrata Cholesterol-TEG addition at the 5’end of siRNA allows significant increase of its uptake by hemocytes from Biomphalaria glabrata, the schistosomiasis vector snail. Under review in PeeJ. Schistosoma mansoni lysine specific demethylase (SmLSD1) is a druggable target involved in parasite survival, oviposition and stem cell proliferation." Thesis, Perpignan, 2020. https://theses-public.univ-perp.fr/2020PERP0036.pdf.
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La bilharziose est une maladie touchant 230 millions de personnes dans le monde (source OMS). Cette parasitose est provoquée par le schistosome, un vers plat parasite nommé Schistosoma mansoni. Avant de pénétrer dans l’organisme humain par la peau, ce parasite se développe chez un escargot d’eau douce, Biomphalaria glabrata, qui lui sert d’hôte intermédiaire. Nous avons dans ce contexte identifié et étudié deux protéines appartenant à la famille des pore formingtoxins (PFTs), que nous avons appelé Biomphalysine et Glabralysine. Les PFTs sont des effecteurs très connus dans le monde procaryote pour favoriser leurs pathogénicités. Ces protéines sont produites de manière soluble par les organismes, pour se fixer et s’agréger sur les membranes cellulaires cible, ce qui a pour conséquence de créer un pore lytique. Cette super famille de protéine se divise en deux sous familles, alpha et beta, classées en fonction de la modalité de formation du pore. Des études antérieures ont permis de caractériser pour la première fois des ß-PFT chez le mollusque Biomphalaria glabrata, ces protéines ont montré un rôle clef dans l’immunité du mollusque, notamment la capacité de lier au parasite et de le tuer. Cette découverte a pu ouvrir le champ à l’investigation de protéines similaire chez le mollusque et chez le parasite avec lequel il interagie. Ce projet de thèse a pour objectif, au travers d’étude génomique, transcriptomique et protéomique de caractériser et de comprendre la fonction des différentes « pore forming toxins » présentes chez le mollusque Biomphalaria glabrata et chez le parasite Schistosoma mansoni. Grace à des données collectées avant et durant le projet de thèse, nous avons pu caractériser 23 variants apparentés à la famille des Biomphalysines. Cette famille multigénique sans intron, semble avoir été acquise au travers de transfert horizontal de gênes. Par homologie avec les biomphalysines, nous avons pu caractériser 5 gènes codants pour un deuxième groupe de ß-PFT chez Biomphalaria glabrata, que nous avons appelé les Glabralysines. Ces protéines constituent une famille à part entière, proche des toxines cry de Bacillus thuringiensis, et qui d’un point de vue structural partage des homologies avec la Biomphalysine. Elles sont en effet, produites par les cellules immunitaires du mollusque et induites lors de l’infection par le parasite Schistosoma mansoni. Finalement, nous avons aussipu découvrir deux gènes codants pour des toxines, nommées Schistolysines, de la famille des ß-PFT chez le parasite Schistosoma mansoni. Ces protéines semblent être répandues chez les parasites et jouer des rôles essentiels dans leur développement, la reproduction et peut être la nutrition. Nous montrons que ces protéines sont exclusivement retrouvées au stade adulte du parasite et devraient donc jouer un rôle dans l’interaction avec l’hôte humain ou dans l’implantation ou l’exploitation de cet hôte. Ces différentes approches, dans le contexte de l’interaction entre l’hôte et son parasite permettront potentiellement d’identifier de nouvelles stratégies de lutte ou de contrôle de la maladie sur le terrain. Les résultats générés dans ce travail pourront permettre également d’étudier le rôle de ces molécules dans l’interaction avec d’autres pathogènes ou leur lien avec d’autres pathologies et plus particulièrement leur utilisation dans le développement de nouveaux traitements contre le cancer par exemple<br>Bilharzia is a disease affecting 230 million people worldwide (source WHO). This parasitosis is caused by schistosome, a parasitic flatworm, and in particular by Schistosoma mansoni, responsible for intestinal bilharzia in Africa and tropical America. Before entering the human body through the skin, this parasite develops in a freshwater snail, Biomphalaria glabrata, which serves as an intermediate host. In this context, we have identified and studied two proteins belonging to the pore forming toxins (PFTs) family, which we have called Biomphalysin and Glabralysin. Pore forming toxins are effectors well known in the prokaryotic world to promote their pathogenicity. These proteins are produced in a soluble way by organisms, to bind and aggregate on the target cell membranes, resulting in the creation of a lytic pore. This protein superfamily is divided into two families, alpha and beta, classified according to the pore formation modality. Previous studies have characterized for the first time §-PFTs in the mollusc Biomphalaria glabrata, these proteins have shown a key role in the immunity of the mollusc, including the ability to bind and kill the parasite. This discovery may have opened the field to the investigation of similar proteins in the mollusc and in the parasite with which it interacts. This thesis project aims, through genomic, transcriptomic and proteomic studies, to characterize and understand the function of different "pore forming toxins" present in the mollusc Biomphalaria glabrata and in the parasite Schistosoma mansoni. Thanks to data collected before and during the thesis project, we were able to characterize 23 variants related to the Biomphalysin family. This multigenic family, without intron, seems to have been acquired through horizontal gene transfer. By homology with the Biomphalysins, we were able to characterize 5 genes coding for a second group of §-PFT in Biomphalaria glabrata, which we called Glabralysins. These proteins constitute a family in their own right, close to the Cry toxins of Bacillus thuringiensis, and which structurally share homologies with the Biomphalysin. They are indeed produced by the immune cells of the mollusc and induced during the infection by the parasite Schistosoma mansoni. Finally, we were also able to discover two genes coding for toxins, called Schistolysins, of the §-PFT family in the parasite Schistosoma mansoni. These proteins seem to be widespread in parasites and play essential roles in their development, in reproduction and hypothetically in nutrition. We show that these proteins are found exclusively in the adult stage of the parasite and should therefore play a role in the interaction with the human host or in the implantation or exploitation of this host. These different approaches, in the context of the interaction between the host and its parasite, will potentially lead to the identification of new strategies for the control or management of the disease in the field. The results generated in this work could also allow to study the role of these molecules in the interaction with other pathogens or their link with other pathologies and more particularly their use in the development of new cancer treatments for example
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Seung, Edward. "CD40-CD154 Blockade Facilitates Induction of Allogeneic Hematopoietic Chimerism and Transplantation Tolerance: A Dissertation." eScholarship@UMMS, 2003. https://escholarship.umassmed.edu/gsbs_diss/103.
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Allogeneic hematopoietic chimerism leading to central tolerance has significant therapeutic potential. Establishment of hematopoietic chimerism created by stem cell transplantation has been shown to prevent and cure a number of autoimmune diseases and induce the most robust and long-lasting form of transplantation tolerance known. However, the realization of the vast clinical potential of hematopoietic chimerism for induction of transplantation tolerance has been impeded by the toxicity of the host conditioning regimen and the development of graft-versus-host disease (GVHD). This thesis describes the development of stem cell transplantation protocols that 1) reduce the host conditioning regimen; and 2) abrogate the development of GVHD. When applied to the treatment of autoimmune diabetic NOD mice, a model of type 1 diabetes, stem cell transplantation was able to 3) prevent autoimmune recurrence; and 4) permit curative pancreatic islet transplantation.
I first describe a tolerance-based stem cell transplantation protocol that combines sub-lethal irradiation with transient blockade of the CD40-CD154 costimulatory pathway using an anti-CD154 antibody. With this protocol, I established hematopoietic chimerism in BALB/c mice transplanted with fully allogeneic C57BL/6 bone marrow. All chimeric mice treated with anti-CD154 antibody remained free of graft vs.host disease (GVHD) and accepted donor-origin but not third party skin allografts. It was similarly possible to create allogeneic hematopoietic chimerism in NOD/Lt mice with spontaneous autoimmune diabetes. Pancreatic islet allografts transplanted into chimeric NOD/Lt mice were resistant not only to allorejection but also to recurrence of autoimmunity. I conclude that it is possible to establish robust allogeneic hematopoietic chimerism in sub-lethally irradiated mice without subsequent GVHD by blocking the CD40-CD154 costimulatory pathway using as few as two injections of anti-CD154 antibody. I also conclude that chimerism created in this way generates donor-specific allograft tolerance and reverses the predisposition to recurrent autoimmune diabetes in NOD/Lt mice, enabling them to accept curative islet allografts.
In order to further reduce the impediments associated with the implementation of allogeneic hematopoietic chimerism as a therapeutic modality, I adapted a costimulation blockade-based protocol developed for solid organ transplantation for use in stem cell transplantation. The protocol combines a donor-specific transfusion (DST) with anti-CD154 antibody to induce peripheral transplantation tolerance. When applied to stem cell transplantation, administration of DST, anti-CD154 antibody, and allogeneic bone marrow led to hematopoietic chimerism and central tolerance with no myeloablation (i.e. no radiation) and no GVHD in 3 different strains of mice. The development of donor-specific tolerance in this system was shown to involve deletion of both peripheral host alloreactive CD8+ T cells and nascent intrathymic alloreactive CD8+ T cells. In the absence of large numbers of host alloreactive CD8+ T cells, the cell transfusion that precedes transplantation need not be of donor-origin, suggesting that both allo-specific and non-allo-specific mechanisms regulate engraftment. Agents that interfere with peripheral transplantation tolerance partially impair establishment of chimerism. I conclude that robust allogeneic hematopoietic chimerism and central tolerance can be established in the absence of host myeloablative conditioning using a peripheral transplantation tolerance protocol.
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Tavares, Lucas Alves. "O envolvimento da proteína adaptadora 1 (AP-1) no mecanismo de regulação negativa do receptor CD4 por Nef de HIV-1." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/17/17136/tde-06012017-113215/.
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O Vírus da Imunodeficiência Humana (HIV) é o agente etiológico da Síndrome da Imunodeficiência Adquirida (AIDS). A AIDS é uma doença de distribuição mundial, e estima-se que existam atualmente pelo menos 36,9 milhões de pessoas infectadas com o vírus. Durante o seu ciclo replicativo, o HIV promove diversas alterações na fisiologia da célula hospedeira a fim de promover sua sobrevivência e potencializar a replicação. A rápida progressão da infecção pelo HIV-1 em humanos e em modelos animais está intimamente ligada à função da proteína acessória Nef. Dentre as diversas ações de Nef está a regulação negativa de proteínas importantes na resposta imunológica, como o receptor CD4. Sabe-se que esta ação resulta da indução da degradação de CD4 em lisossomos, mas os mecanismos moleculares envolvidos ainda são totalmente elucidados. Nef forma um complexo tripartite com a cauda citosólica de CD4 e a proteína adaptadora 2 (AP-2), em vesículas revestidas por clatrina nascentes, induzindo a internalização e degradação lisossomal de CD4. Pesquisas anteriores demonstraram que o direcionamento de CD4 aos lisossomos por Nef envolve a entrada do receptor na via dos corpos multivesiculares (MVBs), por um mecanismo atípico, pois, embora não necessite da ubiquitinação de carga, depende da ação de proteínas que compõem os ESCRTs (Endosomal Sorting Complexes Required for Transport) e da ação de Alix, uma proteína acessória da maquinaria ESCRT. Já foi reportado que Nef interage com subunidades dos complexos AP-1, AP-2, AP-3 e Nef não parece interagir com subunidades de AP-4 e AP-5. Entretanto, o papel da interação de Nef com AP-1 e AP-3 na regulação negativa de CD4 ainda não está totalmente elucidado. Ademais, AP-1, AP-2 e AP-3 são potencialmente heterogêneos devido à existência de isoformas múltiplas das subunidades codificadas por diferentes genes. Todavia, existem poucos estudos para demonstrar se as diferentes combinações de isoformas dos APs são formadas e se possuem propriedades funcionais distintas. O presente trabalho procurou identificar e caracterizar fatores celulares envolvidos na regulação do tráfego intracelular de proteínas no processo de regulação negativa de CD4 induzido por Nef. Mais especificamente, este estudo buscou caracterizar a participação do complexo AP-1 na modulação negativa de CD4 por Nef de HIV-1, através do estudo funcional das duas isoformas de ?-adaptina, subunidades de AP-1. Utilizando a técnica de Pull-down demonstramos que Nef é capaz de interagir com ?2. Além disso, nossos dados de Imunoblot indicaram que a proteína ?2-adaptina, e não ?1-adaptina, é necessária no processo de degradação lisossomal de CD4 por Nef e que esta participação é conservada para degradação de CD4 por Nef de diferentes cepas virais. Ademais, por citometria de fluxo, o silenciamento de ?2, e não de ?1, compromete a diminuição dos níveis de CD4 por Nef da membrana plasmática. A análise por imunofluorêsncia indireta também revelou que a diminuição dos níveis de ?2 impede a redistribuição de CD4 por Nef para regiões perinucleares, acarretando no acúmulo de CD4, retirados por Nef da membrana plasmática, em endossomos primários. A depleção de ?1A, outra subunidade de AP-1, acarretou na diminuição dos níveis celulares de ?2 e ?1, bem como, no comprometimento da eficiente degradação de CD4 por Nef. Além disso, foi possível observar que, ao perturbar a maquinaria ESCRT via super-expressão de HRS (uma subunidade do complexo ESCRT-0), ocorreu um acumulo de ?2 em endossomos dilatados contendo HRS-GFP, nos quais também detectou-se CD4 que foi internalizado por Nef. Em conjunto, os resultados indicam que ?2-adaptina é uma importante molécula para o direcionamento de CD4 por Nef para a via ESCRT/MVB, mostrando ser uma proteína relevante no sistema endo-lisossomal. Ademais, os resultados indicaram que as isoformas ?-adaptinas não só possuem funções distintas, mas também parecem compor complexos AP-1 com diferentes funções celulares, já que apenas a variante AP-1 contendo ?2, mas não ?1, participa da regulação negativa de CD4 por Nef. Estes estudos contribuem para o melhor entendimento dos mecanismos moleculares envolvidos na atividade de Nef, que poderão também ajudar na melhor compreensão da patogênese do HIV e da síndrome relacionada. Em adição, este trabalho contribui para o entendimento de processos fundamentais da regulação do tráfego de proteínas transmembrana no sistema endo-lisossomal.<br>The Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease which has a global distribution, and it is estimated that there are currently at least 36.9 million people infected with the virus. During the replication cycle, HIV promotes several changes in the physiology of the host cell to promote their survival and enhance replication. The fast progression of HIV-1 in humans and animal models is closely linked to the function of an accessory protein Nef. Among several actions of Nef, one is the most important is the down-regulation of proteins from the immune response, such as the CD4 receptor. It is known that this action causes CD4 degradation in lysosome, but the molecular mechanisms are still incompletely understood. Nef forms a tripartite complex with the cytosolic tail of the CD4 and adapter protein 2 (AP-2) in clathrin-coated vesicles, inducing CD4 internalization and lysosome degradation. Previous research has demonstrated that CD4 target to lysosomes by Nef involves targeting of this receptor to multivesicular bodies (MVBs) pathway by an atypical mechanism because, although not need charging ubiquitination, depends on the proteins from ESCRTs (Endosomal Sorting Complexes Required for Transport) machinery and the action of Alix, an accessory protein ESCRT machinery. It has been reported that Nef interacts with subunits of AP- 1, AP-2, AP-3 complexes and Nef does not appear to interact with AP-4 and AP-5 subunits. However, the role of Nef interaction with AP-1 or AP-3 in CD4 down-regulation is poorly understood. Furthermore, AP-1, AP-2 and AP-3 are potentially heterogeneous due to the existence of multiple subunits isoforms encoded by different genes. However, there are few studies to demonstrate if the different combinations of APs isoforms are form and if they have distinct functional properties. This study aim to identify and characterize cellular factors involved on CD4 down-modulation induced by Nef from HIV-1. More specifically, this study aimed to characterize the involvement of AP-1 complex in the down-regulation of CD4 by Nef HIV-1 through the functional study of the two isoforms of ?-adaptins, AP-1 subunits. By pull-down technique, we showed that Nef is able to interact with ?2. In addition, our data from immunoblots indicated that ?2- adaptin, not ?1-adaptin, is required in Nef-mediated targeting of CD4 to lysosomes and the ?2 participation in this process is conserved by Nef from different viral strains. Furthermore, by flow cytometry assay, ?2 depletion, but not ?1 depletion, compromises the reduction of surface CD4 levels induced by Nef. Immunofluorescence microscopy analysis also revealed that ?2 depletion impairs the redistribution of CD4 by Nef to juxtanuclear region, resulting in CD4 accumulation in primary endosomes. Knockdown of ?1A, another subunit of AP-1, resulted in decreased cellular levels of ?1 and ?2 and, compromising the efficient CD4 degradation by Nef. Moreover, upon artificially stabilizing ESCRT-I in early endosomes, via overexpression of HRS, internalized CD4 accumulates in enlarged HRS-GFP positive endosomes, where co-localize with ?2. Together, the results indicate that ?2-adaptin is a molecule that is essential for CD4 targeting by Nef to ESCRT/MVB pathway, being an important protein in the endo-lysosomal system. Furthermore, the results indicate that ?-adaptins isoforms not only have different functions, but also seem to compose AP-1 complex with distinct cell functions, and only the AP-1 variant comprising ?2, but not ?1, acts in the CD4 down-regulation induced by Nef. These studies contribute to a better understanding on the molecular mechanisms involved in Nef activities, which may also help to improve the understanding of the HIV pathogenesis and the related syndrome. In addition, this work contributes with the understanding of primordial process regulation on intracellular trafficking of transmembrane proteins.
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YADAV, DIVYANSHI. "TO STUDY INTERACTIONS BETWEEN MONOCLONAL ANTIBODY AND CHO HOST CELL PROTEINS TO DESIGN THE WASH PROCESS FOR MAB PURIFICATION." Thesis, 2017. http://dspace.dtu.ac.in:8080/jspui/handle/repository/15879.
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Today, recombinant protein therapies represent a substantial focus of the pharmaceutical industry. Most therapeutic proteins are produced in host cells of non-human origin, including Escherichia coli, yeast, and various mammalian cell lines e.g. Chinese Hamster Ovary Cell Lines. A major focus of all therapeutic protein purification process is to reduce components of host organism, including host cell proteins (HCPs), to levels considered as adequate in the final formulated drug product. HCPs can pose potential safety risks for patients, including immune reactions, decreased product stability, adjuvant activity, and (theoretically) protein-specific biological activity.(Schenauer, Flynn, & Goetze, 2012)
With rapidly growing cases and increased number of cancer, autoimmune diseases, Alzheimer’s disease has become the most common death-causing diseases worldwide. Recent studies indicate that mAb is effective in treatment of these diseases and with limited number of treatment options people need to rely on these medicines. Thus, the purity of these medicines becomes an important factor. If these medicines are not pure the HCP might induce antigenic reactions in the patient also these HCPs if proteolytic may degrade the desired amount of mAb to be effective as dose, thus making the medicine ineffective over a period. Thus here, we report results of the studies relating to the most interactive HCPs which isolated along with mAb and studied their interactions to design the wash process accordingly. Using the proteome of Chinese Hamster, the hotspot for the proteins were found using Aggrescan. Structure prediction was done using threading software’s Bhageerath, Pyre 2, Multicom-Raptor-X, Robetta and I-TASSER. The model generated was further validated by Independent servers Prochek, Verify-3d, Errat and with meta servers such as Protsav and SAVES. The models were refined using 3D refine and galaxy refine.
The best models were docked with mAb using cluspro to identify the interactions between HCP and mAb then find the amino acid interaction profile using PDB-Sum and further work on developing modified wash process to break these bonds and obtain pure mAb to ensure effective treatment by functionally characterizing the proteins.
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Lum, Mabel Yuen Teng. "Identification of host cell proteins involved in Shigella flexneri pathogenesis." Thesis, 2014. http://hdl.handle.net/2440/87369.
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Shigella flexneri is the etiological agent of bacillary dysentery (shigellosis). It is transmitted via the faecal-oral route and is a significant human pathogen due to the high morbidity among children <5 years in developing countries. The key pathogenic features of Shigella include cell death induction in myeloid immune cells and circumventing cell death in colonic epithelial cells, the site of bacterial infection. Shigella also interact with host proteins to initiate de novo actin synthesis to facilitate its intra- and intercellular spread to disseminate in the host. In this thesis, the role of three host proteins: myosin IIA, dynamin II, and dynamin-related protein 1 (Drp1) during Shigella cell-to-cell spreading was examined. The myosin IIA specific kinase, myosin like chain kinase (MLCK), was previously shown to be important for Shigella plaque formation. Myosin IIA and MLCK have also been implicated in septin caging of non-motile Shigella which are targeted for degradation. Chemical inhibition and siRNA knockdown of myosin IIA reduced Shigella plaque formation. Curiously HeLa cells infected with Shigella mutants defective in cell-to-cell spreading have significantly reduced myosin IIA levels when quantified by immunofluorescence microscopy. Dynamin II and Drp1 are members of the dynamin superfamily. Both proteins have self-assembly driven GTPase activation. Dynamin II is important for clathrin-mediated endocytosis and pinches the budding clathrin-coated vesicle, and Drp1 is essential for mitochondrial fission. It was hypothesized that Shigella protrusion formation into adjacent host cells resembles endocytic and exocytic processes, and components of these processes may facilitate Shigella dissemination. When dynamin II GTPase was inhibited with dynasore and dynamin II was knocked down with siRNA, Shigella cell-to-cell spreading was significantly reduced. The in vivo efficacy of dynasore was tested in a murine Sereny model. No significant reduction in inflammation was observed but mice were protected against weight loss during infection. Further experimentation suggested dynasore protected mice against cytotoxic effects from the three secretion system (TTSS) effectors expressed by Shigella during infection. Drp1 was investigated in this thesis as dynasore also inhibits the GTPase of this mitochondrial fission protein. Mitochondrial fission is important in maintaining mitochondrial dynamics and also in events downstream of intrinsic apoptosis and programmed necrosis pathways activation. Loss of mitochondrial function in Shigella-induced epithelial cell death has been reported previously. Hence the role of Drp1 in Shigella plaque formation and HeLa death was examined with the Drp1-specific inhibitor, Mdivi-1, and siRNA knockdown. HeLa cell death was significantly reduced; suggesting loss of mitochondrial function observed previously may now be attributed to Drp1 and subsequent Drp1-mediated mitochondrial fission. The impairment in Shigella cell-to-cell spreading in the absence of Drp1 suggests maintaining an intact mitochondrial network is essential for Shigella lateral spread since loss of Drp1 function would result in excessive mitochondrial fusion, leading to formation of netlike or perinuclear structures. The outcomes of this thesis highlight the importance of host proteins during different stages of Shigella infection. By improving our understanding on the host and bacteria interaction, future work on novel approaches to prevent Shigella dissemination can be developed.<br>Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2014