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1
Nayyar, Ashima. "Yeast flocculation : understanding cell surface structure-function relationships in industrial yeast strains". Thesis, Abertay University, 2015. https://rke.abertay.ac.uk/en/studentTheses/cec13693-e667-4426-ba6c-6873e5c2b642.
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Adhesion properties of microorganisms are crucial for many essential biological processes such as sexual reproduction, tissue or substrate invasion, biofilm formation and cell-cell aggregation. One of such controlled forms of cellular adhesion in yeast that occurs preferentially in the liquid environments is a process of asexual aggregation of cells which is also referred to as flocculation. The timing during growth and the causes of onset of yeast flocculation are of commercial interest to the brewing industry, as flocculation can determine the degree of attenuation of the wort. Early or premature flocculation is one common causes of ‘hung’ or ‘stuck’ fermentations giving rise to sweeter beer whereas a lack or delay in flocculation can cause filtration difficulties and some problems in obtaining a bright sparkling beer; in addition, the presence of excess yeast in beer during ageing can cause off flavours due to yeast autolysis. Despite this commercial interest, limited information is available about the onset of flocculation and the various factors that may be responsible in the process. In particular, what are the signals that trigger flocculation? Adhesion properties applicable in improving yeast biotechnology are dependent directly or indirectly on characteristics of cellular surfaces, usually the outer layer of the cell wall. Change in the structure and or composition of the cell wall leads to changes in the microbial adhesion properties. Exploring more into the cell wall and studying the nanoscale structure of the yeast cell wall would thus be beneficial to augment our understanding of flocculation.
2
Proszynski, Tomasz. "Protein sorting and cell surface polarity in yeast". Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2005. http://nbn-resolving.de/urn:nbn:de:swb:14-1131974045019-73555.
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The studies presented here were focused on the understanding of the principles for protein sorting from the Golgi to the cell surface. As a marker protein we used Fus1p, a type I plasma membrane protein that is O-glycosylated on the extracellular domain and plays a role in cell fusion during yeast mating. Additionally, we analyzed mechanisms responsible for asymmetric distribution of Fus1p in mating cells. We demonstrated that the glycans attached to the protein act as a sorting determinant for protein transport to the cell surface. In cells lacking PMT4, encoding a mannosyltransferase involved in the initial step of O-glycosylation, Fus1p was not glycosylated and accumulated in late Golgi structures. A similar defect in exocytosis was observed when a Fus1p mutant lacking the O-glycosylated domain was expressed in wild-type cells, however, the cell surface delivery could be rescued if the 33 amino acid portion of the Fus1p ectodomain, containing 15 potentially glycosylated sites was added to the protein. It was previously well documented in epithelial cells that different types of protein glycosylation and association with lipid rafts play a role of determinants for protein delivery to the apical plasma membrane. However, otherwise the machinery responsible for cargo sorting to the apical membrane is poorly understood. Our finding that also in yeast, protein glycosylation can function as a sorting determinant provides a new possibility to investigate underlying mechanisms...
3
Foster, Alexander J. "Cell surface analysis of the basidiomycete yeast cryptococcus neoformans". Thesis, Aston University, 2004. http://publications.aston.ac.uk/11011/.
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Cell surface properties of the basidiomycete yeast Cryptococcus neoformans were investigated with a combination of novel and well proven approaches. Non-specific cell adhesion forces, as well as exposed carbohydrate and protein moieties potentially associated with specific cellular interaction, were analysed. Experimentation and analysis employed cryptococcal cells of different strains, capsular status and culture age. Investigation of cellular charge by particulate microelectrophoresis revealed encapsulated yeast forms of C. neoformans manifest a distinctive negative charge regardless of the age of cells involved; in turn, the neutral charge of acapsulate yeasts confirmed that the polysaccharide capsule, and not the cell wall, was responsible for this occurrence. Hydrophobicity was measured by MATH and HICH techniques, as well as by the attachment of polystyrene microspheres. All three techniques, where applicable, found C. neoformans yeast to be consistently hydrophilic; this state varied little regardless of strain and culture age. Cell surface carbohydrates and protein were investigated with novel fluorescent tagging protocols, flow cytometry and confocal microscopy. Cell surface carbohydrate was identified by controlled oxidation in association with biotin hydrazide and fluorescein-streptavidin tagging. Marked amounts of carbohydrate were measured and observed on the cell wall surface of cryptococcal yeasts. Furthermore, tagging of carbohydrates with selective fluorescent lectins supported the identification, measurement and observation of substantial amounts of mannose, glucose and N-acetyl-glucosamine. Cryptococcal cell surface protein was identified using sulfo-NHS-biotin with fluorescein-streptavidin, and then readily quantified by flow cytometry.
4
George, Ellen. "The influence of brewing yeast physiology on cell surface properties". Thesis, Oxford Brookes University, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318535.
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5
Murai, Toshiyuki. "Studies on genetic display of hydrolytic enzymes on yeast cell surface". Kyoto University, 1998. http://hdl.handle.net/2433/182354.
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6
Shiraga, Seizaburo. "Studies on modification of functions of hydrolytic enzymes by yeast cell surface engineering". 京都大学 (Kyoto University), 2005. http://hdl.handle.net/2433/144560.
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Kyoto University (京都大学)0048
新制・課程博士
博士(工学)
甲第11889号
工博第2582号
新制||工||1362(附属図書館)
23669
UT51-2005-N723
京都大学大学院工学研究科合成・生物化学専攻
(主査)教授 森 泰生, 教授 今中 忠行, 教授 青山 安宏
学位規則第4条第1項該当
7
Zou, Wen. "STUDIES ON THE NEW FUNCTIONAL YEAST STRAINS CONSTRUCTED AND SCREENED BY CELL SURFACE ENGINEERING". 京都大学 (Kyoto University), 2001. http://hdl.handle.net/2433/150264.
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8
Hennig, Stefan. "Utilization of yeast pheromones and hydrophobin-based surface engineering for novel whole-cell sensor applications". Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-223271.
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Whole-cell sensors represent an emerging branch in biosensor development since they obviate the need for enzyme/antibody purification and provide the unique opportunity to assess global parameters such as genotoxicity and bioavailability. Yeast species such as Saccharomyces cerevisiae are ideal hosts for whole-cell sensor applications. However, current approaches almost exclusively rely on analyte-induced expression of fluorescent proteins or luciferases that imply issues with light scattering and/or require the supply of additional substrates. In this study, the yeast α-factor mating pheromone, a peptide pheromone involved in cell-cell communication in Saccharomyces cerevisiae, was utilized to create the whole-cell sensor read-out signal, in particular by employing engineered sensor cells that couple the response to a user-defined environmental signal to α-factor secretion.
Two novel immunoassays - relying on hydrophobin-based surface engineering - were developed to quantify the α-factor. Hydrophobins are amphiphilic fungal proteins that self-assemble into robust monolayers at hydrophobic surfaces. Two recombinant hydrophobins, either lacking (EAS) or exposing the α-factor pheromone (EAS-α) upon self-assembly, were used to functionalize polystyrene supports. In a first approach (competitive immunoassay), pheromone-specific antibodies initially bound to the functionalized surface (due to the α-factor exposed by the hydrophobin layer) were competitively detached by soluble α-factor. In a second approach, the antibodies were first premixed with pheromone-containing samples and subsequently applied to functionalized surfaces, allowing for the attachment of antibodies that still carried available binding sites (inverse immunoassay).
Both immunoassays enabled quantitative assessment of the yeast pheromone in a unique but partially overlapping dynamic range and allowed for facile tuning of the assay sensitivity by adjustment of the EAS-α content of the hydrophobin layer. With a limit of detection of 0.1 nM α-factor, the inverse immunoassay proved to be the most sensitive pheromone quantification assay currently available. Due to the high stability of hydrophobin monolayers, functionalized surfaces could be reused for multiple consecutive measurements. Favorably, both immunoassays proved to be largely robust against the changes in the sample matrix composition, allowing for pheromone quantification in complex sample matrices such as yeast culture supernatants.
Hence, these immunoassays could also be applied to study the pheromone secretion of wild-type and engineered Saccharomyces cerevisiae strains. Additionally, a proof-of-concept whole-cell sensor for thiamine was developed by combining the hydrophobin-based immunoassays with engineered sensor cells of Schizosaccharomyces pombe modulating the secretion of the α-factor pheromone in response to thiamine. Since this read-out strategy encompasses intrinsic signal amplification and enables flexible choice of the transducer element, it could contribute to the development of miniaturized, portable whole-cell sensors for on-site application.
9
Chetty, Bronwyn Jean. "Improvement of cell-surface adhered cellulase activities in recombinant strains of Saccharomyces cerevisiae engineered for consolidated bioprocessing". University of Western Cape, 2021. http://hdl.handle.net/11394/8357.
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>Magister Scientiae - MScConsolidated bioprocessing (CBP), in which a single organism in a single reactor is responsible for the conversion of pretreated lignocellulosic biomass to bioethanol, remains an attractive option for production of commodity products if an organism fit for this process can be engineered. The yeast Saccharomyces cerevisiae requires engineered cellulolytic activity to enable its use in CBP production of second generation bioethanol. Current recombinant yeast strains engineered for this purpose must overcome the drawback of generally low secretion titres. A promising strategy for directly converting lignocellulose to ethanol is by displaying heterologous cellulolytic enzymes on the cell surface by means of the glycosylphosphatidylinositol (GPI) or similar anchoring systems. Recently, a strain producing cell-adhered enzymes in a ratio-optimized manner was created that showed significant crystalline cellulose hydrolysis.
10
Hennig, Stefan, Gerhard Rödel i Kai Ostermann. "Hydrophobin-Based Surface Engineering for Sensitive and Robust Quantification of Yeast Pheromones". Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-214807.
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Detection and quantification of small peptides, such as yeast pheromones, are often challenging. We developed a highly sensitive and robust affinity-assay for the quantification of the α-factor pheromone of Saccharomyces cerevisiae based on recombinant hydrophobins. These small, amphipathic proteins self-assemble into highly stable monolayers at hydrophilic-hydrophobic interfaces. Upon functionalization of solid supports with a combination of hydrophobins either lacking or exposing the α-factor, pheromone-specific antibodies were bound to the surface. Increasing concentrations of the pheromone competitively detached the antibodies, thus allowing for quantification of the pheromone. By adjusting the percentage of pheromone-exposing hydrophobins, the sensitivity of the assay could be precisely predefined. The assay proved to be highly robust against changes in sample matrix composition. Due to the high stability of hydrophobin layers, the functionalized surfaces could be repeatedly used without affecting the sensitivity. Furthermore, by using an inverse setup, the sensitivity was increased by three orders of magnitude, yielding a novel kind of biosensor for the yeast pheromone with the lowest limit of detection reported so far. This assay was applied to study the pheromone secretion of diverse yeast strains including a whole-cell biosensor strain of Schizosaccharomyces pombe modulating α-factor secretion in response to an environmental signal.
11
Hennig, Stefan, Gerhard Rödel i Kai Ostermann. "Hydrophobin-Based Surface Engineering for Sensitive and Robust Quantification of Yeast Pheromones". Multidisciplinary Digital Publishing Institute (MDPI), 2016. https://tud.qucosa.de/id/qucosa%3A30004.
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Detection and quantification of small peptides, such as yeast pheromones, are often challenging. We developed a highly sensitive and robust affinity-assay for the quantification of the α-factor pheromone of Saccharomyces cerevisiae based on recombinant hydrophobins. These small, amphipathic proteins self-assemble into highly stable monolayers at hydrophilic-hydrophobic interfaces. Upon functionalization of solid supports with a combination of hydrophobins either lacking or exposing the α-factor, pheromone-specific antibodies were bound to the surface. Increasing concentrations of the pheromone competitively detached the antibodies, thus allowing for quantification of the pheromone. By adjusting the percentage of pheromone-exposing hydrophobins, the sensitivity of the assay could be precisely predefined. The assay proved to be highly robust against changes in sample matrix composition. Due to the high stability of hydrophobin layers, the functionalized surfaces could be repeatedly used without affecting the sensitivity. Furthermore, by using an inverse setup, the sensitivity was increased by three orders of magnitude, yielding a novel kind of biosensor for the yeast pheromone with the lowest limit of detection reported so far. This assay was applied to study the pheromone secretion of diverse yeast strains including a whole-cell biosensor strain of Schizosaccharomyces pombe modulating α-factor secretion in response to an environmental signal.
12
Watanabe, Yukio. "Molecular breeding of yeast Saccharomyces cerevisiae for effective ammonia production from food processing wastes". Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263698.
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13
Qian, Jiang. "Cell Wall/Surface Proteome of Candida albicans: its Application in Rapid Identification of Yeast Species by Mass Signature and Characterization by in vitro and in vivo Chemical Labelings". ScholarWorks@UNO, 2010. http://scholarworks.uno.edu/td/1115.
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Candida albicans is an opportunistic fungal pathogen that may cause mucutaneous infection and/or disseminated candidasis if the host defense system is impaired (such as those in HIV patients). Cell surface of C. albicans is the frontier where initial interplay between host-pathogen takes place and therefore is of great importance in understanding the mechanism of hostpathogen interaction. MALDI-TOF-MS analysis of intact fungal cells yielded mass signatures for rapid species differentiation, strain grouping and yeast morphogenesis monitoring. Cell surface biotinylations at low temperature (4°C), enzymatic digestion of the intact fungal cell surface proteins ("whole cell shaving"), biotin-avidin affinity enrichment of biotinylated peptides, liquid chromatography mass spectrometry (LC-MS) based proteomic approach were employed for unambiguous identification of cell wall/cell wall associated proteins and the exposed peptide segments of these proteins. SILAC (Stable Isotope Labeling by Amino acids in Cell Culture) based CWP quantification analyses were performed to monitor CWP accumulation level change in response to hyphae induction. Information on surface exposed peptide segments and regulation of cell wall/surface protein during morphogenesis provided new candidates to the pool of potential peptide targets for protective vaccine development. A New type of "fluorous" (fluorinated alkane) affinity gained popularity due to its low level nonspecific protein/peptide binding. Fluorous labeling reagents that target primary amine groups in proteins/peptides were synthesized and characterized. The acid labile linker in the labeling reagents allows cleavage of the bulky fluorous tag moiety and the long oligo ethylene glycol (OEG) spacer after fluorous affinity purification. Upon collision induced decomposition, the labeled peptide ion yielded a characteristic fragment that could be retrieved from the residual portion of fluorous affinity tag, and serve as a marker to indicate that the relevant peptide had been successfully labeled. Results showed that both the protein/peptide labeling and affinity enrichment/separation process were highly efficient.
14
Takagi, Toshiyuki. "Studies on breeding of yeast Saccharomyces cerevisiae for effective macroalgae utilization based on the metabolism of marine bacterium". 京都大学 (Kyoto University), 2017. http://hdl.handle.net/2433/225665.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第20440号
農博第2225号
新制||農||1049(附属図書館)
学位論文||H29||N5061(農学部図書室)
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 植田 充美, 教授 小川 順, 教授 渡邊 隆司
学位規則第4条第1項該当
15
Hennig, Stefan [Verfasser], Gerhard [Akademischer Betreuer] [Gutachter] Rödel i Tilo [Gutachter] Pompe. "Utilization of yeast pheromones and hydrophobin-based surface engineering for novel whole-cell sensor applications / Stefan Hennig ; Gutachter: Gerhard Rödel, Tilo Pompe ; Betreuer: Gerhard Rödel". Dresden : Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://d-nb.info/1130092992/34.
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16
Hennig, Stefan [Verfasser], Gerhard [Akademischer Betreuer] Rödel i Tilo [Gutachter] Pompe. "Utilization of yeast pheromones and hydrophobin-based surface engineering for novel whole-cell sensor applications / Stefan Hennig ; Gutachter: Gerhard Rödel, Tilo Pompe ; Betreuer: Gerhard Rödel". Dresden : Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2017. http://d-nb.info/1130092992/34.
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17
Isogawa, Danya. "Studies on the active site of chitosanase from Paenibacillus fukuinensis and its functional modification for utilizing chitosan". Kyoto University, 2014. http://hdl.handle.net/2433/188769.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第18331号
農博第2056号
新制||農||1022(附属図書館)
学位論文||H26||N4838(農学部図書室)
31189
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 植田 充美, 教授 三上 文三, 教授 小川 順
学位規則第4条第1項該当
18
Davì, Valeria. "Dynamique de la paroi cellulaire dans la régulation de la morphogenèse et de la croissance cellulaire". Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS295/document.
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Les cellules dans la nature se développent dans un large éventail de formes, suivant divers modèles de croissance. Malgré l'importance de ces processus fondamentaux, la façon dont les cellules régulent leur croissance et leur morphogenèse est encore mal comprise. Dans cette thèse, j'ai exploré ces aspects, avec une approche principalement biomécanique, en concentrant mes investigations sur des cellules à paroi à croissance de pointe et en exploitant en particulier la levure fissipare Schyzosaccharomyces pombe. J'ai d'abord développé de nouvelles méthodes pour mesurer les paramètres mécaniques clés de la paroi cellulaire in vivo et à grande échelle, ce qui a permis les premières observations de la dynamique des parois cellulaires. Ceci a révélé que la paroi cellulaire est plus souple et très variable au niveau des pôles de croissance, et presque stable et plus rigide dans les sites non cultivés. Au cours de l'allongement, il existe une interaction entre la mécanique des parois et la croissance cellulaire, dont le contrôle actif permet l'expansion cellulaire tout en préservant l'intégrité des cellules. De plus, j'ai observé qu'il existe une forte corrélation entre la mécanique des parois cellulaires et la morphologie cellulaire, et des perturbations des propriétés de la paroi affectent directement l'établissement et la maintenance de la forme. Ensemble, mes résultats montrent que la régulation de la paroi est fondamentale dans la détermination de la dynamique cellulaire dans les cellules à parois épaissies. Globalement, cela suggère que l'observation dynamique de la mécanique de surface cellulaire est essentielle pour une compréhension complète des processus multifactoriels et complexes comme la croissance et la morphogenèseCells in nature develop in a wide range of forms, following diverse growth patterns. Despite the importance of these fundamental processes, how cells regulate their growth and morphogenesis is still poorly understood. In this thesis, I explored these processes, focusing my investigations on tip growing walled cells and in particular, by exploiting the fission yeast Schyzosaccharomyces pombe, adopting a mainly biomechanical approach. To this aim, I first developed novel methods to measure key cell wall mechanical parameters in vivo and in large scale, which allowed the very first observations of cell wall dynamics. This revealed that the cell wall is softer and highly variable at growing poles, and almost stable and stiffer at non-growing sites. During elongation, there is an interplay between wall mechanics and cell growth, whose active control allows cell expansion while preserving cell integrity. In addition, I observed that there is a strong correlation between cell wall mechanics and cell morphology, and ectopic perturbations of wall properties directly affect shape establishment and maintenance. Together my results show that the regulation of wall mechanics is fundamental in the determination of cell dynamics in tip growing walled cells. Moreover, this suggests that dynamic observation of cell surface mechanics is crucial for a complete understanding of multifactorial and complex processes as growth and morphogenesis
19
bhardwaj, vinay. "Label-free surface-enhanced Raman spectroscopy-linked immunosensor assay (SLISA) for environmental surveillance". FIU Digital Commons, 2015. http://digitalcommons.fiu.edu/etd/2321.
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The contamination of the environment, accidental or intentional, in particular with chemical toxins such as industrial chemicals and chemical warfare agents has increased public fear. There is a critical requirement for the continuous detection of toxins present at very low levels in the environment. Indeed, some ultra-sensitive analytical techniques already exist, for example chromatography and mass spectroscopy, which are approved by the US Environmental Protection Agency for the detection of toxins. However, these techniques are limited to the detection of known toxins. Cellular expression of genomic and proteomic biomarkers in response to toxins allows monitoring of known as well as unknown toxins using Polymerase Chain Reaction and Enzyme Linked Immunosensor Assays. However, these molecular assays allow only the endpoint (extracellular) detection and use labels such as fluorometric, colorimetric and radioactive, which increase chances of uncertainty in detection. Additionally, they are time, labor and cost intensive. These technical limitations are unfavorable towards the development of a biosensor technology for continuous detection of toxins. Federal agencies including the Departments of Homeland Security, Agriculture, Defense and others have urged the development of a detect-to-protect class of advanced biosensors, which enable environmental surveillance of toxins in resource-limited settings.
In this study a Surface-Enhanced Raman Spectroscopy (SERS) immunosensor, aka a SERS-linked immunosensor assay (SLISA), has been developed. Colloidal silver nanoparticles (Ag NPs) were used to design a flexible SERS immunosensor. The SLISA proof-of-concept biosensor was validated by the measurement of a dose dependent expression of RAD54 and HSP70 proteins in response to H2O2 and UV. A prototype microchip, best suited for SERS acquisition, was fabricated using an on-chip SLISA to detect RAD54 expression in response to H2O2. A dose-response relationship between H2O2 and RAD54 is established and correlated with EPA databases, which are established for human health risk assessment in the events of chemical exposure. SLISA outperformed ELISA by allowing RISE (rapid, inexpensive, simple and effective) detection of proteins within 2 hours and 3 steps. It did not require any label and provided qualitative information on antigen-antibody binding. SLISA can easily be translated to a portable assay using a handheld Raman spectrometer and it can be used in resource-limited settings. Additionally, this is the first report to deliver Ag NPs using TATHA2, a fusogenic peptide with cell permeability and endosomal rupture release properties, for rapid and high levels of Ag NPs uptake into yeast without significant toxicity, prerequisites for the development of the first intracellular SERS immunosensor.
20
Kuroda, Kouichi. "Studies on cell-surface-engineered yeasts for bioremediation and their applications". 京都大学 (Kyoto University), 2005. http://hdl.handle.net/2433/145392.
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Kyoto University (京都大学)0048
新制・課程博士
博士(工学)
甲第11234号
工博第2445号
新制||工||1328(附属図書館)
22843
UT51-2004-U439
京都大学大学院工学研究科合成・生物化学専攻
(主査)教授 森 泰生, 教授 青山 安宏, 教授 村上 正浩
学位規則第4条第1項該当
21
Linciano, Sara <1993>. "Copper-catalyzed azide-alkyne click chemistry-based modification of peptides displayed on the surface of yeast cells". Master's Degree Thesis, Università Ca' Foscari Venezia, 2018. http://hdl.handle.net/10579/13710.
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Short circulating half-life and poor plasma stability and solubility remain key challenges in the field of therapeutic peptide; to overcome these limitations we decided to take advantage of serum albumin, a protein that has the intrinsic capability to bind and transport a large variety of molecules, including drugs. Albumin is not only the major transporter of drugs, but it is also the main fatty acids carrier to tissues. Inspired by the evidence that long fatty acid are able to bind albumin, we thought to increase the stability and half-life of therapeutic peptides by chemically conjugating long fatty acids chain to them. Towards this goal, we applied yeast display technology encoding non-canonical amino-acids. During the first part of the project, we optimized the conditions for the conjugation of peptides displayed on the surface of yeast cells. To assess the efficacy and bio-viability of the cicloaddition reaction, we used alkyne biotin and a peptide bearing an azide group. During the second part of the work, we applied the identified conditions to conjugate an alkyne fatty acid to a peptide and measured its binding affinity against albumin. Most of the collected data have been obtained using flow cytometer technique, through which is possible to gain insights about the cell morphology and aggregation as well as the efficiency of the chemical reaction and the peptide binding affinity
22
Paul, Terry R. "Influence of iron deprivation and sub-inhibitory concentrations of antifungal antibiotics on surface antigens of candida albicans yeast cells". Thesis, Aston University, 1988. http://publications.aston.ac.uk/12520/.
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This study examined the effect of iron deprivation and sub-inhibitory concentrations of antifungal agents on yeast cell surface antigen recognition by antibodies from patients with Candida infections. Separation of cell wall surface proteins by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and immunological detection by immunoblotting, revealed that antigenic profiles of yeasts were profoundly influenced by the growth environment. Cells grown under iron-depleted conditions expressed several iron-regulated proteins that were recognized by antibodies from patient sera. An attempt to characterize these proteins by lectin blotting with concanavalin A revealed that some could be glycoprotein in nature. Furthermore, these proteins which were located within cell walls and on yeast surfaces, were barely or not expressed in yeasts cultivated under iron-sufficient conditions. The magnitude and heterogeneity of human antibody responses to these iron-regulated proteins were dependent on the type of Candida infection, serum antibody class and yeast strain. Hydroxamate-type siderophores were also detected in supernatants of iron depleted yeast cultures. This evidence suggests that Candida albicans expresses iron-regulated proteins/glycoproteins in vitro which may play a role in siderophore-mediated iron uptake in Candida albicans. Sequential monitoring of IgG antibodies directed against yeast surface antigens during immunization of rabbits revealed that different antigens were recognized particularly during early and later stages of immunization in iron-depleted cells compared to iron-sufficient cells. In vitro and in vivo adherence studies demonstrated that growth phase, yeast strain and growth conditions affect adhesion mechanisms. In particular, growth under iron-depletion in the presence of sub-inhibitory concentrations of polyene and azole antifungals enhanced the hydrophobicity of C.albicans. Growth conditions also influenced MICs of antifungals, notably that of ketoconazole. Sub-inhibitory concentrations of amphotericin B and fluconazole had little effect on surface antigens, whereas nystatin induced profound changes in surface antigens of yeast cells. The effects of such drug concentrations on yeast cells coupled with host defence mechanisms may have a significant affect on the course of Candida infections.
23
Chung, I.-Ming. "A study of the interactions between cells (yeasts, erythrocytes and fibroblasts) and various surfaces in liquid media, using atomic force microscopy". Thesis, Imperial College London, 2004. http://hdl.handle.net/10044/1/11268.
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24
Sivelle, Coline. "Conception et production d’anticorps anti-TNFa non immunogènes pour le traitement des maladies inflammatoires". Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS603.
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L’efficacité des anticorps anti-TNFα peut être particulièrement affectée par leur immunogénicité. Elle se traduit par la production d’anticorps dirigés contre la protéine thérapeutique (ADA) et induisant des effets indésirables. Pour l’adalimumab (Humira®), qui est l’anti-TNFα le plus utilisé, ce phénomène est observé chez plus de 30% des patients pour certaines pathologies. Les épitopes T responsables de cette immunogénicité sont principalement localisés dans les régions CDR impliquées dans l’interaction avec le TNFα. L’objectif de ce travail est de retirer les séquences épitopes T de l’anticorps sans altérer sa fonctionnalité. Afin de maitriser ce problème d’immunogénicité, ces travaux de thèse proposent d’utiliser le Yeast Surface Display (YSD) afin de contrôler la fonctionnalité de l’anticorps tout au long du processus de mutagénèse. Pour cela, une première étape de comparaison des différents formats d’expression d’anticorps en YSD a permis de définir le format d’affichage à utiliser pour la suite du projet. La stratégie de déimmunisation adoptée ensuite est basée sur la suppression des épitopes T. Dans un premier temps, elle réunit une approche de mutagénèse exhaustive (DMS) et l’utilisation d’algorithme afin d’identifier les substitutions délétères pour l’interaction HLA II/épitope T, mais neutre pour la fonction de l’anticorps. Dans un second temps, ces substitutions sont combinées dans des banques pré-enrichies en mutants fonctionnels. Des mutants ayant une immunogénicité potentielle réduite ont ensuite été sélectionnés à partir de ces banques. Plusieurs mutants de l’adalimumab à l’immunogénicité réduite selon l’algorithme de prédiction ont été identifiés et caractérisés. Ils possèdent tous une affinité augmentée pour le TNFα se traduisant par une activité au moins doublée par rapport à l’adalimumabEfficacy of anti-TNFα antibodies is well known to be affected by their immunogenicity. Some patients developp anti-drug antibodies (ADA) which can elicit adverse effects and neutralize the therapeutic protein. Adalimumab (Humira®), which is the most used anti-TNFα, is reported to be immunogenic for more than 30% of patients in some diseases. T-cell epitopes that account for its immunogenicity are mostly carried by regions implied in the interaction with TNFα. In the present work, we propose to remove T-cell epitopes from the antibody sequence while maintaining its functionality.To undertake this issue, this PhD project uses Yeast Surface Display (YSD) to monitor the affinity of the biologic during the mutagenesis process. To do so, a comparison of different expression formats of antibody in YSD has been performed in order to define the format that will be used for the deimmunization method. Then, the strategy chosen to reduce immunogenicity is based on T-cell epitopes removal. First, it merges deep mutational scanning and in silico HLA II binding prediction to identify substitutions deleterious for HLA II/T-cell epitopes interaction while neutral for the function of the biologic. Secondly, these substitutions were combined to obtain libraries pre-enriched with functional sequences. Mutants with reduced immunogenic potential were then selected from these libraries. Several mutants of adalimumab with reduced immunogenicity potential according to HLA II binding prediction were identified and caracterized. All of them show an increased affinity for TNFα associated with an improvement of activity of at least two fold in comparison to adalimumab
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Mikoliunaite, Lina, Raul D. Rodriguez, Evgeniya Sheremet, Vladimir Kolchuzhin, Jan Mehner, Arunas Ramanavicius i Dietrich R. T. Zahn. "The substrate matters in the Raman spectroscopy analysis of cells". Universitätsbibliothek Chemnitz, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:ch1-qucosa-178060.
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Raman spectroscopy is a powerful analytical method that allows deposited and/or immobilized cells to be evaluated without complex sample preparation or labeling. However, a main limitation of Raman spectroscopy in cell analysis is the extremely weak Raman intensity that results in low signal to noise ratios. Therefore, it is important to seize any opportunity that increases the intensity of the Raman signal and to understand whether and how the signal enhancement changes with respect to the substrate used. Our experimental results show clear differences in the spectroscopic response from cells on different surfaces. This result is partly due to the difference in spatial distribution of electric field at the substrate/cell interface as shown by numerical simulations. We found that the substrate also changes the spatial location of maximum field enhancement around the cells. Moreover, beyond conventional flat surfaces, we introduce an efficient nanostructured silver substrate that largely enhances the Raman signal intensity from a single yeast cell. This work contributes to the field of vibrational spectroscopy analysis by providing a fresh look at the significance of the substrate for Raman investigations in cell research.
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Prószyński, Tomasz J. [Verfasser]. "Protein sorting and cell surface polarity in yeast / von Tomasz J. Prószyński". 2005. http://d-nb.info/977677419/34.
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Haass, Friederike A. "Identification of yeast proteins necessary for cell surface function of a potassium channel". Diss., 2007. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3289302.
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Potter, Greg. "An Examination Of Cell Wall Properties Affecting Brewing Yeast Flocculation". 2014. http://hdl.handle.net/10222/43745.
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Flocculation, the process whereby yeast cells attach in groups and sediment to the top or bottom of a fermenter, is industrially important in many fermentation batch operations. These batch operations include wine, distilled spirits, cider, bio-ethanol and production of commercial yeast metabolites. In the case of brewing yeast, it has been determined that flocculation occurs due to three forces called hydrophobic interactions, zymolectin binding and to a lesser extent, surface charge neutralization. This project sought to more closely study hydrophobic interactions and zymolectin binding.
Earlier studies had shown that certain hydrophobic carboxylic acids, 3-OH oxylipins, formed in brewing yeast at flocculation onset. Therefore, these compounds showed potential as an indicator of overall cell surface hydrophobicity, and it was believed that flocculation level, cell surface hydrophobicity and oxylipin level would increase in unison in the yeast cell during brewing fermentations. During lab scale fermentations in shaker flasks and in a miniature fermentation assay setup, both flocculation level and cell surface hydrophobicity increased coincidently. However, 3-OH oxylipins could not be isolated from whole cells or cell wall isolates grown in the shaker flasks or whole cells grown in the miniature fermentation assay at detection limits approximated as 50 ng/0.5 g wet yeast. Due to their minute levels in brewing yeast cells, it was proposed that 3-OH oxylipins may mediate flocculation and aggregation via a quorum sensing mechanism instead of by increasing cell surface hydrophobicity.
A disagreement exists in the literature where certain researchers believe zymolectin activity is induced, while others believe it is constitutive. The second part of this study attempted to address this by measuring zymolectin density during lab scale fermentations with a flow cytometer. Because of flow cytometry’s capacity for multiparametric analysis, large amounts of data were produced which gave information on not only zymolectin density, but also cell size and cellular complexity. Upon statistical analysis of the data, it was not possible to either refute or confirm the claim that zymolectin activity is induced or constitutive. However, the results suggested there could have been a population of cells with fewer zymolectins, and this certainly warrants further investigation. During the lab scale fermentations, cell size measured by a flow cytometer appeared to be correlated with manual measures of cell size. Furthermore, cell size tended towards uniformity during the fermentation which has also been observed in similar studies employing flow cytometry. Conversely, the cellular complexity of the yeast in this study did not change as in other studies by this may have been due either to strain differences or the methods used herein.
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Hennig, Stefan. "Utilization of yeast pheromones and hydrophobin-based surface engineering for novel whole-cell sensor applications". Doctoral thesis, 2016. https://tud.qucosa.de/id/qucosa%3A29235.
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Whole-cell sensors represent an emerging branch in biosensor development since they obviate the need for enzyme/antibody purification and provide the unique opportunity to assess global parameters such as genotoxicity and bioavailability. Yeast species such as Saccharomyces cerevisiae are ideal hosts for whole-cell sensor applications. However, current approaches almost exclusively rely on analyte-induced expression of fluorescent proteins or luciferases that imply issues with light scattering and/or require the supply of additional substrates. In this study, the yeast α-factor mating pheromone, a peptide pheromone involved in cell-cell communication in Saccharomyces cerevisiae, was utilized to create the whole-cell sensor read-out signal, in particular by employing engineered sensor cells that couple the response to a user-defined environmental signal to α-factor secretion.
Two novel immunoassays - relying on hydrophobin-based surface engineering - were developed to quantify the α-factor. Hydrophobins are amphiphilic fungal proteins that self-assemble into robust monolayers at hydrophobic surfaces. Two recombinant hydrophobins, either lacking (EAS) or exposing the α-factor pheromone (EAS-α) upon self-assembly, were used to functionalize polystyrene supports. In a first approach (competitive immunoassay), pheromone-specific antibodies initially bound to the functionalized surface (due to the α-factor exposed by the hydrophobin layer) were competitively detached by soluble α-factor. In a second approach, the antibodies were first premixed with pheromone-containing samples and subsequently applied to functionalized surfaces, allowing for the attachment of antibodies that still carried available binding sites (inverse immunoassay).
Both immunoassays enabled quantitative assessment of the yeast pheromone in a unique but partially overlapping dynamic range and allowed for facile tuning of the assay sensitivity by adjustment of the EAS-α content of the hydrophobin layer. With a limit of detection of 0.1 nM α-factor, the inverse immunoassay proved to be the most sensitive pheromone quantification assay currently available. Due to the high stability of hydrophobin monolayers, functionalized surfaces could be reused for multiple consecutive measurements. Favorably, both immunoassays proved to be largely robust against the changes in the sample matrix composition, allowing for pheromone quantification in complex sample matrices such as yeast culture supernatants.
Hence, these immunoassays could also be applied to study the pheromone secretion of wild-type and engineered Saccharomyces cerevisiae strains. Additionally, a proof-of-concept whole-cell sensor for thiamine was developed by combining the hydrophobin-based immunoassays with engineered sensor cells of Schizosaccharomyces pombe modulating the secretion of the α-factor pheromone in response to thiamine. Since this read-out strategy encompasses intrinsic signal amplification and enables flexible choice of the transducer element, it could contribute to the development of miniaturized, portable whole-cell sensors for on-site application.