Browsing by Subject "Antibody"
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Initiation of lignification in Norway spruce xylem detected by immunolabeling and Raman spectroscopy (2021)Wood development is a significant process with both financial as well as natural perspectives. Trees and wood are of highly significance in Finland where a huge part of the gross national income devises from the forestry area. Ecologically and commercially the Norway spruce (Picea abies) is one of the most common tree species in Europe. It covers about 30% of Finland's forest area. Norway spruce is frequently used in research to study many phenomena related specifically to the wood formation and lignification. The principal objective of my thesis work was to reveal an unknown step in the lignification process in developing xylem of Norway spruce, i.e. the initiation site(s) for lignification. To achieve this goal, the aim was to investigate the chemical identity of possible lignification initiation sites in the middle lamellae and cell corners of developing Norway spruce xylem, and to answer the question where in the cell wall soluble monolignols first emerge and lead to the start of lignin formation (polymerization). I was approaching this goal with immunolabeling technique for confocal microscopy and Raman spectroscopy to unravel this initiation site of lignification by using specific monoclonal antibodies for cell wall compounds and comparing the results with the initial lignin deposition sites. To detect the location/distribution of some important polysaccharides and lignin substructure for lignification initiation, monoclonal antibodies i.e. LM10, LM11, LM15, LM24 and antibody Dibenzodioxocin or DBD were applied for confocal microscopy and some monolignol specific spectra were applied for Raman microscopy. The xylan was detected by LM10 in secondary cell wall abundantly and few are in primary cell wall of Norway spruce. The LM11 against arabinoxylan was determined more in primary cell walls but less in secondary cell wall. The location of xyloglucan was identified in the middle lamellae, primary and secondary cell wall of Norway spruce by LM15. The LM24 against glycosylated xyloglucan was found in secondary cell walls, abundantly in cell corners but few in primary cell wall. The primary antibody Dibenzodioxocin or DBD for the lignin substructure revealed that these were present in the mature cells of secondary cell walls (S2 and S3 layers). The lignin substructures DBD were not found in youngest cells where secondary cell walls are absent. The developing xylem of Norway spruce was subjected Raman microscopy and which revealed the locations of cinnamyl alcohol, coniferyl alcohol and coniferyl aldehyde. The cinnamyl alcohol was abundantly found at cell corner and middle lamellae in most developing part of xylem. The coniferyl alcohol was determined only in developing xylem cell corners. The coniferyl aldehyde was observed at cell corners, middle lamella and primary cell walls of developing xylem. The coniferyl aldehyde was located more in mature cells than younger cells. So, the Confocal and Raman microscopy images revealed the possible bindings of monolignols to polysaccharide in young cell corners, cell wall layers and middle lamellae.
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INITIATION OF LIGNIFICATION IN NORWAY SPRUCE XYLEM DETECTED BY IMMUNOLABELING AND RAMAN SPECTROSCOPY (2021)Wood development is a significant process with both financial as well as natural perspectives. Trees and wood are of highly significance in Finland where a huge part of the gross national income devises from the forestry area. Ecologically and commercially the Norway spruce (Picea abies) is one of the most common tree species in Europe. It covers about 30% of Finland's forest area. Norway spruce is frequently used in research to study many phenomena related specifically to the wood formation and lignification. The principal objective of my thesis work was to reveal an unknown step in the lignification process in developing xylem of Norway spruce, i.e. the initiation site(s) for lignification. To achieve this goal, the aim was to investigate the chemical identity of possible lignification initiation sites in the middle lamellae and cell corners of developing Norway spruce xylem, and to answer the question where in the cell wall soluble monolignols first emerge and lead to the start of lignin formation (polymerization). I was approaching this goal with immunolabeling technique for confocal microscopy and Raman spectroscopy to unravel this initiation site of lignification by using specific monoclonal antibodies for cell wall compounds and comparing the results with the initial lignin deposition sites. To detect the location/distribution of some important polysaccharides and lignin substructure for lignification initiation, monoclonal antibodies i.e. LM10, LM11, LM15, LM24 and antibody Dibenzodioxocin or DBD were applied for confocal microscopy and some monolignol specific spectra were applied for Raman microscopy. The xylan was detected by LM10 in secondary cell wall abundantly and few are in primary cell wall of Norway spruce. The LM11 against arabinoxylan was determined more in primary cell walls but less in secondary cell wall. The location of xyloglucan was identified in the middle lamellae, primary and secondary cell wall of Norway spruce by LM15. The LM24 against glycosylated xyloglucan was found in secondary cell walls, abundantly in cell corners but few in primary cell wall. The primary antibody Dibenzodioxocin or DBD for the lignin substructure revealed that these were present in the mature cells of secondary cell walls (S2 and S3 layers). The lignin substructures DBD were not found in youngest cells where secondary cell walls are absent. The developing xylem of Norway spruce was subjected Raman microscopy and which revealed the locations of cinnamyl alcohol, coniferyl alcohol and coniferyl aldehyde. The cinnamyl alcohol was abundantly found at cell corner and middle lamellae in most developing part of xylem. The coniferyl alcohol was determined only in developing xylem cell corners. The coniferyl aldehyde was observed at cell corners, middle lamella and primary cell walls of developing xylem. The coniferyl aldehyde was located more in mature cells than younger cells. So, the Confocal and Raman microscopy images revealed the possible bindings of monolignols to polysaccharide in young cell corners, cell wall layers and middle lamellae.
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(2022)COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has thus far claimed over six million lives. Vaccines against SARS-CoV-2 have successfully mitigated severe disease and eased the burden on healthcare systems. Neutralizing antibodies play crucial roles both in vaccine derived immunity, and as drugs widely utilized in monoclonal antibody therapy. Neutralizing antibodies primarily target the spike protein, where most of the neutralizing epitopes are located in the receptor binding domain (RBD). Elucidating the sites of vulnerability to neutralization on SARS-CoV-2 can facilitate the development of therapeutics. 7A12 is a newly-discovered IgG antigen-binding fragment (Fab) isolated from a COVID-19 patient in Finland that can neutralize SARS-CoV-2 by targeting the spike protein. In this thesis, a complex of the Fab 7A12 with SARS-CoV-2 spike ectodomain trimer was studied using cryogenic electron microscopy (cryo-EM) single-particle analysis to elucidate the epitope of 7A12 and to gain insight into the neutralization mechanism of 7A12. Cryo-EM data of the complex revealed that Fab 7A12 can bind to both “open” and “closed” conformations of RBD. Rigid-body fitting of the spike trimer and Fab 7A12 models into the cryo-EM density indicates that 7A12 recognizes an epitope in the RBD which is mainly located outside the ACE2 binding site. Together, these results suggest that the 7A12 epitope belongs to class III of SARS-CoV-2 neutralizing epitopes located in the RBD, indicating that 7A12 could neutralize by sterically hindering ACE2 and by preventing the adjacent RBD from changing to ”up” conformation. Furthermore, our results revealed an overlap of 7A12 epitope with the putative binding site of heparan sulfate, a host factor of SARS-CoV-2 infection, which might contribute to neutralization. 7A12-RBD interface mapping delineated the residues comprising the epitope, which do not include mutants found in Beta, Gamma, and Delta variants, while four mutants were found in Omicron within the epitope indicating that 7A12 is likely to neutralize Beta, Gamma, and Delta variants of concern.
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(2022)COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has thus far claimed over six million lives. Vaccines against SARS-CoV-2 have successfully mitigated severe disease and eased the burden on healthcare systems. Neutralizing antibodies play crucial roles both in vaccine derived immunity, and as drugs widely utilized in monoclonal antibody therapy. Neutralizing antibodies primarily target the spike protein, where most of the neutralizing epitopes are located in the receptor binding domain (RBD). Elucidating the sites of vulnerability to neutralization on SARS-CoV-2 can facilitate the development of therapeutics. 7A12 is a newly-discovered IgG antigen-binding fragment (Fab) isolated from a COVID-19 patient in Finland that can neutralize SARS-CoV-2 by targeting the spike protein. In this thesis, a complex of the Fab 7A12 with SARS-CoV-2 spike ectodomain trimer was studied using cryogenic electron microscopy (cryo-EM) single-particle analysis to elucidate the epitope of 7A12 and to gain insight into the neutralization mechanism of 7A12. Cryo-EM data of the complex revealed that Fab 7A12 can bind to both “open” and “closed” conformations of RBD. Rigid-body fitting of the spike trimer and Fab 7A12 models into the cryo-EM density indicates that 7A12 recognizes an epitope in the RBD which is mainly located outside the ACE2 binding site. Together, these results suggest that the 7A12 epitope belongs to class III of SARS-CoV-2 neutralizing epitopes located in the RBD, indicating that 7A12 could neutralize by sterically hindering ACE2 and by preventing the adjacent RBD from changing to ”up” conformation. Furthermore, our results revealed an overlap of 7A12 epitope with the putative binding site of heparan sulfate, a host factor of SARS-CoV-2 infection, which might contribute to neutralization. 7A12-RBD interface mapping delineated the residues comprising the epitope, which do not include mutants found in Beta, Gamma, and Delta variants, while four mutants were found in Omicron within the epitope indicating that 7A12 is likely to neutralize Beta, Gamma, and Delta variants of concern.
Now showing items 1-4 of 4