Dosen, Ina

At leve og arbejde i svampeinficerede bygninger kan være skadelig - både med hensyn til helbredet og økonomien. Dette gælder især for hypersensitive, astmatikere og allergiske personer, der ofte oplever forværring af deres tilstand, når de befinder sig i et vådt, svampeinficeret indeklima. Mennesker som ellers er sunde kan også opleve negative helbredspåvirkninger såsom udslæt, hovedpine, svimmelhed og kronisk træthed. Under deres vækst på byggematerialer producere og frigiver svampene mange forskellige komponenter. Disse komponenter er mistænkt for at forårsage negative helbreds påvirkninger, men deres årsagssammenhæng er endnu ikke blevet dokumenteret. Svampene producere biomasse i form af mycelium og sporer sammen med en række sekundær metabolitter og andre bioaktive stoffer. Når svampebiomassen tørrer ind bliver hele, levedygtige sporer, fragmenterede sporer og myceliedele, der indeholder sekundær metabolitter og bioaktive stoffer, frigivet fra byggematerialet.Mangfoldigheden af forskellige svampearter i indeklimaet er så stor som antallet af bioaktive stoffer de producere. Dette PhD studium fokuserer på svampearter karakteriseret som såkaldte tertiære kolonisatorer, det vil sige Stachybotrys og Chaetomium spp. Både Stachybotrys og Chaetomium spp. kræver høj vandaktivitet for optimal vækst (aw ~ 0,98), som under indeklimaomstændigheder ofte er ensbetydende med alvorlige vandskader snarere end kun højt niveau af kondensering. Derfor er tilstedeværelsen af disse arter og/eller deres metabolitter i indeklimaet en god indikator af en gammel eller ny vandskade. Endvidere er sekundær metabolitter produceret af Stachybotrys og Chaetomium spp. kendte mykotoksiner, hvorfor risikoen for at forårsage negativ indvirkning på helbredet forøges. Derfor var et af hovedformålene med dette PhD studium at udvikle og optimere metoder til kvalitativ og semikvantitativ analyse af sekundær metabolitter og andre bioaktive stoffer produceret af Stachybotrys og Chaetomium spp.Den primære analytiske teknik anvendt til dette formål var væskekromatografi koblet til masse-spektrometri. Udnyttelse af avanceret teknologi såsom massespektrometri med høj resolution muliggjorde brugen af standardiseret MS/HRMS biblioteker og databaser over kemiske stoffer til metabolitprofilering af Stachybotrys og Chaetomium spp. Det præliminære arbejde i dette PhD studium var derfor at lave et MS/HRMS bibliotek, som indeholdte alle internt tilgængelige sekundær metabolitter (~1500). Til metabolitprofileringsformål, blev MS/HRMS biblioteket suppleret med databaser over arts- og slægtsspecifikke sekundære metabolitter beskrevet i litteraturen såvel som foreløbigt identificeret stoffer. Metabolitprofilering af Stachybotrys og Chaetomium spp. blev udført ved brug af agarkulturer. Derefter blev de fundne sekundære metabolitter bestemt i ekstrakter af kunstigt inficeret byggematerialer og senere i materialer fra naturligt inficeret bygninger.Resultaterne for Chaetomium spp. viste, at indeklimastammer har betydeligt forskellige metabolitprofiler i modsætning til ikke-indeklima reference stammer. Undersøgelse af Chaetomium spp. på kunstigt inficeret byggematerialer viste en præference for træbaseret materialer for disse arter. Almindeligt forekommende Chaetomium metabolitter, såsom chaetoglobosins, viste sig at være C. globosum-specifik, hvorimod cochliodones and chaetoglobin A var til stede i alle Chaetomium-forurenet indeklimaprøver og er derfor kandidater for indeklimaspecifikke Chaetomium biomarkører. Endvidere var dette første gang at cochliodones blev observeret i indeklima Chaetomium arter. Til kvantificeringsformål blev en semi-kvantitativ UHPLC-DAD-QTOFMS metode udviklet og semi-valideret, hvorved muliggjordes en estimering af mængden af Chaetomium metabolitter på byggematerialer. Afslutningsvis blev de beskrevet analytiske værktøjer anvendt til analyse af naturligt inficerede byggematerialer, hvor tilstedeværelsen af alle tidligere fundne metabolitter blev bekræftet.Resultaterne for Stachybotrys spp. viste ingen signifikant forskel i metabolitprofiler fundet in vitro og in vivo. Undersøgelse af Stachybotrys spp. på kunstigt inficeret byggematerialer bekræftede tilstedeværelsen af denne slægt på gips i sammenligning med træbaseret materialer. Metabolitter tilhørende macrocyclic trichothecene-, atranone- og spirocyclic drimane-gruppen blev fundet på både kunstigt og naturligt inficeret byggematerialer. Spirocyclic drimaner viste sig at være gode kandidater til indeklimaspecifikke Stachybotrys-biomarkøre, da de både blev produceret af alle indeklima Stachybotrys arter såvel som i størst mængde. De bedste ioniserings stoffer fra Stachybotrys blev udvalgt og direkte overført til UHPLC-QqQ instrumentet hvorved følsomheden af analysen forøgedes af de screenede metabolitter. Metoden var kvantitativ for størstedelen af de screenede metabolitter, hvorved muliggjordes en estimering af mængderne der kan findes på byggematerialer. Afslutningsvis blev metoden anvendt til analyse af støvprøver indsamlet i en vandskadet børnehave. Som et resultat heraf blev flere macrocyclic trichothecener og spirocyclic drimaner for første gang detekteret og kvantificeret i støv.De analytiske værktøjer, der er udviklet og optimeret under dette PhD studium, blev med succes anvendt til analysen af sekundære metabolitter og bioaktive stoffer produceret af Stachybotrys og Chaetomium spp. Denne metodologi repræsenterer et signifikant fremskridt i bestemmelsen af svampemetabolitter i indeklima prøver, heriblandt støvprøver. Som sådan kan dette PhD studium have en indflydelse på vores forståelse af forholdet mellem eksponering for skimmelsvamp og sygdom. Endvidere er den præsenteret metodologi anvendelig til stoffer produceret af andre almindeligt forekommende svampearter i indeklimaet.Living and working in fungi-ridden buildings can be detrimental for occupants both in terms of the effects on their health and in an economic sense. This is especially true for hypersensitive, asthmatic and allergic individuals who often experience exacerbation of their conditions when present in wet, fungi contaminated indoor environment. Otherwise healthy people may also experience negative health effects, such as skin rashes, headaches, dizziness and chronic fatigue. During their growth on building materials, indoor fungi produce and release many different kinds of components. These components are suspected of causing adverse health effects, however that causality has yet to be documented. Fungi produce biomass in the form of mycelium and spores both of which contain an array of secondary metabolites and bioactive compounds. When fungal biomass dries up, whole viable spores, fragmented spores and mycelium parts containing secondary metabolites and bioactive compounds are released from the building material.Not only is the variety of different fungal species present in the indoor environment large, the number of bioactive compounds they produce is also broad. This PhD study focused on fungal species characterized as so-called tertiary colonizers, namely Stachybotrys spp. and Chaetomium spp. Both Stachybotrys spp. and Chaetomium spp. require high water activity for optimal growth (aw ~ 0.98), which, for the indoor environment, often translates into serious water ingress rather than a high level of condensation. Thus, presence of these species and/or their metabolites in indoor environment is a good indicator of water damage, whether old or new. Furthermore, secondary metabolites produced by Stachybotrys spp. and Chaetomium spp. are known mycotoxins, thereby increasing likelyhood of causing negative health impact. With this in mind, a prime goal of this PhD study was to develop and optimize methods for qualitative and semi-quantitative analysis of secondary metabolites and bioactive compounds produced by Stachybotrys spp. and Chaetomium spp.The main analytical technique used for this purpose was liquid chromatography coupled to mass spectrometry. Utilizing advanced technology such as high resolution mass spectrometry enabled use of standardized MS/HRMS libraries and compound databases in metabolite profiling of Stachybotrys spp. and Chaetomium spp. Thus, the preliminary work performed in this PhD study was MS/HRMS library building, which contained all in-house available secondary metabolites (~1500). For metabolite profiling purposes, MS/HRMS library was supplemented with species- and genus-specific compound databases containing all secondary metabolites described in literature and not included in the library, as well as tentatively identified compounds. Metabolite profiling of Stachybotrys spp. and Chaetomium spp. was performed in pure agar cultures.Thereafter, mapped secondary metabolites were screened for in extracts of artificially inoculated building materials and materials from naturally infected buildings.Work performed on Chaetomium spp. showed that indoor strains have substantially different metabolite profiles in comparison to non-indoor reference strains. The study of Chaetomium spp. on artificially inoculated building materials revealed a preference for wood-based materials in these species. Commonly screened Chaetomium metabolites such as chaetoglobosins, were shown to be C. globosum specific, whilst cochliodones and chaetoglobin A were present in all Chaetomium contaminated indoor samples and thus are candidates for indoor specific Chaetomium biomarkers. Furthermore, this was the first time cochliodones were reported by indoor Chaetomium species. For the purpose of quantification a semi-quantative UHPLC-DAD-QTOFMS method was developed and semi-validated, enabling estimation of quantities for Chaetomium metabolites on different building materials. Finally, the aforementioned analytical tools were applied to the analysis of naturally contaminated building materials, where presence of all previously mapped metabolites was confirmed.Work done on Stachybotrys spp showed no significant difference in metabolite profiles obtained in vitro and in vivo. Concurrently the study of Stachybotrys spp. on artificially inoculated building materials confirmed a preference of this genus for gypsum in comparison to wood-based materials. Metabolites belonging to macrocyclic trichothecene, atranone and spirocyclic drimane groups of compounds were found on both artificially and naturally infected building materials. Spirocyclic drimanes were shown to be good candidates for indoor specific Stachybotrys biomarkers, as they were both produced in high quantities by all indoor Stachybotrys species. The best ionizing Stachybotrys compounds were chosen and their detection was directly transferred to the UHPLC-QqQ instrument, thereby increasing the sensitivity of the analysis for the screened metabolites. The method was quantitative for the majority of the screened metabolites, enabling for estimation of the amounts found on the samples building materials. Finally, the method was used for analysis of dust samples collected in a water-damaged kindergarten. As a result, several macrocyclic trichothecens and spirocyclic drimanes were detected and quantified in the dust for the first time.The analytical tools developed and optimized during this PhD study were successfully applied in analysis of secondary metabolites and bioactive compounds produced by Stachybotrys spp. and Chaetomium spp. This methodology represents a significant advance in the detection of fungal metabolites in indoor samples, including dust samples. As such, this PhD study could have an impact on our understanding of the relationship between mould exposure and sickness. Furthermore, the presented methodology is applicable to compounds produced by other commonly found indoor species

Online Research Database In Technology

LC-MS based analysis of secondary metabolites from <i>Chaetomium</i> and <i>Stachybotrys</i> growth in indoor environments

http://orbit.dtu.dk/ws/files/127587138/LC_MS_based_analysis.pdf

LC-MS based analysis of secondary metabolites from <i>Chaetomium</i> and <i>Stachybotrys</i> growth in indoor environments

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