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Ensuring Access to Safe and Nutritious Food for All Through the Transformation of Food Systems
Pollution-induced community tolerance in freshwater biofilms â from molecular mechanisms to loss of community functions
Exposure to herbicides poses a threat to aquatic biofilms by affecting their community structure, physiology and function. These changes render biofilms to become more tolerant, but on the downside community tolerance has ecologic costs. A concept that addresses induced community tolerance to a pollutant (PICT) was introduced by Blanck and WĂ€ngberg (1988). The basic principle of the concept is that microbial communities undergo pollution-induced succession when exposed to a pollutant over a long period of time, which changes communities structurally and functionally and enhancing tolerance to the pollutant exposure. However, the mechanisms of tolerance and the ecologic consequences were hardly studied up to date. This thesis addresses the structural and functional changes in biofilm communities and applies modern molecular methods to unravel molecular tolerance mechanisms.
Two different freshwater biofilm communities were cultivated for a period of five weeks, with one of the communities being contaminated with 4 ÎŒg L-1 diuron. Subsequently, the communities were characterized for structural and functional differences, especially focusing on their crucial role of photosynthesis. The community structure of the autotrophs was assessed using HPLC-based pigment analysis and their functional alterations were investigated using Imaging-PAM fluorometry to study photosynthesis and community oxygen profiling to determine net primary production. Then, the molecular fingerprints of the communities were measured with meta-transcriptomics (RNA-Seq) and GC-based community metabolomics approaches and analyzed with respect to changes in their molecular functions. The communities were acute exposed to diuron for one hour in a dose-response design, to reveal a potential PICT and uncover related adaptation to diuron exposure. The combination of apical and molecular methods in a dose-response design enabled the linkage of functional effects of diuron exposure and underlying molecular mechanisms based on a sensitivity analysis.
Chronic exposure to diuron impaired freshwater biofilms in their biomass accrual. The contaminated communities particularly lost autotrophic biomass, reflected by the decrease in specific chlorophyll a content. This loss was associated with a change in the molecular fingerprint of the communities, which substantiates structural and physiological changes. The decline in autotrophic biomass could be due to a primary loss of sensitive autotrophic organisms caused by the selection of better adapted species in the course of chronic exposure. Related to this hypothesis, an increase in diuron tolerance has been detected in the contaminated communities and molecular mechanisms facilitating tolerance have been found. It was shown that genes of the photosystem, reductive-pentose phosphate cycle and arginine metabolism were differentially expressed among the communities and that an increased amount of potential antioxidant degradation products was found in the contaminated communities. This led to the hypothesis that contaminated communities may have adapted to oxidative stress, making them less sensitive to diuron exposure. Moreover, the photosynthetic light harvesting complex was altered and the photoprotective xanthophyll cycle was increased in the contaminated communities. Despite these adaptation strategies, the loss of autotrophic biomass has been shown to impair primary production. This impairment persisted even under repeated short-term exposure, so that the tolerance mechanisms cannot safeguard primary production as a key function in aquatic systems.:1. The effect of chemicals on organisms and their functions .............................. 1
1.1 Welcome to the anthropocene .......................................................................... 1
1.2 From cellular stress responses to ecosystem resilience ................................... 3
1.2.1 The individual pursuit for homeostasis ....................................................... 3
1.2.2 Stability from diversity ................................................................................. 5
1.3 Community ecotoxicology - a step forward in monitoring the effects of chemical
pollution? ................................................................................................................. 6
1.4 Functional ecotoxicological assessment of microbial communities ................... 9
1.5 Molecular tools â the key to a mechanistic understanding of stressor effects
from a functional perspective in microbial communities? ...................................... 12
2. Aims and Hypothesis ......................................................................................... 14
2.1 Research question .......................................................................................... 14
2.2 Hypothesis and outline .................................................................................... 15
2.3 Experimental approach & concept .................................................................. 16
2.3.1 Aquatic freshwater biofilms as model community ..................................... 16
2.3.2 Diuron as model herbicide ........................................................................ 17
2.3.3 Experimental design ................................................................................. 18
3. Structural and physiological changes in microbial communities after chronic
exposure - PICT and altered functional capacity ................................................. 21
3.1 Introduction ..................................................................................................... 21
3.2 Methods .......................................................................................................... 23
3.2.1 Biofilm cultivation ...................................................................................... 23
3.2.2 Dry weight and autotrophic index ............................................................. 23
3.2.4 Pigment analysis of periphyton ................................................................. 23
3.2.4.1 In-vivo pigment analysis for community characterization ....................... 24
3.2.4.2 In-vivo pigment analysis based on Imaging-PAM fluorometry ............... 24
3.2.4.3 In-vivo pigment fluorescence for tolerance detection ............................. 26
3.2.4.4 Ex-vivo pigment analysis by high-pressure liquid-chromatography ....... 27
3.2.5 Community oxygen metabolism measurements ....................................... 28
3.3 Results and discussion ................................................................................... 29
3.3.1 Comparison of the structural community parameters ............................... 29
3.3.2 Photosynthetic activity and primary production of the communities after
selection phase ................................................................................................. 33
3.3.3 Acquisition of photosynthetic tolerance .................................................... 34
3.3.4 Primary production at exposure conditions ............................................... 36
3.3.5 Tolerance detection in primary production ................................................ 37
3.4 Summary and Conclusion ........................................................................... 40
4. Community gene expression analysis by meta-transcriptomics ................... 41
4.1 Introduction to meta-transcriptomics ............................................................... 41
4.2. Methods ......................................................................................................... 43
4.2.1 Sampling and RNA extraction................................................................... 43
4.2.2 RNA sequencing analysis ......................................................................... 44
4.2.3 Data assembly and processing................................................................. 45
4.2.4 Prioritization of contigs and annotation ..................................................... 47
4.2.5 Sensitivity analysis of biological processes .............................................. 48
4.3 Results and discussion ................................................................................... 48
4.3.1 Characterization of the meta-transcriptomic fingerprints .......................... 49
4.3.2 Insights into community stress response mechanisms using trend analysis
(DRomicâs) ......................................................................................................... 51
4.3.3 Response pattern in the isoform PS genes .............................................. 63
4.5 Summary and conclusion ................................................................................ 65
5. Community metabolome analysis ..................................................................... 66
5.1 Introduction to community metabolomics ........................................................ 66
5.2 Methods .......................................................................................................... 68
5.2.1 Sampling, metabolite extraction and derivatisation................................... 68
5.2.2 GC-TOF-MS analysis ............................................................................... 69
5.2.3 Data processing and statistical analysis ................................................... 69
5.3 Results and discussion ................................................................................... 70
5.3.1 Characterization of the metabolic fingerprints .......................................... 70
5.3.2 Difference in the metabolic fingerprints .................................................... 71
5.3.3 Differential metabolic responses of the communities to short-term exposure
of diuron ............................................................................................................ 73
5.4 Summary and conclusion ................................................................................ 78
6. Synthesis ............................................................................................................. 79
6.1 Approaches and challenges for linking molecular data to functional
measurements ...................................................................................................... 79
6.2 Methods .......................................................................................................... 83
6.2.1 Summary on the data ............................................................................... 83
6.2.2 Aggregation of molecular data to index values (TELI and MELI) .............. 83
6.2.3 Functional annotation of contigs and metabolites using KEGG ................ 83
6.3 Results and discussion ................................................................................... 85
6.3.1 Results of aggregation techniques ........................................................... 85
6.3.2 Sensitivity analysis of the different molecular approaches and endpoints 86
6.3.3 Mechanistic view of the molecular stress responses based on KEGG
functions ............................................................................................................ 89
6.4 Consolidation of the results â holistic interpretation and discussion ............... 93
6.4.1 Adaptation to chronic diuron exposure - from molecular changes to
community effects.............................................................................................. 93
6.4.2 Assessment of the ecological costs of Pollution-induced community
tolerance based on primary production ............................................................. 94
6.5 Outlook ............................................................................................................ 9
Nonparametric Two-Sample Test for Networks Using Joint Graphon Estimation
This paper focuses on the comparison of networks on the basis of statistical
inference. For that purpose, we rely on smooth graphon models as a
nonparametric modeling strategy that is able to capture complex structural
patterns. The graphon itself can be viewed more broadly as density or intensity
function on networks, making the model a natural choice for comparison
purposes. Extending graphon estimation towards modeling multiple networks
simultaneously consequently provides substantial information about the
(dis-)similarity between networks. Fitting such a joint model - which can be
accomplished by applying an EM-type algorithm - provides a joint graphon
estimate plus a corresponding prediction of the node positions for each
network. In particular, it entails a generalized network alignment, where
nearby nodes play similar structural roles in their respective domains. Given
that, we construct a chi-squared test on equivalence of network structures.
Simulation studies and real-world examples support the applicability of our
network comparison strategy.Comment: 25 pages, 6 figure
Norsk rÄ kumelk, en kilde til zoonotiske patogener?
The worldwide emerging trend of eating ânaturalâ foods, that has not been
processed, also applies for beverages. According to Norwegian legislation, all
milk must be pasteurized before commercial sale but drinking milk that has
not been heat-treated, is gaining increasing popularity. Scientist are warning
against this trend and highlights the risk of contracting disease from milkborne
microorganisms. To examine potential risks associated with drinking
unpasteurized milk in Norway, milk- and environmental samples were
collected from dairy farms located in south-east of Norway. The samples
were analyzed for the presence of specific zoonotic pathogens; Listeria
monocytogenes, Campylobacter spp., and Shiga toxin-producing Escherichia
coli (STEC). Cattle are known to be healthy carriers of these pathogens, and
Campylobacter spp. and STEC have a low infectious dose, meaning that
infection can be established by ingesting a low number of bacterial cells. L.
monocytogenes causes one of the most severe foodborne zoonotic diseases,
listeriosis, that has a high fatality rate. All three pathogens have caused milk
borne disease outbreaks all over the world, also in Norway.
During this work, we observed that the prevalence of the three examined
bacteria were high in the environment at the examined farms. In addition, 7%
of the milk filters were contaminated by STEC, 13% by L. monocytogenes and
4% by Campylobacter spp. Four of the STEC isolates detected were eaepositive,
which is associated with the capability to cause severe human
disease. One of the eae-positive STEC isolates were collected from a milk
filter, which strongly indicate that Norwegian raw milk may contain potential
pathogenic STEC.
To further assess the possibilities of getting ill by STEC after consuming raw
milk, we examined the growth of the four eae-positive STEC isolates in raw milk at different temperatures. All four isolates seemed to have ability to multiply in raw milk at 8°C, and one isolate had significant growth after 72 hours. Incubation at 6°C seemed to reduce the number of bacteria during the
first 24 hours before cell death stopped. These findings highlight the
importance of stable refrigerator temperatures, preferable < 4°C, for storage
of raw milk.
The L. monocytogenes isolates collected during this study show genetic
similarities to isolates collected from urban and rural environmental
locations, but different clones were predominant in agricultural
environments compared to clinical and food environments. However, the
results indicate that the same clone can persist in a farm over time, and that
milk can be contaminated by L. monocytogenes clones present in farm
environment.
Despite testing small volumes (25 mL) of milk, we were able to isolate both
STEC and Campylobacter spp. directly from raw milk. A proportion of 3% of
the bulk tank milk and teat milk samples were contaminated by
Campylobacter spp. and one STEC was isolated from bulk tank milk. L
monocytogenes was not detected in bulk tank milk, nor in teat milk samples.
The agricultural evolvement during the past decades have led to larger
production units and new food safety challenges. Dairy cattle production in
Norway is in a current transition from tie-stall housing with conventional
pipeline milking systems, to modern loose housing systems with robotic
milking. The occurrence of the three pathogens in this project were higher in
samples collected from farms with loose housing compared to those with tiestall
housing.
Pasteurization of cowâs milk is a risk reducing procedure to protect
consumers from microbial pathogens and in most EU countries, commercial
distribution of unpasteurized milk is legally restricted. Together, the results
presented in this thesis show that the animal housing may influence the level
of pathogenic bacteria in the raw milk and that ingestion of Norwegian raw
cowâs milk may expose consumers to pathogenic bacteria which can cause
severe disease, especially in children, elderly and in persons with underlying
diseases. The results also highlight the importance of storing raw milk at low
temperatures between milking and consumption.Ă
spise mat som er mindre prosessert og mer «naturlig» er en pÄgÄende
trend i Norge og i andre deler av verden. Interessen for Ă„ drikke melk som
ikke er varmebehandlet, sÄkalt rÄ melk, er ogsÄ Þkende. I Norge er det pÄbudt
Ă„ pasteurisere melk fĂžr kommersielt salg for Ă„ beskytte forbrukeren mot
sykdomsfremkallende mikroorganismer. Fagfolk advarer mot Ä drikke rÄ
melk, og pÄpeker risikoen for Ä bli syk av patogene bakterier som kan finnes i
melken.
I denne avhandlingen undersĂžker vi den potensielle risikoen det medfĂžrer Ă„
drikke upasteurisert melk fra Norge. I tillegg til Ă„ samle inn tankmelk- og
speneprÞver fra melkegÄrder i sÞrÞst Norge, samlet vi ogsÄ miljÞprÞver fra
de samme gÄrdene for Ä kartlegge forekomst og for Ä identifisere potensielle
mattrygghetsrisikoer i melkeproduksjonen. Alle prĂžvene ble analysert for de
zoonotiske sykdomsfremkallende bakteriene Listeria monocytogenes,
Campylobacter spp., og Shiga toksin-produserende Escherichia coli (STEC).
Kyr kan vĂŠre friske smittebĂŠrere av disse bakteriene, som dermed kan
etablere et reservoar pÄ gÄrdene. Bakteriene kan overfÞres fra gÄrdsmiljÞet
til melkekjeden og dermed utfordre mattryggheten. Disse bakteriene har
forÄrsaket melkebÄrne sykdomsutbrudd over hele verden, ogsÄ i Norge.
Campylobacter spp. og STEC har lav infeksiĂžs dose, som vil si at man kan bli
syk selv om man bare inntar et lavt antall bakterieceller. L. monocytogenes
kan gi sykdommen listeriose, en av de mest alvorlige matbÄrne zoonotiske
sykdommene vi har i den vestlige verden.
Resultater fra denne oppgaven viser en hĂžy forekomst av de tre patogenene i
gÄrdsmiljÞet. I tillegg var 7% av melkefiltrene vi testet positive for STEC, 13%
positive for L. monocytogenes og 4% positive for Campylobacter spp.. Fire av
STEC isolatene bar genet for Intimin, eae, som er ansett som en viktig
virulensfaktor som Ăžker sjansen for alvorlig sykdom. Ett av de eae-positive
isolatene ble funnet i et melkefilter, noe som indikerer at norsk rÄ melk kan
inneholde patogene STEC. For Ă„ videre vurdere risikoen for Ă„ bli syk av STEC
fra rÄ melk undersÞkte vi hvordan de fire eae-positive isolatene vokste i rÄ
melk lagret ved forskjellige temperaturer. For alle isolatene Ăžkte antall
bakterier etter lagring ved 8°C, og for et isolat var veksten signifikant. Etter
lagring ved 6°C ble antallet bakterier redusert de fÞrste 24 timene, deretter
stoppet reduksjonen i antall bakterier. Disse resultatene viser hvor viktig det
er Ä ha stabil lav lagringstemperatur for rÄ melk, helst < 4°C.
L. monocytogenes isolatene som ble samlet inn fra melkegÄrdene viste
genetiske likheter med isolater samlet inn fra urbane og rurale miljĂžer rundt
omkring i Norge. Derimot var kloner som dominerte i landbruksmiljĂžet
forskjellige fra kliniske isolater og isolater fra matproduksjonslokaler. Videre
sÄ man at en klone kan persistere pÄ en gÄrd over tid og at melk kan
kontamineres av L. monocytogenes kloner som er til stede i gÄrdsmiljÞet.
Til tross for smÄ testvolum av tankmelken (25 mL) fant vi bÄde STEC og
Campylobacter spp. i melkeprĂžvene. 3% av tankmelkprĂžvene og
speneprĂžvene var positive for Campylobacter spp. og ett STEC isolat ble
funnet i tankmelk. L. monocytogenes ble ikke funnet direkte i melkeprĂžvene.
Landbruket i Norge er i stadig utvikling der besetningene blir stĂžrre, men
fĂŠrre. Melkebesetningene er midt i en overgang der tradisjonell oppstalling
med melking pÄ bÄs byttes ut med lÞsdriftssystemer og melkeroboter.
Forekomsten av de tre patogenene funnet i denne studien var hĂžyere i
besetningene med lĂžsdrift sammenliknet med besetningene som hadde
melkekyrne oppstallet pÄ bÄs.
Pasteurisering er et viktig forebyggende tiltak for Ă„ beskytte konsumenter fra
mikrobielle patogener, og i de fleste EU-land er kommersielt salg av rÄ melk
juridisk begrenset. Denne studien viser at oppstallingstype kan pÄvirke
nivÄene av patogene bakterier i gÄrdsmiljÞet og i rÄ melk. Inntak av rÄ melk
kan eksponere forbruker for patogene bakterier som kan gi alvorlig sykdom,
spesielt hos barn, eldre og personer med underliggende sykdommer.
Resultatene underbygger viktigheten av Ă„ pasteurisere melk for Ă„ sikre
mattryggheten, og at det er avgjÞrende Ä lagre rÄ melk ved kontinuerlig lave
temperaturer for Ă„ forebygge vekst av zoonotiske patogener
Neuroanatomical and gene expression features of the rabbit accessory olfactory system. Implications of pheromone communication in reproductive behaviour and animal physiology
Mainly driven by the vomeronasal system (VNS), pheromone
communication is involved in many species-specific fundamental innate socio-sexual behaviors such as mating and
fighting, which are essential for animal reproduction and survival. Rabbits are a unique model for studying
chemocommunication due to the discovery of the rabbit mammary pheromone, but paradoxically there has been a
lack of knowledge regarding its VNS pathway. In this work, we aim at filling this gap by approaching the system
from an integrative point of view, providing extensive anatomical and genomic data of the rabbit VNS, as well as
pheromone-mediated reproductive and behavioural studies. Our results build strong foundation for further
translational studies which aim at implementing the use of pheromones to improve animal production and welfare
Machine Learning Research Trends in Africa: A 30 Years Overview with Bibliometric Analysis Review
In this paper, a critical bibliometric analysis study is conducted, coupled
with an extensive literature survey on recent developments and associated
applications in machine learning research with a perspective on Africa. The
presented bibliometric analysis study consists of 2761 machine learning-related
documents, of which 98% were articles with at least 482 citations published in
903 journals during the past 30 years. Furthermore, the collated documents were
retrieved from the Science Citation Index EXPANDED, comprising research
publications from 54 African countries between 1993 and 2021. The bibliometric
study shows the visualization of the current landscape and future trends in
machine learning research and its application to facilitate future
collaborative research and knowledge exchange among authors from different
research institutions scattered across the African continent
Post-Mortem Assessment and Evolutionary Role of the Autopsy
The Chapter is dedicated to the evolutionary role of autopsy, reporting the historical profiles, the state of the art, and prospects for future development of the main related techniques and methods of the ancillary disciplines (like Radiology), involved in historic synergy in the post-mortem assessment, together with the mother discipline Forensic Pathology. A task sustainable through the utilization of the so-called advanced molecular autopsy, a convergence of different skills jointly makes use of the high dimensionality of data generated by new technologies requiring a data mining approach governed by improved bioinformatics and computational biology tools. The evolution of the scientific research and the increased accuracy of the various disciplines will be able to weigh the value of evidence, placed at the disposal of the justice system as truth and proof
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