13,990 research outputs found
Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and autophagy
UFMylation involves the covalent modification of substrate proteins with UFM1 (Ubiquitinâfold modifier 1) and is important for maintaining ER homeostasis. Stalled translation triggers the UFMylation of ERâbound ribosomes and activates C53âmediated autophagy to clear toxic polypeptides. C53 contains noncanonical shuffled ATG8âinteracting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. However, the mechanistic basis of sAIMâmediated ATG8 interaction remains unknown. Here, we show that C53 and sAIMs are conserved across eukaryotes but secondarily lost in fungi and various algal lineages. Biochemical assays showed that the unicellular alga Chlamydomonas reinhardtii has a functional UFMylation pathway, refuting the assumption that UFMylation is linked to multicellularity. Comparative structural analyses revealed that both UFM1 and ATG8 bind sAIMs in C53, but in a distinct way. Conversion of sAIMs into canonical AIMs impaired binding of C53 to UFM1, while strengthening ATG8 binding. Increased ATG8 binding led to the autoactivation of the C53 pathway and sensitization of Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral role of sAIMs in UFMylationâdependent fineâtuning of C53âmediated autophagy activation
Subspecies limits based on morphometry and mitochondrial DNA genomics in a polytypic species, the common grackle, Quiscalus quiscula
Nearctic migratory songbirds have demonstrated low levels of genetic differentiation and weak phylogeographical structure in mitochondrial DNA lineages compared with resident species. The common grackle, Quiscalus quiscula, is a widespread, partially migratory, North American icterid composed of three currently recognized subspecies. In this study, mensural characters (external and skeletal measurements) and the complete mitochondrial genome together with two mitochondrial genes, Cytb and ND2, were used to investigate subspecific differentiation and demographic history of the common grackle. The results showed substantial variation in body size among subspecies, mostly distributed between the âFlorida grackleâ, Quiscalus quiscula quiscula, and the two other subspecies. Analysis of mitochondrial DNA indicated low levels of genetic variation, but we found distinct haplotypes in Florida that form a clade in the phylogenetic tree. This suggests that the nominate subspecies in Florida is a distinct evolutionary unit. The sharing of haplotypes among the other subspecies (Quiscalus quiscula versicolor and Quiscalus quiscula stonei) in the north suggests high levels of gene flow, making the status of these two subspecies equivocal. Gene f low between nominate Q. q. quiscula, Q. q. versicolor and putative Q. q. stonei is probably attributable to historical changes in distribution and abundance following climate change events. We therefore recognize only two subspecies in the common grackle complex
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
Estudo da remodelagem reversa miocĂĄrdica atravĂ©s da anĂĄlise proteĂłmica do miocĂĄrdio e do lĂquido pericĂĄrdico
Valve replacement remains as the standard therapeutic option for aortic
stenosis patients, aiming at abolishing pressure overload and triggering
myocardial reverse remodeling. However, despite the instant hemodynamic
benefit, not all patients show complete regression of myocardial hypertrophy,
being at higher risk for adverse outcomes, such as heart failure. The current
comprehension of the biological mechanisms underlying an incomplete reverse
remodeling is far from complete. Furthermore, definitive prognostic tools and
ancillary therapies to improve the outcome of the patients undergoing valve
replacement are missing. To help abridge these gaps, a combined myocardial
(phospho)proteomics and pericardial fluid proteomics approach was followed,
taking advantage of human biopsies and pericardial fluid collected during
surgery and whose origin anticipated a wealth of molecular information
contained therein.
From over 1800 and 750 proteins identified, respectively, in the myocardium
and in the pericardial fluid of aortic stenosis patients, a total of 90 dysregulated
proteins were detected. Gene annotation and pathway enrichment analyses,
together with discriminant analysis, are compatible with a scenario of increased
pro-hypertrophic gene expression and protein synthesis, defective ubiquitinproteasome system activity, proclivity to cell death (potentially fed by
complement activity and other extrinsic factors, such as death receptor
activators), acute-phase response, immune system activation and fibrosis.
Specific validation of some targets through immunoblot techniques and
correlation with clinical data pointed to complement C3 ÎČ chain, Muscle Ring
Finger protein 1 (MuRF1) and the dual-specificity Tyr-phosphorylation
regulated kinase 1A (DYRK1A) as potential markers of an incomplete
response. In addition, kinase prediction from phosphoproteome data suggests
that the modulation of casein kinase 2, the family of IÎșB kinases, glycogen
synthase kinase 3 and DYRK1A may help improve the outcome of patients
undergoing valve replacement. Particularly, functional studies with DYRK1A+/-
cardiomyocytes show that this kinase may be an important target to treat
cardiac dysfunction, provided that mutant cells presented a different response
to stretch and reduced ability to develop force (active tension).
This study opens many avenues in post-aortic valve replacement reverse
remodeling research. In the future, gain-of-function and/or loss-of-function
studies with isolated cardiomyocytes or with animal models of aortic bandingdebanding will help disclose the efficacy of targeting the surrogate therapeutic
targets. Besides, clinical studies in larger cohorts will bring definitive proof of
complement C3, MuRF1 and DYRK1A prognostic value.A substituição da vĂĄlvula aĂłrtica continua a ser a opção terapĂȘutica de
referĂȘncia para doentes com estenose aĂłrtica e visa a eliminação da
sobrecarga de pressĂŁo, desencadeando a remodelagem reversa miocĂĄrdica.
Contudo, apesar do benefĂcio hemodinĂąmico imediato, nem todos os pacientes
apresentam regressĂŁo completa da hipertrofia do miocĂĄrdio, ficando com maior
risco de eventos adversos, como a insuficiĂȘncia cardĂaca. Atualmente, os
mecanismos biolĂłgicos subjacentes a uma remodelagem reversa incompleta
ainda não são claros. Além disso, não dispomos de ferramentas de
prognóstico definitivos nem de terapias auxiliares para melhorar a condição
dos pacientes indicados para substituição da vålvula. Para ajudar a resolver
estas lacunas, uma abordagem combinada de (fosfo)proteĂłmica e proteĂłmica
para a caracterização, respetivamente, do miocĂĄrdio e do lĂquido pericĂĄrdico
foi seguida, tomando partido de biĂłpsias e lĂquidos pericĂĄrdicos recolhidos em
ambiente cirĂșrgico.
Das mais de 1800 e 750 proteĂnas identificadas, respetivamente, no miocĂĄrdio
e no lĂquido pericĂĄrdico dos pacientes com estenose aĂłrtica, um total de 90
proteĂnas desreguladas foram detetadas. As anĂĄlises de anotação de genes,
de enriquecimento de vias celulares e discriminativa corroboram um cenĂĄrio de
aumento da expressĂŁo de genes pro-hipertrĂłficos e de sĂntese proteica, um
sistema ubiquitina-proteassoma ineficiente, uma tendĂȘncia para morte celular
(potencialmente acelerada pela atividade do complemento e por outros fatores
extrĂnsecos que ativam death receptors), com ativação da resposta de fase
aguda e do sistema imune, assim como da fibrose.
A validação de alguns alvos especĂficos atravĂ©s de immunoblot e correlação
com dados clĂnicos apontou para a cadeia ÎČ do complemento C3, a Muscle
Ring Finger protein 1 (MuRF1) e a dual-specificity Tyr-phosphoylation
regulated kinase 1A (DYRK1A) como potenciais marcadores de uma resposta
incompleta. Por outro lado, a predição de cinases a partir do fosfoproteoma,
sugere que a modulação da caseĂna cinase 2, a famĂlia de cinases do IÎșB, a
glicogénio sintase cinase 3 e da DYRK1A pode ajudar a melhorar a condição
dos pacientes indicados para intervenção. Em particular, a avaliação funcional
de cardiomiĂłcitos DYRK1A+/- mostraram que esta cinase pode ser um alvo
importante para tratar a disfunção cardĂaca, uma vez que os miĂłcitos mutantes
responderam de forma diferente ao estiramento e mostraram uma menor
capacidade para desenvolver força (tensão ativa).
Este estudo levanta vårias hipóteses na investigação da remodelagem reversa.
No futuro, estudos de ganho e/ou perda de função realizados em
cardiomiĂłcitos isolados ou em modelos animais de banding-debanding da
aorta ajudarĂŁo a testar a eficĂĄcia de modular os potenciais alvos terapĂȘuticos
encontrados. AlĂ©m disso, estudos clĂnicos em coortes de maior dimensĂŁo
trarão conclusÔes definitivas quanto ao valor de prognóstico do complemento
C3, MuRF1 e DYRK1A.Programa Doutoral em Biomedicin
Application of advanced fluorescence microscopy and spectroscopy in live-cell imaging
Since its inception, fluorescence microscopy has been a key source of discoveries in cell biology. Advancements in fluorophores, labeling techniques and instrumentation have made fluorescence microscopy a versatile quantitative tool for studying dynamic processes and interactions both in vitro and in live-cells. In this thesis, I apply quantitative fluorescence microscopy techniques in live-cell environments to investigate several biological processes. To study Gag processing in HIV-1 particles, fluorescence lifetime imaging microscopy and single particle tracking are combined to follow nascent HIV-1 virus particles during assembly and release on the plasma membrane of living cells. Proteolytic release of eCFP embedded in the Gag lattice of immature HIV-1 virus particles results in a characteristic increase in its fluorescence lifetime. Gag processing and rearrangement can be detected in individual virus particles using this approach. In another project, a robust method for quantifying Förster resonance energy transfer in live-cells is developed to allow direct comparison of live-cell FRET experiments between laboratories. Finally, I apply image fluctuation spectroscopy to study protein behavior in a variety of cellular environments. Image cross-correlation spectroscopy is used to study the oligomerization of CXCR4, a G-protein coupled receptor on the plasma membrane. With raster image correlation spectroscopy, I measure the diffusion of histones in the nucleoplasm and heterochromatin domains of the nuclei of early mouse embryos. The lower diffusion coefficient of histones in the heterochromatin domain supports the conclusion that heterochromatin forms a liquid phase-separated domain. The wide range of topics covered in this thesis demonstrate that fluorescence microscopy is more than just an imaging tool but also a powerful instrument for the quantification and elucidation of dynamic cellular processes
RNA pull-down-confocal nanoscanning (RP-CONA), a novel method for studying RNA/protein interactions in cell extracts that detected potential drugs for Parkinsonâs disease targeting RNA/HuR complexes
MicroRNAs (miRNAs, miRs) are a class of small non-coding RNAs that regulate gene expression through specific base-pair targeting. The functional mature miRNAs usually undergo a two-step cleavage from primary miRNAs (pri-miRs), then precursor miRNAs (pre-miRs). The biogenesis of miRNAs is tightly controlled by different RNA-binding proteins (RBPs). The dysregulation of miRNAs is closely related to a plethora of diseases. Targeting miRNA biogenesis is becoming a promising therapeutic strategy.
HuR and MSI2 are both RBPs. MiR-7 is post-transcriptionally inhibited by the HuR/MSI2 complex, through a direct interaction between HuR and the conserved terminal loop (CTL) of pri-miR-7-1. Small molecules dissociating pri-miR-7/HuR interaction may induce miR-7 production. Importantly, the miR-7 levels are negatively correlated with Parkinsonâs disease (PD).
PD is a common, incurable neurodegenerative disease causing serious motor deficits. A hallmark of PD is the presence of Lewy bodies in the human brain, which are inclusion bodies mainly composed of an aberrantly aggregated protein named α-synuclein (α-syn). Decreasing α-syn levels or preventing α-syn aggregation are under investigation as PD treatments. Notably, α-syn is negatively regulated by several miRNAs, including miR-7, miR-153, miR-133b and others. One hypothesis is that elevating these miRNA levels can inhibit α-syn expression and ameliorate PD pathologies.
In this project, we identified miR-7 as the most effective α-syn inhibitor, among the miRNAs that are downregulated in PD, and with α-syn targeting potentials. We also observed potential post-transcriptional inhibition on miR-153 biogenesis in neuroblastoma, which may help to uncover novel therapeutic targets towards PD.
To identify miR-7 inducers that benefit PD treatment by repressing α-syn expression, we developed a novel technique RNA Pull-down Confocal Nanoscaning (RP-CONA) to monitor the binding events between pri-miR-7 and HuR. By attaching FITC-pri-miR-7-1-CTL-biotin to streptavidin-coated agarose beads and incubating them in human cultured cell lysates containing overexpressed mCherry-HuR, the bound RNA and protein can be visualised as quantifiable fluorescent rings in corresponding channels in a confocal high-content image system. A pri-miR-7/HuR inhibitor can decrease the relative mCherry/FITC intensity ratio in RP-CONA. With this technique, we performed several small-scale screenings and identified that a bioflavonoid, quercetin can largely dissociate the pri-miR-7/HuR interaction. Further studies proved that quercetin was an effective miR-7 inducer as well as α-syn inhibitor in HeLa cells.
To understand the mechanism of quercetin mediated α-syn inhibition, we tested the effects of quercetin treatment with miR-7-1 and HuR knockout HeLa cells. We found that HuR was essential in this pathway, while miR-7 hardly contributed to the α-syn inhibition. HuR can directly bind an AU-rich element (ARE) at the 3â untranslated region (3â-UTR) of α-syn mRNA and promote translation. We believe quercetin mainly disrupts the ARE/HuR interaction and disables the HuR-induced α-syn expression.
In conclusion, we developed and optimised RP-CONA, an on-bead, lysate-based technique detecting RNA/protein interactions, as well as identifying RNA/protein modulators. With RP-CONA, we found quercetin inducing miR-7 biogenesis, and inhibiting α-syn expression. With these beneficial effects, quercetin has great potential to be applied in the clinic of PD treatment. Finally, RP-CONA can be used in many other RNA/protein interactions studies
A Molecular Approach to the Diagnosis, Assessment, Monitoring and Treatment of Pulmonary Non-Tuberculous Mycobacterial Disease
Introduction: Non-Tuberculous Mycobacteria (NTM) can cause disease of the lungs and sinuses, lymph nodes, joints and central nervous system as well as disseminated infections in immunocompromised individuals. Efforts to tackle infections in NTM are hampered by a lack of reliable biomarkers for diagnosis, assessment of disease activity, and prognostication.
Aims: The broad aims of this thesis are:
1. to develop molecular assays capable of quantifying the 6 most common pathogenic mycobacteria (M. abscessus, M. avium, M. intracellulare, M. malmoense, M. kansasii, M. xenopi) and calculate comparative sensitivities and specificities for each assay.
2. to assess patientsâ clinical course over 12 â 18 months by performing the developed molecular assays against DNA extracted from sputum from patients with NTM infection.
3. to assess dynamic bacterial changes of the lung microbiome in patients on treatment for NTM disease and those who are treatment naâve.
Methods: DNA was extracted from a total of 410 sputum samples obtained from 38 patients who were either:
âą commencing treatment for either M. abscessus or Mycobacterium avium complex.
âą considered colonised with M. abscessus or Mycobacterium avium complex (i.e. cultured NTM but were not deemed to have infection as they did not meet ATS or BTS criteria for disease).
âą Diagnosed with cystic fibrosis (CF) or non-CF bronchiectasis but had never cultured NTM.
For the development of quantitative molecular assays, NTM hsp65 gene sequences were aligned and interrogated for areas of variability. These variable regions enabled the creation of species specific probes. In vitro sensitivity and specificity for each probe was determined by testing each probe against a panel of plasmids containing hsp65
gene inserts from different NTM species. Quantification accuracy was determined by using each assay against a mock community containing serial dilutions of target DNA.
Each sample was tested with the probes targeting: M. abscessus, M. avium and M. intracellulare producing a longitudinal assessment of NTM copy number during each patientâs clinical course.
In addition, a total of 64 samples from 16 patients underwent 16S rRNA gene sequencing to characterise longitudinal changes in the microbiome of both NTM disease and controls.
Results: In vitro sensitivity for the custom assays were 100% and specificity ranged from 91.6% to 100%. In terms of quantification accuracy, there was no significant difference between the measured results of each assay and the expected values when performed in singleplex. The assays were able to accurately determine NTM copy number to a theoretical limit of 10 copies/ÎŒl.
When used against samples derived from human sputum and using culture results as a gold standard, the sensitivity of the assay for M. abscessus was found to be 0.87 and 0.86 for MAC. The specificity of the assay for M. abscessus was 0.95 and 0.62 for MAC. The negative predictive value of the assay for M. abscessus was 0.98 and 0.95 for MAC. This resulted in an AUC of 0.92 for M. abscessus and 0.74 for MAC.
Longitudinal analysis of the lung microbiome using 16SrRNA gene sequencing showed that bacterial burden initially decreases after initiation of antibiotic therapy but begins to return to normal levels over several months of antibiotic therapy. This effect is mirrored by changes in alpha diversity. The decrease in bacterial burden and loss of alpha diversity was found to be secondary to significant changes in specific genera such as Veillonella and Streptococcus. The abundance of other Proteobacteria such as Pseudomonas remain relatively constant.
Conclusion: The molecular assay has shown high in vitro sensitivity and specificity for the detection and accurate quantification of the 6 most commonly pathogenic NTM species. The assays successfully identified NTM DNA from human sputum samples.
A notable association between NTM copy number and the cessation of one or more antibiotics existed (i.e. when one antibiotic was stopped because of patient intolerance, NTM copy number increased, often having been unrecordable prior to this). The qPCR assays developed in this thesis provide an affordable, real time and rapid measurement of NTM burden allowing clinicians to act on problematic results sooner than currently possible.
There was no significant difference between the microbiome in bronchiectasis and cystic fibrosis nor was there a significant difference between the microbiome in patients requiring treatment for NTM and those who did not. Patients receiving treatment experienced an initial decrease in bacterial burden over the first weeks of treatment followed by a gradual increase towards baseline over the next weeks to months. This change was mirrored in measures of alpha diversity. Changes in abundance and diversity were accounted for by decreases in specific bacteria whilst the abundance of other bacteria increased, occupying the microbial niche created. These bacteria (for example Pseudomonas spp) are often associated with morbidity.Open Acces
Understanding novel EGFP-Ubx protein-based film formation
Protein-based materials are currently the subject of intense research interest since they have an extended range of potential applications, such as im-proved bio-membrane biocompatibility for implanted medical devices and the creation of platform materials for novel biosensors. Monomers from Ultrabithorax (Ubx) transcription factor are known to spontaneously self-assemble at an air-water interface to form a monolayer, which has then been used as a basis for forming biopolymeric Ëbers. Here we used the Lang-muir trough technique, Brewster angle microscopy (BAM), ellipsometry and neutron reËectometry (NR) to investigate the inËuences of diËerent exper-imental conditions on EGFP-Ubx monolayer formation and the impact on biopolymeric Ëber structure. We varied protein concentration, buËer prop-erties and waiting times prior to forming biopolymeric Ëbers. Interestingly, we found 3 phases of material formation which brought us to a new protocol for forming Ëbers that reduced protein concentration by 5-fold and wait-ing times by 100-fold. Moreover, an in-house developed MATLAB code was used to analyze SEM images and obtain quantitative structural information about the biopolymeric Ëbers that were correlated directly to the surface Ëlm characteristics measured in the LB trough. These new insights into Ëber formation and structure enhance the usefulness of the Ubx-based biopolymer for biomedical applications
Statistical Learning for Gene Expression Biomarker Detection in Neurodegenerative Diseases
In this work, statistical learning approaches are used to detect biomarkers for neurodegenerative diseases (NDs). NDs are becoming increasingly prevalent as populations age, making understanding of disease and identification of biomarkers progressively important for facilitating early diagnosis and the screening of individuals for clinical trials. Advancements in gene expression profiling has enabled the exploration of disease biomarkers at an unprecedented scale. The work presented here demonstrates the value of gene expression data in understanding the underlying processes and detection of biomarkers of NDs. The value of novel approaches to previously collected -omics data is shown and it is demonstrated that new therapeutic targets can be identified. Additionally, the importance of meta-analysis to improve power of multiple small studies is demonstrated. The value of blood transcriptomics data is shown in applications to researching NDs to understand underlying processes using network analysis and a novel hub detection method. Finally, after demonstrating the value of blood gene expression data for investigating NDs, a combination of feature selection and classification algorithms were used to identify novel accurate biomarker signatures for the diagnosis and prognosis of Parkinsonâs disease (PD) and Alzheimerâs disease (AD). Additionally, the use of feature pools based on previous knowledge of disease and the viability of neural networks in dimensionality reduction and biomarker detection is demonstrated and discussed. In summary, gene expression data is shown to be valuable for the investigation of ND and novel gene biomarker signatures for the diagnosis and prognosis of PD and AD
How to Be a God
When it comes to questions concerning the nature of Reality, Philosophers and Theologians have the answers.
Philosophers have the answers that canât be proven right. Theologians have the answers that canât be proven wrong.
Todayâs designers of Massively-Multiplayer Online Role-Playing Games create realities for a living. They canât spend centuries mulling over the issues: they have to face them head-on. Their practical experiences can indicate which theoretical proposals actually work in practice.
Thatâs todayâs designers. Tomorrowâs will have a whole new set of questions to answer.
The designers of virtual worlds are the literal gods of those realities. Suppose Artificial Intelligence comes through and allows us to create non-player characters as smart as us. What are our responsibilities as gods? How should we, as gods, conduct ourselves?
How should we be gods
- âŠ