13,990 research outputs found

    Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and autophagy

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    UFMylation involves the covalent modification of substrate proteins with UFM1 (Ubiquitin‐fold modifier 1) and is important for maintaining ER homeostasis. Stalled translation triggers the UFMylation of ER‐bound ribosomes and activates C53‐mediated autophagy to clear toxic polypeptides. C53 contains noncanonical shuffled ATG8‐interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. However, the mechanistic basis of sAIM‐mediated 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 UFMylation‐dependent fine‐tuning of C53‐mediated autophagy activation

    Subspecies limits based on morphometry and mitochondrial DNA genomics in a polytypic species, the common grackle, Quiscalus quiscula

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    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 ‘Florida grackle’, 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?

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    The worldwide emerging trend of eating “natural” 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 cow’s 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 cow’s 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.Å spise mat som er mindre prosessert og mer «naturlig» er en pĂ„gĂ„ende trend i Norge og i andre deler av verden. Interessen for Ă„ drikke melk som ikke er varmebehandlet, sĂ„kalt rĂ„ melk, er ogsĂ„ Ăžkende. I Norge er det pĂ„budt Ă„ pasteurisere melk fĂžr kommersielt salg for Ă„ beskytte forbrukeren mot sykdomsfremkallende mikroorganismer. Fagfolk advarer mot Ă„ drikke rĂ„ melk, og pĂ„peker risikoen for Ă„ 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 speneprĂžver fra melkegĂ„rder i sĂžrĂžst Norge, samlet vi ogsĂ„ miljĂžprĂžver fra de samme gĂ„rdene for Ă„ kartlegge forekomst og for Ă„ 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 pĂ„ gĂ„rdene. Bakteriene kan overfĂžres fra gĂ„rdsmiljĂžet til melkekjeden og dermed utfordre mattryggheten. Disse bakteriene har forĂ„rsaket melkebĂ„rne sykdomsutbrudd over hele verden, ogsĂ„ 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 matbĂ„rne zoonotiske sykdommene vi har i den vestlige verden. Resultater fra denne oppgaven viser en hĂžy forekomst av de tre patogenene i gĂ„rdsmiljĂžet. 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 rĂ„ melk kan inneholde patogene STEC. For Ă„ videre vurdere risikoen for Ă„ bli syk av STEC fra rĂ„ melk undersĂžkte vi hvordan de fire eae-positive isolatene vokste i rĂ„ 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 fĂžrste 24 timene, deretter stoppet reduksjonen i antall bakterier. Disse resultatene viser hvor viktig det er Ă„ ha stabil lav lagringstemperatur for rĂ„ melk, helst < 4°C. L. monocytogenes isolatene som ble samlet inn fra melkegĂ„rdene 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 sĂ„ man at en klone kan persistere pĂ„ en gĂ„rd over tid og at melk kan kontamineres av L. monocytogenes kloner som er til stede i gĂ„rdsmiljĂžet. Til tross for smĂ„ testvolum av tankmelken (25 mL) fant vi bĂ„de 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 pĂ„ bĂ„s byttes ut med lĂžsdriftssystemer og melkeroboter. Forekomsten av de tre patogenene funnet i denne studien var hĂžyere i besetningene med lĂžsdrift sammenliknet med besetningene som hadde melkekyrne oppstallet pĂ„ bĂ„s. Pasteurisering er et viktig forebyggende tiltak for Ă„ beskytte konsumenter fra mikrobielle patogener, og i de fleste EU-land er kommersielt salg av rĂ„ melk juridisk begrenset. Denne studien viser at oppstallingstype kan pĂ„virke nivĂ„ene av patogene bakterier i gĂ„rdsmiljĂžet og i rĂ„ melk. Inntak av rĂ„ 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 avgjĂžrende Ă„ lagre rĂ„ melk ved kontinuerlig lave temperaturer for Ă„ forebygge vekst av zoonotiske patogener

    Estudo da remodelagem reversa miocårdica através da anålise proteómica do miocårdio e do líquido pericårdico

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    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 nĂŁo sĂŁo claros. AlĂ©m disso, nĂŁo dispomos de ferramentas de prognĂłstico definitivos nem de terapias auxiliares para melhorar a condição dos pacientes indicados para substituição da vĂĄlvula. 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 glicogĂ©nio 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 (tensĂŁo ativa). Este estudo levanta vĂĄrias hipĂłteses 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 trarĂŁo conclusĂ”es definitivas quanto ao valor de prognĂłstico do complemento C3, MuRF1 e DYRK1A.Programa Doutoral em Biomedicin

    Application of advanced fluorescence microscopy and spectroscopy in live-cell imaging

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    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 Parkinson’s disease targeting RNA/HuR complexes

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    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 Parkinson’s 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 3’ untranslated region (3’-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

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    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 patients’ clinical course over 12 – 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 na ve. Methods: DNA was extracted from a total of 410 sputum samples obtained from 38 patients who were either: ‱ commencing treatment for either M. abscessus or Mycobacterium avium complex. ‱ 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). ‱ 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 patient’s 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

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    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 ˝bers. Here we used the Lang-muir trough technique, Brewster angle microscopy (BAM), ellipsometry and neutron re˛ectometry (NR) to investigate the in˛uences of di˙erent exper-imental conditions on EGFP-Ubx monolayer formation and the impact on biopolymeric ˝ber structure. We varied protein concentration, bu˙er prop-erties and waiting times prior to forming biopolymeric ˝bers. Interestingly, we found 3 phases of material formation which brought us to a new protocol for forming ˝bers 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 ˝bers that were correlated directly to the surface ˝lm characteristics measured in the LB trough. These new insights into ˝ber formation and structure enhance the usefulness of the Ubx-based biopolymer for biomedical applications

    Statistical Learning for Gene Expression Biomarker Detection in Neurodegenerative Diseases

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    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 Parkinson’s disease (PD) and Alzheimer’s 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

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    When it comes to questions concerning the nature of Reality, Philosophers and Theologians have the answers. Philosophers have the answers that can’t be proven right. Theologians have the answers that can’t be proven wrong. Today’s designers of Massively-Multiplayer Online Role-Playing Games create realities for a living. They can’t 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. That’s today’s designers. Tomorrow’s 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
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