1,551 research outputs found
Platelet-activating factor receptor in health and disease.
Background Platelet-activating factor receptor (PAFR) expression has been linked to anthropogenic particulate matter (PM). Traffic-related air pollution (TRAP) now accounts for the majority of this PM. PAFR expression has also been linked to an increased risk of infection from Streptococcus pneumoniae (S. pneumoniae). Children with asthma and sickle cell disease (SCD) have a significantly increased risk of morbidity and mortality from invasive pneumococcal disease (IPD). PAFR expression has not yet been investigated in relation to TRAP-generated PM, nor has constitutive expression been investigated in these children at increased risk of IPD. Methods PM10 was collected from roadside traffic using the Cyclone device. A549 cells were exposed to the collected PM10 and flow cytometry was undertaken to measure PAFR expression by median fluorescence intensity (MFI). Exposed A549 cells also underwent assays to determine bacterial adhesion (colony-forming units, CFU) using D39 S. pneumoniae species. In both experiments, Dulbecco’s phosphate buffered saline (DPBS) was used as a control. In a separate study, children aged 1 – 17 years were recruited into 4 groups: 2 disease groups (children with asthma, and those with SCD); and 2 control groups (healthy children, and children with atopy but not asthma). Nasal epithelial cells were collected and PAFR expression (MFI) measured by flow cytometry. 24-hour PM10 pollution (μg/m3) data were also collected for each participant. Results TRAP-related PM caused a significant increase in PAFR expression in A549 cells when exposed to a concentration of 10 ug/ml (p < 0.05). Bacterial adhesion (CFU) was significantly raised in A549 cells exposed to TRAP PM verses the control wells (p < 0.05). In children, PAFR expression in SCD was notably raised when compared to all other groups (p < 0.001). There was no 7 significant difference in the PAFR expression in those with asthma versus the control groups. 24% of the children within the study demonstrated exposure to PM10 levels above the WHO daily safety limit. Conclusion PAFR expression and subsequent bacterial adhesion is increased following exposure to TRAP. PAFR is shown to be constitutively raised in those with SCD and this may explain some of the reported risk from IPD. Air pollution levels in London remain above safe limits despite public health initiatives trying to decrease them
Single-cell time-series analysis of metabolic rhythms in yeast
The yeast metabolic cycle (YMC) is a biological rhythm in budding yeast (Saccharomyces cerevisiae). It entails oscillations in the concentrations and redox states of intracellular metabolites, oscillations in transcript levels, temporal partitioning of biosynthesis, and, in chemostats, oscillations in oxygen consumption. Most studies on the YMC have been based on chemostat experiments, and it is unclear whether YMCs arise from interactions between cells or are generated independently by each cell. This thesis aims at characterising the YMC in single cells and its response to nutrient and genetic perturbations. Specifically, I use microfluidics to trap and separate yeast cells, then record the time-dependent intensity of flavin autofluorescence, which is a component of the YMC.
Single-cell microfluidics produces a large amount of time series data. Noisy and short time series produced from biological experiments restrict the computational tools that are useful for analysis. I developed a method to filter time series, a machine learning model to classify whether time series are oscillatory, and an autocorrelation method to examine the periodicity of time series data.
My experimental results show that yeast cells show oscillations in the fluorescence of flavins. Specifically, I show that in high glucose conditions, cells generate flavin oscillations asynchronously within a population, and these flavin oscillations couple with the cell division cycle. I show that cells can individually reset the phase of their flavin oscillations in response to abrupt nutrient changes, independently of the cell division cycle. I also show that deletion strains generate flavin oscillations that exhibit different behaviour from dissolved oxygen oscillations from chemostat conditions.
Finally, I use flux balance analysis to address whether proteomic constraints in cellular metabolism mean that temporal partitioning of biosynthesis is advantageous for the yeast cell, and whether such partitioning explains the timing of the metabolic cycle. My results show that under proteomic constraints, it is advantageous for the cell to sequentially synthesise biomass components because doing so shortens the timescale of biomass synthesis. However, the degree of advantage of sequential over parallel biosynthesis is lower when both carbon and nitrogen sources are limiting.
This thesis thus confirms autonomous generation of flavin oscillations, and suggests a model in which the YMC responds to nutrient conditions and subsequently entrains the cell division cycle. It also emphasises the possibility that subpopulations in the culture explain chemostat-based observations of the YMC. Furthermore, this thesis paves the way for using computational methods to analyse large datasets of oscillatory time series, which is useful for various fields of study beyond the YMC
The role of heterosocial perception in men's likelihood to sexually harass
Sexual harassment against women represents sexually aggressive and coercive behaviour that violates women’s dignity and creates an offensive environment, which threatens women’s well-being and ability to prosper in day to day life (Pina, Gannon & Saunders, 2009). Understanding the perceptual characteristics of men with inclinations to sexually harass, through how they perceive women is important in delineating the reasons why some men may engage in the sexual harassment of women. Heterosocial perception is a concept of how an individual perceives another person interacting with the opposite sex. It is typically measured using the Test of Reading Affective Cues (TRAC), a tool encompassing an array of video clips showing a woman interacting with a man, whereby the woman displays a range of affective cues. The perceiver is assessed on their perceptual accuracy when judging the affective cues. Perceptual accuracies of male rapists and male child molesters have been well researched (Lipton, McDonel & McFall, 1987; Stahl & Sacco, 1995), yet male sexual harassment as a singular category has been neglected. The current thesis explores the potential heterosocial perception characteristics of a distinct group of men who are relatively high in the likelihood to engage in sexual harassment of women from scores on Pryor’s (1987) Likelihood to Sexually Harass (LSH) scale focusing on their differences in performance on the TRAC in comparison to those men who are lower in the likelihood to engage in sexual harassment of women.
Five empirical studies are reported in this thesis. Study 1 presents a modernized version of the TRAC and incorporates an analysis to develop it as a research tool, enabling judgements on five affective cues displayed by a woman; friendly, romantic, neutral, bored and rejecting. The tool provides this range of affective cues that were used in later studies to measure differences in heterosocial perception. Study 2 addressed theoretical explanations taken from previous perception research with sexually aggressive men (Malamuth & Brown, xiv 1994) to explain differences in heterosocial perception for men high in LSH. Explanations are given for potential biases evidenced by men high in LSH focusing on Error Management Theory (Haselton & Buss, 2000) arguing that an overperception bias will increase the frequency of falsely inferring a woman’s sexual intent towards sexual pursuit, but considerably reduce the costs of losing a sexual opportunity by falsely inferring that a woman lacked sexual intent. Altogether, study 2 provided support for the misidentification of negative affective cues (negativeness blindness), the overperception of negative affective cues and the romantic overperception bias of friendly affective cues in the perception of men high in LSH.
Study 3 tested the established theoretical link that internal concepts of social power have within men who report sexual aggression and sexual coercion and the subsequent impact on perception. Unexpectedly, power did not exacerbate perceptual inaccuracy for negative affective cues and the romantic overperception bias of friendly affective cues. In study 4, objectification was assessed in its relationship to perception in high LSH men. Instrumental and both specific and general sexual objectification were significantly higher for men high in LSH. Specific sexual objectification was found to negatively mediate romantic categorizations of romantic affective cues, but general sexual objectification was found to positively mediate romantic categorizations of friendly affective cues for men high in LSH. Results also showed that men high in LSH showed poorest perceptual accuracy on bored and rejecting affective cues, and evidenced a greater romantic judgement of friendly affective cues overperception bias. In study 5, the impact of different mental states on perception was assessed, via the use of a cooling system to facilitate self-regulation. A cooling system is a psychological framework proposed for understanding self-control (Metcalfe & Mischel, 1999), and in this study it incorporated techniques of distraction, distancing and empathy enhancement. Results showed that the cooling system was not beneficial in making high LSH xv men’s perceptual judgements more accurate and in making their judgements accurate to the level of low and medium LSH men for negative affective cues. However, cooling did improve perceptual accuracy of friendly affective cues removing the overperception bias to romantic judgements in comparison to the neutral condition. The cooling system was not found to reduce instrumental and sexual objectification for high LSH men. There were differences found on empathy between men high and low and medium on LSH. Differences were found such that men high in LSH showed more state empathy, but less trait empathy than men low and medium in LSH
Parámetros genéticos de los caracteres morfológicos lineales de la raza caprina murciano-granadina y sus relaciones con otros caracteres funcionales
Linear appraisal systems (LAS) are effective strategies for systematically collecting zoometric information from animal populations. Traditionally applied LAS in goats was developed considering the variability and scales found in highly selected breeds. Implementing LAS may reduce time, personnel, and resource needs when performing zoometric large-scale collection. Moreover, selection for zoometrics defines individuals’ productive longevity, endurance, enhanced productive abilities, and consequently, long-term profitability. As a result, traditional LAS may no longer cover the different contexts of goat breeds widespread throughout the world, and departures from normality may be indicative of the different stages of selection at which a certain population can be found. In the first study, an evaluation of the distribution and symmetry properties of twenty-eight zoometric traits was developed. After symmetry analysis was performed, the scale readjustment proposal suggested specific strategies should be implemented such as scale reduction of lower or upper levels, determination of a setup moment to evaluate and collect information from young (up to 2 years) and adult bucks (over 2 years), the addition of upper categories in males due to upper values in the scale being incorrectly clustered together. Thus, the particular analysis of each variable permits determining specific strategies for each trait and serve as a model for other breeds, either selected or in terms of selection. The aim of the second study was to propose a method to optimize and validate LAS in opposition to traditional measuring protocols routinely implemented in Murciano-Granadina goats. The data sample consisted of 41323 LAS and traditional measuring records, belonging to 22727 herdbook registered primipara does, 17111 multipara does, and 1485 bucks. Each record comprised information on 17 linear traits for primipara and multipara does, and 10 traits for bucks. All zoometric parameters were scored on a 9-points scale. Cronbach’s alpha values suggested a high internal consistency of the optimized variable panel. Model fit, variability explanation power, and predictive power (MSE, AIC/AICc, and BIC, respectively) suggested a model comprising zoometric LAS scores performed better than traditional zoometry. Optimization procedures result in reduced models able to capture variability for dairy-related zoometric traits without noticeable detrimental effects on model validity properties. The third study aimed to perform a particular analysis of each variable that permits determining specific strategies for each trait and serves as a model for other breeds. Among the strategies proposed are the reduction/readjustment of the levels in the scale as it happens for limb-related traits, the extension of the scale as it occurs in the stature of males, or the subdivision of the scale used in males into two categories, bucks younger than two years and bucks of two years old and older. Murciano- Granadina goat breed has drifted towards better dairy-linked conformation traits but without losing the grounds of the zoometric basis which confers it with enhanced adaptability to the environment. Hence, such strategies can help to achieve a better understanding of the momentum of selection for dairy-linked zoometric traits in Murciano-Granadina population and their future evolution to enhance the profitability and efficiency of breeding plans. The objective of the fourth study was to evaluate the progress of heritabilities of the traits comprising the linear appraisal system in the Murciano-Granadina breed during the complete decade from December 2011 to December 2021. The estimated values for heritability were obtained from multivariate analyzes using the BLUP methodology and MTDFREML software. For 2021 heritabilities, a simple animal model was applied to records collected from 22727 primiparous goats and 17111 multiparous goats belonging to 85 herds. The model included the linear and quadratic and linear components of the covariates age and days in milk, respectively. The fixed effects considered in the model were herd, reproductive status, calving month, and herd/year interaction. The animal was considered as a random effect. The variables studied included five characteristics related to structure and capacity, two traits related to dairy structure, six related to the mammary system, and three related to legs and feet. The heritabilities for structure and capacity characters progressed from 0.22 to 0.28 including non-convergent variables in June 2012 to values between 0.10 and 0.41 with all variables converging in June 2021. Heritabilities for dairy structure progressed from 0.18 with nonconvergent variables in 2011 to 0.17 to 0.25 in 2021. Heritabilities for mammary system traits progressed from 0.12 to 0, 27 with non-convergent variables in 2012 to between 0.10 and 0.41 in 2021. For legs and feet, heritabilities progressed from 0.16 to 0.17 with non-convergent variables to 0.09 a 0.22. Genetic progress is not only evident in heritability values, but there has been a notable reduction in the standard error of heritabilities from 0.1000 (0.080-0.120) to 0.000 (0.000-0.001) from 2011 to 2021. These results provide evidence of the enhancement in the effectiveness and precision of the linear qualification system applied during the past decade and its successful integration into the breeding program of the Murciano- Granadina breed. The fifth study estimates genetic and phenotypic parameters for zoometric/LAS traits in Murciano-Granadina goats, estimate genetic and phenotypic correlations among all traits, and to determine whether major area selection would be appropriate or if adaptability strategies may need to be followed. Heritability estimates for the zoometric/LAS traits were low to high, ranging from 0.09 to 0.43 and the accuracy of estimation has improved after decades rendering standard errors negligible. Scale inversion of specific traits may need to be performed before major areas selection strategies are implemented. Genetic and phenotypic correlations suggest that negative selection against thicker bones and higher rear insertion heights, indirectly results in the optimization of selection practices in the rest of the traits, especially of those in the structure and capacity and mammary system major areas. The integration and implementation of the strategies proposed within Murciano-Granadina breeding program maximize selection opportunities and the sustainable international competitiveness of the Murciano- Granadina goat in the dairy goat breed panorama. The objective of the sixth study was to develop a discriminant canonical analysis (DCA) tool that permits outlining the role of the individual haplotypes of each component of the casein complex (αS1, β, αS2, and κ-casein) on zoometrics/linear appraisal breeding values. The relationship of the predicted breeding value for 17 zoometric/Linear appraisal traits and αS1, β, αS2, and κ-casein genes haplotypic sequences was assessed. Results suggest that, although a lack of significant differences (P>0.05) was reported across the predictive breeding values of zoometric/linear appraisal traits for αS1, αS2 and κ casein, significant differences were found for β Casein (P0,05) en los valores de cría predichos de los rasgos de zoometría/calificación lineal para la αS1, αS2 y κ-caseína, se encontraron diferencias significativas para la β-caseína (P<0,05), respectivamente. La presencia de secuencias haplotípicas de β-caseína GAGACCCC, GGAACCCC, GGAACCTC, GGAATCTC, GGGACCCC, GGGATCTC y GGGGCCCC, vinculadas a combinaciones diferenciales de mayores cantidades de leche de mayor calidad en términos de su composición, también puede estar relacionada con una mayor valoración zoométrica/lineal de la predicción de los valores de cría. La selección debe realizarse con cuidado, dado que la consideración de animales aparentemente deseables que presentan la secuencia haplotípica GGGATCCC en el gen de la β- caseína, debido a sus valores genéticos predichos positivos para ciertos rasgos de zoometría/calificación lineal, como la altura de la inserción trasera, la calidad ósea , la inserción anterior, la profundidad de ubre, la vista lateral de patas traseras y la vista trasera de patas traseras pueden conducir a una selección indirecta frente al resto de rasgos de zoometría/calificación lineal y a su vez conducir a una selección ineficiente hacia un tipo morfotipo lechero óptimo en cabras Murciano-Granadina. Por el contrario, la consideración de animales que presentan la secuencia haplotípica GGAACCCC implica también considerar animales que aumentan el potencial genético para todos los rasgos de zoometría/calificación lineal, haciéndolos así recomendables como reproductores. La información derivada de los presentes análisis mejorará la selección de individuos reproductores que busquen un tipo lechero bastante deseable, a través de la determinación de las secuencias haplotípicas que presentan en el locus β-caseína. Todos estos estudios persiguen la obtención de un conocimiento más profundo de los caracteres morfológicos lineales de la raza caprina Murciano-Granadina y sus relaciones con otras características funcionales. Esto sienta las bases para estrategias de normalización y mejora de la capacidad productiva y el morfotipo lechero de la cabra Murciano-Granadina y ayudará a alcanzar su consolidación competitiva en el panorama caprino lechero internacional
Ecology of methanotrophs in a landfill methane biofilter
Decomposing landfill waste is a significant anthropogenic source of the potent climate-active gas methane (CH₄). To mitigate fugitive methane emissions Norfolk County Council are trialling a landfill biofilter, designed to harness the methane oxidizing potential of methanotrophic bacteria. These methanotrophs can convert CH₄ to CO₂ or biomass and act as CH₄ sinks.
The most active CH₄ oxidising regions of the Strumpshaw biofilter were identified from in-situ temperature, CH₄, O₂ and CO₂ profiles. While soil CH₄ oxidation potential was estimated and used to confirm methanotroph activity and determine optimal soil moisture conditions for CH₄ oxidation. It was observed that most CH₄ oxidation occurs in the top 60cm of the biofilter (up to 50% of CH4 input) at temperatures around 50ºC, optimal soil moisture was 10-27.5%. A decrease in in-situ temperature following CH₄ supply interruption suggested the high biofilter temperatures were driven by CH₄ oxidation.
The biofilter soil bacterial community was profiled by 16S rRNA gene analysis, with methanotrophs accounting for ~5-10% of bacteria. Active methanotrophs at a range of different incubation temperatures were identified by ¹³CH₄ DNA stable-isotope probing coupled with 16S rRNA gene amplicon and metagenome analysis. These methods identified Methylocella, Methylobacter, Methylocystis and Crenothrix as potential CH₄ oxidisers at the lower temperatures (30ºC/37ºC) observed following system start-up or gas-feed interruption. At higher temperatures typical of established biofilter operation (45ºC/50ºC), Methylocaldum and an unassigned Methylococcaceae species were the dominant active methanotrophs.
Finally, novel methanotrophs Methylococcus capsulatus (Norfolk) and Methylocaldum szegediense (Norfolk) were isolated from biofilter soil enrichments. Methylocaldum szegediense (Norfolk) may be very closely related to or the same species as one of the most abundant active methanotrophs in a metagenome from a 50ºC biofilter soil incubation, based on genome-to-MAG similarity. This isolate was capable of growth over a broad temperature range (37-62ºC) including the higher (in-situ) biofilter temperatures (>50ºC)
Speciation and sex-biased gene expression in the scarce swallowtails
Speciation is the process by which closely related populations of organisms differentiate following reductions in the effective rate of genetic exchange between
them over time. For most speciation events, population genetic data is the only
available information about how reproductive isolation has arisen. We have a poor
understanding of how evolutionary forces and genomic features contribute to reproductive isolation, primarily due to the difficulty of inferring barriers to gene flow. In particular, it is unclear what role genes that are sex-biased in expression and/or
sex-linked play in speciation. In my thesis, I aim to infer the locations of putative
barriers to gene flow to understand to what extent different genomic features, in
particular fast-evolving sex-biased genes, contribute to reproductive isolation between a sister species pair of scarce swallowtail (Iphiclides) butterflies. In my first
research project, I estimate core population genetic parameters across all sister
species pairs of European butterflies and fit simple models of divergence to ask
how well classic phylogeographic hypotheses fit recent diversification events in
this taxonomic group. In my second research project, I infer explicit models of the
speciation process and model effective migration rates along the genome to locate putative barriers to gene flow. I ask whether these barriers to long-term gene
flow are associated with areas of the genome that show a reduction in recent introgression across a hybrid zone. In my third and final research project, I extend the
demographic modeling of speciation in the Iphiclides species pair to the Z chromosome and ask whether barrier regions are associated with sex-biased genes,
as a result of their faster rate of evolution. In summary, my findings suggest that
fast-evolving male-biased genes likely contribute to extensive sex-linked reproductive isolation, as well as paving the way for future research on the population
genetics of European butterflies and the evolutionary genomics of speciation
Discovering circulating protein biomarkers through in-depth plasma proteomics
Plasma, i.e., the liquid component of blood, is one of the most clinically used samples for biomarker measurement. Despite that plasma proteins and metabolites are the most frequently analysed biomarkers in practice, identifying and implementing new circulating protein biomarkers for diagnosis, treatment prediction, prognosis, and disease monitoring has been limited.
This PhD thesis compiles the discovery of systemic alterations in the blood plasma proteome and potential biomarkers related to disease status, prognosis, or treatment through plasma proteomics. We analysed plasma and serum samples with global proteomics by high-resolution isoelectric focusing (HiRIEF) and liquid chromatography coupled with mass-spectrometry (LC-MS/MS), and targeted proteomics by antibody-based proximity extension assays (PEA) in three diseases that would benefit from blood biomarkers: stage IV metastatic cutaneous melanoma (mCM), glioblastoma (GBM), and coronavirus disease 2019 (COVID-19). Specifically:
a.) New treatment options for mCM substantially prolong overall survival (OS), but multiple patients do not respond to treatment or develop treatment resistance, thus having shorter progression free survival (PFS). Corroborated by the presence of multiple metastases, which makes biomarker sampling difficult, circulating proteins derived from the tumour and in response to treatment could serve as predictive and prognostic biomarkers in mCM.
b.) GBM is the most malignant primary brain tumour with limited treatment options and notoriously short OS. Sampling biomarkers for GBM requires an invasive surgical intervention on the skull, which makes GBM a good candidate for circulating protein biomarkers for prognosis and monitoring.
c.) COVID-19 is an inflammation-driven infectious disease that affects multiple organs and systems, thus making the plasma proteome a good source to explore systemic biological processes occurring in COVID-19.
In papers I and II, using HiRIEF LC-MS/MS and PEA, we explored the treatment-driven plasma proteome alterations in mCM patients treated with anti-PD-1 immune checkpoint inhibitors (ICI) and MAPK-inhibitors (MAPKi), respectively, and identified potential treatment predictive and monitoring biomarkers. mCM patients treated with anti-PD-1 ICI had a strong increase in soluble PD-1 levels during treatment, and upregulation of proteins involved in T-cell response. BRAF[V600]-mutated mCM patients treated with MAPKi had deregulation in proteins involved in immune response and proteolysis. CPB1 had the highest increase in patients treated with BRAF- and MEK-inhibitors and was associated with longer PFS. Higher levels of several proteins involved in inflammation before treatment were associated with shorter PFS regardless of ICI or MAPKi treatment.
In paper III, using HiRIEF LC-MS/MS and PEA, we longitudinally analysed the plasma proteome dynamics of GBM patients, collecting plasma samples before surgery and at three timepoints after surgery. Through consensus clustering, based on treatment-naïve plasma protein levels, we identified two patient clusters that differed in median OS. The association between the cluster membership and OS remained consistent after adjustment for age, sex, and treatment. Through machine learning, we identified protein panels that separated the patient clusters and may serve as prognostic biomarkers. The largest alterations in the plasma proteome of GBM patients occurred within two months after surgery, whereas the plasma protein levels at later timepoints had no difference compared to pre- surgery levels. We observed a decrease in glioma-elevated proteins in the blood after surgery, identifying potential monitoring biomarkers.
In paper IV, using HiRIEF LC-MS/MS, we analysed serum proteome alterations in hospitalised COVID-19 patients in comparison to healthy controls, and identified a strong upregulation in inflammatory, interferon-induced, and proteasomal proteins. Several protein groups showed association with clinical parameters of COVID-19 severity, including proteasomal proteins. Serum proteome alterations were traceable to proteome alterations induced in a lung adenocarcinoma cell line (Calu-3) by infection with SARS-CoV-2. Finally, we performed the first meta-analysis of global proteomics studies of the soluble blood proteome in COVID-19, providing estimates of standardised mean differences and summary receiver operating characteristics curves. We demonstrate the high accuracy and precision of HiRIEF LC-MS/MS when compared to the meta-analysis estimates and pinpoint proteins that may serve as biomarkers of COVID-19.
In summary, this thesis postulates that new circulating protein biomarkers would be clinically useful. By combining mass-spectrometry- and antibody-based-proteomics, we demonstrate the potential of in-depth analyses of the plasma proteome in capturing systemic alterations related to treatment, survival, and disease status, pinpointing potentially novel biomarkers that require validation in larger cohorts
Functional connectivity and dendritic integration of feedback in visual cortex
A fundamental question in neuroscience is how different brain regions communicate with each other. Sensory processing engages distributed circuits across many brain areas and involves information flow in the feedforward and feedback direction. While feedforward processing is conceptually well understood, feedback processing has remained mysterious. Cortico-cortical feedback axons are enriched in layer 1, where they form synapses with the apical dendrites of pyramidal neurons. The organization and dendritic integration of information conveyed by these axons, however, are unknown. This thesis describes my efforts to link the circuit-level and dendritic-level organization of cortico-cortical feedback in the mouse visual system. First, using cellular resolution all-optical interrogation across cortical areas, I characterized the functional connectivity between the lateromedial higher visual area (LM) and primary visual cortex (V1). Feedback influence had both facilitating and suppressive effects on visually-evoked activity in V1 neurons, and was spatially organized: retinotopically aligned feedback was relatively more suppressive, while retinotopically offset feedback was relatively more facilitating. Second, to examine how feedback inputs are integrated in apical dendrites, I optogenetically stimulated presynaptic neurons in LM while using 2-photon calcium imaging to map feedback-recipient spines in the apical tufts of layer 5 neurons in V1. Activation of a single feedback-providing input was sufficient to boost calcium signals and recruit branch-specific local events in the recipient dendrite, suggesting that feedback can engage dendritic nonlinearities directly. Finally, I measured the recruitment of apical dendrites during visual stimulus processing. Surround visual stimuli, which should recruit relatively more facilitating feedback, drove local calcium events in apical tuft branches. Moreover, global dendritic event size was not purely determined by somatic activity but modulated by visual stimuli and behavioural state, in a manner consistent with the spatial organization of feedback. In summary, these results point toward a possible involvement of active dendritic processing in the integration of feedback signals. Active dendrites could thus provide a biophysical substrate for the integration of essential top-down information streams, including contextual or predictive processing
Ανάλυση προωτεομικών δεδομένων απο φασματομετρία μάζας και ενσωμάτωσή τους με άλλα κλινικά και μοριακά δεδομένα σε κλινικά δείγματα και καρκινικές σειρές
Οι μοριακοί υπότυποι μιας ασθένειας συχνά συσχετίζονται με διαφορές ως προς την επιβίωση ή πρόοδο της νόσου και άλλοτε ως προς την απόκριση σε συγκεκριμένη θεραπεία. Την τελευταία δεκαετία, μελέτες μοριακής ταξινόμησης του ουροθηλιακού καρκίνου εστιάζουν κυρίως στον διηθητικό τύπο της ασθένειας (~20% των ασθένων στην αρχική διάγνωση) ο οποίος χαρακτηρίζεται από υψηλό κίνδυνο για μετάσταση και χαμηλά ποσοστά πενταετούς επιβίωσης. Οι παραπάνω μελέτες επέτρεψαν την ταυτοποιήση πολλαπλών γενομικών και μεταγραφικών υποτύπων οι οποίοι διαφέρουν ριζικά ως προς το μοριακό τους προφίλ, σχηματίζοντας δύο μεγάλες κατηγορίες: τους basal και τους luminal όγκους. Οι πρώτοι φαίνεται να σχετίζονται με πιο επιθετικούς καρκίνους εμπερικλείοντας όμως ένα σημαντικό ποσοστό ασθενών που ανταποκρίνονται στο βασικό χημειοθεραπευτικό σχήμα. Οι δέυτεροι (luminal) αρχικά προσδιορίστηκαν ως λιγότερο επιθετικοί, επόμενες μελέτες όμως αποκάλυψαν την σημαντική μοριακή ετερογένεια που τους χαρακτηρίζει και που αντανακλάται σε κλινικές παραμέτρους. Σήμερα, πιστέυεται ότι ο διηθητικός καρκίνος της ουροδόχου κύστης ταξινομείται σε 6 βασικούς υποτύπους, αλλά τα δεδομένα που υπάρχουν για να υποστηρίξουν την ένταξη των υποτύπων στην κλινική πράξη είναι ατελή και δεν συμφωνούν μεταξύ τους. Από την άλλη, ο μη διηθητικός τύπος της ασθενεις (~80% των περιπτώσεων στην αρχική διάγνωση) χαρακτηρίζεται από υψηλά ποσοστά υποτροπής και προόδου σε ανώτερο στάδιο καθώς και από σημαντικό δημόσιο οικονομικό κόστος εξαιτίας της αυξημένης συχνότητας παρακολούθησης που απαιτεί. Το μοριακό προφίλ του μη-διηθητικού καρκίνου έχει μελετηθεί σημαντικά λιγότερο από αυτό του διηθητικού, και μέχρι σήμερα υπάρχουν δύο μελέτες που επιχειρούν την ταξινόμησή του σε μοριακούς υποτύπους: η πρώτη στη βάση του μεταγραφώματος, η δέυτερη στη βάση της διακύμνασης αριθμού αντιγράφων. Το πρωτεομικό προφίλ όμως, τόσο του διηθητικού όσο και του μη-διηθητικού καρκίνου της ουροδόχου κύστης, μέχρι και σήμερα έχει μελετηθεί υποτυπωδώς. Σκοπός της παρούσας μελέτης είναι η διερεύνηση της ύπαρξης πρωτεομικών υποτύπων του μη διηθητικού ουροθηλιακού καρκίνου, ο μοριακός χαρακτηρισμός τους, η σχέση τους με προηγούμενα συστήματα ταξινόμησης, καθώς και η ταυτοποίηση απορυθμισμένων πρωτεϊνών και μονοπατιών με δυνητική προγνωστική αξία. Για την εξυπηρέτηση του παραπάνω σκοπού, 117 δείγματα καρκινικού ιστού από ασθενείς που πρωτοδιαγνώσθηκαν με ουροθηλιακό καρκίνο (98 μη-διηθητικό, 19 διηθητικό) συλλέχθησαν και το ολικό πρωτέομά τους απομονώθηκε και αρχικά ποσοτικοποιήθηκε με τη μέθοδο Bradford. Κατόπιν διάσπασης με θρυψίνη, τα πεπτίδια διαχωρίστηκαν σε χρωματογραφική στήλη συνδεδεμένη με φασματογράφο μάζας τύπου Orbitrap. Οι φασματικές πληροφορίες για τα πεπτίδια αναλύθηκαν με το πρόγραμμα Proteome Discoverer θέτοντας FDR (False Discovery Rate) <0.01 και αντιστοιχήθηκαν σε πρωτεινικές ταυτότητες. Η πρωτεϊνική ποσοτικοποίηση έγινε με τη χρήση των τριών πιο άφθονων και μοναδικών πεπτιδίων ανά πρωτεΐνη, ενώ κατόπιν επεξεργασίας τα πρωτεομικά δεδομένα υποβλήθηκαν σε μια σειρά από υπολογιστικές αναλύσεις: μη επιτηρούμενη k-means συσταδοποίηση, ανάλυση κύριων συνιστωσών, ανάλυση για στατιστική σημαντικόντητα πρωτεϊνών, πρωτεϊνικών μονοπατιών, βιολογικών λειτουργιών και γονιδιακής έκφρασης καθώς και στην μοντελοιποίηση ενός μοριακού ταξινομητή Radnom Forest. Μέγιστη σταθερότητα συσταδοποίησης επιτεύχηκε για κ = 3 ομάδες, υποδηλώνοντας την ύπαρξη τριών πρωτεομικών υποτύπων στα δεδομένα. Η ομάδα 1 ήταν η μικρότερη σε μέγεθος (17/98), περιείχε κυρίως καρκίνους υψηλού σταδίου, αλλοίωσης και ρίσκου και παρουσίασε ένα μοριακό φαινότυπο ανοσοδιήθησης με υψηλά επιπέδα των μεταγραφικών παραγόντων STAT1, STAT3 και SND1, καθώς και πρωτεϊνων της αντιγονοπαρουσίασης, υποδηλώνοντας ενεργή ανταλλαγή πληροφοριών μεταξύ του ανοσοποιητικού και των καρκινικών κυττάρων. Παράλληλα, χαρακτηρίζονταν απο υψηλότερες ποσότητες πρωτεϊνών που συμμετέχουν στο κυτταρικό κύκλο, και στη μετάδοση στρεσογόνων σημάτων (αντίδραση μη αναδιπλωμένης πρωτεϊνης και επιδιόρθωση βλαβών του DNA). Η όμαδα 2 συγκέντρωσε ασθενείς με ποικίλα κλινικά χαρακτηριστικά που όμως έφεραν κοινώς, αυξημένες ποσότητες εξωκυττάριων πρωτεϊνών (στρώματος), και χαμηλά επιθηλιακά σήματα. Οι ασθενείς στην ομάδα 3 παρουσίασαν έναν πιο διαφοροποιημένο μοριακό φαινότυπο με υψηλότερα επίπεδα (UPKs και KRT20 κάθως και CDH1) που συμβαδίζει με τα κλινικά χαρακτηριστικά τους αφού οι περισσότεροι διαγιγνώσθηκαν με καρκίνους χαμηλού σταδίου και κινδύνου. Η ανάλυση για ενεργοποιημένα πρωτεϊνικά μονοπάτια έδειξε ότι οι ασθενείς της ομάδας 1 έιχαν ενεργή σηματοδότηση για βιοσυνθετικές διεργασίες, για ιντερφερόνη-γ, και αυξημένη δραστηριότητα των μεταγραφικών παραγόντων MYC και E2F, που ελέγχουν θετικά τον κυτταρικό κύκλο. Από την άλλη οι ασθνενείς της ομάδας 3 σχετίστηκαν με ενεργοποίηση μεταβολικών μονοπατιών όπως αυτό της αποτοξίνωσης μεσολαβούμενο από γλουταθειόνη καθώς και της γλυκογονόλυσης – γλυκόλυσης, αλλά και της απόπτωσης. Συγκρίνοντας το πρωτεομικό προφιλ των ασθένων με μη-διηθητικό καρκίνο με ασθενέις που είχαν διηθητικό καρκίνο χρησιμοποιώντας ανάλυση κύριων συνιστωσών, αποκαλύφθηκε κοντινή σχέση της ομάδας 1 με ασθενείς που έφεραν διηθητικό ουροθηλιακό καρκίνο και αντίστροφα, μακρινή σχέση της ομάδας 3 με τους τελευταίους. Η ομάδα 2 εμφάνισε μεγάλη διασπορά επικαλύπτοντας περιοχές των προηγούμενων δύο ομάδων. Για την επικύρωση των πρωτεομικών αποτελεσμάτων, δεδομένα από μεταγραφικές έρευνες (UROMOL και LUND) αναλύθηκαν αναδρομικά. Στην UROMOL έρευνα επίσης ταυτοποιήθηκαν 3 υπότυποι ο ένας εκ των οποίων συγκέντρωσε τους περισσότερους ασθενείς με πρόδοο σε ανώτερο στάδιο (κακής πρόγνωσης υπότυπος). Συγκριτική ανάλυση μεταξύ των τριών πρωτεομικών ομάδων και των τριών υποτύπων της UROMOL έρευνας με το στατιστικό εργαλείο GSEA, έδειξε στατιστικώς σημαντικές φαινοτυπικές ομοιότητες μεταξύ της πρωτεομικής ομάδας 1 και του υποτύπου «κακής» πρόγνωσης της UROMOL καθώς και μεταξύ της πρωτεομικής ομάδας 3 και του υποτύπου «καλής πρόγνωσης». Χρησιμοποιώντας έναν μη επιτηρούμενο μοριακό ταξινομητή Random Forest, οι υψηλού κινδύνου και χαμηλού κινδύνου φαινότυποι των πρωτεομικών ομάδων 1 και 3, επιβεβαιώθηκαν ύστερα από την ταξινόμηση των ασθενών στους υποτύπους «κακής» και «καλής» πρόγνωσης αντίστοιχα, της UROMOL έρευνας. Στατιστικώς σημαντικες πρωτεΐνες που ξεχωρίζουν αυτές τις δυο ακραίες πρωτεομικές ομάδες αλλά και ταυτόχρονα τον διηθητικό από τον μη διηθητικό καρκίνο βρέθηκαν να διαφέρουν σημαντικά και στο επίπεδο του μεταγραφώματος μεταξύ των ομάδων «κακής» και «καλής» πρόγνωσης σε δύο ανεξάρτητες έρευνες (UROMOL και LUND). Τα παραπάνω μόρια συμμετέχουν σε βιολογικές λειτουργίες-κλειδιά για την ανάπτυξη του μη-διηθητικού καρκίνου, όπως στην επαγωγή αποκρίσεων πρωτεϊνικής σταθερότητας, στη σηματοδότηση κυτοκινών και ιντερφερονών, στην αντιγονοπαρουσίαση, στην επεξεργασία πρώιμων mRNAs, σε μετα-μεταφραστικές τροποποιήσεις αλλά και σε μονοπάτια κυτταρικής αύξησης. Συνολικά, η παρούσα μελέτη ταυτοποιεί τρεις πρωτεομικούς υποτύπους του μη διηθητικού καρκίνου και ακολουθώντας μια σύγκριτική ανάλυση με δύο ανεξάρτητες μεταγραφικές έρευνες, παρέχει ομάδες μορίων που μπορεί να οδηγούν τη πρόοδο του καρκίνου και που χρειάζονται επιπλέον επικύρωση στη κλινική πράξη.DNA/RNA-based classification of Bladder Cancer (BC) supports the existence of multiple molecular subtypes, while investigations at the protein level are scarce. The purpose of this study was to investigate if Non-Muscle Invasive Bladder Cancer (NMIBC) can be stratified to biologically meaningful proteomic groups, to establish associations between the proteomics subtypes and previous transcriptomics classification systems and to characterize the continuum of transcriptomics alterations observed in the different stages of the disease. Subsequently, tissue specimens from 117 patients at primary diagnosis (98 with NMIBC and 19 with MIBC), were processed for high resolution LC-MS/MS analysis. Protein quantification was conducted by utilizing the mean abundance of the top three most abundant unique peptides per protein. The proteomics output was subjected to unsupervised consensus clustering, principal component analysis (PCA), and investigation of subtype-specific features, pathways, and genesets, as well as for the construction and validation of a Random Forest based classifier. NMIBC patients were optimally stratified to 3 proteomic subtypes (classes), differing at size, clinico-pathological and molecular backgrounds: Class 1 (mostly high stage/grade/risk samples) was the smallest in size (17/98) and expressed an immune/inflammatory phenotype, along with features involved in cell proliferation, unfolded protein response and DNA damage response, whereas class 2 (mixed stage/grade/risk composition) presented with an infiltrated/mesenchymal profile. Class 3 was rich in luminal/differentiation markers, in line with its pathological composition (mostly low stage/grade/risk samples). PCA revealed a close proximity of class 1 and conversely, remoteness of class 3 to the proteome of MIBC. Samples from class 2 were distributed in a wider fashion at the rotated space. Comparative analysis with GSEA between the three proteomic classes and the three UROMOL subtypes indicated statistically significant associations between the proteomics class 1 and UROMOL subtype 2 (subtype with a bad prognosis) and also between the proteomics class 3 and UROMOL subtype 1 (subtype with the best prognosis). Utilizing a Random Forest based classifier, the predicted high- and low-risk phenotypes for the proteomic class 1 and class 3, were further supported by their classification into the “progressed” and “non-progressed” subtypes of the UROMOL study, respectively. Statistically significant proteins distinguishing these two extreme classes (1 and 3) and also MIBC from NMIBC samples were found to consistently differ at the mRNA levels between NMIBC “Progressors” and “Non-Progressors” groups of the UROMOL and LUND cohorts. Functional assessment of the observed molecular de-regulations suggested severe pathway alterations at unfolded protein response, cytokine and inferferone-γ signaling, antigen presentation, mRNA processing, post translational modifications and in cell growth/division. Collectively, this study identifies three proteomic NMIBC subtypes and following a cross-omics analysis using transcriptomic data from two independent cohorts, shortlists molecular features potentially driving non-invasive carcinogenesis, meriting further validation in clinical trials
Commercial sharks under scrutiny: baseline genetic distinctiveness supports structured populations of small-spotted catsharks in the Mediterranean Sea
The present study, based on microsatellite markers, describes a population genetic analysis of the small-spotted catshark Scyliorhinus canicula (Linnaeus, 1758), representing one of the most abundant and commonly caught cartilaginous fishes in the Mediterranean Sea and adjacent areas. The analyses were performed to unravel the genetic features (variability, connectivity, sex-biased dispersal) of their relative geographic populations, both at the small (around the coast of Sardinia, Western Mediterranean Sea) and at a larger spatial scale (pan-Mediterranean level and between the Atlantic Ocean and the Mediterranean Sea). Individual clustering, multivariate and variance analyses rejected the hypothesis of genetic homogeneity, with significant genetic differences between the Mediterranean and Atlantic, as well as within the Mediterranean area between the Western and Eastern basins. In details, our results seem to confirm that the Strait of Gibraltar could not represent a complete barrier to the exchange of individuals of small-spotted catshark between the Atlantic Ocean and the Mediterranean Sea. In the latter area, a complex genetic structuring for S. canicula was found. Apart from differences among the Western, Eastern and Adriatic sites, within the Western basin the small-spotted catsharks around Sardinian waters are strongly differentiated from all others (both from the eastern Tyrrhenian Sea and southernmost part of the Algerian basin) and are demographically stable. Several possible mechanisms, both biological and abiotic (e.g., migratory behavior, water fronts and oceanographic discontinuities), are discussed here to explain their peculiar characteristics. Overall, the genetic data presented, both at the local and regional level, could represent a baseline information, useful for the temporal monitoring of populations, and to assess the effects of present or future fishing/management/conservation measures
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