A systems and molecular analysis of G protein-mediated signalling

The ability of cells to respond correctly to signals from their microenvironment is an essential prerequisite of life. Many external signals are detected through G protein-coupled receptor (GPCR) signalling pathways, which control all aspects of eukaryotic physiology. Ligand-bound GPCRs initiate signalling by promoting exchange of GDP for GTP on the Gα subunit of heterotrimeric G proteins, thereby facilitating activation of downstream effectors. Signalling is terminated by the hydrolysis of GTP to GDP through intrinsic GTPase activity of the Gα subunit, in a reaction catalysed by the regulator of G protein signalling (RGS) proteins. Due to the problem of complexity in higher eukaryotic GPCR signalling, the matingresponse in Schizosaccharomyces pombe has been used to study GPCR signalling in isolation. In vivo data from quantitative assays of reporter strains and live-cell uorescence microscopy informs the development of an ordinary differential equation model of the signalling pathway, first described by Smith et al., 2009. The rate of nucleotide exchange on the Gα (Gpa1) is a key molecular mechanism controlling duration and amplitude of signalling response. The in uence of this is investigated through characterisation of Gpa1 nucleotide exchange mutants and perturbation of reaction rate parameters in the computational model. Further, this thesis also presents data relating to the temporal and spatial regulation of Rgs1 (the sole RGS protein for Gpa1). Using an inter-disciplinary approach, evidence is provided to suggest that an interaction between Rgs1 and the C-terminal tail of the GPCR (Mam2) tethers Rgs1 to the plasma membrane to facilitate its function. Finally, quantification of signalling at the single cell level is described. Time-lapse livecell imaging of uorescent reporter cells is optimised and single cell signalling response quantified using image analysis software. Single cell quantification provides greater insight into temporal dynamics, cell-to-cell variability, and highlights the existence of mechanisms for cellular decision-making

Cellular mRNAs access second ORFs using a novel amino acid sequence-dependent coupled translation termination-reinitiation mechanism

Polycistronic transcripts are considered rare in the human genome. Initiation of translation of internal ORFs of eukaryotic genes has been shown to use either leaky scanning or highly structured IRES regions to access initiation codons. Studies on mammalian viruses identified a mechanism of coupled translation termination-reinitiation that allows translation of an additional ORF. Here, the ribosome terminating translation of ORF-1 translocates upstream to reinitiate translation of ORF-2. We have devised an algorithm to identify mRNAs in the human transcriptome in which the major ORF-1 overlaps a second ORF capable of encoding a product of at least 50 aa in length. This identified 4368 transcripts representing 2214 genes. We investigated 24 transcripts, 22 of which were shown to express a protein from ORF-2 highlighting that 3' UTRs contain protein-coding potential more frequently than previously suspected. Five transcripts accessed ORF-2 using a process of coupled translation termination-reinitiation. Analysis of one transcript, encoding the CASQ2 protein, showed that the mechanism by which the coupling process of the cellular mRNAs was achieved was novel. This process was not directed by the mRNA sequence but required an aspartate-rich repeat region at the carboxyl terminus of the terminating ORF-1 protein. Introduction of wobble mutations for the aspartate codon had no effect, whereas replacing aspartate for glutamate repeats eliminated translational coupling. This is the first description of a coordinated expression of two proteins from cellular mRNAs using a coupled translation termination-reinitiation process and is the first example of such a process being determined at the amino acid level

Cosmological Limits on the Neutrino Mass from the Lya Forest

The Lya forest in quasar spectra probes scales where massive neutrinos can strongly suppress the growth of mass fluctuations. Using hydrodynamic simulations with massive neutrinos, we successfully test techniques developed to measure the mass power spectrum from the forest. A recent observational measurement in conjunction with a conservative implementation of other cosmological constraints places upper limits on the neutrino mass: m_nu < 5.5 eV for all values of Omega_m, and m_nu < 2.4 (Omega_m/0.17 -1) eV, if 0.2 < Omega_m <0.5 as currently observationally favored (both 95 % C.L.).Comment: 4 pages, 2 ps figures, REVTex, submitted to Phys. Rev. Let

Parameter identification problems in the modelling of cell motility

We present a novel parameter identification algorithm for the estimation of parameters in models of cell motility using imaging data of migrating cells. Two alternative formulations of the objective functional that measures the difference between the computed and observed data are proposed and the parameter identification problem is formulated as a minimisation problem of nonlinear least squares type. A Levenberg–Marquardt based optimisation method is applied to the solution of the minimisation problem and the details of the implementation are discussed. A number of numerical experiments are presented which illustrate the robustness of the algorithm to parameter identification in the presence of large deformations and noisy data and parameter identification in three dimensional models of cell motility. An application to experimental data is also presented in which we seek to identify parameters in a model for the monopolar growth of fission yeast cells using experimental imaging data. Our numerical tests allow us to compare the method with the two different formulations of the objective functional and we conclude that the results with both objective functionals seem to agree

Interplay in galectin expression predicts patient outcomes in a spatially restricted manner in PDAC

BACKGROUND: Galectins (Gal's) are a family of carbohydrate-binding proteins that are known to support the tumour microenvironment through their immunosuppressive activity and ability to promote metastasis. As such they are attractive therapeutic targets, but little is known about the cellular expression pattern of galectins within the tumour and its neighbouring stromal microenvironment. Here we investigated the cellular expression pattern of Gals within pancreatic ductal adenocarcinoma (PDAC).METHODS: Galectin gene and protein expression were analysed by scRNAseq (n=4) and immunofluorescence imaging (n=19) in fibroblasts and epithelial cells of pancreatic biopsies from PDAC patients. Galectin surface expression was also assessed on tumour adjacent normal fibroblasts and cancer associated primary fibroblasts from PDAC biopsies using flow cytometry.RESULTS: scRNAseq revealed higher Gal-1 expression in fibroblasts and higher Gal-3 and -4 expression in epithelial cells. Both podoplanin (PDPN+, stromal/fibroblast) cells and EpCAM+ epithelial cells expressed Gal-1 protein, with highest expression seen in the stromal compartment. By contrast, significantly more Gal-3 and -4 protein was expressed in ductal cells expressing either EpCAM or PDPN, when compared to the stroma. Ductal Gal-4 cellular expression negatively correlated with ductal Gal-1, but not Gal-3 expression. Higher ductal cellular expression of Gal-1 correlated with smaller tumour size and better patient survival. CONCLUSIONS: In summary, the intricate interplay and cell-specific expression patterns of galectins within the PDAC tissue, particularly the inverse correlation between Gal-1 and Gal-4 in ducts and its significant association with patient survival, highlights the complex molecular landscape underlying PDAC and provides valuable insights for future therapeutic interventions.</p

The \u3cem\u3eChlamydomonas\u3c/em\u3e Genome Reveals the Evolution of Key Animal and Plant Functions

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella

Three-Dimensional Mapping of the Dark Matter

We study the prospects for three-dimensional mapping of the dark matter to high redshift through the shearing of faint galaxies images at multiple distances by gravitational lensing. Such maps could provide invaluable information on the nature of the dark energy and dark matter. While in principle well-posed, mapping by direct inversion introduces exceedingly large, but usefully correlated noise into the reconstruction. By carefully propagating the noise covariance, we show that lensing contains substantial information, both direct and statistical, on the large-scale radial evolution of the density field. This information can be efficiently distilled into low-order signal-to-noise eigenmodes which may be used to compress the data by over an order of magnitude. Such compression will be useful for the statistical analysis of future large data sets. The reconstructed map also contains useful information on the localization of individual massive dark matter halos, and hence the dark energy from halo number counts, but its extraction depends strongly on prior assumptions. We outline a procedure for maximum entropy and point-source regularization of the maps that can identify alternate reconstructions.Comment: 11 pages, 5 figures, submitted to PR

Parasite Transmission in Social Interacting Hosts: Monogenean Epidemics in Guppies

Background: Infection incidence increases with the average number of contacts between susceptible and infected individuals. Contact rates are normally assumed to increase linearly with host density. However, social species seek out each other at low density and saturate their contact rates at high densities. Although predicting epidemic behaviour requires knowing how contact rates scale with host density, few empirical studies have investigated the effect of host density. Also, most theory assumes each host has an equal probability of transmitting parasites, even though individual parasite load and infection duration can vary. To our knowledge, the relative importance of characteristics of the primary infected host vs. the susceptible population has never been tested experimentally. Methodology/Principal Findings: Here, we examine epidemics using a common ectoparasite, Gyrodactylus turnbulli infecting its guppy host (Poecilia reticulata). Hosts were maintained at different densities (3, 6, 12 and 24 fish in 40 L aquaria), and we monitored gyrodactylids both at a population and individual host level. Although parasite population size increased with host density, the probability of an epidemic did not. Epidemics were more likely when the primary infected fish had a high mean intensity and duration of infection. Epidemics only occurred if the primary infected host experienced more than 23 worm days. Female guppies contracted infections sooner than males, probably because females have a higher propensity for shoaling. Conclusions/Significance: These findings suggest that in social hosts like guppies, the frequency of social contact largely governs disease epidemics independent of host density

Adverse childhood experiences are associated with the risk of lung cancer: a prospective cohort study

Background. Strong relationships between exposure to childhood traumatic stressors and smoking behaviours inspire the question whether these adverse childhood experiences (ACEs) are associated with an increased risk of lung cancer during adulthood. Methods. Baseline survey data on health behaviours, health status and exposure to adverse childhood experiences (ACEs) were collected from 17,337 adults during 1995-1997. ACEs included abuse (emotional, physical, sexual), witnessing domestic violence, parental separation or divorce, or growing up in a household where members with mentally ill, substance abusers, or sent to prison. We used the ACE score (an integer count of the 8 categories of ACEs) as a measure of cumulative exposure to traumatic stress during childhood. Two methods of case ascertainment were used to identify incident lung cancer through 2005 follow-up: 1) hospital discharge records and 2) mortality records obtained from the National Death Index. Results. The ACE score showed a graded relationship to smoking behaviors. We identified 64 cases of lung cancer through hospital discharge records (age-standardized risk = 201 × 100,000-1 population) and 111 cases of lung cancer through mortality records (age-standardized mortality rate = 31.1 × 100,000 -1 person-years). The ACE score also showed a graded relationship to the incidence of lung cancer for cases identified through hospital discharge (P = 0.0004), mortality (P = 0.025), and both methods combined (P = 0.001). Compared to persons without ACEs, the risk of lung cancer for those with 6 ACEs was increased approximately 3-fold (hospital records: RR = 3.18, 95%CI = 0.71-14.15; mortality records: RR = 3.55, 95%CI = 1.25-10.09; hospital or mortality records: RR = 2.70, 95%CI = 0.94-7.72). After a priori consideration of a causal pathway (i.e., ACEs smoking lung cancer), risk ratios were attenuated toward the null, although not completely. For lung cancer identified through hospital or mortality records, persons with 6 ACEs were roughly 13 years younger on average at presentation than those without ACEs. Conclusions. Adverse childhood experiences may be associated with an increased risk of lung cancer, particularly premature death from lung cancer. The increase in risk may only be partly explained by smoking suggesting other possible mechanisms by which ACEs may contribute to the occurrence of lung cancer

A prospective study of shoulder pain in primary care: Prevalence of imaged pathology and response to guided diagnostic blocks

<p>Abstract</p> <p>Background</p> <p>The prevalence of imaged pathology in primary care has received little attention and the relevance of identified pathology to symptoms remains unclear. This paper reports the prevalence of imaged pathology and the association between pathology and response to diagnostic blocks into the subacromial bursa (SAB), acromioclavicular joint (ACJ) and glenohumeral joint (GHJ).</p> <p>Methods</p> <p>Consecutive patients with shoulder pain recruited from primary care underwent standardised x-ray, diagnostic ultrasound scan and diagnostic injections of local anaesthetic into the SAB and ACJ. Subjects who reported less than 80% reduction in pain following either of these injections were referred for a magnetic resonance arthrogram (MRA) and GHJ diagnostic block. Differences in proportions of positive and negative imaging findings in the anaesthetic response groups were assessed using Fishers test and odds ratios were calculated a for positive anaesthetic response (PAR) to diagnostic blocks.</p> <p>Results</p> <p>In the 208 subjects recruited, the rotator cuff and SAB displayed the highest prevalence of pathology on both ultrasound (50% and 31% respectively) and MRA (65% and 76% respectively). The prevalence of PAR following SAB injection was 34% and ACJ injection 14%. Of the 59% reporting a negative anaesthetic response (NAR) for both of these injections, 16% demonstrated a PAR to GHJ injection. A full thickness tear of supraspinatus on ultrasound was associated with PAR to SAB injection (OR 5.02; <it>p </it>< 0.05). Ultrasound evidence of a biceps tendon sheath effusion (OR 8.0; <it>p </it>< 0.01) and an intact rotator cuff (OR 1.3; <it>p </it>< 0.05) were associated with PAR to GHJ injection. No imaging findings were strongly associated with PAR to ACJ injection (<it>p </it>≤ 0.05).</p> <p>Conclusions</p> <p>Rotator cuff and SAB pathology were the most common findings on ultrasound and MRA. Evidence of a full thickness supraspinatus tear was associated with symptoms arising from the subacromial region, and a biceps tendon sheath effusion and an intact rotator cuff were associated with an intra-articular GHJ pain source. When combined with clinical information, these results may help guide diagnostic decision making in primary care.</p