Modelling and analysis of macrophage activation pathways

Macrophages are present in virtually all tissues and account for approximately 10% of all body mass. Although classically credited as the scavenger cells of innate immune system, ridding a host of pathogenic material and cellular debris though their phagocytic function, macrophages also play a crucial role in embryogenesis, homeostasis, and inflammation. De-regulation of macrophage function is therefore implicated in the progression of many disease states including cancer, arthritis, and atherosclerosis to name just a few. The diverse range of activities of this cell can be attributed to its exceptional phenotypic plasticity i.e. it is capable of adapting its physiology depending on its environment; for instance in response to different types of pathogens, or specific cocktail of cytokines detected. This plasticity is exemplified by the macrophages capacity to adjust rapidly its transcriptional profile in response to a given stimulus. This includes interferons which are a group of cytokines capable of activating the macrophage by interacting with their cognate receptors on the cell. The different classes of interferons activate downstream signalling cascades, eventually leading to the expression (as well as repression) of hundreds of genes. To begin to fully understand the properties of a dynamic cell such as the macrophage arguably requires a holistic appreciation of its constituents and their interactions. Systems biology investigations aim to escape from a gene-centric view of biological systems. As such this necessitates the development of better ways to order, display, mine and analyse biological information, from our knowledge of protein interactions and the systems they form, to the output of high throughput technologies. The primary objectives of this research were to further characterise the signalling mechanisms driving macrophages activation, especially in response to type-I and type- II interferons, as well as lipopolysaccharide (LPS), using a ‘systems-level’ approach to data analysis and modelling. In order to achieve this end I have explored and developed methods for the executing a ‘systems-level’ analysis. Specifically the questions addressed included: (a) How does one begin to formalise and model the existing knowledge of signalling pathways in the macrophage? (b) What are the similarities and differences between the macrophage response to different types of interferon (namely interferon-β (IFN-β) and interferon-γ (IFN-γ))? (c) How is the macrophage transcriptome affected by siRNA targeting of key regulators of the interferon pathway? (d) To what extent does a model of macrophage signalling aid interpretation of the data generated from functional genomics screens? There is general agreement amongst biologists about the need for high-quality pathway diagrams and a method to formalize the way biological pathways are depicted. In an effort to better understand the molecular networks that underpin macrophage activation an in-silico model or ‘map’ of relevant pathways was constructed by extracting information from published literature describing the interactions of individual constituents of this cell and the processes they modulate (Chapter-2). During its construction process many challenges of converting pathway knowledge into computationally-tractable yet ‘understandable’ diagrams, were to be addressed. The final model comprised 2,170 components connected by 2,553 edges, and is to date the most comprehensive formalised model of macrophage signalling. Nevertheless this still represents just a modest body of knowledge on the cell. Related to the pathway modelling efforts was the need for standardising the graphical depiction of biology in order to achieve these ends. The methods for implementing this and agreeing a ‘standard’ has been the subject of some debate. Described herein (in Chapter-3) is the development of one graphical notation system for biology the modified Edinburgh Pathway Notation (mEPN). By constructing the model of macrophage signalling it has been possible to test and extensively refine the original notation into an intuitive, yet flexible scheme capable of describing a range of biological concepts. The hope is that the mEPN development work will contribute to the on-going community effort to develop and agree a standard for depicting pathways and the published version will provide a coherent guide to those planning to construct pathway diagrams of their biological systems of interest. With a desire to better understand the transcriptional response of primary mouse macrophages to interferon stimulation, genome wide expression profiling was performed and an explorative-network based method applied for analysing the data generated (Chapter-4). Although transcriptomics data pertaining to interferon stimulation of macrophages is not entirely novel, the network based analysis of it provided an alternative approach to visualise, mine and interpret the output. The analysis revealed overlap in the transcriptional targets of the two classes of interferon, as well as processes preferentially induced by either cytokine; for example MHC-Class II antigen processing and presentation by IFN-γ, and an anti-proliferative signature by IFN-β. To further investigate the contribution of individual proteins towards generating the type-I (IFN-β) response, short interfering RNA (siRNA) were employed to repress the expression of selected target genes. However in macrophages and other cells equipped with pathogen detection systems the act of siRNA trasfection can itself induce a type-I interferon response. It was therefore necessary to contend with this autocrine production of IFN-β and optimise an in vitro assay for studying the contribution of siRNA induced gene-knock downs to the interferon response (described in Chapter-5). The final assay design incorporated LPS stimulation of the macrophages, as a means of inducing IFN-β autonomously of the transfection induced type-I response. However genome-wide expression analysis indicated the targeted gene knock-downs did not perturb the LPS response in macrophages on this occasion. The optimisation process underscored the complexities of performing siRNA gene knockdown studies in primary macrophages. Furthermore a more thorough understanding of the transcriptional response of macrophages to stimulation by interferon or by LPS was required. Therefore the final investigations of this thesis (Chapter-6) explore the transcriptional changes over a 24 hour time-course of macrophage activation by IFN-β, IFN-γ, or LPS and the contribution of the macrophage pathway model in interpreting the response to the three stimuli. Taken together the work described in this thesis highlight the advances to be made from a systems-based approach to visualisation, modelling and analysis of macrophage signalling

Moving pathogen genomics out of the lab and into the clinic: what will it take?

Pathogen genomic analysis is a potentially transformative new approach to the clinical and public-health management of infectious diseases. Health systems investing in this technology will need to build infrastructure and develop policies that ensure genomic information can be generated, shared and acted upon in a timely manner

USE OF DIAGRAMS AS INSTRUCTIONAL AIDS IN TEACHING OF GEOMETRICAL CONCEPTS AT SECONDARY SCHOOL LEVEL

Angle is a complex topic defined in a variety of contexts; some define angle to be as a pair of rays coming from a single point, as a rotation about a single point, or in a curve. Due to the multiple definitions of angle students get confused as to what an angle truly consists of. This study paid close attention to the misconceptions high school geometry students’ hold about the concept of angle and how to help them gain a more conceptual understanding of this essential concept with the use of different Diagrams. The main idea behind the conduction of this study is to investigate the effect of use of diagrams as instructional aids on the conceptual understanding of angles. The study hypothesizes that angle can be learned using interactive Diagrams. The results of this study showed that diagrams which based on different A.V aids was shown to be more beneficial for the students who used to clear the concept of angles

Causes of smoking in Pakistan: an analysis of social factors

OBJECTIVE: To determine the factors contributing to the initiation and propagation of smoking in visitors to a major tertiary health center in Karachi, Pakistan. METHODS: Seven major contributing factors to the initiation and propagation of smoking were presented to consenting study participants (n=170) in a questionnaire. Participants were then requested to use their experience and opinion to rate each of the given factors on a scale of 1 to 5 regarding its importance as a causative factor in the initiation and propagation of smoking. Results were analyzed using SPSSv16.0. RESULTS: Preliminary analysis revealed occupational stress relief as the most important factor contributing to smoking with a mean score of 3.25 +/- 1.32. Peer pressure ranked second (Score 3.20 +/- 1.42). Domestic stress relief ranked third with a score of 3.19 +/- 1.32. Smokers gave lower rating than non-smokers to most factors. Younger participants gave higher ratings to peer pressure, and most participants were found to have begun smoking at a young age. CONCLUSIONS: Even though the addictive power of nicotine or stress may appear as a factor in middle aged smokers, the root of their habit lies in the initiation due to peer pressure

The Impact of Workplace Incivility on The Psychological Wellbeing of Employees Through Emotional Exhaustion

Workplace environment has received a lot attention from many authors for research purpose. The present study extends the scope of the workplace incivility literature by adding the dependent variable like psychological wellbeing, prohibitive voice behaviour, intention to leave. I have selected these research variables because of its importance however, the association of these variables with the workplace incivility as independent variable is the unique contribution of the study to the existing body of knowledge. All the dependent variables are also helpful in the career development of employees and make contribution to achieve organization goals. This research will help organizations to study how workplace incivility affects the behaviour of the employees, besides it helps organizations to consider the workplace incivility effects on the employees while making decisions in achieving organization goals. Another significant achievement of the study is the use of emotional exhaustion as a mediator. The mediation role of emotional Exhaustion between workplace incivility and dependent variables like psychological wellbeing, prohibitive voice behaviour and intention to leave reveals much truth through mediation analysis. The use of prohibitive voice behaviour as an independent variable is also uniqueness of this study, it will reveal a lot of facts how prohibitive voice behaviour effects the employees in the organization. This study also uses the mechanism of the Conservation of resource theory to explain and justify the impact of workplace incivility on prohibitive voice behaviour, psychological wellbeing of employees and intention to leave. Although various researchers use the COR theory this study the theory describes how employees have the negative feelings and out of their resources (Knowledge, skills, ability) when dealing with workplace incivility

In vitro Anti-Cancer Effect of Polymeric Nanoparticles Encapsulating Caralluma tuberculata in Cancer Cells

Rapidly evolving drug delivery systems employ therapeutic agents (liposomes, polymers, and nanospheres) to achieve optimum therapeutic and targeted effects with declined side effects to cure chronic diseases like cancer. Nano-formulation of a natural product i.e., Caralluma tuberculata (Ct) extract, has been used as an effective combinational therapy with enhanced biocompatibility owing to its strong anti-oxidant, anti-inflammatory, anti-bacterial, and anti-tumor potential. Ct extract was prepared using three solvents (EtOH, MeOH, and CHCl3) amongst which methanolic Ct extract exhibited the highest percentage yield (9.6%). Qualitative phytochemical screening, thin layer chromatography (TLC), and antioxidant assays (DPPH assay and H2O2 assay) were performed. The percentage free radical scavenging values were found to be 86.25% (IC50=140.1μg/ml) and 88% (IC50=14.22μg/ml) at 1000 μg/ml concentration for both assays respectively. Methanolic Ct extract was then encapsulated in chitosan-tripolyphosphate (CS-TPP) nanoparticles using ionic gelation method with an encapsulation efficiency of 87%. Characterization showed uniform size distribution of 140nm particle size using DLS and encapsulation of Ct extract inside CS-TPP nanoparticles was confirmed by UV spectrophotometry and FTIR. Ct loaded CS-TPP nanoparticles showed less than or equal to 5% hemolytic activity at concentrations of 15.62μg/ml, 31.25μg/ml, 62.5μg/ml, and 125μg/ml, suggesting its safer usage at lower concentration. Rhodamine conjugated Ct loaded CS-TPP nanoparticles showed significant uptake efficiency in breast cancer cells compared to control. Ct extract and the nanoformulation were treated against triple negative breast cancer cell lines (Cal-51) for the evaluation of cytotoxicity exhibiting 30-40% (IC50=122.3μg/ml) and up to 75% (IC50=14.39μg/ml) cytotoxicity respectively. The study paves way for the encapsulation of medicinal plants in polymeric nanoparticles to achieve safer and highly efficient drug delivery systems

Tumor necrosis factor -α, interleukin-10, intercellular and vascular adhesion molecules are possible biomarkers of disease severity in complicated Plasmodium vivax isolates from Pakistan.

Background: Cytokine-mediated endothelial activation pathway is a known mechanism of pathogenesis employed by Plasmodium falciparum to induce severe disease symptoms in human host. Though considered benign, complicated cases of Plasmodium vivax are being reported worldwide and from Pakistan. It has been hypothesized that P.vivax utilizes similar mechanism of pathogenesis, as that of P.falciparum for manifestations of severe malaria. Therefore, the main objective of this study was to characterize the role of cytokines and endothelial activation markers in complicated Plasmodium vivax isolates from Pakistan. Methods and Principle Findings: A case control study using plasma samples from well-characterized groups suffering from P.vivax infection including uncomplicated cases (n=100), complicated cases (n=82) and healthy controls (n=100) were investigated. Base line levels of Tumor necrosis factor-α (TNF-α), Interleukin-6 (IL-6), Interleukin-10 (IL-10), Intercellular adhesion molecule-1 (ICAM-1), Vascular adhesion molecule-1(VCAM-1) and Eselectin were measured by ELISA. Correlation of cytokines and endothelial activation markers was done using Spearman’s correlation analysis. Furthermore, significance of these biomarkers as indicators of disease severity was also analyzed. The results showed that TNF-α, IL-10, ICAM-1and VCAM-1 were 3-fold, 3.7 fold and 2 fold increased between uncomplicated and complicated cases. Comparison of healthy controls with uncomplicated cases showed no significant difference in TNF-α concentrations while IL-6, IL-10, ICAM-1, VCAM-1 and E-selectin were found to be elevated respectively. In addition, significant positive correlation was observed between TNF-α and IL-10/ ICAM-1, IL-6 and IL-10, ICAM-1 and VCAM-1.A Receiver operating curve (ROC) was generated which showed that TNF-α, IL-10, ICAM-1 and VCAM-1 were the best individual predictors of complicated P.vivax malaria. Conclusion: The results suggest that though endothelial adhesion molecules are inducible by pro-inflammatory cytokine TNF-α, however, cytokine-mediated endothelial activation pathway is not clearly demonstrated as a mechanism of pathogenesis in complicated P.vivax malaria cases from Pakistan

A logic-based diagram of signalling pathways central to macrophage activation.

BACKGROUND: The complex yet flexible cellular response to pathogens is orchestrated by the interaction of multiple signalling and metabolic pathways. The molecular regulation of this response has been studied in great detail but comprehensive and unambiguous diagrams describing these events are generally unavailable. Four key signalling cascades triggered early-on in the innate immune response are the toll-like receptor, interferon, NF-kappaB and apoptotic pathways, which co-operate to defend cells against a given pathogen. However, these pathways are commonly viewed as separate entities rather than an integrated network of molecular interactions. RESULTS: Here we describe the construction of a logically represented pathway diagram which attempts to integrate these four pathways central to innate immunity using a modified version of the Edinburgh Pathway Notation. The pathway map is available in a number of electronic formats and editing is supported by yEd graph editor software. CONCLUSION: The map presents a powerful visual aid for interpreting the available pathway interaction knowledge and underscores the valuable contribution well constructed pathway diagrams make to communicating large amounts of molecular interaction data. Furthermore, we discuss issues with the limitations and scalability of pathways presented in this fashion, explore options for automated layout of large pathway networks and demonstrate how such maps can aid the interpretation of functional studies

Analysis of the transcriptional networks underpinning the activation of murine macrophages by inflammatory mediators

Macrophages respond to the TLR4 agonist LPS with a sequential transcriptional cascade controlled by a complex regulatory network of signaling pathways and transcription factors. At least two distinct pathways are currently known to be engaged by TLR4 and are distinguished by their dependence on the adaptor molecule MyD88. We have used gene expression microarrays to define the effects of each of three variables-LPS dose, LPS versus IFN-beta and -gamma, and genetic background-on the transcriptional response of mouse BMDMs. Analysis of correlation networks generated from the data has identified subnetworks or modules within the macrophage transcriptional network that are activated selectively by these variables. We have identified mouse strain-specific signatures, including a module enriched for SLE susceptibility candidates. In the modules of genes unique to different treatments, we found a module of genes induced by type-I IFN but not by LPS treatment, suggesting another layer of complexity in the LPS-TLR4 signaling feedback control. We also observe that the activation of the complement system, in common with the known activation of MHC class 2 genes, is reliant on IFN-gamma signaling. Taken together, these data further highlight the exquisite nature of the regulatory systems that control macrophage activation, their likely relevance to disease resistance/susceptibility, and the appropriate response of these cells to proinflammatory stimuli