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Dr Gudrun Stenbeck

Dr Gudrun Stenbeck
Lecturer/Work Placement Co-Ordinator

PhD projects for research students

Involvement of the matrix protein SPARC in the dynamic interaction between tumour and host cells

Tumour growth depends on the crosstalk between malignant and surrounding stromal cells (fibroblasts, osteoblasts, endothelial cells and inflammatory cells). Malignant cells secrete soluble proteins that reach neighbouring stromal cells, forcing them to provide a suitable 3D environment for their growth and spreading (metastasis). Different studies, including expression array analysis, identified the matrix protein SPARC as a marker of poor prognosis in different types of cancer.

Growth factors, like TGFbeta and PTHrP, play a role in tumour progression by modulating protein expression. However, little is known of how these external factors modulate the intracellular trafficking machinery to stimulate secretion of proteins such as SPARC.

The main aim of the project is to elucidate how aberrant growth factor signalling alters protein secretion. To investigate the effects of TGFbeta and PTHrP on SPARC secretion, we plan to use tumour cells and primary osteoblastic cells known to naturally express SPARC. We will study secretion of endogenous SPARC by immunoprecipitation as well as by measuring secretion of Green Fluorescent Protein-SPARC chimerae in response to treatment with TGFbeta and PTHrP. TGFbeta and PTHrP signalling most likely stimulates GTP exchange factors (GEF) that are specific for the small GTPases  of the ARF and Rab family. We will therefore study SPARC secretion before and after knockdown of selected GTP-exchange proteins with small interfering RNA.

Differential expression patterns and transport characteristics of aged cells

Ageing is associated with a number of diseases and manifests itself at the organ level (e.g. the morphological changes in the skin) as well as cellular level. Cellular function is dependent on correct delivery of proteins to their destinations, which is especially apparent in the ageing brain where axonal and dendritic transport is impaired. The underlying causes of this impairment most likely are due to changes to intracellular organelles, which are processing the proteins for secretion, i.e. the Golgi apparatus. Fragmentation of the Golgi has been implicated in functional and structural impairments of axons, presynaptic terminals and postsynaptic dendritic spines, which have been observed in neurodegenerative diseases and ageing.

The project aims to analyse key components of the intracellular trafficking machinery in aged cells to identify alterations associated with ageing. Special attention will be given to the analysis of protein lipid modifications in aged cells with the long-term aim of finding intervention possibilities. We have evidence that budding of transport vesicles from the Golgi apparatus in aged cells is altered and will use advanced imaging techniques to establish Golgi transport kinetics in control and aged cells in addition to biochemical Golgi transport assays. Furthermore, we have a system established in our laboratory to purify intracellular organelles and transport vesicles. We will use these purified fractions to analyse alterations in protein modifications by nano liquid chromatography - tandem mass spectrometry (Nano LC-MS/MS).

Nanoparticles in drug delivery

Nanoparticles (NPs) are materials with overall dimensions in the nano scale range (5nm-100nm). Due to their unique physicochemical properties, they have found a wide range of applications and are currently explored as drug and gene delivery vehicles. The mechanism by which NPs are taken up by cells has important implications for their fate and their impact on biological systems. The routes of uptake taken by a specific NP depend on surface charge and shape and determine if NP have inflammatory or toxic effects. Awareness of these effects is an important aspect in the use of NPs in biomedical applications and tailoring the particles for drug delivery ensuring their biosafety.

The aim of the project is to analyse the uptake and intracellular routes of NPs in clinical relevant cell types to establish their potential as drug delivery vehicles. Carbon nanotubes, magnetic particles and gold nanoparticles uptake by immune and cancer cells will be studied by measuring the NPs’ interaction with the cell membrane, their intracellular fate and their effect on signal transduction by high-resolution live cell microscopy, total internal reflection microscopy, Raman spectroscopy and quantitative ELISA. Cytokine release will be measured by microarray and qPCR. Cellular organelles containing NPs will be purified after uptake and their protein content analysed by mass spectrometry. RNAi technology will be employed to verify candidate proteins involved in NP uptake.

Investigating the requisites of increasing the reuse of waste electrical and electronic equipment in the UK

Waste minimisation is key to implementing the circular economy. Circular economy is a concept that promotes the efficient use of resources, by retaining them in the economy for as long as possible, thereby increasing their longevity. This can be achieved by creating loops that feed resources back into the system by extending the service life of a number of goods, via remanufacture, repair and reuse. Reuse of goods, especially of used electrical and electronic equipment is considered to be a particularly important loop as it conserves a range of valuable components (e.g. rare earth metals, glass, plastic, etc.). It can occur via many different routes and may involve many actors.

In the UK reuse of waste electrical and electronic equipment (WEEE) is particularly challenging. This is due to the lack of attention from both policy makers and the waste management industry. Small and medium enterprises that facilitate the recovery of used electrical and electronic equipment, repair and resale have recently been established, demonstrating that reuse could increase in the future. This project, using one of the emerging and successful SMEs, will characterise the input waste electrical and electronic equipment to get an insight into the sources and types and potential reusability of WEEE.

The project will aim to assess the positive and negative impacts of WEEE management via reuse, looking at the potential of maximising value (environmental, economic, social and technical) from different types of electronics and electrical equipment. Using the input-output data from  SMEs the project will try to get an insight into which products can be reused more readily, in order to understand how product design and manufacture affect reusability, and hence the circularity potential of these products. An inventory of WEEE received and reused will be developed, using a number of criteria (e.g. number of components repaired, parts replaced, degree of difficulty, remaining functionality). This inventory will then be used to map inputs against the marketability potential of the outputs after repair, and to understand how to work with local population in order to ensure that most electronics reach SMEs facilitating WEEE reuse, instead of being wasted. 

References:

Iacovidou, E., C. A. Velis, P. Purnell, O. Zwirner, A. Brown, J. Hahladakis, J. Millward-Hopkins and P. T. Williams (2017). Metrics for optimising the multi-dimensional value of resources recovered from waste in a circular economy: A critical review. Journal of Cleaner Production 166: 910-938.

Xu, Y., L. Zhang, C.-H. Yeh and Y. Liu (2018). Evaluating WEEE recycling innovation strategies with interacting sustainability-related criteria. Journal of Cleaner Production 190: 618-629.

Parajuly, K. and H. Wenzel (2017). Potential for circular economy in household WEEE management. Journal of Cleaner Production 151: 272-285.

Differential expression patterns associated with the development of systemic sclerosis

Systemic sclerosis is a chronic autoimmune rheumatic disease, characterised by excessive scarring and fibrosis which causes thickening and tightening of the connective tissues of the skin and damage to major organs, including lungs, heart, kidneys, and the gastrointestinal tract. In collaboration with clinicians at the Royal Free Hospital in London we are using microarray profiling of control and disease tissues to identify changes in protein expression.

The main aim of the project is to establish the defects in membrane traffic and signalling that are at the basis of disease progression in systemic sclerosis. Identification of proteins involved in disease progression will help the development of novel therapeutic targets to halt this debilitating disease.
Microarray analysis and characterisation of proteins that show altered expression levels in diseased samples will be used to identify candidate proteins. These proteins will be further selected by gene ontology profiling and their importance in disease progression tested in our in vitro systems, using novel imaging techniques (e.g. total internal reflection microscopy). 

Involvement of the matrix protein SPARC in the dynamic interaction between tumour and host cells

Tumour growth depends on the crosstalk between malignant and surrounding stromal cells (fibroblasts, osteoblasts, endothelial cells and inflammatory cells). Malignant cells secrete soluble proteins that reach neighbouring stromal cells, forcing them to provide a suitable 3D environment for their growth and spreading (metastasis). Different studies, including expression array analysis, identified the matrix protein SPARC as a marker of poor prognosis in different types of cancer.

Growth factors, like TGFbeta and PTHrP, play a role in tumour progression by modulating protein expression. However, little is known of how these external factors modulate the intracellular trafficking machinery to stimulate secretion of proteins such as SPARC.

The main aim of the project is to elucidate how aberrant growth factor signalling alters protein secretion. To investigate the effects of TGFbeta and PTHrP on SPARC secretion, we plan to use tumour cells and primary osteoblastic cells known to naturally express SPARC. We will study secretion of endogenous SPARC by immunoprecipitation as well as by measuring secretion of Green Fluorescent Protein-SPARC chimerae in response to treatment with TGFbeta and PTHrP. TGFbeta and PTHrP signalling most likely stimulates GTP exchange factors (GEF) that are specific for the small GTPases  of the ARF and Rab family. We will therefore study SPARC secretion before and after knockdown of selected GTP-exchange proteins with small interfering RNA.

Effects on the genome of new treatments for the premature ageing disease Hutchinson-Gilford Progeria Syndrome

HGPS is a premature ageing syndrome that affects children leading to their premature death in their teens through heart attacks and strokes. There have been a few drugs that have been given to the children through clinical trials and more planned. However, not very much is known about how these drugs affect the genome biology. We have investigated some of the drugs in use for the treatment of progeria and have mixed effects on the genome of these drugs. It is obvious that some new better treatments are required. 

The project will involve the testing of newly proposed drugs on genome behaviour in HGPS cells. These assays will include chromosome positioning, gene and chromosome movement, genomic instability, the epigenome, association with specific structures and transcription profiles. A range of techniques will be used such as super-resolution bioimaging, fluorescence in situ hybridisation, real-time PCR, chromatin immuno-precipitation and sequencing. Furthermore, you will be part of a team working on new treatments using state-of-the-art applications involving nanotechnology and gene therapy that will also form part
of your PhD thesis.