In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts

Background Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult. Methods We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state. Results In vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma. Conclusion We have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in vivo. Induction of MMP gene expression in both the epithelial tumour cells and surrounding stromal cells is associated with increased metastatic potential. Our data demonstrate the contribution of the stroma to epithelial MMP gene expression, and highlight the complexity of the role of MMPs in the stromal-epithelial interactions within breast carcinoma.

Quantitative proteomic approach to study subcellular localization of membrane proteins

As proteins within cells are spatially organized according to their role, knowledge about protein localization gives insight into protein function. Here, we describe the LOPIT technique (localization of organelle proteins by isotope tagging) developed for the simultaneous and confident determination of the steady-state distribution of hundreds of integral membrane proteins within organelles. The technique uses a partial membrane fractionation strategy in conjunction with quantitative proteomics. Localization of proteins is achieved by measuring their distribution pattern across the density gradient using amine-reactive isotope tagging and comparing these patterns with those of known organelle residents. LOPIT relies on the assumption that proteins belonging to the same organelle will co-fractionate. Multivariate statistical tools are then used to group proteins according to the similarities in their distributions, and hence localization without complete centrifugal separation is achieved. The protocol requires approximately 3 weeks to complete and can be applied in a high-throughput manner to material from many varied sources.

Continuous appearance-based localisation and mapping

This thesis presents a novel approach to mobile robot navigation using visual information towards the goal of long-term autonomy. A novel concept of a continuous appearance-based trajectory is proposed in order to solve the limitations of previous robot navigation systems, and two new algorithms for mobile robots, CAT-SLAM and CAT-Graph, are presented and evaluated. These algorithms yield performance exceeding state-of-the-art methods on public benchmark datasets and large-scale real-world environments, and will help enable widespread use of mobile robots in everyday applications.

Dealing with shadows : capturing intrinsic scene appearance for image-based outdoor localisation

In outdoor environments shadows are common. These typically strong visual features cause considerable change in the appearance of a place, and therefore confound vision-based localisation approaches. In this paper we describe how to convert a colour image of the scene to a greyscale invariant image where pixel values are a function of underlying material property not lighting. We summarise the theory of shadow invariant images and discuss the modelling and calibration issues which are important for non-ideal off-the-shelf colour cameras. We evaluate the technique with a commonly used robotic camera and an autonomous car operating in an outdoor environment, and show that it can outperform the use of ordinary greyscale images for the task of visual localisation.

Low cost localisation for agricultural robotics

This paper presents a pose estimation approach that is resilient to typical sensor failure and suitable for low cost agricultural robots. Guiding large agricultural machinery with highly accurate GPS/INS systems has become standard practice, however these systems are inappropriate for smaller, lower-cost robots. Our positioning system estimates pose by fusing data from a low-cost global positioning sensor, low-cost inertial sensors and a new technique for vision-based row tracking. The results first demonstrate that our positioning system will accurately guide a robot to perform a coverage task across a 6 hectare field. The results then demonstrate that our vision-based row tracking algorithm improves the performance of the positioning system despite long periods of precision correction signal dropout and intermittent dropouts of the entire GPS sensor.

Combining multiple sensor modalities for a localisation robust to smoke

This paper proposes an approach to obtain a localisation that is robust to smoke by exploiting multiple sensing modalities: visual and infrared (IR) cameras. This localisation is based on a state-of-the-art visual SLAM algorithm. First, we show that a reasonably accurate localisation can be obtained in the presence of smoke by using only an IR camera, a sensor that is hardly affected by smoke, contrary to a visual camera (operating in the visible spectrum). Second, we demonstrate that improved results can be obtained by combining the information from the two sensor modalities (visual and IR cameras). Third, we show that by detecting the impact of smoke on the visual images using a data quality metric, we can anticipate and mitigate the degradation in performance of the localisation by discarding the most affected data. The experimental validation presents multiple trajectories estimated by the various methods considered, all thoroughly compared to an accurate dGPS/INS reference.

Sensor Network Based AUV Localisation

The operation of Autonomous Underwater Vehicles (AUVs) within underwater sensor network fields provides an opportunity to reuse the network infrastructure for long baseline localisation of the AUV. Computationally efficient localisation can be accomplished using off-the-shelf hardware that is comparatively inexpensive and which could already be deployed in the environment for monitoring purposes. This paper describes the development of a particle filter based localisation system which is implemented onboard an AUV in real-time using ranging information obtained from an ad-hoc underwater sensor network. An experimental demonstration of this approach was conducted in a lake with results presented illustrating network communication and localisation performance.

Scene signatures : localised and point-less features for localisation

This paper is about localising across extreme lighting and weather conditions. We depart from the traditional point-feature-based approach as matching under dramatic appearance changes is a brittle and hard thing. Point feature detectors are fixed and rigid procedures which pass over an image examining small, low-level structure such as corners or blobs. They apply the same criteria applied all images of all places. This paper takes a contrary view and asks what is possible if instead we learn a bespoke detector for every place. Our localisation task then turns into curating a large bank of spatially indexed detectors and we show that this yields vastly superior performance in terms of robustness in exchange for a reduced but tolerable metric precision. We present an unsupervised system that produces broad-region detectors for distinctive visual elements, called scene signatures, which can be associated across almost all appearance changes. We show, using 21km of data collected over a period of 3 months, that our system is capable of producing metric localisation estimates from night-to-day or summer-to-winter conditions.

Redundant sensing for localisation in outdoor industrial environments

We describe our experiences with automating a large fork-lift type vehicle that operates outdoors and in all weather. In particular, we focus on the use of independent and robust localisation systems for reliable navigation around the worksite. Two localisation systems are briefly described. The first is based on laser range finders and retro-reflective beacons, and the second uses a two camera vision system to estimate the vehicle’s pose relative to a known model of the surrounding buildings. We show the results from an experiment where the 20 tonne experimental vehicle, an autonomous Hot Metal Carrier, was conducting autonomous operations and one of the localisation systems was deliberately made to fail.

Is one-parameter scaling theory of localisation consistent? Comment on Schuh's comment

It is maintained that the one-parameter scaling theory is inconsistent with the physics of Anderson localisation.

Note on the one-parameter scaling theory of localisation

The necessary and sufficient condition for the existence of the one-parameter scale function, the /Munction, is obtained exactly. The analysis reveals certain inconsistency inherent in the scaling theory, and tends to support Motts’ idea of minimum metallic conductivity.

Note on the one-parameter scaling theory of localisation

The necessary and sufficient condition for the existence of the one-parameter scale function, the /Munction, is obtained exactly. The analysis reveals certain inconsistency inherent in the scaling theory, and tends to support Motts’ idea of minimum metallic conductivity.

A potent nonporphyrin class of photodynamic therapeutic agent: Cellular localisation, cytotoxic potential and influence of hypoxia

We have developed a totally new class of nonporphyrin photodynamic therapeutic agents with a specific focus on two lead candidates azadipyrromethene (ADPM)01 and ADPM06. Confocal laser scanning microscopy imaging showed that these compounds are exclusively localised to the cytosolic compartment, with specific accumulation in the endoplasmic reticulum and to a lesser extent in the mitochondria. Light-induced toxicity assays, carried out over a broad range of human tumour cell lines, displayed EC50 values in the micro-molar range for ADPM01 and nano-molar range for ADPM06, with no discernable activity bias for a specific cell type. Strikingly, the more active agent, ADPM06, even retained significant activity under hypoxic conditions. Both photosensitisers showed low to nondeterminable dark toxicity. Flow cytometric analysis revealed that ADPM01 and ADPM06 were highly effective at inducing apoptosis as a mode of cell death. The photophysical and biological characteristics of these PDT agents suggest that they have potential for the development of new anticancer therapeutics. © 2005 Cancer Research UK.

Expression, Enzymatic Activities and Subcellular Localization of Hepatitis E Virus and Semliki Forest Virus Replicase Proteins

Viruses are submicroscopic, infectious agents that are obligate intracellular parasites. They adopt various types of strategies for their parasitic replication and proliferation in infected cells. The nucleic acid genome of a virus contains information that redirects molecular machinery of the cell to the replication and production of new virions. Viruses that replicate in the cytoplasm and are unable to use the nuclear transcription machinery of the host cell have developed their own transcription and capping systems. This thesis describes replication strategies of two distantly related viruses, hepatitis E virus (HEV) and Semliki Forest virus (SFV), which belong to the alphavirus-like superfamily of positive-strand RNA viruses. We have demonstrated that HEV and SFV share a unique cap formation pathway specific for alphavirus-like superfamily. The capping enzyme first acts as a methyltransferase, catalyzing the transfer of a methyl group from S-adenosylmethionine to GTP to yield m7GTP. It then transfers the methylated guanosine to the end of viral mRNA. Both reactions are virus-specific and differ from those described for the host cell. Therefore, these capping reactions offer attractive targets for the development of antiviral drugs. Additionally, it has been shown that replication of SFV and HEV takes place in association with cellular membranes. The origin of these membranes and the intracellular localization of the components of the replication complex were studied by modern microscopy techniques. It was demonstrated that SFV replicates in cytoplasmic membranes that are derived from endosomes and lysosomes. According to our studies, site for HEV replication seems to be the intermediate compartment which mediates the traffic between endoplasmic reticulum and the Golgi complex. As a result of this work, a unique mechanism of cap formation for hepatitis E virus replicase has been characterized. It represents a novel target for the development of specific inhibitors against viral replication.