Biochemical and immunological roles of heat shock proteins in human cancer

Found in every cell of every organism, heat shock proteins (hsps) participate in a wide range of cellular processes and primarily function as molecular chaperones that mediate the activity of other cellular proteins. Hsps are required for a range of fundamental mechanisms used by cancer cells and they have consequently been identified as valid targets in the treatment of cancer. It was the aim of this thesis to further investigate these roles in breast cancer and melanoma using novel approaches from a biochemical and immunological perspective. In a preliminary study, breast cancer tissues (n = 30) were demonstrated by Western immunoblotting to widely express hsps 90 and 70. Two-dimensional gel electrophoresis indicated that a number of proteins were differentially expressed in tumour and healthy breast tissue from the same patient. These results suggest that a number of possibly unidentified proteins may play important roles in breast cancer and thus have use as therapeutic targets or biomarkers. The role of hsp90 and associated client proteins in breast cancer was further investigated by non-denaturing immunoprecipitation followed by elution with geldanamycin, a specific inhibitor of hsp90. Geldanamycin-sensitive hsp90 client proteins were observed in seven of 11 protein extracts from breast cancer patients and one healthy individual. Immunoprecipitation, Western immunoblotting and LC-MS identified hsps 40, 56/FKBP52, 60, 70, 105 and lumican as potential hsp90 client proteins. These proteins may thus assist breast cancer progression alongside hsp90. In one patient sample, a cancer-specific group of proteins was identified, while in all experiments geldanamycin resistance was observed. The results of this study may have relevance for the future of breast cancer research and clinical treatment

Fast Raman spectral mapping of highly fluorescing samples by time-gated spectral multiplexed detection

We present a time-gated Raman micro-spectroscopy technique suitable for fast Raman mapping of samples eliciting large laser-induced fluorescence backgrounds. To achieve the required time resolution for effective fluorescence rejection, a picosecond pulsed laser and a single-photon avalanche diode were used. A module consisting of a spectrometer, digital micromirror device, and two prisms was used for high-resolution spectral filtering and multiplexing, which is required for a high chemical specificity and short integration times. With this instrument, we demonstrated time-gated Raman imaging of highly fluorescent samples, achieving acquisition times as short as 3 min for 40×40 pixel resolution images

Promoting Spiritual Wellness on a College Campus through Community Based Participatory Research

This case study describes an initiative to promote spiritual wellness on a public state college campus and demonstrates evidence of the effectiveness of taking a community-based approach. We employed the community readiness model to develop an initiative to promote spiritual wellness in a Midwestern state university. We recruited informants through purposeful sampling and conducted interviews, both pre- and post-initiative launch. Baseline data was used to inform initiative efforts. The community’s level of readiness to promote spiritual wellness increased from stage three, vague awareness, at baseline to stage six, initiation, at follow up. Although these findings are specific to our community, this communitybased participatipatory research approach may be an effective way of developing appropriate strategies to promoting spiritual practices throughout higher education

The BRAF pseudogene functions as a competitive endogenous RNA and induces lymphoma in vivo

SummaryResearch over the past decade has suggested important roles for pseudogenes in physiology and disease. In vitro experiments demonstrated that pseudogenes contribute to cell transformation through several mechanisms. However, in vivo evidence for a causal role of pseudogenes in cancer development is lacking. Here, we report that mice engineered to overexpress either the full-length murine B-Raf pseudogene Braf-rs1 or its pseudo “CDS” or “3′ UTR” develop an aggressive malignancy resembling human diffuse large B cell lymphoma. We show that Braf-rs1 and its human ortholog, BRAFP1, elicit their oncogenic activity, at least in part, as competitive endogenous RNAs (ceRNAs) that elevate BRAF expression and MAPK activation in vitro and in vivo. Notably, we find that transcriptional or genomic aberrations of BRAFP1 occur frequently in multiple human cancers, including B cell lymphomas. Our engineered mouse models demonstrate the oncogenic potential of pseudogenes and indicate that ceRNA-mediated microRNA sequestration may contribute to the development of cancer

Modeling Magnification and Anisotropy in the Primate Foveal Confluence

A basic organizational principle of the primate visual system is that it maps the visual environment repeatedly and retinotopically onto cortex. Simple algebraic models can be used to describe the projection from visual space to cortical space not only for V1, but also for the complex of areas V1, V2 and V3. Typically a conformal (angle-preserving) projection ensuring local isotropy is regarded as ideal and primate visual cortex is often regarded as an approximation of this ideal. However, empirical data show systematic deviations from this ideal that are especially relevant in the foveal projection. The aims of this study were to map the nature of anisotropy predicted by existing models, to investigate the optimization targets faced by different types of retino-cortical maps, and finally to propose a novel map that better models empirical data than other candidates. The retino-cortical map can be optimized towards a space-conserving homogenous representation or a quasi-conformal mapping. The latter would require a significantly enlarged representation of specific parts of the cortical maps. In particular it would require significant enlargement of parafoveal V2 and V3 which is not supported by empirical data. Further, the recently published principal layout of the foveal singularity cannot be explained by existing models. We suggest a new model that accurately describes foveal data, minimizing cortical surface area in the periphery but suggesting that local isotropy dominates the most foveal part at the expense of additional cortical surface. The foveal confluence is an important example of the detailed trade-offs between the compromises required for the mapping of environmental space to a complex of neighboring cortical areas. Our models demonstrate that the organization follows clear morphogenetic principles that are essential for our understanding of foveal vision in daily life

Effect of Culture at Low Oxygen Tension on the Expression of Heat Shock Proteins in a Panel of Melanoma Cell Lines

Tumours are commonly hypoxic and this can be associated with aggressive tumour type, metastasis and resistance to therapy. Heat shock proteins (hsps) are induced in response to hypoxia, provide cancer cells with protection against tumour-associated stressors and chaperone oncoproteins that drive tumour proliferation. This study examined the effect of different oxygen concentrations on the expression of hsps in melanoma cell lines.Melanoma cell lines were cultured in 2% and 20% O(2). Expression of Hsp90, Hsp70, Hsp60, Hsp40 and Hsp32 proteins were determined by flow cytometry.Growth rates and viability were reduced in the majority of cell lines by culture in 2% O(2). Hsp expression was different in 2% compared to 20% O(2) and changes in Hsp90 expression correlated with cell line generation time (P<0.005) and viability (P<0.01). Greater total hsp expression correlated with improved viability in 2% but not 20% O(2) (P<0.05). Relative expression of the different hsps was consistent across cell lines and each correlated with the others (P = 0.0001) but not with Hsp32. Hsp expression was inversely correlated with cell line adhesion to laminin as well as collagen type IV and Breslow depth of the original primary tumour tissue (P<0.05), but not with Clark level or patient survival. All five hsps were identified on the cell surface.Culture in 2% O(2) variably altered hsp expression in a panel of melanoma cell lines. Hsp expression was associated with certain cell line characteristics and clinical parameters of the originating tumour

Non-Invasive Mapping of the Gastrointestinal Microbiota Identifies Children with Inflammatory Bowel Disease

Background: Pediatric inflammatory bowel disease (IBD) is challenging to diagnose because of the non-specificity of symptoms; an unequivocal diagnosis can only be made using colonoscopy, which clinicians are reluctant to recommend for children. Diagnosis of pediatric IBD is therefore frequently delayed, leading to inappropriate treatment plans and poor outcomes. We investigated the use of 16S rRNA sequencing of fecal samples and new analytical methods to assess differences in the microbiota of children with IBD and other gastrointestinal disorders. Methodology/Principal Findings: We applied synthetic learning in microbial ecology (SLiME) analysis to 16S sequencing data obtained from i) published surveys of microbiota diversity in IBD and ii) fecal samples from 91 children and young adults who were treated in the gastroenterology program of Children’s Hospital (Boston, USA). The developed method accurately distinguished control samples from those of patients with IBD; the area under the receiver-operating-characteristic curve (AUC) value was 0.83 (corresponding to 80.3% sensitivity and 69.7% specificity at a set threshold). The accuracy was maintained among data sets collected by different sampling and sequencing methods. The method identified taxa associated with disease states and distinguished patients with Crohn’s disease from those with ulcerative colitis with reasonable accuracy. The findings were validated using samples from an additional group of 68 patients; the validation test identified patients with IBD with an AUC value of 0.84 (e.g. 92% sensitivity, 58.5% specificity). Conclusions/Significance: Microbiome-based diagnostics can distinguish pediatric patients with IBD from patients with similar symptoms. Although this test can not replace endoscopy and histological examination as diagnostic tools, classification based on microbial diversity is an effective complementary technique for IBD detection in pediatric patients.Natural Sciences and Engineering Research Council of Canada (Award NSERC PGS D)National Institutes of Health (U.S.) (1-R21-A1084032-01A1

Hsps 70 and 105 associate with a group of hsp90 client proteins that are selectively found in human breast cancer

This study investigated the interaction of heat shock proteins (hsps) and their client proteins in tumour and normal human breast tissue. The objective of the study was to discover hsp interactions specific to cancer cells. Hsps have been observed to be over-expressed in a range of human cancers. Of which hsps 70, 90 and others have been noted to be up-regulated in breast cancer. Up-regulation of these hsps has been shown to predict patient prognosis and response to therapies [1, 2]. The majority of hsp90's client proteins are signal transduction proteins. In addition, hsp90 is essential for cellular survival and has been proposed to participate in all 6 "hallmarks of cancer" [3]. Hsp90 inhibitors are under intense investigation and are currently in phase III clinical trials for the treatment of cancer. Hsp90 is known to associate with other hsps in the assembly and function of chaperone complexes. These experiments examined the hsp chaperone complexes and the effect of geldanamycin (an hsp90 inhibitor) on these complexes in human breast cancer