The role of small heat shock proteins in mutant superoxide dismutase-linked familial amyotrophic lateral sclerosis

The mechanisms by which mutations in the gene encoding superoxide dismutase 1 (S0D1) lead to amyotrophic lateral sclerosis (ALS) remain incompletely understood. Mutant SODI inclusions are observed in both ALS patients and animal models of the disease. Chaperone proteins have been shown to reduce mutant S0D1 inclusion formation in both cell and animal systems and, up-regulation of heat shock proteins (HSPs) in a mouse model of ALS increases their life expectancy. The results presented in this thesis are based on an investigation into the role of small heat shock proteins (sHSPs) in mutant SODI inclusion formation, using a model HEK293 cell system. Over-expression of yellow fluorescent protein (YFP)-tagged G85R mutant SODI in HEK293 cells and subsequent treatment with proteasome inhibitor leads to mutant S0D1-inclusion formation, as shown by immunofluorescence (IMF) microscopy. Using this model of mutant S0D1- inclusion formation, we demonstrate that over-expression of sHSPs decreases the proportion of insoluble mutant SODI present within these cells. Mutations in these sHSPs prevent this function, and further increase the proportion of insoluble mutant S0D1. These mutant sHSPs also cause an increase in the insolubility of normally soluble proteins, such as wild-type SODI. Similar results were observed in Neuro 2a cells, where over-expression of sHSPs caused the phenotype of the mutant SODI inclusions to change, from dense, tight structures to more diffuse ones. We have shown that sHSPs decrease the amount of insoluble mutant SODI in HEK2S3 cells, supporting reports that chaperone proteins prevent mutant SODI-inclusion formation and are beneficial in a mouse model Gf AL

New paradigms for BRCA1/BRCA2 testing in women with ovarian cancer: results of the Genetic Testing in Epithelial Ovarian Cancer (GTEOC) study.

BACKGROUND: Over recent years genetic testing for germline mutations in BRCA1/BRCA2 has become more readily available because of technological advances and reducing costs. OBJECTIVE: To explore the feasibility and acceptability of offering genetic testing to all women recently diagnosed with epithelial ovarian cancer (EOC). METHODS: Between 1 July 2013 and 30 June 2015 women newly diagnosed with EOC were recruited through six sites in East Anglia, UK into the Genetic Testing in Epithelial Ovarian Cancer (GTEOC) study. Eligibility was irrespective of patient age and family history of cancer. The psychosocial arm of the study used self-report, psychometrically validated questionnaires (Depression Anxiety and Stress Scale (DASS-21); Impact of Event Scale (IES)) and cost analysis was performed. RESULTS: 232 women were recruited and 18 mutations were detected (12 in BRCA1, 6 in BRCA2), giving a mutation yield of 8%, which increased to 12% in unselected women aged <70 years (17/146) but was only 1% in unselected women aged ≥70 years (1/86). IES and DASS-21 scores in response to genetic testing were significantly lower than equivalent scores in response to cancer diagnosis (p<0.001). Correlation tests indicated that although older age is a protective factor against any traumatic impacts of genetic testing, no significant correlation exists between age and distress outcomes. CONCLUSIONS: The mutation yield in unselected women diagnosed with EOC from a heterogeneous population with no founder mutations was 8% in all ages and 12% in women under 70. Unselected genetic testing in women with EOC was acceptable to patients and is potentially less resource-intensive than current standard practice.This work was supported by Target Ovarian Cancer grant number T005MT.This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by the BMJ Group

Remarkable kinetic stability of α-thiocarbamoyl substituted 4-methoxybenzylcations

4-Methoxybenzyl cations bearing α-(N,N-dimethylcarbamoyl) and α-(N,N-dimethylthiocarbamoyl) substituents have been generated photochemically upon irradiation of precursors with pentafluorobenzoate or 4-methoxybenzoate leaving groups. The ions have been observed with flash photolysis in 40:60 acetonitrile:water and in 50:50 methanol:water, and rate constants were measured for their decay in solvent alone and for their capture by azide ion. The cations so studied and their lifetimes in 40% acetonitrile are 6, ArC+H-CONMe2, 0.6 μs; 2, ArC+H-CSNMe2, 7 ms; and 4, ArC+(CH3)-CSMe2, 6 ms, where Ar = 4-MeOC6H4. The cation 4 reacts with solvent by elimination of a proton from the α-methyl group, and the rate constant for solvent addition must be less than 1 s-1. The CSNMe2 substituted cations are 105-107-fold longer lived than analogs where the thioamide group has been replaced with an α-methyl. The UV-visible absorption spectra of these two cations also show significant differences from those of typical 4-methoxybenzyl cations. Thus, both the lifetimes and spectra point to a strong interaction of the benzylic centre with the thioamide group. Key words: flash photolysis, thiocarbamoyl stabilized carbocation, photosolvolysis. </jats:p