CD95R-Fc coated Microparticle Delivery to Cancer Cells

PhD thesisTriple-negative breast cancers lack the three main breast cancer biomarkers; oestrogen-, progesterone- or HER2-receptors and chemotherapy is the only established treatment option for patients. Chemotherapies are associated with physically debilitating, off-target toxicities and tumours often develop resistance to cytotoxic compounds. Nanomedicine has the potential to improve chemotherapy efficacy and has enabled the development of small, therapeutic carriers such as microparticles (MPs). These can be made from biodegradable polymers, loaded with (sometimes poorly soluble) chemotherapeutic agents and their surfaces coated in tumour targeting proteins. CD95L has been reported to be upregulated in cancer cells identifying it a promising target. In this study, the CD95 receptor, CD95R, was fused to the Fc region of human IgG1, and baculoviral-mediated expression was optimised in Sf21 and Hi5 insect cells. The protein was purified by affinity chromatography and used to coat microparticles. CD95R-coated MPs improved the uptake into triple-negative, MDA-MB-231 cells, ostensibly via a mechanism involving CD95L. Anti-CD95L antibodies were validated following expression of CD95L-Myc-DDK in otherwise naïve HEK293T cells. The expression of supposed target ligand CD95L was then tested in MDA-MB-231 cells and found to be undetectable. Furthermore, the CD95R-coated particles were efficiently taken up by non-cancerous primary fibroblasts. The reasons for the enhanced uptake of CD95R-coated MPs were explored in MDA-MB-231 cells. Differences in the surface charge of the coated-MPs, characterised by dynamic light scattering (DLS) and electrophoresis, were ruled out as a cause. However, inhibition of the common endocytic pathways did suggest that coated-MP uptake likely occurs via macropino- or phagocytosis. The use of this system for targeted cancer treatment or for wider applications are discussed

Structural insight into the TRIAP1/PRELI-like domain family of mitochondrial phospholipid transfer complexes

The composition of the mitochondrial membrane is important for its architecture and proper function. Mitochondria depend on a tightly regulated supply of phospholipid via intra-mitochondrial synthesis and by direct import from the endoplasmic reticulum. The Ups1/PRELI-like family together with its mitochondrial chaperones (TRIAP1/Mdm35) represent a unique heterodimeric lipid transfer system that is evolutionary conserved from yeast to man. Work presented here provides new atomic resolution insight into the function of a human member of this system. Crystal structures of free TRIAP1 and the TRIAP1–SLMO1 complex reveal how the PRELI domain is chaperoned during import into the intermembrane mitochondrial space. The structural resemblance of PRELI-like domain of SLMO1 with that of mammalian phoshatidylinositol transfer proteins (PITPs) suggest that they share similar lipid transfer mechanisms, in which access to a buried phospholipid-binding cavity is regulated by conformationally adaptable loops

Do two and three year old children use an incremental first-NP-as-agent bias to process active transitive and passive sentences? : A permutation analysis

We used eye-tracking to investigate if and when children show an incremental bias to assume that the first noun phrase in a sentence is the agent (first-NP-as-agent bias) while processing the meaning of English active and passive transitive sentences. We also investi-gated whether children can override this bias to successfully distinguish active from passive sentences, after processing the remainder of the sentence frame. For this second question we used eye-tracking (Study 1) and forced-choice pointing (Study 2). For both studies, we used a paradigm in which participants simultaneously saw two novel actions with reversed agent-patient relations while listening to active and passive sentences. We compared English-speaking 25-month-olds and 41-month-olds in between-subjects sentence struc-ture conditions (Active Transitive Condition vs. Passive Condition). A permutation analysis found that both age groups showed a bias to incrementally map the first noun in a sentence onto an agent role. Regarding the second question, 25-month-olds showed some evidence of distinguishing the two structures in the eye-tracking study. However, the 25-month-olds did not distinguish active from passive sentences in the forced choice pointing task. In contrast, the 41-month-old children did reanalyse their initial first-NP-as-agent bias to the extent that they clearly distinguished between active and passive sentences both in the eye-tracking data and in the pointing task. The results are discussed in relation to the development of syntactic (re)parsing