High-resolution μCT of a mouse embryo using a compact laser-driven X-ray betatron source

High-resolution microcomputed tomography with benchtop X-ray sources requires long scan times because of the heat load limitation on the anode. We present an alternative, high-brightness plasma-based X-ray source that does not suffer from this restriction. A demonstration of tomography of a centimeter-scale complex organism achieves equivalent quality to a commercial scanner. We will soon be able to record such scans in minutes, rather than the hours required by conventional X-ray tubes

Annexin A6-Induced Alterations in Cholesterol Transport and Caveolin Export from the Golgi Complex

Annexin A6 (AnxA6) belongs to a family of Ca2+-dependent membrane-binding proteins and is involved in the regulation of endocytic and exocytic pathways. We previously demonstrated that AnxA6 regulates receptor-mediated endocytosis and lysosomal targeting of low-density lipoproteins and translocates to cholesterol-enriched late endosomes (LE). As cholesterol modulates the membrane binding and the cellular location of AnxA6, but also affects the intracellular distribution of caveolin, we investigated the localization and trafficking of caveolin in AnxA6-expressing cells. Here, we show that cells expressing high levels of AnxA6 are characterized by an accumulation of caveolin-1 (cav-1) in the Golgi complex. This is associated with a sequestration of cholesterol in the LE and lower levels of cholesterol in the Golgi and the plasma membrane, both likely contributing to retention of caveolin in the Golgi apparatus and a reduced number of caveolae at the cell surface. Further strengthening these findings, knock down of AnxA6 and the ectopic expression of the Niemann–Pick C1 protein in AnxA6-overexpressing cells restore the cellular distribution of cav-1 and cholesterol, respectively. In summary, this study demonstrates that elevated expression levels of AnxA6 perturb the intracellular distribution of cholesterol, which indirectly inhibits the exit of caveolin from the Golgi complex

Establishing a genotoxic predictive test of toxicities and response to irinotecan chemotherapy in metastatic colorectal cancer

Irinotecan is a topoisomerase I inhibitor prescribed to treat metastatic colorectal cancer. Its use is limited by the heterogeneity in its toxicities and clinical response which are currently unpredictable for the most commonly prescribed doses. Presented in this thesis are the design, conduct and mechanistic analysis of the first prospective clinical study performed to assess whether DNA damage induced in peripheral blood lymphocytes (PBLs), following irinotecan exposure, is a predictive biomarker of this drug’s effect. PBLs were isolated from patients before and after receiving irinotecan based chemotherapy. A predictive test of irinotecan effect may have improved the management in 11 of the 42 patients recruited who experienced severe toxicities and 7 who gained no clinical benefit. Irinotecan did not induce DNA damage that could be measured using the alkaline comet assay in PBLs in vivo. An ex vivo method was subsequently developed using mitogenic stimulation of PBLs prior to treatment with SN-38 (the active metabolite of irinotecan) to induce DNA damage that could then be detected. Results demonstrated the presence of a wide inter-individual variation in the DNA damage levels. Several factors including intrinsic assay variability, cell cycle disturbance, apoptosis and DNA repair were investigated and purported to account for these variations. DNA damage did not correlate with the presence of the UGT1A1*28 polymorphism (associated with slow glucuronidation of SN-38) nor with toxicities to treatment. Liquid chromatography-mass spectrometry analysis of metabolites extracted from PBLs, demonstrated that glucuronidation of SN-38 was not occurring ex vivo, thus providing a potential explanation for these absent associations. However, DNA damage was weakly associated with tumour response and significantly correlated with progression free survival (PFS). In conclusion, there was no evidence that this model predicted normal tissue toxicities to irinotecan treatment; but associations, warranting further investigation, of DNA damage with response and survival were demonstrated

Establishing a genotoxic predictive test of toxicities and response to irinotecan chemotherapy in metastatic colorectal cancer

Irinotecan is a topoisomerase I inhibitor prescribed to treat metastatic colorectal cancer. Its use is limited by the heterogeneity in its toxicities and clinical response which are currently unpredictable for the most commonly prescribed doses. Presented in this thesis are the design, conduct and mechanistic analysis of the first prospective clinical study performed to assess whether DNA damage induced in peripheral blood lymphocytes (PBLs), following irinotecan exposure, is a predictive biomarker of this drug’s effect. PBLs were isolated from patients before and after receiving irinotecan based chemotherapy. A predictive test of irinotecan effect may have improved the management in 11 of the 42 patients recruited who experienced severe toxicities and 7 who gained no clinical benefit. Irinotecan did not induce DNA damage that could be measured using the alkaline comet assay in PBLs in vivo. An ex vivo method was subsequently developed using mitogenic stimulation of PBLs prior to treatment with SN-38 (the active metabolite of irinotecan) to induce DNA damage that could then be detected. Results demonstrated the presence of a wide inter-individual variation in the DNA damage levels. Several factors including intrinsic assay variability, cell cycle disturbance, apoptosis and DNA repair were investigated and purported to account for these variations. DNA damage did not correlate with the presence of the UGT1A1*28 polymorphism (associated with slow glucuronidation of SN-38) nor with toxicities to treatment. Liquid chromatography-mass spectrometry analysis of metabolites extracted from PBLs, demonstrated that glucuronidation of SN-38 was not occurring ex vivo, thus providing a potential explanation for these absent associations. However, DNA damage was weakly associated with tumour response and significantly correlated with progression free survival (PFS). In conclusion, there was no evidence that this model predicted normal tissue toxicities to irinotecan treatment; but associations, warranting further investigation, of DNA damage with response and survival were demonstrated.EThOS - Electronic Theses Online ServiceHope Against CancerGBUnited Kingdo

‘Why nurses are leaving the profession...lack of support from managers’: what nurses from an e-cohort study said

The shortage of nurses is known. We explored nurses' reasons for leaving the profession based on responses from an e-newsletter of the Nurses and Midwives e-cohort Study. Qualitative content analysis of data from email responses (n=66) showed 'Lack of support' as a social work value describing their manager's lack of support, unsupportive relationships within their work group, and a health-care system putting business principles before care resulting in job dissatisfaction and nurse turnover. These findings are examples of a 'complex'/'wicked' problem and as such will resist simple solutions to the presenting issues. The dialogic process such as Open Space Technology and Talking Circles should be considered as potentially suitable to the needs of nurses

Plasma membrane-associated annexin A6 reduces Ca2+ entry by stabilizing the cortical actin cytoskeleton

The annexins are a family of Ca(2+)- and phospholipid-binding proteins, which interact with membranes upon increase of [Ca(2+)](i) or during cytoplasmic acidification. The transient nature of the membrane binding of annexins complicates the study of their influence on intracellular processes. To address the function of annexins at the plasma membrane (PM), we fused fluorescent protein-tagged annexins A6, A1, and A2 with H- and K-Ras membrane anchors. Stable PM localization of membrane-anchored annexin A6 significantly decreased the store-operated Ca(2+) entry (SOCE), but did not influence the rates of Ca(2+) extrusion. This attenuation was specific for annexin A6 because PM-anchored annexins A1 and A2 did not alter SOCE. Membrane association of annexin A6 was necessary for a measurable decrease of SOCE, because cytoplasmic annexin A6 had no effect on Ca(2+) entry as long as [Ca(2+)](i) was below the threshold of annexin A6-membrane translocation. However, when [Ca(2+)](i) reached the levels necessary for the Ca(2+)-dependent PM association of ectopically expressed wild-type annexin A6, SOCE was also inhibited. Conversely, knockdown of the endogenous annexin A6 in HEK293 cells resulted in an elevated Ca(2+) entry. Constitutive PM localization of annexin A6 caused a rearrangement and accumulation of F-actin at the PM, indicating a stabilized cortical cytoskeleton. Consistent with these findings, disruption of the actin cytoskeleton using latrunculin A abolished the inhibitory effect of PM-anchored annexin A6 on SOCE. In agreement with the inhibitory effect of annexin A6 on SOCE, constitutive PM localization of annexin A6 inhibited cell proliferation. Taken together, our results implicate annexin A6 in the actin-dependent regulation of Ca(2+) entry, with consequences for the rates of cell proliferation

Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and in HuH7 cells.

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters

High-resolution μCT of a mouse embryo using a compact laser-driven X-ray betatron source

In the field of X-ray microcomputed tomography (µCT) there is a growing need to reduce acquisition times at high spatial resolution (approximate micrometers) to facilitate in vivo and high-throughput operations. The state of the art represented by synchrotron light sources is not practical for certain applications, and therefore the development of high-brightness laboratoryscale sources is crucial. We present here imaging of a fixed embryonic mouse sample using a compact laser–plasma-based X-ray light source and compare the results to images obtained using a commercial X-ray µCT scanner. The radiation is generated by the betatron motion of electrons inside a dilute and transient plasma, which circumvents the flux limitations imposed by the solid or liquid anodes used in conventional electron-impact X-ray tubes. This X-ray source is pulsed (duration 1010 photons per pulse), small (diameter 15 keV. Stable X-ray performance enabled tomographic imaging of equivalent quality to that of the µCT scanner, an important confirmation of the suitability of the laserdriven source for applications. The X-ray flux achievable with this approach scales with the laser repetition rate without compromising the source size, which will allow the recording of high-resolution µCT scans in minutes