Circadian clocks, glucocorticoids and the gated inflammatory response

In mammals endogenous, self sustained oscillators, known as circadian clocks, have evolved as a result of day night cycles, with a period close to 24 hours, and are involved in many physiological processes; such as sleep wake cycles, metabolic and hormonal activity. The suprachiasmatic nucleus (SCN), is the central oscillator, and is synchronised to the external environment by light, via the eye. It has been demonstrated that peripheral clocks, too, contain the circadian oscillator, with tissues such as the lung, liver, heart and kidney as well as many isolated cell types remaining rhythmic, in culture, for many days. However, these peripheral oscillators require a signal from the central oscillator in order to co-ordinate a synchronised time. Leading candidates in the relay of this information are the circulating glucocorticoid hormones corticosterone (rodents) or cortisol (man), which are known to have potent effects on the peripheral clock, both in-vivo and in-vitro. Further to this, glucocorticoids have been used for many decades to suppress the symptoms of inflammation, a by product of many human diseases.This thesis aims to address the temporal regulation of the peripheral clock by the endogenous glucocorticoid, corticosterone, using a transgenic mouse harbouring a luciferase conjugated clock reporter, and circadian reporter cell lines. It also aims to address the relative contribution of the two closely related nuclear hormone receptors, the glucocorticoid and mineralocorticoid receptors. A further aim of the work with glucocorticoid signalling was to design a flow-though culture system, in order to address the effects of the endogenous pulsatile release of glucocorticoids on the peripheral oscillator. This thesis also aims to characterise the inflammatory response in relation to its circadian characteristics; its relationship with corticosterone and the effect of inflammation on the central clock components. Finally, this thesis aims to investigate a potential input/output of the clock, a member of the family of C/EBP transcription factors, C/EBP alpha, and whether it is under endogenous circadian control and regulated by glucocorticoids.Work in this thesis has shown that glucocorticoids dynamically regulate the peripheral clock at all phases of the circadian cycle and that this regulation occurs mainly through the glucocorticoid receptor; yet the mineralocorticoid receptor does have a function in the immediate response to glucocorticoid administration. Furthermore, as a result of the initial temporal profile after corticosterone addition, on the clock protein PERIOD2, I have shown transient regulation of the clock through Caveolin-1 based signalling. There is also a significant circadian component to the inflammatory response, which appears, at least in part, to be REV-ERB alpha mediated, and the inflammatory response also has profound effects on circadian gene expression in the periphery. A functional flow-through system was designed and a working model produced, albeit with technical difficulties, to address glucocorticoid pulsing and circadian timing but much more work is needed for effects to be fully understood. C/EBP alpha appears not to be under circadian regulation nor under direct glucocorticoid regulation, at least in peripheral models used here.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Visualizing the triheteromeric N-methyl-D-aspartate receptor subunit composition

N-methyl-D-aspartate receptors (NMDARs) are one of three ligand-gated ionotropic channels that transduce the effects of neurotransmitter glutamate at excitatory synapses within the central nervous system. Their ability to influx Ca2+ into cells, unlike mature AMPA or kainate receptors, implicates them in a variety of processes ranging from synaptic plasticity to cell death. Many of the receptor’s capabilities, including binding glutamate and regulating Ca2+ influx, have been attributed to their subunit composition, determined putatively using cell biology, electrophysiology and/or pharmacology. Here, we show that subunit composition of synaptic NMDARs can also be readily visualized in acute brain slices (rat) using highly specific antibodies directed against extracellular epitopes of the subunit proteins and high-resolution confocal microscopy. This has helped confirm the expression of triheteromeric t-NMDARs (containing GluN1, GluN2, and GluN3 subunits) at synapses for the first time and reconcile functional differences with diheteromeric d-NMDARs (containing GluN1 and GluN2 subunits) described previously. Even though structural information about individual receptors is still diffraction limited, fluorescently tagged receptor subunit puncta coalesce with precision at various magnifications and/or with the postsynaptic density (PSD-95) but not the presynaptic active zone marker Bassoon. These data are particularly relevant for identifying GluN3A-containing t-NMDARs that are highly Ca2+ permeable and whose expression at excitatory synapses renders neurons vulnerable to excitotoxicity and cell death. Imaging NMDAR subunit proteins at synapses not only offers firsthand insights into subunit composition to correlate function but may also help identify zones of vulnerability within brain structures underlying neurodegenerative diseases like Temporal Lobe Epilepsy

Gene-based outcome prediction in multiple cohorts of pediatric T-cell acute lymphoblastic leukemia: a Children's Oncology Group study

<p>Abstract</p> <p>Background</p> <p>Continuous complete clinical remission in T-cell acute lymphoblastic leukemia (T-ALL) is now approaching 80% due to the implementation of aggressive chemotherapy protocols but patients that relapse continue to have a poor prognosis. Such patients could benefit from augmented therapy if their clinical outcome could be more accurately predicted at the time of diagnosis. Gene expression profiling offers the potential to identify additional prognostic markers but has had limited success in generating robust signatures that predict outcome across multiple patient cohorts. This study aimed to identify robust gene classifiers that could be used for the accurate prediction of relapse in independent cohorts and across different experimental platforms.</p> <p>Results</p> <p>Using HG-U133Plus2 microarrays we modeled a five-gene classifier (5-GC) that accurately predicted clinical outcome in a cohort of 50 T-ALL patients. The 5-GC was further tested against three independent cohorts of T-ALL patients, using either qRT-PCR or microarray gene expression, and could predict patients with significantly adverse clinical outcome in each. The 5-GC featured the interleukin-7 receptor (<it>IL-7R</it>), low-expression of which was independently predictive of relapse in T-ALL patients. In T-ALL cell lines, low <it>IL-7R </it>expression was correlated with diminished growth response to IL-7 and enhanced glucocorticoid resistance. Analysis of biological pathways identified the NF-κB and Wnt pathways, and the cell adhesion receptor family (particularly integrins) as being predictive of relapse. Outcome modeling using genes from these pathways identified patients with significantly worse relapse-free survival in each T-ALL cohort.</p> <p>Conclusions</p> <p>We have used two different approaches to identify, for the first time, robust gene signatures that can successfully discriminate relapse and CCR patients at the time of diagnosis across multiple patient cohorts and platforms. Such genes and pathways represent markers for improved patient risk stratification and potential targets for novel T-ALL therapies.</p

Qualitative analysis of how patients decide that they want risk-reducing mastectomy, and the implications for surgeons in responding to emotionally-motivated patient requests

Objective Contemporary approaches to medical decision-making advise that clinicians should respect patients’ decisions. However, patients’ decisions are often shaped by heuristics, such as being guided by emotion, rather than by objective risk and benefit. Risk-reducing mastectomy (RRM) decisions focus this dilemma sharply. RRM reduces breast cancer (BC) risk, but is invasive and can have iatrogenic consequences. Previous evidence suggests that emotion guides patients’ decision-making about RRM. We interviewed patients to better understand how they made decisions about RRM, using findings to consider how clinicians could ethically respond to their decisions. Methods Qualitative face-to-face interviews with 34 patients listed for RRM surgery and two who had decided against RRM. Results Patients generally did not use objective risk estimates or, indeed, consider risks and benefits of RRM. Instead emotions guided their decisions: they chose RRM because they feared BC and wanted to do ‘all they could’ to prevent it. Most therefore perceived RRM to be the ‘obvious’ option and made the decision easily. However, many recounted extensive post-decisional deliberation, generally directed towards justifying the original decision. A few patients deliberated before the decision because fears of surgery counterbalanced those of BC. Conclusion Patients seeking RRM were motivated by fear of BC, and the need to avoid potential regret for not doing all they could to prevent it. We suggest that choices such as that for RRM, which are made emotionally, can be respected as autonomous decisions, provided patients have considered risks and benefits. Drawing on psychological theory about how people do make decisions, as well as normative views of how they should, we propose that practitioners can guide consideration of risks and benefits even, where necessary, after patients have opted for surgery. This model of practice could be extended to other medical decisions that are influenced by patients’ emotions

A network analysis to identify mediators of germline-driven differences in breast cancer prognosis.

Identifying the underlying genetic drivers of the heritability of breast cancer prognosis remains elusive. We adapt a network-based approach to handle underpowered complex datasets to provide new insights into the potential function of germline variants in breast cancer prognosis. This network-based analysis studies ~7.3 million variants in 84,457 breast cancer patients in relation to breast cancer survival and confirms the results on 12,381 independent patients. Aggregating the prognostic effects of genetic variants across multiple genes, we identify four gene modules associated with survival in estrogen receptor (ER)-negative and one in ER-positive disease. The modules show biological enrichment for cancer-related processes such as G-alpha signaling, circadian clock, angiogenesis, and Rho-GTPases in apoptosis

HER3 and downstream pathways are involved in colonization of brain metastases from breast cancer

Introduction: Metastases to the brain from breast cancer have a high mortality, and basal-like breast cancers have a propensity for brain metastases. However, the mechanisms that allow cells to colonize the brain are unclear.Methods: We used morphology, immunohistochemistry, gene expression and somatic mutation profiling to analyze 39 matched pairs of primary breast cancers and brain metastases, 22 unmatched brain metastases of breast cancer, 11 non-breast brain metastases and 6 autopsy cases of patients with breast cancer metastases to multiple sites, including the brain.Results: Most brain metastases were triple negative and basal-like. the brain metastases over-expressed one or more members of the HER family and in particular HER3 was significantly over-expressed relative to matched primary tumors. Brain metastases from breast and other primary sites, and metastases to multiple organs in the autopsied cases, also contained somatic mutations in EGFR, HRAS, KRAS, NRAS or PIK3CA. This paralleled the frequent activation of AKT and MAPK pathways. in particular, activation of the MAPK pathway was increased in the brain metastases compared to the primary tumors.Conclusions: Deregulated HER family receptors, particularly HER3, and their downstream pathways are implicated in colonization of brain metastasis. the need for HER family receptors to dimerize for activation suggests that tumors may be susceptible to combinations of anti-HER family inhibitors, and may even be effective in the absence of HER2 amplification (that is, in triple negative/basal cancers). However, the presence of activating mutations in PIK3CA, HRAS, KRAS and NRAS suggests the necessity for also specifically targeting downstream molecules.Ludwig Institute of Cancer ResearchNational Breast Cancer FoundationUniv Queensland, Clin Res Ctr, Brisbane, Qld 4029, AustraliaQueensland Inst Med Res, Brisbane, Qld 4006, AustraliaUniversidade Federal de São Paulo, EPM, Dept Anat Patol, BR-04024000 São Paulo, BrazilGriffith Univ, Brisbane, Qld 4011, AustraliaUniv Queensland, Ctr Magnet Resonance, Brisbane, Qld 4072, AustraliaEijkman Inst, Jakarta 10430, IndonesiaInst Nacl Canc, Dept Patol, BR-20230130 Rio de Janeiro, BrazilLab Salomao & Zoppi, Dept Patol, BR-04104000 São Paulo, BrazilCharles Univ Prague, Fac Med, Dept Pathol, Plzen 30605, Czech RepublicUniv Sydney, Inst Clin Pathol & Med Res, Sydney W Area Hlth Serv, Sydney, NSW 2145, AustraliaUniv Sydney, Westmead Millennium Inst, Sydney W Area Hlth Serv, Sydney, NSW 2145, AustraliaPeter MacCallum Canc Ctr, Dept Pathol, Melbourne, Vic 3002, AustraliaUniv Queensland, Queensland Brain Inst, Brisbane, Qld 4072, AustraliaRoyal Brisbane & Womens Hosp, Brisbane, Qld 4029, AustraliaUniversidade Federal de São Paulo, EPM, Dept Anat Patol, BR-04024000 São Paulo, BrazilWeb of Scienc