Pygoscelid penguin populations throughout the Antarctic Peninsula region have changed rapidly in recent decades. Ongoing climate change is thought to underpin these changes through bottom-up effects on habitat suitability and prey availability, ultimately affecting penguin behavior, survival and reproduction. To quantify winter behavior and energetic requirements required to support winter activity and to estimate population-level consequences of variation in survival rates under conditions of rapid climate change, this dissertation investigates two projects, each using a different penguin species. Daily activity and energetic demands during winter were estimated for gentoo penguins (Pygoscelis papua) using data from archival temperature tags. Foraging trip frequencies ranged from 0.85 to 1.0 trips day⁻¹ and total trip durations were positively correlated with day length. Mean daily food requirements, based on a mixed diet of fish and krill (Euphausia superba) were estimated at 0.70 ± 0.12 kg day⁻¹. Early winter foraging trips matched day length better than late winter foraging trips, suggesting that individuals maximized foraging time during the early winter period to recover body mass following the breeding season and molt. The attenuated response of foraging trip durations to increasing day length in late winter may be related to differences in local resource availability or individual behaviors prior to the upcoming breeding season. To investigate population-level consequences of variation in survival rates, data from long-term mark-recapture studies of Adélie penguins (Pygoscelis adeliae) were integrated in a stochastic population model to estimate the risk of local extirpation. No trends in survival rates were evident, and variability in survival rates was poorly explained by the selected suite of environmental covariates. Stochastic projections based on the extant variability of survival rates suggests that small increases in the frequency of years with poor survival result in a rapid increase in risk of near-term local extirpation. Compared to other populations of Adélie penguins around the Antarctic continent, survivorship and population growth rates were lowest in the northern Antarctic Peninsula region. Despite no simple correlations with environmental indices, it is readily apparent that Adélie penguins are vulnerable to the rapid environmental changes that are occurring in the Antarctic Peninsula region

The Ca(2+)-responsive phosphatase calcineurin/protein phosphatase 2B dephosphorylates the transcription factor NFATc3. In the myocardium activation of NFATc3 down-regulates the expression of voltage-gated K(+) (Kv) channels after myocardial infarction (MI). This prolongs action potential duration and increases the probability of arrhythmias. Although recent studies infer that calcineurin is activated by local and transient Ca(2+) signals the molecular mechanism that underlies the process is unclear in ventricular myocytes. Here we test the hypothesis that sequestering of calcineurin to the sarcolemma of ventricular myocytes by the anchoring protein AKAP150 is required for acute activation of NFATc3 and the concomitant down-regulation of Kv channels following MI. Biochemical and cell based measurements resolve that approximately 0.2% of the total calcineurin activity in cardiomyocytes is associated with AKAP150. Electrophysiological analyses establish that formation of this AKAP150-calcineurin signaling dyad is essential for the activation of the phosphatase and the subsequent down-regulation of Kv channel currents following MI. Thus AKAP150-mediated targeting of calcineurin to sarcolemmal micro-domains in ventricular myocytes contributes to the local and acute gene remodeling events that lead to the down-regulation of Kv currents.

Rationale

Increased contractility of arterial myocytes and enhanced vascular tone during hyperglycemia and diabetes mellitus may arise from impaired large-conductance Ca(2+)-activated K(+) (BKCa) channel function. The scaffolding protein A-kinase anchoring protein 150 (AKAP150) is a key regulator of calcineurin (CaN), a phosphatase known to modulate the expression of the regulatory BKCa β1 subunit. Whether AKAP150 mediates BKCa channel suppression during hyperglycemia and diabetes mellitus is unknown.

Objective

To test the hypothesis that AKAP150-dependent CaN signaling mediates BKCa β1 downregulation and impaired vascular BKCa channel function during hyperglycemia and diabetes mellitus.

Methods and results

We found that AKAP150 is an important determinant of BKCa channel remodeling, CaN/nuclear factor of activated T-cells c3 (NFATc3) activation, and resistance artery constriction in hyperglycemic animals on high-fat diet. Genetic ablation of AKAP150 protected against these alterations, including augmented vasoconstriction. d-glucose-dependent suppression of BKCa channel β1 subunits required Ca(2+) influx via voltage-gated L-type Ca(2+) channels and mobilization of a CaN/NFATc3 signaling pathway. Remarkably, high-fat diet mice expressing a mutant AKAP150 unable to anchor CaN resisted activation of NFATc3 and downregulation of BKCa β1 subunits and attenuated high-fat diet-induced elevation in arterial blood pressure.

Conclusions

Our results support a model whereby subcellular anchoring of CaN by AKAP150 is a key molecular determinant of vascular BKCa channel remodeling, which contributes to vasoconstriction during diabetes mellitus.

Detection of DNA copy number aberrations by shallow whole-genome sequencing (WGS) faces many challenges, including lack of completion and errors in the human reference genome, repetitive sequences, polymorphisms, variable sample quality, and biases in the sequencing procedures. Formalin-fixed paraffin-embedded (FFPE) archival material, the analysis of which is important for studies of cancer, presents particular analytical difficulties due to degradation of the DNA and frequent lack of matched reference samples. We present a robust, cost-effective WGS method for DNA copy number analysis that addresses these challenges more successfully than currently available procedures. In practice, very useful profiles can be obtained with ∼0.1× genome coverage. We improve on previous methods by first implementing a combined correction for sequence mappability and GC content, and second, by applying this procedure to sequence data from the 1000 Genomes Project in order to develop a blacklist of problematic genome regions. A small subset of these blacklisted regions was previously identified by ENCODE, but the vast majority are novel unappreciated problematic regions. Our procedures are implemented in a pipeline called QDNAseq. We have analyzed over 1000 samples, most of which were obtained from the fixed tissue archives of more than 25 institutions. We demonstrate that for most samples our sequencing and analysis procedures yield genome profiles with noise levels near the statistical limit imposed by read counting. The described procedures also provide better correction of artifacts introduced by low DNA quality than prior approaches and better copy number data than high-resolution microarrays at a substantially lower cost.

Background

Primary breast cancer (BC) patients with extensive axillary lymph-node involvement have a limited prognosis. The Arbeitsgemeinschaft fuer Gynaekologische Onkologie (AGO) trial compared intense dose-dense (idd) adjuvant chemotherapy with conventionally scheduled chemotherapy in high-risk BC patients. Here we report the final, 10-year follow-up analysis.

Patients and methods

Enrolment took place between December 1998 and April 2003. A total of 1284 patients with 4 or more involved axillary lymph nodes were randomly assigned to receive 3 courses each of idd sequential epirubicin, paclitaxel and cyclophosphamide (iddEPC) q2w or standard epirubicin/cyclophosphamide followed by paclitaxel (EC → P) q3w. Event-free survival (EFS) was the primary end point.

Results

A total of 658 patients were assigned to receive iddEPC and 626 patients were assigned to receive EC → P. The median duration of follow-up was 122 months. EFS was 47% (95% CI 43% to 52%) in the standard group and 56% (95% CI 52% to 60%) in the iddEPC group [hazard ratio (HR) 0.74, 95% CI 0.63-0.87; log-rank P = 0.00014, one-sided]. This benefit was independent of menopausal, hormone receptor or HER2 status. Ten-year overall survival (OS) was 59% (95% CI 55% to 63%) for patients in the standard group and 69% (95% CI 65% to 73%) for patients in the iddEPC group (HR = 0.72, 95% CI 0.60-0.87; log-rank P = 0.0007, two-sided). Nine versus two cases of secondary myeloid leukemia/myelodysplastic syndrome were observed in the iddEPC and the EC → P arm, respectively.

Conclusion

The previously reported OS benefit of iddEPC in comparison to conventionally dosed EC → P has been further increased and achieved an absolute difference of 10% after 10 years of follow-up.

Endocrine release of insulin principally controls glucose homeostasis. Nutrient-induced exocytosis of insulin granules from pancreatic β-cells involves ion channels and mobilization of Ca(2+) and cyclic AMP (cAMP) signalling pathways. Whole-animal physiology, islet studies and live-β-cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L-type Ca(2+) currents, and attenuates cytoplasmic accumulation of Ca(2+) and cAMP in β-cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock-in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven-residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity.

Temozolomide (TMZ) increases the overall survival of patients with glioblastoma (GBM), but its role in the clinical management of diffuse low-grade gliomas (LGG) is still being defined. DNA hypermethylation of the O (6) -methylguanine-DNA methyltransferase (MGMT) promoter is associated with an improved response to TMZ treatment, while inactivation of the DNA mismatch repair (MMR) pathway is associated with therapeutic resistance and TMZ-induced mutagenesis. We previously demonstrated that TMZ treatment of LGG induces driver mutations in the RB and AKT-mTOR pathways, which may drive malignant progression to secondary GBM. To better understand the mechanisms underlying TMZ-induced mutagenesis and malignant progression, we explored the evolution of MGMT methylation and genetic alterations affecting MMR genes in a cohort of 34 treatment-naïve LGGs and their recurrences. Recurrences with TMZ-associated hypermutation had increased MGMT methylation compared to their untreated initial tumors and higher overall MGMT methylation compared to TMZ-treated non-hypermutated recurrences. A TMZ-associated mutation in one or more MMR genes was observed in five out of six TMZ-treated hypermutated recurrences. In two cases, pre-existing heterozygous deletions encompassing MGMT, or an MMR gene, were followed by TMZ-associated mutations in one of the genes of interest. These results suggest that tumor cells with methylated MGMT may undergo positive selection during TMZ treatment in the context of MMR deficiency.

Excited states in (38,40,42) Si nuclei have been studied via in-beam γ-ray spectroscopy with multinucleon removal reactions. Intense radioactive beams of ^{40}S and (44)S provided at the new facility of the RIKEN Radioactive Isotope Beam Factory enabled γ-γ coincidence measurements. A prominent γ line observed with an energy of 742(8) keV in (42) Si confirms the 2(+) state reported in an earlier study. Among the γ lines observed in coincidence with the 2^{+} → 0+ transition, the most probable candidate for the transition from the yrast 4(+) state was identified, leading to a 4(1)+) energy of 2173(14) keV. The energy ratio of 2.93(5) between the 2(1)+ and 4(1)(+) states indicates well-developed deformation in (42) Si at N = 28 and Z = 14. Also for 38,40)Si energy ratios with values of 2.09(5) and 2.56(5) were obtained. Together with the ratio for (42)Si, the results show a rapid deformation development of Si isotopes from N = 24 to N = 28.

Obesity is caused by a prolonged positive energy balance1,2. Whether reduced energy expenditure stemming from reduced activity levels contributes is debated3,4. Here we show that in both sexes, total energy expenditure (TEE) adjusted for body composition and age declined since the late 1980s, while adjusted activity energy expenditure increased over time. We use the International Atomic Energy Agency Doubly Labelled Water database on energy expenditure of adults in the United States and Europe (n = 4,799) to explore patterns in total (TEE: n = 4,799), basal (BEE: n = 1,432) and physical activity energy expenditure (n = 1,432) over time. In males, adjusted BEE decreased significantly, but in females this did not reach significance. A larger dataset of basal metabolic rate (equivalent to BEE) measurements of 9,912 adults across 163 studies spanning 100 years replicates the decline in BEE in both sexes. We conclude that increasing obesity in the United States/Europe has probably not been fuelled by reduced physical activity leading to lowered TEE. We identify here a decline in adjusted BEE as a previously unrecognized factor.

Southern Ocean ecosystems are under pressure from resource exploitation and climate change^1,2. Mitigation requires the identification and protection of Areas of Ecological Significance (AESs), which have so far not been determined at the ocean-basin scale. Here, using assemblage-level tracking of marine predators, we identify AESs for this globally important region and assess current threats and protection levels. Integration of more than 4,000 tracks from 17 bird and mammal species reveals AESs around sub-Antarctic islands in the Atlantic and Indian Oceans and over the Antarctic continental shelf. Fishing pressure is disproportionately concentrated inside AESs, and climate change over the next century is predicted to impose pressure on these areas, particularly around the Antarctic continent. At present, 7.1% of the ocean south of 40°S is under formal protection, including 29% of the total AESs. The establishment and regular revision of networks of protection that encompass AESs are needed to provide long-term mitigation of growing pressures on Southern Ocean ecosystems.