Discovery of novel molecular and biochemical predictors of response and outcome in diffuse large B-cell lymphoma

PhDDiscovery of Novel Molecular and Biochemical Predictors of Response and Outcome in Diffuse Large B-cell Lymphoma Diffuse large B-cell lymphoma (DLBCL) is the commonest form of non-Hodgkin lymphoma and responds to treatment with a 5-year overall survival (OS) of 40-50%. Predicting outcome using the best available method, the International Prognostic Index (IPI), is inaccurate and unsatisfactory. This thesis describes research undertaken to discover, explore and validate new molecular and biochemical predictors of response and long-term outcome with the aims of improving on the inaccurate IPI and of suggesting novel therapeutic approaches. Two strategies were adopted: a rational and an empirical approach. The rational strategy used gene expression profiling to identify transcriptional signatures that correlated with outcome to treatment and from which a model of 13-genes accurately predict long-term OS. Two components of the 13-gene model, PKC and PDE4B, were studied using inhibitors in lymphoma cell-lines and primary cell cultures. PKC inhibition using SC-236 proved to be cytostatic and cytotoxic in the cell-lines examined and to a lesser extent in primary tumours. PDE4 inhibition using piclamilast and rolipram had no effect either alone or in combination with chemotherapy. The empirical approach investigated the trace element selenium in presentation serum and found that it was a biochemical predictor of response and outcome to treatment. In an attempt to provide evidence of a causal relationship as an explanation for the associations between presentation serum selenium, response and outcome, two selenium compounds, methylseleninic acid (MSA) and selenodiglutathione (SDG) were studied in vitro in the same lymphoma cell-lines and primary cell cultures. Both MSA and SDG exhibited cytostatic and cytotoxic activity and caspase-8 and caspase-9 driven apoptosis. For SDG reactive oxygen species generation was important for its activity in three of the four cell-lines. In conclusion, molecular and biochemical predictors of response and survival were discovered in DLBCL that led to viable targets for drug intervention being validated in vitro

Moonlight drives ocean-scale mass vertical migration of zooplankton during the Arctic winter

The creation of the pan-Arctic archive of ADCP data was supported by the UK Natural Environment Research Council (NERC) (Panarchive: NE/H012524/1 and SOFI: NE/F012381/1) as was mooring work in Svalbard (Oceans 2025 and Northern Sea Program). Moorings were also supported by the Research Council of Norway (NFR) projects: Circa (214271), Cleopatra (178766), Cleopatra II (216537), and Marine Night (226471).In extreme high-latitude marine environments that are without solar illumination in winter, light-mediated patterns of biological migration have historically been considered non-existent [1]. However, diel vertical migration (DVM) of zooplankton has been shown to occur even during the darkest part of the polar night, when illumination levels are exceptionally low [2 and 3]. This paradox is, as yet, unexplained. Here, we present evidence of an unexpected uniform behavior across the entire Arctic, in fjord, shelf, slope and open sea, where vertical migrations of zooplankton are driven by lunar illumination. A shift from solar-day (24-hr period) to lunar-day (24.8-hr period) vertical migration takes place in winter when the moon rises above the horizon. Further, mass sinking of zooplankton from the surface waters and accumulation at a depth of ∼50 m occurs every 29.5 days in winter, coincident with the periods of full moon. Moonlight may enable predation of zooplankton by carnivorous zooplankters, fish, and birds now known to feed during the polar night [4]. Although primary production is almost nil at this time, lunar vertical migration (LVM) may facilitate monthly pulses of carbon remineralization, as they occur continuously in illuminated mesopelagic systems [5], due to community respiration of carnivorous and detritivorous zooplankton. The extent of LVM during the winter suggests that the behavior is highly conserved and adaptive and therefore needs to be considered as “baseline” zooplankton activity in a changing Arctic ocean [6, 7, 8 and 9].Publisher PDFPeer reviewe

Loss of buoyancy control in the copepod Calanus finmarchicus

A mechanism is demonstrated that could explain large-scale aggregations of lipid-rich copepods in the surface waters of marine environments. Laboratory experiments establish that changes in salinity and temperature induce lipid-mediated buoyancy instability that entrains copepods in surface waters. Reduced hydrostatic pressure associated with forced ascent of copepods at fjordic sills, shelf breaks and seamounts would also reduce the density of the lipid reserves, forcing copepods and particularly those in diapause to the surface. We propose that salinity, temperature and hydrodynamics of the physical environment, in conjunction with the biophysical properties of lipids, explain periodic high abundances of lipid-rich copepods in surface waters

Evidence for oscillating circadian clock genes in the copepod Calanus finmarchicus during the summer solstice in the high Arctic

The circadian clock provides a mechanism for anticipating environmental cycles and is synchronized by temporal cues such as daily light/dark cycle or photoperiod. However, the Arctic environment is characterized by several months of Midnight Sun when the sun is continuously above the horizon and where sea ice further attenuates photoperiod. To test if the oscillations of circadian clock genes remain in synchrony with subtle environmental changes, we sampled the copepod Calanus finmarchicus, a key zooplankter in the north Atlantic, to determine in situ daily circadian clock gene expression near the summer solstice at a southern (74.5° N) sea ice-free and a northern (82.5° N) sea ice-covered station. Results revealed significant oscillation of genes at both stations, indicating the persistence of the clock at this time. While copepods from the southern station showed oscillations in the daily range, those from the northern station exhibited an increase in ultradian oscillations. We suggest that in C. finmarchicus, even small daily changes of solar altitude seem to be sufficient to entrain the circadian clock and propose that at very high latitudes, in under-ice ecosystems, tidal cues may be used as an additional entrainment cue

Calanus finmarchicus seasonal cycle and diapause in relation to gene expression, physiology, and endogenous clocks: Calanus finmarchicus seasonal rhythmicity

The copepod Calanus finmarchicus plays a crucial role in the north Atlantic food web. Its seasonal life cycle involves reproduction and development in surface waters before overwintering in diapause at depth. Although diapause has been studied for more than a century, the factors responsible for the initiation and termination of it are still unclear. Endogenous clocks have been identified as potent tools for photoperiod measurement and seasonal rhythmicity in many terrestrial species, but knowledge of these remains scarce in the marine realm. Focusing on the dominant CV copepodid stage, we sampled a population of C. finmarchicus from a Scottish sea loch to characterize population dynamics, several physiological parameters, and diel and seasonal expression rhythms of 35 genes representing different metabolic pathways, including the circadian clock machinery. This generated a detailed overview of the seasonal cycle of C. finmarchicus including the most extensive field dataset on circadian clock gene expression in a marine species to date. Gene expression patterns revealed distinct gene clusters upregulated at different phases of the copepod's seasonal cycle. While diel clock cycling was restricted to the active spring/summer phase, many clock genes exhibited the highest expression during diapause. Our results provide new insights into diapause on physiological and genetic levels. We suggest that photoperiod, in interaction with internal and external factors (lipid content, temperature, food availability) and the endogenous clock mechanism, plays an important role in the timing of diapause in C. finmarchicus

Circadian Clock Involvement in Zooplankton Diel Vertical Migration

Biological clocks are a ubiquitous ancient and adaptive mechanism enabling organisms to anticipate environmental cycles and to regulate behavioral and physiological processes accordingly [1]. Although terrestrial circadian clocks are well understood, knowledge of clocks in marine organisms is still very limited [2, 3, 4, 5]. This is particularly true for abundant species displaying large-scale rhythms like diel vertical migration (DVM) that contribute significantly to shaping their respective ecosystems [6]. Here we describe exogenous cycles and endogenous rhythms associated with DVM of the ecologically important and highly abundant planktic copepod Calanus finmarchicus. In the laboratory, C. finmarchicus shows circadian rhythms of DVM, metabolism, and most core circadian clock genes (clock, period1, period2, timeless, cryptochrome2, and clockwork orange). Most of these genes also cycle in animals assessed in the wild, though expression is less rhythmic at depth (50–140 m) relative to shallow-caught animals (0–50 m). Further, peak expressions of clock genes generally occurred at either sunset or sunrise, coinciding with peak migration times. Including one of the first field investigations of clock genes in a marine species [5, 7], this study couples clock gene measurements with laboratory and field data on DVM. While the mechanistic connection remains elusive, our results imply a high degree of causality between clock gene expression and one of the planet’s largest daily migrations of biomass. We thus suggest that circadian clocks increase zooplankton fitness by optimizing the temporal trade-off between feeding and predator avoidance, especially when environmental drivers are weak or absent [8]

Photoperiodism and overwintering in boreal and sub-Arctic Calanus finmarchicus populations

The copepod Calanus finmarchicus, a key species in the North Atlantic, generally spends the non-productive season by descending into deep waters and entering diapause, a physiological state characterized by reduced metabolism and arrested development. In the open ocean, overwintering depths are below 600 m, where temperature and light conditions are favourable to initiate diapause. However, C. finmarchicus has also been reported diapausing in areas with shallow water depth such as fjords, coastal waters and shelf seas. In these environments, the temperature and light conditions are different, and it has been hypothesized that under such conditions C. finmarchicus may remain active throughout winter. Here, we investigated changes in the swimming activity of C. finmarchicus from shallow fjords in the eastern North Atlantic during overwintering in response to ambient photoperiod. We conducted monthly experiments with populations from 2 fjords from different latitudes (sub-Arctic Ramfjord, 69°N and boreal Loch Etive, 56°N), measuring the locomotor activity of individual C. finmarchicus stage CVs exposed to a natural light:dark cycle. At both locations, peaks in activity in response to the light cycle were observed to shift from nocturnal during the early overwintering phase to diurnal during mid and late overwintering phase, with a minimal intensity observed during the mid-overwintering phase. In Ramfjord, activity and rhythmicity were generally lower than in Loch Etive. We conclude that C. finmarchicus remains active throughout its overwintering period when in shallow (<200 m) locations but down-regulates its locomotor activity during the main overwintering phase, which we describe as a winter resting state as distinct from classical diapause