Pathways Project Final Report Volume 1: Field Investigation and Catchment Conceptual Models

viii
Executive Summary
The Pathways Project field studies were targeted at improving the understanding of contaminant transport along different hydrological pathways in Irish catchments, including their associated impacts on water quality and river ecology. The contaminants of interest were phosphorus, nitrogen and sediment. The working Pathways conceptual model included overland flow, interflow, shallow groundwater flow, and deep groundwater flow. This research informed the development of a set of Catchment Management Support Tools (CMSTs) comprising an Exploratory Tool, Catchment Characterization Tool (CCT) and Catchment Modelling Tool (CMT) as outlined in Pathways Project Final Reports Volumes 3 and 4.
In order to inform the CMST, four suitable study catchments were selected following an extensive selection process, namely the Mattock catchment, Co. Louth/Meath; Gortinlieve catchment, Co. Donegal; Nuenna catchment, Co. Kilkenny and the Glen Burn catchment, Co. Down. The Nuenna catchment is well drained as it is underlain by a regionally important karstified limestone aquifer with permeable limestone tills and gravels, while the other three catchments are underlain by poorly productive aquifers and low permeability clayey tills, and are poorly drained.
All catchments were instrumented, and groundwater, surface and near-surface water and aquatic ecology were monitored for a period of two years. Intensive water quality sampling during rainfall events was used to investigate the pathways delivering nutrients. The proportion of flow along each pathway was determined using chemical and physical hydrograph separation techniques, supported by numerical modelling.
The outcome of the field studies broadly supported the use of the initial four-pathway conceptual model used in the Pathways CMT (time-variant model). The artificial drainage network was found to be a significant contributing pathway in the poorly drained catchments, at low flows and during peak flows in wet antecedent conditions. The transition zone (TZ), i.e. the broken up weathered zone at the top of the bedrock, was also found to be an important pathway. It was observed to operate in two contrasting hydrogeological scenarios: in groundwater discharge zones the TZ can be regarded as being part of the shallow groundwater pathway, whereas in groundwater recharge zones it behaves more like interflow.
In the catchments overlying poorly productive aquifers, only a few fractures or fracture zones were found to be hydraulically active and the TZ, where present, was the main groundwater pathway. In the karstified Nuenna catchment, the springs, which are linked to conduits as well as to a diffuse fracture network, delivered the majority of the flow. These findings confirm the two-component groundwater contribution from bedrock but suggest that the size and nature of the hydraulically active fractures and the nature of the TZ are the dominant factors at the scale of a stream flow event.
Diffuse sources of nitrate were found to be typically delivered via the subsurface pathways, especially in the TZ and land drains in the poorly productive aquifer catchments, and via the bedrock groundwater in the Nuenna. Phosphorus was primarily transported via overland flow in both particulate and soluble forms. Where preferential flow paths existed in the soil and subsoil, soluble P, and to a lesser extent particulate P, were also transported via the TZ and in drains and ditches. Arable land was found to be the most important land use for
ix
the delivery of sediment, although channel bank and in-stream sources were the most significant in the Glen Burn catchment. Overland flow was found to be the predominant transport sediment pathway in the poorly productive catchments. These findings informed the development of the transport and attenuation equations used in the CCT and CMT. From an assessment of the relationship between physico-chemical and biological conditions, it is suggested that in the Nuenna, Glen Burn and Gortinlieve catchments, a relationship may exist between biological water quality and nitrogen concentrations, as well as with P. In the Nuenna, there was also a relationship between macroinvertebrate status and alkalinity.
Further research is recommended on the transport and delivery of phosphorus in groundwater, the transport and attenuation dynamics in the TZ in different hydrogeological settings and the relationship between macroinvertebrates and co-limiting factors. High resolution temporal and spatial sampling was found to be important for constraining the conceptual understanding of nutrient and sediment dynamics which should also be considered in future studies.

PCR amplification of short tandem repeat sequences allows serial studies of chimaerism/engraftment following BMT in rodents

Animal models of bone marrow transplantation (BMT) allow evaluation of new experimental treatment strategies. One potential strategy involves the treatment of donor marrow with ultra-violet B light to allow transplantation across histocompatibility boundaries without an increase in graft rejection or graft-versus-host disease. A major requirement for a new experimental protocol, particularly if it involves manipulation of the donor marrow, is that the manipulated marrow gives rise to long-term multilineage engraftment. DNA based methodologies are now routinely used by many centres to evaluate engraftment and degree of chimaerism post-BMT in humans. We report the adaptation of this methodology to the serial study of engraftment in rodents. Conditions have been defined which allow analysis of serial tail vein samples using PCR of short tandem repeat sequences (STR-PCR). These markers have been used to evaluate the contribution of ultraviolet B treated marrow to engraftment following BMT in rodents without compromising the health of the animals under study. Chimaerism data from sequential tail vein samples and bone marrow from selected sacrificed animals showed excellent correlation, thus confirming the validity of this approach in analysing haemopoietic tissue. Thus the use of this assay may facilitate experimental studies in animal BMT.

The dual EGFR/HER2 inhibitor lapatinib synergistically enhances the antitumor activity of the histone deacetylase inhibitor panobinostat in colorectal cancer models

As key molecules that drive progression and chemoresistance in gastrointestinal cancers, epidermal growth factor receptor (EGFR) and HER2 have become efficacious drug targets in this setting. Lapatinib is an EGFR/HER2 kinase inhibitor suppressing signaling through the RAS/RAF/MEK (MAP/ERK kinase)/MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase)/AKT pathways. Histone deacetylase inhibitors (HDACi) are a novel class of agents that induce cell cycle arrest and apoptosis following the acetylation of histone and nonhistone proteins modulating gene expression and disrupting HSP90 function inducing the degradation of EGFR-pathway client proteins. This study sought to evaluate the therapeutic potential of combining lapatinib with the HDACi panobinostat in colorectal cancer (CRC) cell lines with varying EGFR/HER2 expression and KRAS/BRAF/PIK3CA mutations. Lapatinib and panobinostat exerted concentration-dependent antiproliferative effects in vitro (panobinostat range 7.2-30 nmol/L; lapatinib range 7.6-25.8 μmol/L). Combined lapatinib and panobinostat treatment interacted synergistically to inhibit the proliferation and colony formation in all CRC cell lines tested. Combination treatment resulted in rapid induction of apoptosis that coincided with increased DNA double-strand breaks, caspase-8 activation, and PARP cleavage. This was paralleled by decreased signaling through both the PI3K and MAPK pathways and increased downregulation of transcriptional targets including NF-κB1, IRAK1, and CCND1. Panobinostat treatment induced downregulation of EGFR, HER2, and HER3 mRNA and protein through transcriptional and posttranslational mechanisms. In the LoVo KRAS mutant CRC xenograft model, the combination showed greater antitumor activity than either agent alone, with no apparent increase in toxicity. Our results offer preclinical rationale warranting further clinical investigation combining HDACi with EGFR and HER2-targeted therapies for CRC treatment.