In this thesis, novel Case-Based Reasoning (CBR) methods were developed to be included in CBRDP (Case-Based Reasoning Dose Planner) -an adaptive decision support system for radiotherapy dose planning. CBR is an artificial intelligence methodology which solves new problems by retrieving solutions to previously solved similar problems stored in a case base. The focus of this research is on dose planning for prostate cancer patients. The records of patients successfully treated in the Nottingham University Hospitals NHS Trust, City Hospital Campus, UK, were stored in a case base and were exploited using case-based reasoning for future decision making. After each successful run of the system, a group based Simulated Annealing (SA) algorithm automatically searches for an optimal/near optimal combination of feature weights to be used in the future retrieval process of CBR. A number of research issues associated with the prostate cancer dose planning problem and the use of CBR are addressed including: (a) trade-off between the benefit of delivering a higher dose of radiation to cancer cells and the risk to damage surrounding organs, (b) deciding when and how much to violate the limitations of dose limits imposed to surrounding organs, (c) fusion of knowledge and experience gained over time in treating patients similar to the new one, (d) incorporation of the 5 years Progression Free Probability and success rate in the decision making process and (e) hybridisation of CBR with a novel group based simulated annealing algorithm to update knowledge/experience gained in treating patients over time. The efficiency of the proposed system was validated using real data sets collected from the Nottingham University Hospitals. Experiments based on a leave-one-out strategy demonstrated that for most of the patients, the dose plans generated by our approach are coherent with the dose plans prescribed by an experienced oncologist or even better. This system may play a vital role to assist the oncologist in making a better decision in less time; it incorporates the success rate of previously treated similar patients in the dose planning for a new patient and it can also be used in teaching and training processes. In addition, the developed method is generic in nature and can be used to solve similar non-linear real world complex problems.

After the introduction of clean water, vaccination is thought to be the most effective public health tool ever introduced, responsible for preventing millions of cases of disease, disability and death each year. Unfortunately there remain a number of important human diseases for which we have no vaccine, particularly parasitic diseases, such as leishmaniasis, which primarily affect poor communities in tropical regions. There are many complex reasons why we have failed to develop effective vaccines for parasitic diseases, but there is hope that with our improved understanding of the immune system alongside the development of a new generation of vaccines, we will soon develop new vaccines which are effective enough to prevent such diseases. Toll-like receptors (TLRs) are major targets for adjuvants and have been shown to be crucial for defence against a number of infections. TLR2 recognises bacterial lipopeptides in a heterodimer with either TLR1 or TLR6, and its function has been linked to protection against various bacterial infections and to the efficacy of the BCG vaccine. TLR2 has been shown to recognise surface glycoconjugates of Leishmania parasites in vitro, particularly lipophosphoglycan (LPG). In this study, in vivo experimental infections show that TLR2 has a protective role in controlling cutaneous leishmaniasis (CL), as shown by increased lesion sizes and parasite burdens in TLR2-/- mice infected with L. major and L. mexicana. Furthermore, it appears that LPG is not the major mediator of TLR2 activation during infection with L. mexicana, as parasites lacking LPG also resulted in exacerbated disease in TLR2-/- mice. Mice lacking TLR2 co-receptors TLR1 and TLR6 did not show increased susceptibility to infection, suggesting either mono-TLR2 function or alternative co-receptor involvement. Infected TLR2-/- mice show a skewed Th2 immune response to Leishmania, as demonstrated by elevated IL-4, IL-13 and IL-10 production by draining lymph node (DLN) cells in response to antigen. These results suggest that TLR2 is involved in promoting protective immune responses to Leishmania parasites during primary infection in vivo, and is a potential target for protective and therapeutic vaccine adjuvants. Paradoxically, however, TLR2-targeting lipopeptides Pam2 and Pam3 were ineffective adjuvants for use in a whole-cell vaccine to protect against CL, as whole-cell autoclaved L. major (ALM) vaccines containing lipopeptides resulted in exacerbated disease upon challenge when compared to unvaccinated controls and in contrast to effective vaccination when CpG adjuvants were used. The ratio of antigen specific IgG1:IgG2c antibody isotypes, which is a marker of the type of adaptive immune response (Th1 or Th2), was elevated in mice that received vaccines containing lipopeptide adjuvants, suggesting that these adjuvants drive non-protective Th2 responses to Leishmania. In a Th2-dependent vaccine model using Brugia malayi, the use of Pam2 as an adjuvant resulted in an enhanced protective phenotype with similar efficacy to the Th2-driving adjuvant Alum. Thus, in the context of CL infection TLR2 has a protective role in late-stage primary infections with L. major and L. mexicana, yet when targeted with lipopeptide adjuvants in whole-cell vaccines promotes exacerbated disease in challenge infections, through driving Th2 immune responses. Lipopeptides that target TLR2, such as Pam2, are therefore more appropriate for use as adjuvants in vaccines where Th2 protective immunity is required.

Background: Phylogenetic characterisation of local HIV-1 epidemics can be used to understand emerging transmission networks. We analysed subtype-unassigned sequences in the UK HIV Drug Resistance Database (UK HIV DRD) to map the emergence and origin of genetically diverse recombinant strains using phylogenetic analyses, near full-length sequencing of plasma HIV-1 and CD4 cell count decline. Methods: 55,556 genotyped pol sequences (protease amino acids 1-99, RT amino acids 1-234) were analysed for evidence of recombination. Near full-length single genome sequencing of plasma HIV-1 was performed on stored specimens from six patients with a putative A1/D novel recombinant strain. Recombination and phylogenetic analyses were performed using RIP, PhyML, jpHMM, Simplot, SCUEAL and FastTree v2.3.1. Evolutionary analysis of putative novel recombinants was performed using Bayesian Evolutionary Analysis by Sampling Trees (BEAST). Geographic screening for additional instances of recombinant structures was performed using HIV BLAST and the Los Alamos National HIV database. Demographic data were available for 50/72 patients. CD4 cell count decline was assessed using linear regression. Results: The proportion of subtype-unassigned HIV-1 strains in the UK HIV DRD increased significantly during 1999-2008 (p=<0.01). 2,030 putative B-recombinant sequences were analysed for evidence of transmission of novel recombinant strains; 15 novel recombinant clusters comprising 94 individuals were identified. The proportion of intravenous drug users (IVDU), males and people of white ethnicity was significantly higher among novel recombinant clusters than among people infected with pure subtypes and recognised CRFs. Geographic screening showed co-circulation of novel strains in seven countries over three continents and import and export of novel strains from the UK was identified. Near full-length sequencing of six plasma specimens showed five patients sharing an identical A1/D strain; this was registered as CRF50_A1D and 67 further instances in the UK HIV DRD were identified. Geographic analysis showed close relation of component subtype A1 and D regions to East African strains; monophyletic clustering indicated a single introduction of this variant into the UK. Time scaled analysis showed a time to most recent common ancestor of approximately 1992. Demographic data showed the earliest CRF50 transmissions were confined to men who have sex with men (MSM), with subsequent transmissions to heterosexuals and IVDUs. Analysis of the sixth, complex, sequence demonstrated onward recombination of CRF50 with a subtype B strain (median divergence year 2000). CD4 cell count analysis suggested infections with CRF50 progressed in a similar manner to subtype B infections. Conclusions: Significant increasing genetic diversity of HIV-1 in the UK is linked to both UK and international transmissions among multiple exposure routes. These findings highlight the changing dynamics of HIV transmission in the UK and the converging of the two previously distinct MSM and heterosexual epidemics.

Neutrophils have capacity to cause tissue damage in chronic inflammatory disease. In rheumatoid arthritis (RA) they infiltrate joints, secrete proteases and reactive oxygen species (ROS), and express cytokines. RA neutrophils express NAMPT which is a highly conserved, pleiotropic protein, that catalyses the rate-limiting step of the NAD salvage pathway, but also has cytokine-like activity. NAMPT is elevated in inflammation and exerts pro-inflammatory effects on neutrophils. The aim of this research was to determine the role of NAMPT, as a signalling molecule and as an enzyme, in regulating neutrophil activities of pathological importance. Neutrophils were isolated from the blood of healthy donors and either stimulated with NAMPT or else NAMPT was inhibited (with FK866), in the presence and absence of pro-inflammatory cytokines in vitro. Assays for specific neutrophil functions such as production of ROS, bacterial killing and apoptosis were performed, and expression of cytokines was also examined. Transcriptome sequencing of neutrophils treated with FK866 and TNFα in combination, was carried out to investigate the wider impact of NAMPT inhibition on neutrophil gene expression. NAMPT expression was also examined in control, cytokine treated and RA patient neutrophils. NAMPT had little capacity to prime neutrophils, although it did delay neutrophil apoptosis and stabilise the anti-apoptotic protein Mcl-1. NAMPT inhibition in neutrophils, depleted NAD(P)/H and had effects on neutrophil functions and gene expression. Notably, FK866 decreased ROS production but did not affect the ability of neutrophils to kill bacteria. NAMPT-inhibited neutrophils exhibited decreased activation of signalling pathways and a diminished response to cytokines; transcriptome analysis showed that FK866 decreased TNFα-induced expression of many genes. NAMPT expression was not dynamically regulated in control neutrophils, but in RA patient neutrophils, NAMPT mRNA correlated with TNFα expression in a cohort of patients, and NAMPT protein was elevated in synovial fluid neutrophils compared to those from paired blood. Thus, NAMPT is elevated in activated inflammatory neutrophils and correlates with other markers of inflammation in RA, suggesting that it may be a marker of inflammation in these cells. Also, as a NAD biosynthetic enzyme, NAMPT regulates neutrophil functions and gene products central to RA disease pathology, without affecting bacterial killing capacity. This suggests that inhibition of NAMPT may potentially have the capacity to decrease neutrophil mediated tissue-damage observed in chronic inflammation, without adversely compromising host defence, and thus may be a future treatment option for diseases such as RA.

As key regulators of cell survival, proliferation and immune responses, the NFκB family of transcription factors, which signal via the classical and alternative activation pathways, play important roles in intestinal physiology. The pro-inflammatory function of the classical NFκB activation pathway has previously been demonstrated in various animal models of intestinal inflammation. Persistent activation of this pathway has also been detected in the colonic mucosa of patients with inflammatory bowel diseases (IBD). Conversely, several studies in transgenic mice have shown that disruption of the classical NFκB activation pathway specifically in intestinal epithelial cells renders these cells more susceptible to undergoing apoptosis and also results in increased susceptibility to developing colitis. However, the specific roles of individual members of the NFκB family and in particular the role of the alternative NFκB activation pathway in regulating the susceptibility of the intestine to apoptosis, inflammation and colitis associated colon cancer have not been well defined. We therefore hypothesised that individual members of the NFκB family of proteins which signal via the classical and alternative activation pathways specifically regulate intestinal epithelial cell proliferation, apoptosis and colonic inflammation, consequently modulating susceptibility to developing inflammation associated colon cancer. Most of these aims were addressed using transgenic mice with germline deletions of c-Rel, NFκB1, and NFκB2, along with their wild-type counterparts. Intestinal epithelial apoptosis was induced using either γ-irradiation or irinotecan administration. Colitis was induced using dextran sulphate sodium (DSS) and colitis associated cancer by a combination of azoxymethane (AOM) and DSS. In vitro studies were performed in HCT116 colon carcinoma cells. Disruption of classical NFκB signalling by deletion of NFκB1 but not c-Rel sensitised intestinal epithelial cells to undergo apoptosis following γ-irradiation and irinotecan administration. Deleting either c-Rel or NFκB1 also exacerbated the severity of experimental acute and chronic DSS-induced colitis in mice. However, only c-Rel-null mice showed an increase in AOM/DSS induced colonic tumours. This was associated with c-Rel, but not NFκB1 deletion resulting in persistent colonic epithelial mitosis and increased crypt regeneration following DNA damage. Disruption of the alternative NFκB activation pathway by either deleting NFκB2 (in vivo) or suppressing its expression (in vitro) also increased the susceptibility of intestinal epithelial cells to undergo apoptosis following DNA damaging stimuli. Conversely and intriguingly however, deleting NFκB2 also protected mice from developing colitis and colitis associated colon cancer. Specific members of the NFκB family therefore play different roles in regulating the intestinal responses to various types of cellular injury. NFκB2 in particular appears to be essential for developing colitis and its associated cancer. Pharmacological inhibition of NFκB2 may therefore be a promising novel therapeutic strategy for IBD, whereas inhibition of c-Rel signalling may increase susceptibility to developing colitis and its associated colon cancer.

The intestinal epithelium represents a critical component of the gut barrier and is composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions. This epithelium prevents the entrance of harmful microorganisms, antigens and toxins from the gut lumen into the circulation. Small intestinal homeostasis is maintained by the rate of shedding of senescent enterocytes from the villus tip exactly matching the rate of generation of new cells in the crypt. However, in various localised and systemic inflammatory conditions, intestinal homeostasis may be disturbed as a result of increased IEC shedding. Such pathological IEC shedding may cause transient gaps to develop in the epithelial barrier and result in increased intestinal permeability. Although pathological IEC shedding has been implicated in the pathogenesis of inflammatory bowel disease, understanding of the underlying mechanisms remains limited. This thesis describes the development of a murine model to study this phenomenon, as IEC shedding in this species is morphologically analogous to humans. IEC shedding was induced by systemic lipopolysaccharide (LPS) administration in wild-type C57BL/6 mice, and mice deficient in TNF-receptor 1 (Tnfr1-/-), Tnfr2-/-, Nuclear Factor kappa B1 (Nfkb1-/-) or Nfkb2-/-. IEC apoptosis and cell shedding was quantified using immunohistochemistry for active Caspase-3 and gut lumen to systemic circulation permeability was assessed by measuring plasma fluorescence following fluorescein isothiocyanate-dextran gavage. LPS at doses ≥0.125mg/kg induced rapid villus IEC apoptosis and cell shedding which was maximal at 1.5h. This coincided with significant villus shortening, fluid exudation into the gut lumen and diarrhoea. A significant increase in gut to circulation permeability was observed at 5h. TNFR1 was essential for LPS-induced IEC apoptosis and shedding and the fate of the IEC was also dependent on NFκB, with signalling via NFκB1 favouring cell survival, and via NFκB2 favouring apoptosis. This model will enable investigation of the importance and regulation of pathological IEC apoptosis and cell shedding in intestinal and systemic diseases.

Introduction and Aims: Epilepsy is a neurological disorder and is a heterogeneous condition both in terms of cause and prognosis. Prognostic factors identify patients at varying degrees of risk for specific outcomes which facilitates treatment choice and aids patient counselling. Few prognostic models based on prospective cohorts or randomised controlled trial data have been published in epilepsy. Patients with epilepsy can be loosely categorised as having had a first seizure, being newly diagnosed with epilepsy, having established epilepsy or frequent unremitting seizures despite optimum treatment. This thesis concerns modelling prognostic factors for these patient groups, for outcomes including seizure recurrence, seizure remission and treatment failure. Methods: Methods for modelling prognostic factors are discussed and applied to several examples including eligibility to drive following a first seizure and following withdrawal of treatment after a period of remission from seizures. Internal and external model validation techniques are reviewed. The latter is investigated further in a simulation study, the results of which are demonstrated in a motivating example. Mixture modelling is introduced and assessed to better predict whether a patient would achieve remission from seizures immediately, at a later time point, or whether they may never achieve remission. Results: Multivariable models identified a number of significant factors. Future risk of a seizure was therefore obtained for various patient subgroups. The models identified that the chance of a second seizure was below the risk threshold for driving, set by the DVLA, after six months, and the risk of a seizure following treatment withdrawal after a period of remission from seizures was below the risk threshold after three months. Selected models were found to be internally valid and the simulation study indicated that concordance and a variety of imputation methods for handling covariates missing from the validation dataset were useful approaches for external validation of prognostic models. Assessing these methods for a selected model indicated that the model was valid in independent datasets. Mixture modelling techniques begin to show an improved prognostic model for the frequently reported outcome time to 12-month remission. Conclusions: The models described within this thesis can be used to predict outcome for patients with first seizures or epilepsy aiding individual patient risk stratification and the design and analysis of future epilepsy trials. Prognostic models are not commonly externally validated. A method of external validation in the presence of a missing covariate has been proposed and may facilitate validation of prognostic models making the evidence base more transparent and reliable and instil confidence in any significant findings.

Of the environmental factors which influence body weight, nutrients have the most impact. Saturated fatty acids (SFAs) are a macronutrient, which induce obesity, characterised by metabolic dysfunction and altered feeding behaviour. Currently, there are no effective pharmaceutical treatments for obesity. Nutraceutical intervention, including consumption of polyunsaturated fatty acids (PUFAs), represents a promising alternative. PUFAs oppose the effects of SFAs, attenuating weight gain by enhancing satiety. However, in studies using human and rodent models findings are difficult to interpret, due to variation in protocols. The primary aim of this project was to create a rational model of chronic PUFA consumption. Obesity is underpinned by alterations in hypothalamic neuronal plasticity including impairment of neurogenesis. These changes occur in response to nutrients or by regulation of appetite-related hormones. In contrast, dietary restriction stimulates neurogenesis, and evidence has suggested that PUFAs enhance hypothalamic neurogenesis. The secondary aim of this project was to pilot methods for stimulating and observing neurogenesis in hypothalamus of rat. These two lines of enquiry were pursued to begin addressing the complex research question of whether PUFAs exert beneficial effects on body weight by stimulating hypothalamic neurogenesis. Wistar rats fed commercially formulated diets equally enriched with high concentrations of SFAs and PUFAs, from lard and fish oil, respectively, for two months showed no difference in energy intake or body weight. Both diets induced leptin and insulin resistance, but PUFAs reduced triglyceride concentrations. Hence, PUFAs improved lipid metabolism independently of induced obese phenotype. The meal pattern signatures associated with each diet were also similar; however, PUFA-fed animals demonstrated enhanced diurnal satiety. Closer examination of the diet compositions revealed the overlap of results was likely due to the presence of SFAs in the PUFA diet. This led to reformulation, using fatty acids of greater purity. The chosen sources were coconut oil (SFAs), and a commercial preparation of omega-3 PUFAs. A shorter-term, preliminary investigation involving three weeks’ dietary exposure was conducted. Energy intake was again similar between SFA- and omega-3-fed animals but weight gain was attenuated and adiposity reduced by omega-3 feeding. However, the enhanced satiety previously observed was not borne out. A rise in concentration of brain-derived neurotrophic factor, known to be associated with beneficial effects of PUFA intake, in the final study week suggests that studies of longer duration may be required to fully assess the effects of dietary PUFAs. Concurrent pilot work showed that hypothalamic cell proliferation could be stimulated in response to simple enrichment (play tube) introduced to the cage. However, a full study failed to repeat these findings, supporting the notion that neurogenesis is subject to many influences, including age, species, strain and stress, the degrees of influence of which would have to be determined in a series of systematically controlled studies. Failure to stimulate cell proliferation in PUFA-fed rats suggested further that change in dietary fatty acid composition is not a powerful enough intervention to stimulate neurogenesis, when used alone in Wistar rats. In conclusion, application of appropriate controls for dietary energy content and composition show that benefits to body weight metabolism of long-term consumption of diets highly enriched with PUFAs, and, in particular, omega-3 fatty acids can be successfully modelled in rat. However, further work is required to determine the precise timeline of their emergence and underlying mechanisms.

The development of adult intensive care in England and Wales has been researched using primary and secondary literary sources and the oral history of participants in intensive care in England and Wales from soon after its inception until the present (2011). The development of theory has been inductive. In 1952 Bjørn Ibsen, a Danish anaesthetist, treated patients with bulbo-spinal poliomyelitis (polio) with a regime based on intubation of the trachea (windpipe) and intermittent positive-pressure ventilation (IPPV) of the lungs. This resulted in a reduction of mortality of about fifty percent in patients in Copenhagen with this condition. This dramatic result is considered to have initiated the development of modern intensive care throughout the world. The significance of this event is re-evaluated: IPPV was not new. It was in common use in anaesthesia in 1952. It was not more successful than treatment in advanced negative-pressure cabinet respirators in use in the United States. The use of IPPV in paralytic polio was not inevitable. Nevertheless, IPPV did have advantages that made it generally preferable in intensive care. It was applicable to a wider range of conditions which cause respiratory failure, the equipment required was much less expensive and generally available than advanced cabinet respirators and it allowed unrestricted access to the patient. Ibsen’s contribution was to bring an anaesthetic technique into use in an infectious disease unit and later into intensive care units. He also extended the applicability of IPPV by combining it with the use of relaxant drugs. The development of respiratory support techniques in the 40 years before 1952 is described and it is argued that some of these earlier practitioners should be credited with having practised what would later be called intensive care. For a decade after the introduction of IPPV in paralytic polio and tetanus its use in the United Kingdom was largely by infectious disease physicians, sometimes in collaboration with anaesthetists. The development of the specialty of anaesthesia to a point where anaesthetists were able to become the major participants in intensive care after polio and tetanus had become rare after 1963 is described. Intensive care is not simply a matter of connecting a patient to a mechanical respirator: The part played by nurses in the US and the UK in establishing intensive care units is stressed. Oral histories have shown that medical participation was for many years often by one or two enthusiastic clinicians whose work in intensive care was not given financial or sessional recognition and was in addition to full-time work in their base specialties Funding of units, apart from a contribution for nurses’ salaries, was largely from local or regional budgets, often supported by charitable donations. Since the 1980s intensive care has received government funding and has been recognised nationally and internationally as an independent specialty and full-time medical cover for ICUs has been provided. However, these processes are (in 2011) not complete and further development may be anticipated.

The normal structure and function of the heart, the common pathological changes that cause abnormal function and the interventions proposed to improve or restore its function are fundamentally based on cardiac anatomy. Therefore in all these areas a detailed and accurate understanding of 3D structure is essential. However there is still disparity over some aspects of the form and function of the healthy heart. Furthermore, in heart failure (HF) the transition from compensated to decompensated HF is poorly understood, and details of ventricular, and particularly atrial, remodelling and their effects on cardiac function are yet to be fully elucidated. In addition little is known on how the 3D morphology of the cardiac conduction system is affected in disease, and further knowledge is required on the structural substrates for arrhythmogenesis associated with HF. Here we have developed contrast enhanced micro-CT for soft tissue imaging, allowing non-invasive high resolution (~5 µm attainable) differentiation of multiple soft tissue types including; muscle, connective tissue and fat. Micro-CT was optimised for imaging of whole intact mammalian hearts and from these data we reveal novel morphological and anatomical detail in healthy hearts and in hearts after experimental HF (volume and pressure overload). Remodelling of the myocardium in HF was dramatic with significant hypertrophy and dilatation observed in both atria and ventricles. The atria showed a 67% increase in myocardial volume, with the left atrium showing a 93% increase. The pectinate muscle: wall thickness ratio was significantly increased in both atria (p=<0.05), and total cavity volume of the atria was increased by 119% (p=<0.05). An 84% (p=<0.05) and 28% (p=<0.01) increase in myocardial volume was seen in the right and left ventricles (RV and LV) respectively. The LV cavity increased to 231% of control (p=<0.001). Regional remodelling occurred in the LV with the base and mid-wall of the heart undergoing eccentric hypertrophy and the apex undergoing mixed hypertrophy. Longitudinal in vivo analysis by echocardiography revealed progressive loss of function pre termination (41% reduction in fractional shortening, p=< 0.001). We have imaged the major regions of the cardiac conduction system (CCS) in single intact hearts and we present the data as high resolution 3D renderings. This is the first time that such data has been shown for any species. In HF all regions of the CCS underwent dramatic morphological changes, with all regions undergoing hypertrophy and stretch. The sinoatrial node showed an 85% increase in volume (p=0.08) and 51% increase in surface area (p=<0.05). The atrioventricular node increased in both volume (52% p=< 0.05) and 3D length (14% p=< 0.05). The free running Purkinje fibre network showed an 85% increase in volume (p=<0.05); 3D filament analysis revealed the free running length was increased by 62% (p=<0.05), and was strongly correlated with the increase in LV cavity volume. Applying a novel technique for the extraction of 3D fibre orientation from micro-CT data we showed that significant changes occur in HF. The technique was first validated in skeletal muscle and then applied to the heart. Fibre orientation in the ventricles was consistent with previous findings, and novel insight into the complex and heterogeneous fibre orientation of the atria and their accompanying muscle bands was obtained. In HF significant changes in fibre orientation were seen in regions of dilatation; in the LV fibres became more vertical at the endocardium and this coincided with a reduction in the transverse angle; in the transmural mid-wall of both the LV and interventricular septum the percentage of circumferential fibres was reduced, and fibres became more disordered. Based on changes in morphology and 3D fibre orientation we present the idea of regional specific compensated and decompensated HF in the presence of volume and pressure overload. Data presented here provides new information on remodelling of the heart in HF, giving insight into the mechanisms underlying the contractile and electrical pathologies associated with HF.