Dissertationen zum Thema „Pathophysiology“

Osteoarthritis (OA) is a multifactorial disease characterized by a progressive degeneration and eventual failure of the synovial joint functionality. Although it has been traditionally considered as an exclusive disease of the articular cartilage, nowadays it is considered as a whole joint disease. Therefore, the progression of the disease involves articular cartilage degeneration, osteochondral bone sclerosis and synovial membrane hypertrophy. Articular cartilage is a connective tissue resistant to tensile and shears strength that is composed of water (>70%) and a dense extracellular matrix (ECM) that encompasses the unique cellular type of the cartilage, the chondrocytes. The major component of the ECM is the collagen network consisting mainly of type II collagen fibers and type IX and XI collagen macromolecules attached to the surface of the fiber. Non-collagenous components such as GAGs, aggregated proteoglycans (mainly aggrecan) and small leucine rich proteoglycans (SLRP’s) are also binding the collagen fiber. Cartilage is a no innervated and also an avascular tissue, thus gets its nutrients by diffusion from the synovial fluid. Due to this condition, chondrocytes live in a hypoxic environment, and intracellular survival factors, such as hypoxia-inducible factor 1α, are required for maintenance of homeostasis and adaptation to the mechanical environment. Under physiological conditions, the collagen network and proteoglycan content is maintained by the chondrocytes. However, local and systemic risk factors could lead chondrocytes to fail to maintain the ECM and thus the cartilage tissue is progressively degraded. To this point, the three general objectives of this thesis are: 1. Collagenase-3 (COL3) also known as MMP13, is a matrix metalloproteinase abnormally over-expressed in pathological processes. Several COL3 transcripts are expressed in human chondrocytes although their role in OA is still unknown. This study aimed to characterize the presence of two non-canonical COL3 isoforms, named COL3-DEL (deleted form) and COL3-9B (exon 9 added form) in human OA cartilage, and to analyse their proteolytic activity. 2. Opticin (OPTC), a SLRP known to play a role in the assembly of the fibrillar collagens and the structural stability of the extracellular matrix, was previously demonstrated to be produced and degraded in osteoarthritic (OA) human cartilage. Here, we further investigated the OPTC role in OA cartilage by the study of the in vivo effect of OPTC deficiency in mouse model 3. Chondroitin sulfate (CS) is a Symptomatic Slow acting Drug against Osteoarthritis (SySADOA) with anti-inflammatory effects. In this study, we tried to unveil the mechanism of action on osteoarthritic synovial membrane. The general discussion of this thesis is: OA is a heterogeneous disease that encloses multiple phenotypes. In order to develop new diagnostic and prognostic tools and eventually advance in the discovery of successful treatments, clearly defining the different phenotypes of OA is of great importance. In this line, the findings comprised in this thesis reveal two different approaches to identify patient’s subgroups. On one hand, and as described in chapter 1, the presence of a new discovered collagenase-3 (COL3) isoform (COL3-DEL) resulting from a mutation of the canonical COL3, could be used as an indicator of differential extracellular matrix degradation in human articular cartilage. On the other hand, results from chapter 2 suggest that the compositions of the members of SLRP super-family in the human extracellular matrix of the articular cartilage could be applied as a new tool for OA prognosis classification. Finally, the controversy regarding the efficacy of systemic treatment with nutraceuticals – including chondroitin sulfate - may arrive to an end if new tools are used to predict which patients are best suited for a given drug. Importantly, further work remains to be done to understand how to integrate these findings into a final and comprehensive concept that could explain the patient’s heterogeneity and the differential prognosis of OA disease.
L’artrosi (OA) és la malaltia articular més comuna i es caracteritza, principalment, per la destrucció del cartílag articular. El principal paper del cartílag articular és el de suportar les forces de tensió i compressió a les que es troba sotmès i que recau principalment en els seus components: el col·lagen i els proteoglicans. Durant el metabolisme normal del cartílag hi ha un equilibri entre la síntesi i degradació d’aquest components, però a l’artrosi, aquest equilibri es trenca i el metabolisme catabòlic supera l'anabòlic. Durant el desenvolupament de l'OA, els condròcits expressen la proteasa més involucrada en la degradació del cartílag, la MMP-13, que a diferència d’altres MMPs, en humans presenta 3 transcrits diferents. Malauradament, tot i la seva elevada prevalença, l'OA es troba orfe d’una bona tècnica diagnòstica, ja que actualment es realitza per mitjà de tècniques radiològiques que presenten l'inconvenient de que la malaltia només es reflecteix quan l’articulació es troba molt afectada. Actualment, els tractaments farmacològics més utilitzats són analgèsics, AINEs, i els nutracèutics, anomenats SYSADOA per les seves sigles en anglès (Symptomatic Slow Acting Drugs for Osteoarthritis) d’entre els que destaca el sulfat de glucosamina i el condroitin sulfat. Amb aquests antecedents previs, els objectius de la tesis són: 1. Demostrar la presència de 3 isoformes de MMP-13 en humans • Clonació (sistema Bac-to-bac) i purificació de les 3 isoformes en cèl·lules d’insecte (Sf9). • Producció d’anticossos policlonals específics de cada isoforma al Servei de Producció d’Anticossos de la UAB • Cerca de les isoformes en pacients artròsics per WB. • Comprovació de l’activitat de cada isoforma en front a diferents components matricials. 2. Estudiar l'efecte "in vivo" de la deleció del gen de l'Opticina en un model murí d'artrosi. • Confirmar l'expressió de l' OPTC al cartílag articular dels ratolins. • Induïr OA mitjançant el mètode de destabilització del menisc medial (DMM) a ratolins Optc-/- i Optc +/+ de 10 setmanes d'edat. • Analitzar l'efecte de la deficiència de l'OPTC al desenvolupament de l'OA, evaluant la degradació del cartílag i la hipertròfia de la membrana sinovial, deu setmanes després de l'inducció de l'OA per DMM. • Comparar l'expressió de marcadors pro-inflamatoris, catabòlics i anabòlics entre ratolins Optc-/- i Optc+/+. • Analitzar la producció al cartílag de differents SLRPs. • Analitzar l'organització i l'ultraestructura de les fibres de colàgen. 3. Estudiar l'efecte anti-angiogenic del condroitin sulfat en sinoviòcits sota condicions d'hipòxia i d'hipòxia més inflamació. • Mimetitzar condicions inflamatòries i d'hipòxia "in vitro" en sinoviòcits humans. • Estudiar l'expressió i la producció de mediadors angiogènics per sinoviòcits artròsics sota condicions d'hipòxia i inflamació. • Estudiar l'efecte del pretractament del CS en cultius de sinoviòcits humans artròsics • Estudiar l'interacció entre els sinoviòcits artròsics i les cèl·lules endotelials sota hipòxia i inflamació, amb o sense pretractament de CS. L'artrosi és una malaltia que engloba diferents fenotips, però tots comparteixen una sèrie de característiques com la degeneració del cartílag articular, inflamació de la membrana sinovial i esclerosi de l'ós subcondral. Aquestes característiques resulten en un conjunt de símptomes que impedeixen la correcte mobilitat del pacient i creen dolor que pot arribar a ser crònic. Alhora de desenvolupar noves eines de diagnòstic i prognòstic i eventualment avançar en el descobriment de tractaments exitosos, definir clarament els diferents fenotips de l'artrosi és de gran interès. En aquesta línia, els descobriments compresos en aquesta tesis revelen dues possibles maneres d'identificar subgrups de pacients. Per una part, com es descriu al capítol 1, la presència d'una nova isoforma de la colagenasa-3 podria utilitzar-se com a un indicador de diferencies en la degradació matricial del cartíleg humà articular. Per una altre banda, els resultats del capítol 2 suggereixen que la composició dels membres de la famíla de SLRP a la matriu extracel·lular del cartíleg podria aplicar-se com a una nova eina de classificació del prognòstic de la malaltia artròsic. Finalment, la controversia sobre l'eficàcia del tractament amb nutracèutics com el condroitin sulfat podria arribar a solucionar-se si es descobreixen noves eines per predir quins pacients poden respondre millor a un medicament donat. És important tenir en compte que s'ha de continuar investigant per entendre com integrar aquests resultats en un concepte final que pogués explicar l'heterogeneïtat dels pacients i les diferències en el prognòstic de l'artrosi.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2003.
Includes bibliographical references (leaves 146-166).
Lymph node metastases have a negative impact on cancer survival, but the mechanisms for lymphatic metastasis are not well understood. The universal finding in solid tumors of an absence of functional lymphatic vessels seems paradoxical, as cancer cells do travel through lymphatics in order to disseminate. In order to address some of these issues, this thesis proposes two etiologies for the absence of functional lymphatic vessels in solid tumors. The first hypothesis addresses whether Vascular Endothelial Growth Factor-C (VEGF-C), a lymphangiogenic factor, was sufficient to induce lymphatic function in tumors. The overexpression of VEGF-C in tumors leads to an increase in lymph node metastasis as well as structures that positively stain for lymphatic markers, but does not induce functional lymphatics within the tumor. Thus VEGF-C is not sufficient to grow functional lymphatic vessels in tumors. The second hypothesis addresses whether mechanical forces generated by the proliferation of cancer cells in a confined space compress lymphatic vessels in tumors. The mechanical forces inside of the tumor were reduced by the selective killing of human cancer cells grown in mice by Diphtheria Toxin. Tumor cell death leads to an increase in the fraction of lymphatics with open lumen. In addition, lymphatic vessels with open lumen are surrounded by a lower cellular density than collapsed vessels. Thus, relieving solid stress allows lymphatic vessels to open. However, function was not restored in these vessels. This is presumably due to the inability of the lymphatic vessels to completely open along its entire length, leaving focal areas of lymphatic collapse. Compressive forces are common to all growing tumors, giving credence to the mechanical etiology of the absence of functional lymphatic vessels in tumors, regardless of tumor type or organ site.
(cont.) These findings lead to an interesting question: Does cancer treatment in humans relieve the mechanical compression allowing lymphatic and blood vessels to open? Furthermore, would the resumption of function of compressed blood and lymphatic vessels lead to a paradoxical increase in metastasis? These questions require further investigation.
by Timothy P. Padera.
Ph.D.

Pathophysiology of Canine Fucosidosis by Jessica L Fletcher Understanding processes involved in the onset and progression of fucosidosis, a childhood neurodegenerative lysosomal storage disease, is critical to the development of effective treatment. Fucosidosis is caused by deficiency of the lysosomal hydrolase α-L-fucosidase due to inherited mutations in the FUCA1 gene. Canine fucosidosis, the only preserved animal model, was used to characterise preclinical pathophysiology, to establish potential markers of disease progression and test their capacity to measure responses to early therapy. Statistical interrogation of clinical data revealed that clinical signs are consistent from 12 months, and that rapid decline is signalled by sensory dysfunction from 18 months of age. Regression analysis against existing neuropathological data identified apoptosis and neuroinflammation as significant contributors to clinical dysfunction. Gene expression profiling of the frontal cortex at 16 weeks identified neuroinflammation to be prominent in fucosidosis pathogenesis (33% of differentially expressed genes) with both neuroinflammatory and mitochondrial-mediated pathways contributing to apoptotic cell death. Myelin genes were significantly downregulated, prompting investigation of oligodendrocyte populations in preclinical disease. Mature oligodendrocytes were reduced by 45-67%. Intracisternal enzyme replacement therapy from 8-16 weeks improved oligodendrocyte survival by 21%, and 29% of Purkinje neurons demonstrated reduced vulnerability to apoptosis. Neuroinflammatory cytokines and chemokines were investigated in cerebrospinal fluid to identify candidate biomarkers. MCP-1/CCL2 and KC/CXCL1 demonstrated potential, with significant increases with disease progression, and reductions with enzyme replacement therapy. Overall, these findings provide tools for disease management and a revised model of pathophysiology which can be used as a framework for future investigations into fucosidosis pathogenesis and therapy.

Ageing is a natural process, which is characterised by progressive decline in physiological functions and increased susceptibility to disease and death. Brain is particularly susceptible to structural and functional changes, which is more evident in disorders associated with ageing such as Alzheimer disease (AD). Copper is necessary for the protection against oxidative stress, energy production and neurotransmitter processing in the brain. However, higher copper levels can increase oxidative stress, resulting in neuronal damage. In order to avoid copper induced cytotoxicity, cells have to regulate copper levels through distribution into three intracellular pathways. By identifying changes in the copper pathways in the healthy and AD brain and by estimating the effects of copper chelation or supplementation in model cell line a better understanding of copper function in the brain will be obtained. In order to accomplish that copper, activity and protein levels of cytochrome c oxidase (COX) and superoxide dismutase (SOD) were measured in the healthy, AD brain and in HEK293 cell treated with copper chelators or supplemented with copper. Copper concentration was significantly decrease by more than 40% in healthy ageing brain and in the AD brain. Copper loss did not seem to affect the activity or protein level of the COX and SOD, since their levels were significantly increased in the ageing and AD brain. On the other hand, cells treated with copper chelators for three days faced a more than 75% decrease in intracellular copper concentration, which led to a more than 85% inhibition of the COX and SOD activity. Copper levels should be regulated properly in order to meet body’s metabolic demands and avoid cytotoxicity. Brain seems to have a mechanism where its energy demands have to be fulfilled even under low copper concentrations. Whereas, the prolonged and severe copper loss can dramatically affect the energy production and antioxidant defence systems which could be fatal to the cells.

In under a century, melioidosis, the infection due to Burkholderia pseudomallei, has emerged from Whitmore’s series of glanders-like infections amongst the morphia addicts in Burma to a major cause of mortality in northeastern Thailand and northern Australia. Also endemic in other parts of south-east Asia, melioidosis may have varied presentations ranging from severe, overwhelming infection to chronic, low grade disease. Observational evidence had suggested that granulocyte colony stimulating factor (G-CSF), a naturally occurring substance produced by the body in response to infection, may have been useful in reducing the high mortality associated with the more severe forms of this infection. Other observations linked the occurrence of this disease to various environmental factors, such as contamination of drinking water and the annual rainfall. This thesis explores and attempts to quantify these associations. There are three parts to this thesis. In the first part, I reviewed the epidemiology and management of patients with melioidosis. The use of G-CSF and meropenem was associated with a fall in mortality, although other factors may have at least partially contributed to this effect. In the second part, I progressed towards a clinical trial of G-CSF. There was no other evidence supporting the use of G-CSF in severe sepsis and ethical issues precluded a trial in Darwin. There was not evidence from laboratory models of G-CSF action in melioidosis to support the use of G-CSF in patients, although there remained some doubt regarding the applicability of such models to human disease. I examined clinical methods to identify patients at high risk of death from melioidosis. A simple scoring system based on clinical and laboratory parameters was developed and externally validated. However, clinical definitions of severe sepsis appeared to be better predictors of mortality. A clinical trial based on clinical definitions was commenced in Thailand. In the final part, I explored the question of whether different strains or B. pseudomallei or different environmental conditions caused different patterns of infection. There was no evidence that strain types of this bacterium determine the pattern or severity of disease, but weather conditions appeared to influence the distribution of disease in northern Australia.

Thesis (Ph. D.) University of Missouri-Columbia, 2006.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on July 31, 2007) Includes bibliographical references.

This thesis explores the prevalence, nature and pathogenesis of injuries in boxing. Following an introductory chapter and literature review (Chapters 1 and 2 respectively); Chapter 3 examines injuries in the GB boxing squad from 2005 to 2009. There were a total of 66 boxers on the squad during this period 61% were injured, a total of 297 injuries were recorded. The injury rate in competition was at least 460 times higher than in training, and most injuries were new rather than recurrent (246 v 51). The incidence of concussion is comparatively low compared to other studies in amateur boxing (5 in 5 years). Hand and wrist injuries were the most frequent (23.2%). Chapter 4 describes the nature of hand and wrist injuries in more detail. ‘Boxers’ knuckle’, skiers thumb, Bennett’s fracture and carpometacarpal instability were the most frequent hand and wrist injuries and also took the longest time to recover compared to all other hand and wrist injuries that occurred. These injuries occur significantly more frequently in competition than in training (347 injuries per 1,000 hours in competition less than 0.5 per 1000 hours in training). Chapter 5 describes efforts to identify and validate a means to measure the pressure at each knuckle, given that ‘boxers’ knuckle’ was found to be such a debilitating injury. This does differentiate between the proportion of knuckle impact forces (PKIF) displayed during punching and no punching but displays very poor test-re-test reliability. This method might allow the impact of changes in the hand wraps or the gloves to be measured. Chapters 6 and 7 deal with head injury in boxing. Head guards were removed from amateur boxers in 2013. The effect of this removal on boxers’ health was investigated by reviewing the number of bouts stopped due to blows to the head both with and without head guards (Chapter 6). To improve the quality of this analysis, an examination of video from championships with and without head guards (Chapter 7) was carried out. A significant decrease in observable signs of concussion (p < 0.05) and a significant increase in cuts (p < 0.001) was observed when the head guards were removed. This work will have implications for the protection of boxers’ hands and the use of head guards in other contact sports.

Normal pressure hydrocephalus (NPH), a CSF circulation disorder, is important as a reversible cause of gait and cognitive disturbance in an aging population. The inconsistent response to CSF shunting is usually attributed to difficulties in differential diagnosis or co-morbidity. Improving outcome depends on an increased understanding of the pathophysiology of NPH. Specifically, this thesis examines the contribution of, and inter-relationship between, the brain parenchyma and CSF circulation in the pathophysiology of NPH. Of the four core studies of the thesis, the first quantifies the characteristics of the CSF circulation and parenchyma in NPH using CSF infusion studies to measure the resistance to CSF absorption and brain compliance. The second study assesses cerebral blood flow (CBF) was using O15-labelled positron emission tomography (PET) with MR co-registration. By performing CSF infusion studies in the PET scanner, CBF at baseline CSF pressure and at a higher equilibrium pressure is measured. Regional changes and autoregulatory capacity are assessed. The final study examines the microstructural integrity of the parenchyma using MR diffusion tensor imaging. These studies confirm the importance of the inter-relationship of the brain parenchyma and CSF circulation. NPH symptomatology and its relationship to the observed regional CBF reductions in the basal ganglia and thalamus are discussed. Regional CBF reductions with increased CSF pressure and the implications for autoregulatory capacity in NPH are considered. The reduction in CBF when CSF was increased was most striking in the periventricular regions. In addition, periventricular structures demonstrated increased diffusivity and decreased anisotropy. The relationship between these changes and mechanisms such as transependymal CSF passage are reviewed. The findings of this thesis support a role of both the CSF circulation and the brain parenchyma in the pathophysiology of NPH. The results have implications for the approach to the management of patients with NPH.

Normal pressure hydrocephalus (NPH), a CSF circulation disorder, is important as a reversible cause of gait and cognitive disturbance in an aging population. The inconsistent response to CSF shunting is usually attributed to difficulties in differential diagnosis or co-morbidity. Improving outcome depends on an increased understanding of the pathophysiology of NPH. Specifically, this thesis examines the contribution of, and inter-relationship between, the brain parenchyma and CSF circulation in the pathophysiology of NPH. Of the four core studies of the thesis, the first quantifies the characteristics of the CSF circulation and parenchyma in NPH using CSF infusion studies to measure the resistance to CSF absorption and brain compliance. The second study assesses cerebral blood flow (CBF) was using O15-labelled positron emission tomography (PET) with MR co-registration. By performing CSF infusion studies in the PET scanner, CBF at baseline CSF pressure and at a higher equilibrium pressure is measured. Regional changes and autoregulatory capacity are assessed. The final study examines the microstructural integrity of the parenchyma using MR diffusion tensor imaging. These studies confirm the importance of the inter-relationship of the brain parenchyma and CSF circulation. NPH symptomatology and its relationship to the observed regional CBF reductions in the basal ganglia and thalamus are discussed. Regional CBF reductions with increased CSF pressure and the implications for autoregulatory capacity in NPH are considered. The reduction in CBF when CSF was increased was most striking in the periventricular regions. In addition, periventricular structures demonstrated increased diffusivity and decreased anisotropy. The relationship between these changes and mechanisms such as transependymal CSF passage are reviewed. The findings of this thesis support a role of both the CSF circulation and the brain parenchyma in the pathophysiology of NPH. The results have implications for the approach to the management of patients with NPH.

This thesis describes a series of studies involving healthy subjects, carefully selected patients with functional movement disorders and organic movement disorders, in which different aspect of the mechanism underlying functional movement disorders were explored: 1. The presence of physical precipitating factors at onset of functional movement disorder by using semistructured interviews. I found that most patients with functional movement disorder have a clear physical event prior to the onset of functional symptoms. 2. The presence of a “jumping to conclusions” reasoning style that may predispose patients with functional movement disorder to accept new hypothesis on the basis of less evidence. They requested less evidence that healthy controls to make a judgement, which is here suggested to influence the manner in which they process novel sensory data occurring during triggering events. 3. The role of attention in symptoms production by using different motor tasks in which the predictability of movements as well as the effect of explicit and implicit strategies in motor control were manipulated. Motor impairment in patients with functional movement disorder was found to be related to the employment of explicit strategies or when pre-planning movements is possible. 4. The intensity and duration of tremor in patients with functional tremor in a real life situation using accelerometers. They were found to fail to perceive 6 that tremor is not present most of the time compared with patients with organic tremor. 5. Finally, I explored the phenomenon of the sensory attenuation using a force-matching task as a measure of sense of agency for movement in these patients. Patients with functional movement disorders have an abnormal sensory attenuation for movement, which may help to explain the lack of agency for the abnormal movement. These results contribute to the understanding of the mechanisms underlying functional movement disorders and by extension, other functional neurological symptoms, and demonstrate that they are amenable to neuroscientific study.

The value of a nursing course revision for the purpose of improved student learning and outcomes is discussed in this descriptive, quasi-experimental study. An instructor of a nursing pathophysiology course devised and implemented the study to determine if outcomes would differ between two groups of first semester baccalaureate nursing students taking the required pathophysiology course. The revisions, which were applied and then evaluated, demonstrated improvement in student learning.

Aims of the thesis: To study the mechanisms of chemokine production that may occur during hepatic disease, the role of chemokines in hepatic injury and repair following paracetamol poisoning and hepatic expression of chemokines in patients with liver disease. In addition, the roles of tumour necrosis factor alpha (TNF) and stem cell factor (SCF) were also studied following paracetamol poisoning. Results: Both CXC and CC chemokines were produced during monocyte adhesion with human hepatoma cell lines. IL-8 production was dependent on proinflammatory cytokine production. In contrast, CC chemokine production appeared to be dependent on free radical activation of NF-κB. Direct TNF stimulated IL-8 production was mediated by TNF RI receptors via a protein kinase C pathway and was inhibited by dexamethasone. Both CXC and CC chemokines were detectable in human liver biopsies in patients with hepatitis C, hepatic allograft rejection and alcoholic hepatitis. Hepatic CXC and CC chemokines were also induced following paracetamol poisoning, inhibition of MIP2 and MIP1 alpha increased 3 day mortality and augmenting MIP2 expression was associated with accelerated hepatic regeneration. Hepatic TNF expression was not induced following paracetamol poisoning and inhibiting TNF was not associated with protection from hepatic injury. SCF was present within the liver at high concentration and located in both hepatocytes and bile ducts. Paracetamol poisoning was associated with reduced hepatic SCF concentrations and inhibiting SCF was associated with delayed hepatic regeneration. Conclusions: Both adhesion mediated and direct proinflammatory cytokine stimulation induce hepatic chemokine production. Chemokines are expressed in liver tissue from patients with hepatitis and therefore may be implicated in the pathogenesis of hepatic inflammation.

Hepcidin, the critical iron regulatory factor, is a small peptide produced by the hepatocytes in response to increased body iron and inflammation. Circulating hepcidin controls both intestinal iron absorption and the release of iron from macrophages into plasma via a negative iron feedback system. I developed a novel competitive immunoassay for hepcidin using a polyclonal antibody produced against synthetic hepcidin. I validated the immunoassay and determined it was able to discriminate between healthy controls and selected disease groups. I compared the immunoassay against another established method of measuring hepcidin. I established that plasma hepcidin has a diurnal rhythm and that plasma hepcidin increases in response to intravenous iron in anaemic patients. Elevated levels of hepcidin in renal failure may have a role in the erythropoietin resistance observed in renal anaemia. In haemodialysis patients, hepcidin levels were significantly elevated, but there was no correlation with inflammatory markers. Elevated hepcidin was associated with anemia, but erythropoietin dose was negatively correlated with hepcidin, suggesting that erythropoietin suppresses hepcidin levels. This was confirmed in patients when hepcidin levels significantly decreased after erythropoietin treatment. The association between plasma hepcidin and other iron parameters were also examined in healthy controls after erythropoietin administration and venesection. Profound hepcidin suppression was observed after an erythropoietin dose, with peak levels reduced by 73.2%, and then gradually recovering over the following two weeks. A similar but more gradual change in hepcidin was observed after reducing hematocrit by removal of 250 mL blood. The studies suggested that the marrow–hepcidin axis is regulated by factors other than those specifically investigated. In summary, I have developed and validated a novel immunoassay for hepcidin which will allow further investigation of the vital role of this peptide in iron homeostasis and human physiology.

Owing to its strategic position in the liver sinusoid, pathologic and morphologic alterations of the Liver Sinusoidal Endothelial Cell (LSEC) have far-reaching repercussions for the whole liver and systemic metabolism. LSECs are perforated with fenestrations, which are pores that facilitate the transfer of lipoproteins and macromolecules between blood and hepatocytes. Loss of LSEC porosity is termed defenestration, which can result from loss of fenestrations and/ or decreases in fenestration diameter. Gram negative bacterial endotoxin (Lipopolysaccharide, LPS) has marked effects on LSEC morphology, including induction LSEC defenestration. Sepsis is associated with hyperlipidemia, and proposed mechanisms include inhibition of tissue lipoprotein lipase and increased triglyceride production by the liver. The LSEC has an increasingly recognized role in hyperlipidemia. Conditions associated with reduced numbers of fenestrations such as ageing and bacterial infections are associated with impaired lipoprotein and chylomicron remnant uptake by the liver and consequent hyperlipidemia. Given the role of the LSEC in liver allograft rejection and hyperlipidemia, changes in the LSEC induced by LPS may have significant clinical implications. In this thesis, the following major hypotheses are explored: 1. The Pseudomonas aeruginosa toxin pyocyanin induces defenestration of the LSEC both in vitro and in vivo 2. The effects of pyocyanin on the LSEC are mediated by oxidative stress 3. Defenestration induced by old age and poloxamer 407 causes intrahepatocytic hypoxia and upregulation of hypoxia-related responses 4. Defenestration of the LSEC seen in old age can be exacerbated by diabetes mellitus and prevented or ameliorated by caloric restriction commencing early in life

Doctor of Philosophy(PhD)
Owing to its strategic position in the liver sinusoid, pathologic and morphologic alterations of the Liver Sinusoidal Endothelial Cell (LSEC) have far-reaching repercussions for the whole liver and systemic metabolism. LSECs are perforated with fenestrations, which are pores that facilitate the transfer of lipoproteins and macromolecules between blood and hepatocytes. Loss of LSEC porosity is termed defenestration, which can result from loss of fenestrations and/ or decreases in fenestration diameter. Gram negative bacterial endotoxin (Lipopolysaccharide, LPS) has marked effects on LSEC morphology, including induction LSEC defenestration. Sepsis is associated with hyperlipidemia, and proposed mechanisms include inhibition of tissue lipoprotein lipase and increased triglyceride production by the liver. The LSEC has an increasingly recognized role in hyperlipidemia. Conditions associated with reduced numbers of fenestrations such as ageing and bacterial infections are associated with impaired lipoprotein and chylomicron remnant uptake by the liver and consequent hyperlipidemia. Given the role of the LSEC in liver allograft rejection and hyperlipidemia, changes in the LSEC induced by LPS may have significant clinical implications. In this thesis, the following major hypotheses are explored: 1. The Pseudomonas aeruginosa toxin pyocyanin induces defenestration of the LSEC both in vitro and in vivo 2. The effects of pyocyanin on the LSEC are mediated by oxidative stress 3. Defenestration induced by old age and poloxamer 407 causes intrahepatocytic hypoxia and upregulation of hypoxia-related responses 4. Defenestration of the LSEC seen in old age can be exacerbated by diabetes mellitus and prevented or ameliorated by caloric restriction commencing early in life

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, life-threatening condition caused by an expansion of a clone of haemopoietic stem cells (HSC) harboring a somatic mutation of the PIG-A gene. PNH blood cells are deficient in glycophosphatidylinositollinked (GPI) proteins rendering PNH erythrocytes susceptible to complement attack which leads to intravascular haemolysis and an increased tendency to develop thromboses. The survival of 79 consecutive patients treated with eculizumab was compared to both age and sex matched normal controls and to 30 similar patients managed in the era immediately prior to anti-complement therapy. The survival of those treated with eculizumab was no different that of the control group and was significantly better than the group with PNH that did not receive eculizumab (p=0.3). Transfusion requirements and documented thromboses were reduced with eculizumab. Sixty-six percent of transfusion dependent patients became transfusion independent and thrombotic events reduced from 5.6 to 0.8 events per 100 years. Eleven women were monitored through 15 pregnancies whilst on eculizumab. There was 1 first trimester miscarriage and 15 healthy babies born. There were no maternal deaths observed and no thrombotic events occurred in the pregnancy or the postpartum period. Eculizumab did not appear to cross the placenta or be expressed in breast milk. Thirty-five patients were evaluated for PIG-M mutations to see if this mutation was prevalent in PNH. No PIG-M promoter mutations were identified. Two genes were evaluated to see if secondary mutations affecting them could account for clonal expansion in PNH. Thirty-six patients underwent JAK2 V617F mutation analysis with 1 patient shown to have a JAK2 mutation. Forty-two patients were evaluated for increased HMGA2 levels by 2 different PCR methods. There was an overall reduction in HMGA2 expression in PNH patients as compared to normal controls. An in vitro model of the bone marrow in PNH was developed and 18 PNH bone marrow samples were evaluated using this model. Colony forming assays (CFA) showed an increase in colony formation when T-cells were removed from the PNH bone marrow samples. This improvement was reversed when the T-cells were added back to the experiments. This work supports an immune mechanism for the expansion of the PNH clone.

Post-operative peritoneal adhesions are fibrous bands of tissue joining together organs occurring in the majority of patients following laparotomy, leading to complications such as abdominal pain, infertility in women and intestinal obstruction. However the structure and pathogenesis of adhesions is still not clear. It is proposed that persistence of fibrin between damaged tissues due to impaired plasminogen activator activity induces adhesion formation. This thesis aims to: (a) assess the structure of peritoneal adhesions in humans and in an experimental murine model (b) characterise and assess the growth of nerve fibres in adhesions and (c) to elucidate the role of fibrinolysis in adhesion formation in animals. Human peritoneal adhesions were collected from patients undergoing laparotomy, whereas murine adhesions were generated in a standardised murine model. Adhesion samples were processed and nerve fibres were characterized histologically, immunohistochemically, and ultrastructurally. In mice two-weeks post-operatively adhesions contained nerve fibres which were synaptophysin, calcitonin-gene-related- peptide, and substance-P-immunoreactive. At 4-weeks, nerve fibres originated from both the caecum and abdominal wall and by acetylcholinesterase-histochemistry were found traversing the entire adhesion. Nerve fibres were similarly present in all human adhesion specimens assessed. Ultrastructural analysis showed both myelinated and non myelinated nerve fibres were present in human and mouse adhesions. To elucidate the role of fibrinolysis a novel approach was taken using mice deficient in tissue plasminogen activator (t-PA) or urokinase plasminogen activators (u- PA) and inducing adhesion formation either by surgery or following an inflammatory episode. Mice deficient in t-PA were significantly more susceptible to adhesion formation following chronic inflammatory episode compared with u-PA deficient and wild-type mice. This thesis provides the first direct evidence that sensory nerve fibres grow into peritoneal adhesions suggesting that they may be capable of conducting pain sensation. Furthermore the persistence of fibrin due to decreased t-PA activity plays a major role in peritoneal adhesion formation.

Alzheimer’s disease (AD) is a devastating neurodegenerative disease with growing prevalence in our society. Patients suffering from this debilitating disorder also develop neuropsychiatric symptoms. Depression is one of the most frequently conveyed comorbidity; moreover, depression is also a risk factor associated with AD development. There is a complex interplay between the neurobiology of depression and AD, but their concomitant disease mechanisms remain largely unknown. Retraction of axons and dendrites has been reported to be a common occurrence in both illnesses, proposing the involvement of cytoskeletal dysfunction. Tau is a microtubule-associated protein that undergoes aberrant processing to form neurofibrillary tangles in neurodegenerative diseases such as AD. However, the role of tau in depression has not been well studied. The elucidation of pathophysiological mechanisms in depression is important to provide a more holistic understanding of AD pathogenesis. This study proposes the potential participation of tau phosphorylation in the pathogenesis of depression. In addition, this study will also investigate tau modifications under concomitant models of depression and AD. Primary cultures of hippocampal neurons were exposed to independent and cotreatments of corticosterone and β-amyloid (Aβ), to induce in vitro models of depression and AD, respectively. Sprague Dawley rats were subcutaneously injected with corticosterone for 14 days to induce an in vivo model of depression. Tau phosphorylation, aggregation and interaction with microtubules were examined. Results demonstrated that in both in vitro and in vivo models of corticosterone-induce depression, tau underwent increased phosphorylation at residues S396 and S404. Phosphorylated tau showed decreased interactions with microtubules and increased vulnerability to aggregate. Furthermore, the in vivo model of depression illustrated an altered localization of tau in the CA3 region of the hippocampus. Co-treatment of corticosterone and Aβ exacerbated aberrant tau phosphorylation and aggregation. In conclusion, this study provides evidence for the role of tau in depression, suggesting the occurrence of abnormal tau phosphorylation as an early event in the pathogenesis. Additionally, the pathophysiology of depression and AD may involve similar mechanisms in tau phosphorylation and aggregation. This study provides insight into the neurobiological linkages between depression and AD, and emphasizes the importance of tau-targeted interventions in neuropsychiatric disorders.
published_or_final_version
Anatomy
Master
Master of Philosophy

La sarna sarcóptica es una enfermedad parasitaria causada por el ácaro Sarcoptes scabiei. Afecta a mamíferos de todo el mundo, incluyendo al ser humano. En la fauna salvaje se considera una enfermedad emergente, pudiendo causar graves consecuencias poblacionales. La cabra montés (Capra pyrenaica) es un ungulado de montaña endémico de la Península Ibérica. Desde finales de los años 80, las poblaciones de cabra montés del sur y del este peninsular se han visto afectadas por esta parasitosis, con mortalidades variables que han llegado a superar el 90%. La mayoría de los estudios sobre la sarna en la cabra montés se han centrado en la epidemiología y los efectos poblacionales, por lo que no se conoce totalmente la fisiopatología y la patogenia de esta enfermedad en esta especie. En los dos primeros estudios de esta tesis, se analizaron las proteínas de fase aguda (PFA) (Estudio I) y se validó una prueba para la detección de inmunoglobulinas G (IgG) frente S. scabiei (Estudio II) en cabras monteses en libertad, tanto sanas como afectadas por sarna sarcóptica. En el Estudio I, se observó el aumento de las concentraciones de la proteína amiloide sérica tipo A y de la alfa-1 glicoproteína ácida, aunque en menor medida, en función de la extensión de las lesiones causadas por la sarna sarcóptica. Por el contrario, la concentración de haptoglobina no varió entre las cabras monteses sanas y las infestadas. Debido a la falta de un método diagnóstico de laboratorio efectivo, en el Estudio II se evaluaron tres ensayos por inmunoabsorción ligada a enzimas (ELISAs) para detectar IgG frente a S. scabiei en cabra montés, validando uno de los tres ELISAs que mostró una elevada especificidad y sensibilidad, al emplear el sistema de avidina-biotina. Los Estudios III y IV se llevaron a cabo con cabras monteses que presentaban diferentes alelos del gen DRB1 del complejo mayor de histocompatibilidad (MHC) clase II, infestadas experimentalmente con S. scabiei. Aunque todas las cabras infestadas desarrollaron lesiones compatibles con sarna sarcóptica, la evolución clínica varió desde lesiones extensas que afectaron la mayor parte de la superficie corporal hasta lesiones leves y recuperación clínica de la enfermedad (Estudio III). Sin embargo, estas diferencias clínicas no parecieron estar relacionadas con diferencias en el MHC. En las cabras monteses que desarrollaron cuadros clínicos graves de sarna se observó anemia, posiblemente relacionada con la inflamación causada por el ácaro, así como neutrofilia y linfopenia, probablemente debidas a las infecciones secundarias facilitadas por la sarna sarcóptica. La concentración de IgG también aumentó en función de la gravedad de las lesiones. Finalmente, en el Estudio IV se estudió la respuesta genómica de las cabras monteses frente a la infestación experimental con S. scabiei. En las cabras monteses con cuadros clínicos graves se observó un aumento de la expresión génica de vías relacionadas con la inmunidad y la inflamación, reflejo de la respuesta inmune generalizada, exacerbada e ineficaz inducida por el ácaro y de la respuesta frente las infecciones secundarias. Asimismo, las cabras monteses que se recuperaron mostraron un aumento de la expresión de genes relacionados con la presentación de antígeno y de activación de linfocitos T en la piel. Como resumen, la sarna sarcóptica produce cambios tanto sistémicos como locales, causando un aumento de PFA y anticuerpos, así como alteraciones hematológicas y en la expresión génica local y sistémica. Aunque las causas de las diferencias encontradas en la evolución clínica no han podido ser completamente dilucidadas, la inmunidad celular local cutánea puede ser clave en el control de la infestación. La detección de IgG mediante ELISA puede ser útil como método diagnóstico efectivo de la sarna sarcóptica en cabra montés, mientras que las PFA son un indicador pronóstico.
Sarcoptic mange is a parasitic skin disease caused by the burrowing mite Sarcoptes scabiei. It affects mammals worldwide, including humans. Sarcoptic mange in wildlife is considered an emerging disease, and can cause severe population declines. Iberian ibex (Capra pyrenaica) is a medium-sized mountain ungulate endemic to the Iberian Peninsula. Since the end of the ‘80s, the Iberian Ibex populations of Southern and Eastern Spain have been affected by mange, suffering variables mortalities reported to reach up to 90%. Most of the studies on sarcoptic mange in Iberian ibex have focused on the epidemiology and the population consequences of the diseases, thus existing a lack of knowledge about the pathophysiology and pathogenesis of this disease in this species. The two first studies of this thesis analysed the acute phase proteins (APP) (Study I) and validated a test for the detection of immunoglobulins G (IgG) against S. scabiei (Study II) in free-ranging Iberian ibexes, both healthy and affected by sarcoptic mange. In the Study I, an increase of serum amyloid protein type A (SAA) and in lower magnitude of alpha-1 acid glicoprotein (AGP) concentrations was observed, in correlation with the extent of the skin lesions caused by sarcoptic mange. Conversely, haptoglobin (Hp) concentration was not different between the healthy and infested ibexes. Since there is not an effective laboratory diagnostic method, in the Study II three enzyme-linked immunosorbent assays were evaluated for IgG detection against S. scabiei in Iberian ibex, and one of the three showed high specificity and sensitivity by using the avidin-biotin system, which allowed it to be validated. The Studies III and IV were carried out on Iberian ibexes with different alleles of the DRB1 gen of the major histocompatibility complex (MHC) class II, experimentally infested with S. scabiei. Although all the infested ibexes developed lesions compatible with sarcoptic mange, the clinical evolution varied from extensive lesions affecting most of the body surface to mild lesions and clinical recovering of the disease (Study III). However, such clinical differences seemed unrelated to MHC differences. The severely affected ibexes showed anaemia, possibly related to the inflammation caused by the mite, as well as neutrophilia and lymphopenia, probably due to secondary infections favoured by sarcoptic mange. Immunoglobulin G concentration also increased in agreement with the severity of the lesions. Finally, the Study IV addressed the genomic response of Iberian ibexes to the experimental infestation with S. scabiei. The severely affected Iberian ibexes showed an increase in the gene expression of pathways related to immunity and inflammation, agreeing with the exacerbated and non-effective generalized immune response induced by the mite and the response to secondary infections. Moreover, the Iberian ibexes that recovered showed an increase in the local skin expression of genes related with antigen presentation and T-lymphocytes activation. To summarize, sarcoptic mange induces both systemic and local changes in the Iberian Ibex, causing an increase in APP and antibodies, as well as haematological and local and systemic gene expression disorders. Although the causes of the differences found in the clinical evolution have not been completely elucidated, local skin cellular immunity may be key in controlling the infestation. Immunoglobulin G detection by ELISA can be a useful and effective diagnostic tool for sarcoptic mange in Iberian Ibex, while APP are a prognostic indicator.

This thesis investigates the physiology underlying central axon development and the pathophysiology of ischaemic injury to both immature and mature central axons, using the murine optic nerve model. A central theme to the work is that voltage gated Ca2+ channels (VGCCs) are involved in both axon development and injury. First, electrophysiological and immunohistochemical techniques were used to describe the expression of L-type and P/Q-type VGCCs at early developmental time points. Following this, two strains of mice, leaner and ducky2J, in which P/Q-type VGCC function is compromised, were used to ascertain what the function of this channel subtype might be. Malformation of nodes of Ranvier, disrupted targeting of nodal proteins and alterations in axon morphology, some subtle, some significant, are reported. These findings imply that P/Q-type VGCCs play an important role in regulating central axon development. Subsequent to this I investigated the hypothesis that VGCCs expressed during development contribute to ischaemic injury of developing axons. Simulation of ischaemia, by withdrawal of oxygen and glucose (OGD), resulted in irreversible injury and a minor role in the injury pathway could be ascribed to VGCCs; the major pathways are reported as being Ca2+ influx via ionotropic glutamate receptors. The final data offered in this thesis regard the ischaemic injury of mature, myelinated axons. N-methyl-D-aspartate (NMDA) receptor antagonism conferred a small but significant degree of protection against irreversible injury, indicating a change in the modality of OGD-mediated injury over time.

Barrett’s oesophagus is a common condition in which the normal stratified squamous oesophageal epithelium is replaced by metaplastic reflux-induced glandular (“columnar”) mucosa (Jankowski, Barr, Wang et al. 2010;Playford 2005). Over the last three decades, the incidences of oesophageal adenocarcinoma (OA) and Barrett’s oesophagus have risen to the point that OA is now common in the United Kingdom, with Scotland having one of the highest rates in the world (Jankowski, Provenzale, & Moayyedi 2002). Unfortunately most cancers present at an advanced stage with five year survival less than 30% (Holmes and Vaughan 2007). Barrett’s oesophagus is associated with malignant progression via a recognised metaplasia-dysplasia-carcinoma sequence (Jankowski, Wright, Meltzer et al. 1999). The premalignant nature of Barrett’s oesophagus has powered intense clinical interest in the hope of eventually having an impact on the earlier diagnosis and treatment of dysplasia, and ultimately the prognosis of oesophageal adenocarcinoma. Despite years of research interest, Barrett’s oesophagus remains an enigmatic condition. The exact incidence is unknown, and it is recognised that not all patients with Barrett’s oesophagus will progress to adenocarcinoma. Current strategies aim to ascertain the presence of dysplasia, the current gold standard marker of malignant progression. However although Barrett’s mucosa is visible at endoscopy, the presence of dysplasia is difficult to diagnose as these areas tend to be focal and inconspicuous to the naked eye. Current systematic biopsy regimes are recommended, but can be fraught with sampling errors. Furthermore, the molecular mechanisms underlying Barrett’s metaplasia and progression to dysplasia remain unclear. Molecular risk biomarkers have been sought with modest success, and at present dysplasia remains the most reliable clinical marker. However dysplasia itself is not without limitations: focal dysplasia can be difficult to ascertain, with many biopsies sometimes necessary to detect it reliably (Abela, Going, Mackenzie et al. 2008). Inter-observer variability may cause over or under diagnosis, especially regarding LGD (Flejou 2005). Moreover, although patients with HGD are at elevated risk of progression to OA, few studies provide reliable data on rates of progression from HGD to OA, with estimates varying between 16-59% at five years (Reid, Blount, Feng et al. 2000;Schnell, Sontag, Chejfec et al. 2001;Shaheen and Richter 2009;Spechler SJ 2011). There is a real need, therefore, to be able to identify and treat those patients at greatest risk of malignant transformation, and reassure those at low risk. Without an improved molecular understanding of Barrett's metaplasia and progression to neoplasia, clinically useful prognostic biomarkers (allowing appropriate targeting of surveillance and therapy) will be delayed. The current challenges associated with Barrett’s oesophagus are 1) to accurately determine the rate of malignant progression of Barrett’s oesophagus and identify clinical risk factors, 2) to improve the endoscopic detection of dysplasia and early neoplasia allowing earlier diagnosis and treatment and, 3) to understand the molecular mechanisms involved in the initiation of Barrett’s metaplasia, and the pathways involved in disease progression. In an attempt to improve the care of patients with Barrett’s oesophagus within the West of Scotland, my thesis will address each of the main challenges associated with this puzzling condition at clinical, endoscopic and molecular levels. The hypotheses of my thesis are threefold - a) Patients with Barrett's oesophagus in the West of Scotland have high rates of progression to high grade dysplasia and oesophageal adenocarcinoma. b) The WavSTAT optical biopsy system will be able to correctly identify non-dysplastic and dysplastic Barrett's oesophagus. c) The Wnt signalling pathway is upregulated in Barrett's oesophagus and dysplasia. The aims of my thesis are as follows: 1)To present a general overview of the Barrett’s literature highlighting current clinical challenges 2)To examine the incidence of dysplasia and oesophageal adenocarcinoma in the West of Scotland by analysing a cohort of patients undergoing surveillance endoscopy 3)To review the current endoscopic imaging adjuncts for the diagnosis of Barrett’s oesophagus and dysplasia, and assess the role of optical biopsy forceps in determining the presence of dysplasia 4)To evaluate the role of Wnt signalling in Barrett’s oesophagus, from metaplasia to carcinoma in a mouse model, with complementary human studies Chapter 1 introduces the reader to Barrett’s oesophagus and highlights current areas of clinical challenge and debate. A universal definition of Barrett’s oesophagus does not exist and Chapter 2 explores the need for the presence of intestinal metaplasia in the diagnosis of Barrett’s oesophagus. Chapters 3 and 4 present original data from a West of Scotland Barrett’s oesophagus database, specifically analysing rates of dysplasia and adenocarcinoma and cause of death. This study suggests patients with Barrett’s oesophagus in the West of Scotland are at high risk of disease progression with almost 10% of patients dying from oesophageal adenocarcinoma. The results highlight the importance of a comprehensive surveillance in our “high risk” population - an ideal niche for future chemopreventative and molecular studies. In an attempt to improve the diagnosis of dysplasia in our West of Scotland population, Chapter 5 reviews current endoscopic imaging adjuncts used in research and clinical practice while Chapter 6 presents original data from a pilot study assessing the use of innovative optical biopsy forceps in the endoscopic diagnosis of dysplasia. While this technology is in its infancy and further changes in the algorithm are required, the optical forceps could be a promising tool for ongoing surveillance in high risk Barrett’s patients. Chapter 7 summarises the role of biomarkers in Barrett’s oesophagus, reviewing the literature and highlighting the lack of clinically useful markers of disease progression to date. The Wnt signalling pathway plays an important role in normal oesophageal (and intestinal) development, yet when aberrantly activated leads to carcinogenesis. To date, very little is known about the role of Wnt signalling in Barrett’s oesophagus. Chapter 8 presents the results of a mouse model of upregulated Wnt signalling and the interesting finding of dysplasia within the oesophageal mucosa. Chapter 9 therefore translates these results to the human population by assessing the role of Wnt signalling in Barrett’s metaplasia and dysplasia by immunohistochemical analysis of a panel of markers. The results suggest Wnt signalling is upregulated in Barrett’s dysplasia, particularly in high grade, and this may have a future role as a biomarker. Chapter 10 summarises the main findings of the thesis, and presents future directions.