Toward Optimization of Medical Therapies with a Little Help from Knowledge Management

This chapter emphasizes the importance of identifying and managing knowledge from Informally Structured Domains, especially in the medical field, where very short and repeated serial measurements are often present. This information is made up of attributes of both patients and their treatments that influence their state of health and usually includes measurements of various parameters taken at different times during the duration of treatment and usually after the application of the therapeutic resource. The chapter communicates the use of the KDSM methodology through a case study and the importance of paying attention to the characteristics of the domain to perform appropriate knowledge management in the domain

Identification of vascular endothelial growth factor receptor 3 (VEGFR3) as an in vitro and in vivo substrate of the Alzheimer's Disease linked protease BACE2

The protease ß-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) is a key drug target in Alzheimer’s disease (AD). It catalyzes the first step in the generation of the pathogenic amyloid ß (Aß) peptide and its inhibition is therefore a promising approach to prevent or delay the onset of AD. To date however, most inhibitory compounds do not discriminate between BACE1 and its close non-amyloidogenic homologue BACE2 and therefore may lead to undesired off target effects, resulting from BACE2 biology. Therefore, future compounds require a higher selectivity for BACE1 and a biomarker is required to confirm unimpaired in vivo BACE2 activity. To replace a long lasting depigmentation assay, which is the current standard for in vivo BACE2 activity monitoring, the blood plasma of BACE2 knockout mice (B2KO) was screened and the tyrosine kinase receptor Vascular Endothelial Growth Factor 3 (VEGFR3) was identified as a putative BACE2 substrate. Subsequently, VEGFR3 was thoroughly validated as an in vitro and in vivo BACE2 substrate and the BACE2 cleavage site was determined. In direct comparison to the pigmentation readout, plasma VEGFR3 performed superior and displayed higher sensitivity and lower variance. Importantly, reduction of VEGFR3 was also detectable in the plasma of BACE inhibitor treated non-human primates (NHP) and clinical trial participants, highlighting potential for applicability in the clinical context. To test whether BACE2 cleavage may be a novel mechanism to control VEGFR3 function, downstream events of VEGFR3 signaling were monitored in primary lymphatic endothelial cells (LECs). Impairment of BACE2 dependent VEGFR3 processing was accompanied by increased activation of the VEGFR3 dependent pathways AKT and ERK and resulted into enhanced transcription of the VEGFR3 inducible genes (FOXC2) and Delta-like 4 (DLL4). As a consequence, alterations in the morphological structure and drainage efficiency of lymphatic vessels and cannot be excluded in the periphery and central nervous system (CNS). Future developments in the BACE inhibitor field need to consider these implications and plasma VEGFR3 levels may be used to control for possible of target effects from BACE2 inhibition

Paraneoplastic Antibodies

Paraneoplastic neurological syndromes (PNS) are remote effects of cancer. These are not caused by invasion of the tumor or its metastasis nor by other direct effects of the tumor or its treatment. PNS are rare, affecting less than 0.1% of all cancer patients. PNS have a subacute course, leaving the patient severely disabled in weeks to months. In most cases PNS precede the diagnosis of cancer. The discovery of paraneoplastic antineuronal antibodies facilitated the diagnosis. ‘Well characterized onconeural antibodies’ are defined by recognizable patterns on rat brain immunohistochemistry and positive immunoblotting on recombinant antigen proteins. These are exclusively found in patients with cancer and include anti-Hu, Yo, CV2, Ri, Ma2 and amphiphysin. These antigens represent intracellular proteins, so in PNS damage is caused by cellular immune responses, explaining the poor response to immune modulating treatment and poor prognosis. We identified the anti-Tr antigen as the transmembrane protein Delta/ Notch-like epidermal growth factor-related receptor (DNER). More recently a still growing number of autoantibodies directed against synaptic or neuronal cell-surface antigens has been identified, including mGluR1, NMDA, AMPA and GABA receptors. These autoantibodies have direct access to their target antigen and are potentially pathogenic. The associated clinical syndromes may be paraneoplastic or may represent an autoimmune encephalitis (without underlying tumor). Patients harboring autoantibodies directed against synaptic or neuronal cell-surface antigens respond favorably to immunotherapy with a good outcome in up to 80%. This thesis focuses on paraneoplastic antineuronal antibodies and includes studies on new methods of autoantibody detection, identification of novel paraneoplastic antigen(s) and the description of clinical syndromes associated with newly detected paraneoplastic antibodies

Dopamiini retseptoritele ligandi sidumise uurimist võimaldavate katsesüsteemide arendamine

Väitekirja elektrooniline versioon ei sisalda publikatsioonePolüsahhariidid, nukleiinhapped ja valgud on eluks vajalikud biopolümeerid. Inimese organismis on neist kõige enim valke, mis on ühtlasi ka kõige mitmekülgsemad biomolekulid, seda nii oma struktuuri kui ka funktsiooni poolest. Valgud vastutavad mitmete eluks vajalike funktsioonide läbiviimise eest. Näiteks ensüümvalgud kiirendavad organismis toimuvaid keemilisi reaktsioone. Lisaks on valgud olulised ainete transportijad, pakuvad mehaanilist tuge ja immuunkaitset ning osalevad närvisignaali ülekandes. Käesolevas töös keskenduti retseptorvalkude, täpsemalt G-valguga seotud retseptorite uurimisele. Need valgud paiknevad raku membraanis, kus need vahendavad närvisignaali ülekannet raku väliskeskkonnast raku sisemusse. Signaaliülekande toimumiseks peab retseptoriga seostuma teatud tüüpi keemiline ühend, mida nimetatakse ligandiks. Ligandi seostumine retseptorile põhjustab retseptori struktuuri muutumise, mis omakorda mõjutab raku sees olevaid signaalmolekule. Dopamiini retseptorid on G-valguga seotud retseptorite perekonda kuuluvad valgud, mis vahendavad inimese organismis mitmeid olulisi funktsioone. Häired dopamiinergilises signaaliülekandes võivad põhjustada mitmesuguseid haigusi, millest tuntuimad on skisofreenia ja Parkinsoni tõbi. Seetõttu on dopamiini retseptorid olulised ravimite sihtmärgid ning detailsed teadmised dopamiini retseptorite toimimise kohta on äärmiselt vajalikud. Antud töö käigus keskenduti katsesüsteemide arendamisele, eesmärgiga iseloomustada ligandide seostumist erinevatele dopamiini retseptoritele. Katseid viidi läbi nii natiivsete retseptoritega (koeproovid) kui ka erinevate rekombinantsete dopamiini retseptoritega, mille saamiseks kasutati erinevaid ekspressioonisüsteeme (imetajarakud, putukarakud, pungunud bakuloviirused). Retseptorite ja ligandide vaheliste interaktsioonide iseloomustamiseks rakendati mitmesuguseid, põhiliselt fluorestsentsil põhinevaid meetodeid.Biopolymers, like polysaccharides, nucleic acids and proteins are essential for all living organisms. Besides water, proteins are the most abundant type of molecules in the human body. Proteins are also the most versatile of all biomolecules, performing many functions required for life. Some proteins have catalytic activity and function as enzymes, others serve as structural elements, signal receptors, or transporters that carry specific substances into or out of cells. This thesis focuses on the receptor proteins, specifically to G protein-coupled receptors that are located in the cell’s plasma membrane. These proteins are responsible for detecting signals outside the cell by binding various chemical compounds, called ligands. Ligand binding to a receptor can initiate a conformational change in the structure of the protein, which in turn leads to a chain of biochemical events inside the cell. Dopamine receptors, belonging to the family of G protein-coupled receptors, mediate several important functions in the human body. Abnormal dopaminergic signaling can lead to several neurological and psychiatric disorders, such as schizophrenia and Parkinson’s disease. Therefore, dopamine receptors are relevant drug targets and acquiring further information about the signal transduction process mediated by dopamine receptors is essential. This thesis focused on the development of novel assay systems to characterize ligand binding to different subtypes of dopamine receptors. Experiments were carried out with native receptors (tissue homogenates) and with various recombinant protein expression systems (mammalian and insect cells, budded baculovirus particles). Additionally, several methods were implemented to study receptor-ligand interactions with the emphasis on fluorescence-based methods

The Impact of glucose and glucoregulation on memory

The effect of glucose on memory has been investigated for in excess of 25 years, with some consensus generated amongst the literature indicating that glucose has a facilitating effect. However, the robustness of the glucose effect has been questioned, with a considerable body of evidence reporting no glucose facilitation of memory. It has been suggested that glucoregulatory control may be a key mediating factor of the glucose effect. Glucoregulatory control and cognitive functioning are intrinsically linked, with cognitive impairments a common feature in populations presenting with poor glucoregulatory control such as diabetics, Alzheimer‘s disease sufferers, schizophrenics and the elderly. Although again the evidence has proven contradictory, with evidence to suggest that both better and poorer glucoregulators are more / less susceptible to the glucose effects on cognition. Verbal declarative memory has been reported to be the most reliably enhanced aspect of memory to benefit from a glucose effect. However, it is not yet clear whether verbal declarative memory as a whole is being facilitated, or whether the different phases of memory (encoding, consolidation, retrieval etc.) are differentially targeted. Consequently the primary aim of this thesis was to evaluate the effect of glucoregulatory control and glucose, on the different phases of verbal declarative memory. This was achieved through the use of novel paradigms employed previously within the cognitive sciences literature. Chapter 2 addressed a secondary aim of this thesis; investigating the current gap in the literature pertaining to the effect of glucose administration on cognition in children. Chapter 3 investigated the types of recognition (recollection and familiarity) that were made subsequent to a glucose load, using the ?remember/know‘ paradigm. Chapter 4 investigated encoding efficiency during the item method directed forgetting paradigm, in which participants actively attempt to forget specific stimuli through cessation of encoding. In chapters 5 and 6 the potential mediation of inhibition processes was explored, with both semantically related (Retrieval Induced Forgetting paradigm) and orthographically similar but semantically unrelated stimuli (Memory Blocking Effect paradigm). The tentative evidence presented in this thesis indicates that glucoregulatory control may mediate the glucose facilitation effect during the encoding phase, with better regulators seemingly benefiting from greater encoding benefits than poorer following glucose. Glucose was not observed to influence inhibition processes, or types of recognitions made. However, better glucoregulators exhibited more efficient adaptive inhibition (overcoming inhibition of blocking items to continue searching the lexicon and increased inhibition of semantically related competing stimuli). Administration of glucose did not mediate cognition in children, with the exception of an impairment of performance on a challenging reaction time task following 20 g of glucose. Memory phases are seemingly differentially affected by glucose administration, with the effect mediated by glucoregulatory control. Utilising the paradigms employed here (or similar) to investigate a range of populations presenting with cognitive decline / glucoregulatory control, would further allow the glucose and glucoregulatory effects on the different phases of memory to be further disentangled

Glutamatergic Metabolites and Gray Matter Losses in Schizophrenia: A Longitudinal Study Using In Vivo Proton Magnetic Resonance Spectroscopy

Approximately one in hundred people suffer from schizophrenia. Current medications partially improve the symptoms. There is no cure. Glutamate, an excitatory neurotransmitter, is a possible cause of the schizophrenia symptoms. Excessive glutamate release eventually leads to neurodegeneration. Longitudinal studies are necessary to observe the neurodegenerative process. Seventeen schizophrenia patients and 17 healthy volunteers underwent proton magnetic resonance spectroscopy (MRS) and imaging to measure neurochemical and structural changes in vivo. Metabolite levels were measured from a 1.5cm3 voxel in the anterior cingulate and thalamus using the stimulated echo acquisition mode sequence. Gray matter (GM) was assessed with voxel-based morphometry and ANALYZE. Total glutamatergic metabolite (tGL), N-acetylaspartate (NAA), and GM were significantly decreased in schizophrenia over 80 months. Reduced tGL and NAA levels were significantly correlated with GM changes. tGL loss was negatively correlated with social functioning. Significantly decreased tGL levels were possibly associated with GM loss in the spectroscopy voxel. Metabolite signal-to-noise ratio, but not quantification, was decreased as a function of MR system age. These findings demonstrate the feasibility of long-term MRS studies and implications for the pathophysiology of schizophrenia. tGL and GM losses were consistent with neurodegeneration but the effects of an early neurodevelopmental lesion or the effects of chronic medication cannot be ruled out. Structural and metabolite changes in these patients implicate glutamate as a possible target of medication in this disorder. The association between tGL loss and social functioning suggests it might be possible to arrest deterioration with pharmaceuticals that target glutamate