Stem and progenitor cells are an attractive cell source for the treatment of degenerative diseases due to their potential to differentiate into multiple cell types and provide large cell yields. Thus far, however, clinical applications have been limited due to inefficient differentiation into desired cell types with sufficient yields for adequate tissue repair and regeneration. The ability to spontaneously aggregate in suspension makes embryonic stem cells (ESCs) amenable to large-scale culture techniques for the production of large yields of differentiating cell spheroids (termed embryoid bodies or EBs); however, the introduction of hydrodynamic conditions may alter differentiation profiles within EBs and should be methodically examined. The work presented here employs a novel, laboratory-scale hydrodynamic culture model to systematically interrogate the effects of ESC culture hydrodynamics on cardiomyocyte differentiation through the modulation of a developmentally-relevant signaling pathway. The fluidic environment was defined using computational fluid dynamic modeling, and the effects of hydrodynamic conditions on EB formation, morphology and structure were assessed. Additionally, EB differentiation was examined through gene and protein expression, and indicated that hydrodynamic conditions modulate differentiation patterns, particularly cardiogenic lineage development. This work illustrates that mixing conditions can modulate common signaling pathways active in ESC differentiation and suggests that differentiation may be regulated via bioprocessing parameters and bioreactor design.

Current approaches to treat neurodegenerative disease provide only mild symptomatic relief but do not modify the natural history of these conditions. A large body of evidence suggests that mitochondrial dysfunction is a key event in the pathophysiology of neurodegeneration. Supporting and improving mitochondrial function has a big potential as a strategy for neuroprotection. The goal of this dissertation was to test whether interventions that target mitochondrial function are effective at preventing neurodegeneration induced by mitochondrial failure in vivo. A rodent model of optic neuropathy induced by the mitochondrial toxin rotenone was used to test the neuroprotective effects of methylene blue (MB) and near-infrared light (NIL), two interventions with mechanisms of action localized to mitochondria. This work also tested the effects of memantine, an NMDA receptor blocker, to further characterize the relationship between excitotoxicity and mitochondrial dysfunction. Neuroprotective effects were evaluated via behavioral testing of visual function and histopathological analysis of the retina. The neurochemical effects of MB, NIL and memantine were analyzed in vitro and in vivo with indicators of oxidative stress, cell respiration and catalytic activity of respiratory enzymes, including NADH dehydrogenase and cytochrome oxidase. MB, a diaminophenothiazine with potent antioxidant and unique redox properties, prevented the changes in visual function and the retinal histopathology induced by rotenone. In vitro, MB increased oxygen consumption and prevented the increases in oxidative stress in brain tissue induced by rotenone. NIL prevented the behavioral impairment and the decrease in retinal and visual pathway metabolic activity, retinal nerve fiber layer thickness and ganglion cell layer cell density induced by rotenone in a dose-dependent manner. Whole-brain cytochrome oxidase and superoxide dismutase activities were also increased in NIL-treated subjects in a dose-dependent manner, suggesting an in vivo transcranial effect of NIL. Finally, uncompetitive NMDA receptor blockade with memantine displayed neuroprotection against rotenone-induced neurodegeneration in a dose-response manner, and this effect was associated with a decrease in retinal oxidative stress and a long-term increase in neuronal energy metabolism capacity. These data constitute a proof-of-principle that interventions that target the mitochondria and support the function of the respiratory chain are effective at preventing neurodegeneration in vivo. / text

A high frequency of retinitis pigmentosa (RP) is found in Northern Sweden. In an inventory of autosomal recessive RP patients in Västerbotten County, a great number of cases with a unique phenotype was noticed, denoted Bothnia Dystrophy (BD). The aim of the study was to describe the phenotype, to determine the chromosomal location, and to identify the gene. Patients typically show night blindness from early childhood. Symptoms of defect macular function with a decrease of visual acuity can appear in early adulthood. The retinal fundus shows irregular white spots in a central, and parafoveal pattern along the arcades. Centrally areolar maculopathy develops and round circular atrophies are observed in the periphery. The disease was shown to be associated with a missense mutation in the RLBP1 gene resulting in an amino acid substitution (R234W) in the cellular retinaldehyde-binding protein (CRALBP). The R234W mutation was found in a homozygous state in 61 patients affected with BD. Ten patients were heterozygous for the R234W mutation, and presented a similar phenotype. No additional mutations in the coding sequence or exon-intron junctions were found. CRALBP is localised in retinal pigment epithelium (RPE), and Müller cells of the retina. In the RPE, CRALBP functions as a carrier protein for endogenous retinoids. Dark adaptometry and electrophysiologic testing showed an initial loss of rod function followed by a progressive reduction of the cone responses in older ages. A compromised rod function, dysfunction of the Müller cells, and indications of a disturbed function of the inner retina were found. With prolonged dark adaptation, a gradual increase in retinal sensitivity to light and an improvement of the ERG components occurred. The findings indicate a prolonged synthesis of photopigments, retardation of the visual process in the retinal pigment epithelium and a loss of retinal cells probably starting at a relative early age in BD. To evaluate the subjective visual function in BD patients, a battery of objective tests of visual function and composite score of the 25-item NEI-Visual Function Questionnaire (VFQ-25) were analyzed. We found that weighted distance logMAR visual acuity (WVA), had the strongest association with subjective visual function, and that there was a considerable loss of subjective and objective visual function with increasing age in BD patients. The prevalence of BD is as high as 1:3600 in Västerbotten County. The possibility that recycling of retinoids localized in the RPE might be impaired in BD might give future therapeutic possibilities. Due to the large and clinically well-characterized set of patients with this disease, they constitute a suitable study group.

The aim of the study was to assess the feasibility of CT based 3D analysis of degenerated aortic bioprostheses to make easier their morphological assessment. This could be helpful during regular follow-up and for case selection, improved planning and mapping of valve-in-valve procedure. The challenge was represented by leaflets enhancement because of highly noised CT images. Contrast-enhanced ECG-gated CT scan was performed in patients with degenerated aortic bioprostheses before reoperation (in-vivo images). Different methods for noise reduction were tested and proposed. 3D reconstruction of bioprostheses components was achieved using stick based region segmentation methods. After reoperation, segmentation methods were applied to CT images of the explanted prostheses (exvivo images). Noise reduction obtained by improved stick filter showed best results in terms of signal to noise ratio comparing to anisotropic diffusion filters. All segmentation methods applied to the best phase of in-vivo images allowed 3D bioprosthetic leaflets reconstruction. Explanted bioprostheses CT images were also processed and used as reference. Qualitative analysis revealed a good concordance between the in-vivo images and the bioprostheses alterations. Results from different methods were compared by means of volumetric criteria and discussed. A first approach for spatiotemporal visualization of 3D+T images of valve bioprosthesis has been proposed. Volume rendering and motion compensation techniques were applied to visualize different phases of CT data. Native valve was also considered. ECG-gated CT images of aortic bioprostheses need a preprocessing to reduce noise and artifacts in order to enhance prosthetic leaflets. Stick based methods seems to provide an interesting approach for the morphological characterization of degenerated bioprostheses.

Age related macular degeneration (AMD) is a public health concern in an aging society. The retinal pigment epithelium (RPE) layer of the eye is a principal site of pathogenesis for AMD. Morphological characteristics of the cells in the RPE layer can be used to discriminate age and disease status of individuals. In this thesis three genotypes of mice of various ages are used to study the predictive abilities of these characteristics. The disease state is represented by two mutant genotypes and the healthy state by the wild-type. Classification analysis is applied to the RPE morphology from the different spatial regions of the RPE layer. Variable reduction is accomplished by principal component analysis (PCA) and classification analysis by the k-nearest neighbor (k-NN) algorithm. In this way the differential ability of the spatial regions to predict age and disease status by cellular variables is explored.

Darbo uždaviniai: 1. Nustatyti pradinės AGDD paplitimą vidutinio amžiaus (40 – 64 metų) pacientų, sergančių IŠL grupėje bei atsitiktinėje to paties amžiaus Kauno miesto gyventojų imtyje. 2. Palyginti pacientų, sergančių tik IŠL ir IŠL bei pradinės AGDD klinikinius duomenis. 3. Nustatyti MMP-2 (-735 C/T), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T) genotipų dažnį bei genotipų derinių įtaką AGDD susiformavimui. 4. Nustatyti MMP-2 (-735 C/T), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T) genotipų dažnį esant minkštoms ir kietoms drūzoms, sergant AGDD. 5. Nustatyti MMP-2 (-735 C/T), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T) genotipų dažnį bei genotipų derinių įtaką AGDD ir IŠL drauge bei tik IŠL pasireiškimui. 6. Nustatyti funkcinio kontrastinio jautrumo tyrimo rodmenis pacientams, sergantiems pradine lengva ir pradine vidutine AGDD bei spalvų juslės pokyčius sergantiems pradine AGDD, ir oftalmologiškai sveikiems pacientams. / The goals were as follows: 1. To determine the prevalence of AMD in patients with IHD and compare with the prevalence in a random sample of Kaunas population (at 40-64 yrs old). 2. To compare the main clinical characteristics of the patients exhibiting early AMD and IHD together with the patients with IHD alone. 3. To determine the frequency of the genotypes of the matrix metalloproteinases (MMP-2 (-735 C/T), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T)), and genotype combinations that have an influence on the development of early AMD. 4. To determine the frequency of the genotypes of the matrix metallo¬proteinases (MMP-2 (-735 C/T), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T)), in early AMD patients with soft or hard drusen. 5. To determine the frequency of the genotypes of the matrix metallo¬proteinases (MMP-2 (-735 C/T), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T)), and the influence of genotype combi¬na¬tions on the development of AMD and IHD together, and only on IHD development. 6. To determine the results of functional acuity contrast sensitivity test in patients with early mild and early intermediate AMD, and color contrast sensitivity in patients with AMD, and in ophthalmologically healthy patients.

Didėjant vyresnių žmonių populiacijai amžinė geltonosios dėmės degeneracija (AGDD) yra vis dažnesnė vyresnio nei 50 metų amžiaus žmonių negrįžtamo regos netekimo priežastis. AGDD prevencinės priemonės bei gydymo galimybės ribotos, nes ligos etiopatogenezė iki šiol nėra visiškai aiški. Disertacinio darbo metu įvertintos AGDD ir išeminės širdies ligos (IŠL) sąsajos, atsižvelgiant į vainikinių arterijų aterosklerozinius pažeidimus. Pirmą kartą analizuota SCARB1 rs5888 C/T genotipų įtaka AGDD ir IŠL pasireiškimui. Tyrimo rezultatai parodė, kad AGDD ir IŠL, kai vainikinėse arterijose yra aterosklerozinių pažeidimų (IŠLath+), sieja bendri, šių ligų pasireiškimo galimybę didinantys, klinikiniai veiksniai ir oksidacinis stresas. Nustatytas „apsauginis“ SCARB1 rs5888 T/T genotipas, mažinantis AGDD ir IŠLath+, bei „rizikingas“ SCARB1 rs5888 C/T genotipas, didinantis AGDD+IŠLath+ galimybę. Pritaikius matematinės morfologijos metodus, nustatyta, jog sergantiems vėlyvąja AGDD SCARB1 rs5888 „rizikingas“ genetinis variantas susijęs su didesniu centrinės tinklainės dalies pažeidimo plotu. Bendrų AGDD ir IŠL patogenezinių grandžių tyrimas suteikia naujos informacijos apie AGDD etiologiją, patogenezę ir galbūt pasitarnaus efektyvaus gydymo bei prevencijos krypčių kūrimui. Tyrimo metu taikyti morfometriniai geltonosios dėmės pažaidos ploto matavimai gali būti naudojami gydytojų-oftalmologų klinikinėje praktikoje, siekiant tiksliau įvertinti centrinės tinklainės dalies pokyčius dinamikoje... [toliau žr. visą tekstą] / Age-related macular degeneration (ARMD) is the commonest cause of blindness among persons over the age of 50 and its prevalence is likely to increase as a consequence of population ageing. ARMD is a disorder of unknown cause and pathogenesis, therefore current options for ARMD prevention and treatment are limited. In the recent study the associations between ARMD and CAD, according the angiographic findings of atherosclerosis in the coronary arteries, were analyzed. The oxidative stress impact and clinical factors determining susceptibility to ARMD and CADath+, separately and common susceptibility factors for both diseases prediction were ascertained. Analysis of novel genetic biomarker, the rs5888 variant of SCARB1 gene, identified the „protective“ SCARB1 rs5888 TT genotype, associated with the lower risk of ARMD and CADath+, and a „risk-determining“ CT genotype, determining higher ARMD+CADath+ risk. The evaluation of macular lesion area by using the methods of mathematical morphology revealed that in late stage ARMD subjects carriers of SCARB1 rs5888 CT genotype the area of macular lesion was larger than in TT genotype carriers. New information about ARMD and CAD discovered additional knowledge about ARMD etiopathogenesis and might be helpfull in search of new treatments or strategies for ARMD prevention. Evaluation of macular lesion area by mathematical morphology methods used in this study may be useful in ophthalmological practice to monitor the dynamics of ARMD.

This research was designed to address important issues for the effective prescription of, and training in the use of, magnifiers for reading patients with visual impairment. The emphasis was on the development of simple methods of assessment and training that could be easily implemented, at no great cost, by low vision practitioners in clinical practice. To ensure that the results would be widely applicable, the research focused on subjects with age-related macular degeneration (AMD) using stand magnifiers (being the most common cause of low vision and the most commonly prescribed magnifiers respectively). From this research, modifications to the current methods of reading rehabilitation are suggested to more effectively improve low vision reading for the millions of people with low vision around the world. The magnification and reading performance achieved with the magnifier determined by the fixed acuity reserve method was as valid as that achieved with the magnifier determined by the individual acuity reserve method. The fixed acuity reserve is a simpler method to calculate the required magnification, as it requires only near visual acuity and the patient's goal reading task. This method was primarily used to select the appropriate illuminated stand magnifiers for the subjects participating in the subsequent studies and is recommended for use as the starting point in clinical low vision practice. The main study of this thesis was a longitudinal investigation of the benefit of large print reading practice on reading performance with stand magnifiers. Instead of the intensive training programs on magnifier use which have been suggested by previous studies, this study aimed to investigate the effect of simple large print reading practice, under either full or restricted field of view (the latter simulated by a practice stand), on reading rate with stand magnifiers for subjects with AMD. The experimental hypothesis was that reading practice prior to the prescription of stand magnifiers would improve reading performance with the stand magnifiers for subjects with AMD. As previous studies have shown, reading rate reduced when a stand magnifier was first introduced. One week of reading practice on large print, with or without a reduced field of view, gave an improvement in reading rate with the stand magnifier for passages of text (such that the reading rates with and without magnifiers were not significantly different). There was a suggestion that this practice may give a more rapid improvement in reading rate than that achieved by the control subjects who did not do any large print reading practice, but this did not reach statistical significance. Even very brief reading with the stand magnifiers by the control subjects gave some improvement in reading rate. Therefore, home or in-office reading practice on large print or with magnifiers is recommended for patients with AMD before magnifiers are prescribed. Subjects who had neither reading practice nor exposure to the magnifier prior to its prescription required two weeks practice using their stand magnifiers to achieve their maximum reading rate. This suggests that home practice in using stand magnifiers is beneficial and a follow up visit is recommended two weeks after the provision of a magnifier to assess any change in reading rate. If no improvement in the magnifier reading rate is found or the rate is less than the reading rate on large print without a magnifier, further investigations of the patients' vision and/or their magnifier manipulation strategy are necessary. In the last study, a simple method aimed at alleviating difficulties with magnifier manipulation and navigation, the attachment of a line guide to the base of the stand magnifier, was investigated using both objective methods (recording magnifier movements and reading rate measures) and subjective methods (simple questionnaire). Although there was no improvement in the objective measures of reading or navigation performance with the line guide, more than half of the subjects with low vision preferred to have the line guide on their stand magnifiers. This suggests that the objective measures might not be sensitive enough to predict the subjective response, or that other factors that were not measured in this study influenced subjects' preferences in selecting the line guide (e.g., psychological support provided by the line guide in reading orientation). Clinically, the subjective response of patients to the use of low vision aids as well as their motivation are important criteria for success in low vision rehabilitation. There was a tendency for less experienced users to prefer the line guide to assist their use of the stand magnifier for reading. Therefore, a line guide could be offered as a preliminary training aid when stand magnifiers are first prescribed for AMD patients. Possible improvements to the design of the line guide were identified. Further research is required to assess the benefits of this or similar devices for new magnifier users and to understand the difficulties that people with visual impairment have with page navigation in order to determine improved methods of training navigation strategies. The unique contribution of this study to the field of low vision rehabilitation is that the benefit of short-term reading practice, on large print or with magnifiers, as simple, cheap methods of enhancing reading performance with stand magnifiers was demonstrated. The results of this study have led to the development of recommendations for assessing and training AMD patients who are prescribed stand magnifiers.

[Truncated abstract] Sporadic inclusion body myositis (sIBM) is a chronic inflammatory disease that is the most common myopathy in individuals above the age of 50 in the Caucasian population. sIBM is characterised by cytotoxic immune infiltration of skeletal muscle, consisting primarily of CD8+ T-cells and macrophages, as well as a degenerative process, with muscle fibre vacuolation and intracellular filamentous inclusions. The pathogenesis of sIBM is likely to involve a complex interaction between genetic and environmental factors. Whilst the physiological and pathological characteristics of sIBM have been clearly identified, the exact origin and genetic basis of the disease remains unknown. A number of studies show that sIBM is associated with alleles of the major histocompatibility complex (MHC) on chromosome 6p21.3 and specifically with two ancestral haplotypes (AH) in Caucasians – the 8.1AH, defined by HLA-B*0801, HLA-DRB1*0301 and the 35.2AH, defined by HLA-B*3501, HLA-DRB1*0101. Mapping studies subsequently showed that sIBM susceptibility likely originates from a 389kb region of the MHC, spanning from centromeric of PBX2 to telomeric of HLA-DRB1. The central hypothesis of this thesis was that susceptibility to sIBM is conferred by a single allele found within a region defined using the 8.1AH, which is also carried by other haplotypes associated with sIBM. Three patient cohorts from Australia, the U.S.A and Japan were studied. ... Of the 32 alleles genotyped, none were found in all susceptibility haplotypes and one was common, but not unique, to the 8.1AH, 7.2AH and 52.1AH. Five SNPs were also found in two of the three haplotypes, although none were specific to the sIBM susceptibility haplotypes. These data suggest that the 8.1AH is likely to carry an sIBM susceptibility allele independent of the 35.2AH, 7.2AH and 52.1AH. Based on the possible mechanism of action in cellular differentiation and its location within the 8.1AH-defined sIBM susceptibility region reported in 2004, NOTCH4 was a strong candidate for conferring sIBM susceptibility. NOTCH4 coding region polymorphisms were thus investigated in a Caucasian patient cohort to assess any possible role in sIBM susceptibility. While the frequency of some alleles were increased in sIBM patients, the strong linkage disequilibrium throughout the MHC prevented confirmation of any alleles as playing a direct role in sIBM. The 8.1AH-derived sIBM susceptibility region was further refined using recombination mapping. This approach used markers characterised against multiple haplotypes to genotype patients carrying part of the 8.1AH to locate a common, overlapping susceptibility region. Recombination mapping of patients revealed a common overlapping region of the 8.1AH, extending from BTNL2 to HLA-DRB3. The results of the study indicate that 8.1AH-derived susceptibility for sIBM is likely to originate from a 172kb region encompassing HLA-DRA, HLA-DRB3 and part of BTNL2. These genes warrant further investigation in future studies.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.