Under år 2003-2009 utgjorde hypertensiva sjukdomar, såsom eklampsi och preeklampsi, 14,0% av värdens mödradödlighet. Preeklampsi kännetecknas vanligtvis av kliniska observationer av hypertoni och signifikant proteinuri i graviditetens andra trimester. Inom diagnostiken används en riskbedömningsprogramvara som kan beräkna vilken sannolikhetsgrad den havande kvinnan har för att utveckla preeklampsi. Förutom mätning av blodtryck och proteinuri har biomarkören placenta growth factor 1 (PlGF-1) visat ett högt prediktivt värde vid bedömningen. Studien syftade till att kartlägga och fördjupa sig i metoderna som analyserar biomarkören samt verifiera metoden för PlGF på instrumentet Brahms Kryptor compact plus. Fördjupningen baserades på granskning av vetenskapliga artiklar och resulterade i två manuella och tre automatiserade metoder. Metoden Quantikine användes i 47% av artiklarna och konstaterades som studiens golden standard. Vid jämförelse av metoderna sågs en lägre bakgrundsstörning, en högre sensitivitet samt en kortare analystid hos de automatiserade metoderna. Den laborativa verifieringen innefattade bestämning av överensstämmelse med externt laboratorium, beräkning av instrumentets provsmitta mellan höga och låga prov samt kvantifiering av inomserie- och mellanliggande precision. Verifieringen resulterade i en god överensstämmelse (r=0,953, p=0,327) med det externa laboratoriet, en konstaterad provsmitta på 0,04% samt en god precision inom leverantörens angivelser. / Between 2003-2009 hypertensive disorders as eclampsia and preeclampsia constituted 14.0% of the world’s maternal mortality. Preeclampsia characterize as clinical observations of hypertension and significant proteinuria in the second trimester of pregnancy. In diagnostics a risk assessment software is normally used to estimate the probability of developing the disorder. Besides calculating the blood pressure and proteinuria, the placenta growth factor 1 (PlGF-1) has proven to possess a high predictive value. The study’s aim was to chart the different methods used to quantify the biomarker and verify the method for PlGF on Brahms Kryptor compact plus. The recess was based on review of scientific articles and resulted in the findings of two manual and three automated methods. The method Quantikine was used in 47% of the articles and was seen as the golden standard of the study. When comparing the methods a lower signal to noise-ratio, a higher sensitivity and a shorter assay time was observed in the automated methods. The verification contained determination of compliance with an external laboratory, calculation of carry over and quantification of inter-assay and intra-assay precision. The verification resulted in a good compliance (r=0.953, p=0,327) with the external laboratory, a carry over at 0,04% and a good precision within the providers indication.

The dynamics of cell signalling are critical to cell fate decisions. Human Epidermal growth factor Receptors (HERs)-mediated Ras/Raf/MEK/ERK and PI3K/Akt signalling cascades relay extracellular signals from the plasma membrane to targets in the nucleus and cytoplasm and play pivotal roles in cell fate determination including proliferation, differentiation and cell survival. Both pathways, once activated, are further regulated by complex feedback loops which may exert either positive or negative effects on cascade components and can result in signalling oscillation. In this study, heregulin (HRG) - and epidermal growth factor (EGF)- stimulated oscillation of both p-ERK1/2 and p-Akt expression in breast cancer cell lines was demonstrated. The oscillation was cell line dependent and was observed in MCF-7 and MCF-7/HER2-18 cells but not in BT474 cells. The oscillation was augmented by cycloheximide implicating transcriptional involvement. Gene expression analysis identified 29 genes as possible candidates involved in the transcriptional feedback regulation. Apart from the feedback regulation, feedforward regulation was also observed. To expedite the analyses In-cell Western and Reverse Phase Protein Array (RPPA) assays were developed. A scheme of transcriptional feedback loops regulating the oscillation in the ERK1/2 pathway is proposed, including negative feedback loops to ERK1/2 from DUSPs, early positive and late negative feedback loops to MEK1/2 and positive feedback loops to HER-3 from AREG, HB-EGF, CYR61 and CTGF. Two HER-2-targeted inhibitory monoclonal antibodies were investigated – trastuzumab and pertuzumab. Trastuzumab not only inhibited the growth of HER-2 over-expressing MCF-7/HER2- 18 cells and BT474 cells but also that of EGF-driven MCF-7 cells which expressed low/moderate HER-2 levels. Pertuzumab blocked the growth of both MCF-7 and MCF-7/HER2-18 driven by either EGF or HRG. When used in combination with trastuzumab, pertuzumab had much more potent activity in inhibiting cell growth and signalling than either single drug. Trastuzumab and pertuzumab had opposing effects on immediate p-ERK1/2 signalling and trastuzumab’s effects on signalling could be mimicked by the PI3K inhibitor LY294002. PTPN13, a non-receptor type tyrosine protein phosphatase, is a proposed tumour suppressor in breast cancer. This was investigated as a candidate regulator of the signalling oscillation and although not observed as a transcriptional modulator of the oscillation, its high expression level was observed to be associated with cell growth inhibition in MCF-7/HER2-18 cells by trastuzumab. Moreover, immunohistochemical analysis of 121 clinical tumours which had received trastuzumab treatment revealed the correlation between the expression level of PTPN13 and the mutation status of PIK3CA. In conclusion, the observed oscillation may contribute to the elucidation of the complex regulation of signalling pathways, which is vital to the different cell fate decision made through the same core pathway. The synergy between trastuzumab and pertuzumab supports the clinical use of the combination treatment and suggested PI3K/Akt pathway as the major pathway in controlling tumour growth.

Traumatic injuries to the spinal cord (SCI) and peripheral nerve (PNI) affect several thousand people worldwide every year. At present, there is no effective treatment for SCI and despite continuous improvements in microsurgical reconstructive techniques for PNI, many patients are still left with permanent, devastating neurological dysfunction. This thesis investigates the effects of mesenchymal stem cells (MSC) derived from adipose (ASC) and dental (DSC) tissue and chitosan/microRNA-124 polyplex particles on regeneration after spinal cord and peripheral nerve injury in adult rats. Dental stem cells were obtained from apical papilla, dental pulp, and periodontal ligament. ASC and DSC expressed MSC surface markers (CD73, CD90, CD105 and CD146) and various neurotrophic molecules including BDNF, GDNF, NGF, VEGF-A and angiopoietin-1. Growth factor stimulation of the stem cells resulted in increased secretion of these proteins. Both ASC and DSC supported in vitro neurite outgrowth and in contrast to Schwann cells, ASC did not induce activation of astrocytes. Stimulated ASC also showed an enhanced ability to induce capillary-like tube formation in an in vitro angiogenesis assay. In a peripheral nerve injury model, ASC and DSC were seeded into a fibrin conduit, which was used to bridge a 10 mm rat sciatic nerve gap. After 2 weeks, both ASC and DSC promoted axonal regeneration in the conduit and reduced caspase-3 expression in the dorsal root ganglion (DRG). ASC also enhanced GAP-43 and ATF-3 expression in the spinal cord, reduced c-jun expression in the DRG and increased the vascularity of the implant. After transplantation into injured C3-C4 cervical spinal cord, ASC continued to express neurotrophic factors and laminin and stimulated extensive ingrowth of 5HT-positive raphaespinal axons into the trauma zone. In addition, ASC induced sprouting of raphaespinal terminals in C2 contralateral ventral horn and C6 ventral horn on both sides. Transplanted cells also changed the structure and the density of the astroglial scar. Although the transplanted cells had no effect on the density of capillaries around the lesion site, the reactivity of OX42-positive microglial cells was markedly reduced. However, ASC did not enhance recovery of forelimb function. In order to reduce activation of microglia/macrophages and the secondary tissue damage after SCI, the role of microRNA-124 was investigated. In vitro transfection of chitosan/microRNA-124 polyplex particles into rat microglia resulted in the reduction of reactive oxygen species and TNF-α levels and lowered expression of MHC-II. Upon microinjection into uninjured rat spinal cords, particles formed with Cy3-labeled control sequence RNA, were specifically internalized by OX42 positive macrophages and microglia. Alternatively, particles injected in the peritoneum were transported by macrophages to the site of spinal cord injury. Microinjections of chitosan/microRNA-124 particles significantly reduced the number of ED-1 positive macrophages after SCI. In summary, these results show that human MSC produce functional neurotrophic and angiogenic factors, creating a more desirable microenvironment for neural regeneration after spinal cord and peripheral nerve injury. The data also suggests that chitosan/microRNA-124 particles could be potential treatment technique to reduce neuroinflammation.

<p>Heparan sulfate (HS) belongs to the glycosaminoglycan family of polysaccharides and is found attached to protein cores on cell surfaces and in the extracellular matrix. The HS backbone consists of alternating hexuronic acid and glucosamine units and undergoes a number of modification reactions creating HS chains with alternating highly and low modified domains, where high degree of modification correlates with high negative charge. Fibroblast growth factors (FGFs) and their receptors (FRs) both bind to HS, which affect formation of the FGF–FR complexes on the cell surfaces. Activated FRs can trigger several intracellular signaling pathways leading thereby to diverse cellular responses. </p><p>Work presented in this thesis focuses on the effect of HS and its structures on FGF–FR complex formation and FGF-induced signaling. Studies with short, highly modified oligosaccharides and FGF1 and 2 combined with FR1c, 2c, 3c or 4 showed a correlation between the overall degree of modification and amount/stability of FGF–FR complexes. Our findings imply that several HS structures, differently modified but with the same negative charge density are equal in their ability to support complex formation. Co-application of oligosaccharides with FGF2 to HS-deficient cells and investigation of the thereby induced cell signaling confirmed our findings with a cell-free system. The oligosaccharide with the highest modification degree displayed the biggest impact on cell signaling, which was FGF2 concentration dependent. Studies with long HS polysaccharides with preserved high and low modified domains suggest that the proportion between these two types of domains and also the structure of the low modified domains are of importance for the FGF–HS–FR complex formation and cell activation capacity. </p><p>This work illuminates several aspects in how HS structure influences the interplay between FGFs and FRs and contributes to the understanding of what factors affect a cell’s response following FGF stimulation.</p>

Winter wheat has several advantages over spring varieties, higher (25 % more) yield, efficient use of spring moisture, reduction of soil erosion by providing ground cover during the fall and early spring, rapid initial spring growth to out - compete weeds and circumvent the peak of Fusarium head blight infections by flowering early. Winter wheat is planted in early autumn when it germinates and developing seedlings acclimate to cold. The crown survives under snow cover and in spring rapidly grows into a vigorously growing plant for grain to be harvested in summer. However, the harsh Canadian prairie winters require that winter wheat has increased cold hardiness and improved winter survival to reduce losses from sudden cold snaps during winter and spring. Low temperature (LT) tolerance is one of the major components of cold hardiness. Genetic mapping studies have revealed a major quantitative trait locus (Fr-A2) at wheat chromosome 5A which can explain at least 50 % of LT tolerance in wheat. Physical mapping of 5A LT QTL in a hardy winter wheat cv Norstar revealed a cluster of at least 23 C - repeat binding factors (CBF) coinciding with peak of Fr-A2 QTL. The objective of this study is biochemical, and molecular characterization of CBF co - located at Fr-A2 to identify key CBF participating in conferring LT tolerance in winter wheat. A comparative analysis of CBF gene cluster at the Fr-A2 collinear region among Poaceae members showed an expansion in the number of CBF genes with increased LT tolerance. Rice, a cold sensitive member, had only three CBF genes, whereas cold hardy winter wheat cv Norstar has 23 CBF genes. Amino acid sequence - based cluster analysis of complete CBF genes, or their major functional components such as the AP2 - DNA binding domain and C - terminal trans - activation domain, divide Norstar CBF into Pooideae specific clades. However, analyses of Norstar CBF amino acid sequences of different functional groups revealed a shift in clade members. These results suggest divergence of CBF functions which could lead to possible differences / similarity in the regulon activated by a CBF in a specific group. The 15 CBF genes from winter wheat cv Norstar were expressed in E. coli to produce recombinant TrxHisS - CBF fusion proteins in adequate quantities for structural and functional assays. All CBF fusion proteins could be recovered in the E. coli soluble phase of cell extract, except that the CBF17.0 fusion protein could only be recovered with 6 M urea extraction. Eleven of the 15 CBF fusion proteins were very stable in heat (98 oC), 10 % SDS and 6 M urea treatment. The five other CBF members were very labile under native conditions, but were stable in E. coli cell extracts or when extracted under denaturing conditions. Most of the CBF recombinant proteins in denaturing gel electrophoresis migrated slower than expected from their predicted molecular mass, based on amino acid sequence. The slow migration could be associated to their elongated protein structure as determined by dynamic light scattering (DLS). CBF 12.2 and CBF 17.0 were highly resistant to denaturation and retained their secondary structure in these conditions as determined by circular dichroism (CD) spectra. The high stability of these two CBF proteins may be important for cold acclimation or maintenance of cold hardiness in wheat. CBF proteins are transcription factors that bind to the dehydration-responsive element / C-repeat element (DRE / CRT) motif (CCGAC). Ten of the 15 Norstar recombinant CBFs whether purified under native or denaturing conditions showed in vitro binding to the CRT motif. Within hours of cold exposure (4 oC) the native CBF increased their affinity to CRT interaction which could be due to changes in the CBF secondary structures. Some of the CBF for binding preferred the core GGCCGAC motif while others preferred TGCCGAC. Similarly binding assays with truncated CBF revealed that for some CBF proteins, the second signature motif (DSAWR) and remaining C - terminal were not needed, while for others a considerable portion of the C -terminal region was needed for binding. Norstar CBF 12.1 has a memory of cold experience, and upon exposure to cold, has a high and immediate affinity to CRT elements. A homolog CBF12.2 in less cold - hardy winter wheat cv Cappelle - Desprez had a non - functional protein due to a R → Q substitution in a highly conserved residue within the AP2 domain. Several of the cv Norstar CBFs showed increased activity under LT and denaturing conditions, which may be the reason for the greater cold hardiness in Norstar. In conclusion, detailed and extensive analyses of CBF in this study characterized their structure and function relationships, which are important for understanding and improving LT tolerance in plants. The identification of specific CRT binding motifs and two CBFs which were very stable under adverse conditions may be prime candidates for further study to improve LT tolerance in plants.

There is a paucity of research juxtaposing parental education level and teacher emotional support in a single study which examines their relative impacts on students’ academic achievements. Therefore, the first objective of this dissertation is to study the influence of parental education level, in comparison to the influence of teacher emotional support, on students’ math performance, by using more representative data and a rigorous statistical method. The second objective is to identify and examine how some important psychological traits (both affective and cognitive) mediate the effects of social factors on students’ math performance. The third objective is to examine whether those relationships are moderated by gender. Hong Kong’s survey data is extracted from the Program of International Students Assessment (2003) as organized by Organization for Economic Co-operation and Development (OECD), on the math performances of 4,478 students at the age of fifteen. Measurement invariance was first tested, and then followed by Confirmatory Factor Analysis. Two structural models were tested by Structural Equation Modeling using Linear Structural Relations (LISREL) 8.5 which is computer software for SEM. Results indicated that first, parental education level affects children’s math scores by providing home education resources and enhancing children’s math self-efficacy, and second the Self Determination Theory is applicable in supporting the hypothesis that teachers affects their students’ math scores by providing a cooperative learning environment, which in turn, enhances students’ affective and cognitive factors. Three important mediators, namely cooperative learning environment, math self-efficacy, and home education resources are concluded as significant mediating factors upon the effects of parents and teachers on students’ math performance. The perceived support from parents and teachers are not significantly different across gender in Hong Kong. This is consistent with recent studies that differences favoring males in mathematics achievement are disappearing. Theoretical contributions and practical implications are discussed in the final part of the dissertation.

Memoria para optar el título de Bioquímico / El cese de la función ovárica está dado principalmente por la pérdida del número de folículos primordiales. Durante el envejecimiento, existe un decrecimiento en el número de folículos ováricos. En mujeres se ha establecido el periodo de subfertilidad después de los 37 años, cuando quedan menos de 100.000 folículos. En roedores, este período es caracterizado por disminución de la ciclicidad estral, del número de partos y de crías nacidas vivas por cada hembra. En este trabajo, se determinó el período de subfertilidad y el término del período reproductivo en ratas Sprague Dawley (SD). Desde los 8 meses se observaron cambios importantes en ratas, como disminución de estradiol y androstenediona, variación en los niveles de progesterona, aumento de la Noradrenalina (NA) ovárica y adrenal, además, de la disminución de la ciclicidad estral y fertilidad. El contenido intraovárico del factor de crecimiento nervioso (NGF), no cambia durante el período de subfertilidad. Sin embargo, aumenta significativamente a los 16 meses de edad, cuando la rata es infértil. Estos resultados permiten establecer el período de subfertilidad de la rata entre los 8 a 10 meses de edad. Después de esta caracterización se usaron ratas en el período de subfertilidad, para determinar si NGF y su receptor de baja afinidad p75NTR, son parte de un posible mecanismo que participa en este período. Para ello se administró localmente en el ovario una minibomba osmótica que infundió una mezcla de anticuerpo anti NGF con oligonucleótido antisentido contra mRNA de p75NTR . Después de 28 días de perfusión continua, se observó una sobreproducción de NGF intraovárico, el que posiblemente junto con el bloqueo de la traducción de p75NTR provocó: un aumento de androstenediona en plasma, pérdida de la ciclicidad estral, disminución del contenido de NA en médula adrenal, ganglio celíaco y ovario, encontrado al final del período observado. Además, hubo un aumento en el número de folículos saludables antrales y preantrales, y un aumento de estructuras quísticas, folículos tipo III y folículos luteinizados. La administración de la minibomba osmótica con anticuerpo contra NGF y el oligonucleótido antisentido tuvo un mayor efecto en los ovarios de rata de 8 meses que en los de 10 meses de edad y afecta a los dos ovarios por igual, tanto al que está conectado con la minibomba como al ovario contralateral. Los cambios morfológicos observados en ovario de rata de 8 meses de edad sugieren que un incremento en la actividad nerviosa simpática adelanta el envejecimiento reproductivo ovárico, proceso regulado por control neurotrófico intraovárico. / The cessation of reproductive function is mostly due to lost of primordial follicles pool. During aging, there is a decreased in the number of ovarian follicles. In women, the subfertility period was found from 37 years, when the number of follicles are smaller than 100.000. In rodents, this period is characterized for the lost of estrual cycling activity, decrease in the number of pups and deliveries by the female. In the present work, we determined the period of subfertility and the end of the reproductive period in Sprague Dawley (SD) rats. Since 8 month old we found important neuroendocrine changes in rats a such as decrease of estradiol and androstenedione, changes in progesterone, increase in ovarian and adrenal noradrenaline (NA). In addition, there was a decrease of estrual cyclicity and fertility. There were not changes in the nerve growth factor (NGF) at period of subfertility, but NGF was increased at 16 months, when the rat is infertile. Altogether these dates permitted us to define the period of subfertility betwen 8 to 10 months for the Sprague Dawley rat. After this characterization we used rats during the period of subfertility, to determine if NGF and its low affinity p75NTR receptor were involved in the possible mechanisms resulting in the end of ovary function. For that, we used Alzet osmotic minipumps to infuse one rat ovary with a mix of neutralizing antiserum to NGF in conjunction with an antisense oligodeoxynucleotide to mRNA p75 NGFR . After 28 days of continuous perfusion, we observed an overproduction of ovarian protein NGF, which together with the blockade of translation of p75NTR, could be the responsible of the: increase in serum androstenedione, lost of the cyclicity, decrease of NA content from the adrenal medulla, celiac ganglion and ovary, found at the end of observation. In addition, there was an increase in the number of antral and preantral healthy follicles and an increase in cystic structures; follicles type III and luteinizing follicles. The effect of the neutralizing antiserum to NGF with the antisense oligodeoxynucleotide to p75NTR was stronger in the ovary of rats of 8 month old than that in the ovary of rats of 10 month old. In both cases, the effect was the same for contralateral ovary (without miniosmotic pump). The morphological changes found in the ovary of rats of 8 month old suggest that an increasing sympathetic nerve activity accelerated ovarian reproductive aging in a process regulated by intraovarian neurotrophic control

La bilharziose reste un problème de santé majeur. L'équipe du Pr Dessein a montré que les infections élevées étaient déterminées par un locus majeur en 5q31 et que des polymorphismes dans un gène à ce locus,IL13, aggravent l'infection. Notre premier objectif était d'évaluer si des variants d'autres gènes de la voie de l'IL13 intervenaient dans le contrôle de l'infection. Nous avons observé une association entre le SNP rs324013, dans le promoteur de STAT6,et les niveaux d'infection à S. haematobium. Ce polymorphisme a un effet additif avec le polymorphisme IL13rs1800925. Ce SN modifie la fixation de facteurs nucléaires au niveau du promoteur de STAT6. L'équipe du Pr Dessein avait également montré que les fibres hépatiques avancées et sévères étaient déterminées par un autre locus majeur localisé en 6q23. Notre deuxième objectif fut d'évaluer dans le laboratoire du Pr Dessein et en étroite collaboration avec le laboratoire du Pr Li(Yueyang Institute of Parasitic disease)deux gènes candidats(IFNGR1 et CTGF) situés dans cette région chromosomique. Nous avons observé une association entre les deux polyporphismes(rs17066192 er rs673156)localisés dans le promoteur du gène. Nous avons observé une association entre les deux polymorphismes(rs17066192 et rs673156)localisés dans le promoteur du gène IFNGR1 et la fibrose hépatique: le génotype rs673156A/A et rs17066192C/C sont associés à un risque 7.3 fois et 1.5 fois plus élevé, respectivement, de fibrose avancée. Nous avons également montré que les variants rs9402373 et rs12526196 du gène CTGF sont indépendamment associés à la fibrose chez les fermiers et pêcheurs chinois infectés par S.japonicum. Sur la population chinoise d'étude, les risques relatifs associés aux polymorphismes rs9402373 et rs12526196 sont de 2.8 et 3 / Schistosomiasis remains one of the world’s most prevalent diseases. It comprises a group of chronic diseases caused by helminths of the Schistosoma genus. Schistosoma haematobium causes obstructive nephropathy that can be aggravated by urinary bacterial infections. S.japonicum and S.mansoni cause hepatic fibrosis associated with portal blood hypertension, which can be lethal. In previous studies, our laboratory had shown that worm burden in S.haematobium infections were aggravated by IL13 variants and that severe hepatic fibrosis (HF) was controlled by gene(s) located on 6q23. The present study is to further evaluate other IL-13 pathway genes (STAT6) in the control of infection in Malian farmers and to test candidate genes in the 6q23 region in hepatic fibrosis (HF) in S.japonicum infected Chinese fishermen and farmers. First we have developped an improved FTA® technology technique to perform SNP genotyping. This technique allows us to use saliva samples for genotyping SNPs. Subsequently, this improved FTA® technology was used in our study on HF.Our work on a Malian sample infected with S. haematobium indicated that a polymorphism (rs324013) in the promoter of STAT6 gene was associated with the control of S. haematobium infection levels and has an additive effect with IL13rs1800925, a polymorphism previously associated with infection in this same population. Both SNPs modify the binding of nuclear factors to the promoter regions of their respective genes. Thus, both SNPs may play a crucial role in controlling S. haematobium infection levels. In order to study HF in S.japonicum infections, we have participated actively in the study that recruited of a large sample of Chinese fishermen and farmers who had been exposed to the infection for most of their life. HF was evaluated by ultrasound and covariates that could affect HF were evaluated by interviews. Then, we tested two genes (IFNGR1, CTGF) of the 6q23 region that were good candidates for the control of HF on these samples. Both genes encode molecules that were shown in animal and human studies to have strong effect on extracellular matrix proteins deposition and turnover. We found that two polymorphisms (rs17066192 and rs673156) in IFNGR1 promoter were associated with HF: the rs673156A/A genotype was associated with a 7.3-fold increased risk of advanced HF; and rs17066192C/C genotype with a 1.5-fold increased risk of HF. These results must now be confirmed in another population sample. We also found that variants of CTGF rs9402373 and rs12526196 were independently associated with HF in Chinese fishermen and farmers, in Sudanese, and in Brazilians infected with either S. japonicum or S. mansoni. Our results provide additional evidence for a protective role of IL-13 in schistosome infections, and they also demonstrate that TGFβ / CTGF pathway plays a key role in HF and should be targeted by chemotherapy. Ongoing studies evaluate whether CTGF variants could be used in the prognosis of the HF caused by schistosomes and also by other infectious agents.

<p>Age-related macular degeneration is one of the leading causes of vision loss in the world. While identification of various environmental risk factors including but not limited to smoking, ethnicity, and diet have been reported to contribute to the complex etiology of AMD, age and genetics remain the largest susceptibility factors in its pathogenesis. Initially, with the identification of the common Y402H variant in CFH, approximately 35% of the genetic determinants of AMD had been identified with the majority remaining unknown. Therefore, we set forth to A) identify additional AMD susceptibility genes that contribute to AMD through the use to next generation sequencing technologies and B) to assess associated alleles for pathogenicity in the attempt to interpret their functional contributions to AMD outcome as observed via patient serum and zebrafish analysis. In doing such, we have identified both common and rare variants that contribute to the heritability of AMD. Additionally, we report one of the first instances of a rare variant significantly increasing disease onset and a gene with increased rare mutational burden in AMD patients. All together adding to our understanding of the genetics of AMD and potentially leading to putative therapeutic targets.</p> / Dissertation

Als Therapieversuch bei Plexusläsionen wird die Replantation ausgerissener Vorderwurzelfasern durchgeführt. Voraussetzung für die erfolgreiche Regeneration von Motoneuronaxonen sind 1. Überleben einer ausreichenden Anzahl von Motoneuronen 2. erfolgreiche Wiederherstellung der Kontuität ausgerissener Axone mit dem Rückenmark und 3. funktionelle Hochwertigkeit regenerierter Axone. Neurotrophe Faktoren können Überleben und Regenerationsfähigkeit von Motoneuronen fördern. Gegenstand der vorliegenden Arbeit war die Analyse des Einflusses von CNTF und BDNF auf die Regeneration von Motoneuronaxonen nach Ausriss und Replantation im Segment C7 nach einer Überlebenszeit von 3 Wochen bzw. 6 Monaten. Vervollständigt wurden diese Untersuchungen durch detaillierte morphologische Analysen von Spinalganglien, durchtrennter Hinterwurzel und verletztem Hinterhorn. In verschiedenen Gruppen von adulten Kaninchen wurden CNTF, BDNF, oder beide Faktoren auf die ventrolaterale Replantationsstelle appliziert, Kontrollen wurden ohne Faktor belassen (n>5). Die Überlebenszeit der Versuchstiere lag bei 3 Wochen (n=3 Kontrollen) und 6 Monaten (n=27). Aus dem perfundiertem Gewebe wurden Semidünnschnitte durch Vorderwurzel/Spinalganglien und Kryostatserienschnitte durch das Segment C7 angefertigt. DiI-Fluoreszenztracing, Markscheidenfärbung, eine modifizierte Klüver-Barrera-Färbung der Kryostatschnitte sowie eine Touloidinblaufärbung der Semidünnschnitte ermöglichte die morphologische und morphometrische Analyse des Gewebes. Die Anzahl der überlebenden Motoneurone lag nach sechs Monaten bei allen Versuchsgruppen bei etwa 30%. Fluoreszenz-Tracing und Markscheidenfärbungen von Serienschnitten zeigten, dass Axone sowohl über die ursprünglichen ventralen Austrittstellen als auch über die ventrolaterale Replantationsstelle das Rückenmark verließen und im Bereich des Spinalganglions eine kompakte Vorderwurzel bildeten. Ventral austretende Axone zeigten signifikant größere Durchmesser als lateral austretende. Ausmaß und Art der Regeneration waren interindividuell unterschiedlich, die besten Ergebnisse zeigte die Replantation nah am ursprünglichen Austrittsort der Vorderwurzel. Unterschiede zwischen den Gruppen waren nicht deutlich. In Semidünnschnitten durch die regenerierte Vorderwurzel fanden sich nach drei Wochen kaum intakte, myelinisierte Axone, nach sechs Monaten war die Zahl der Axone auf etwa 45% der Zahl der gesunden Seite angestiegen. Regenerierte Axone waren dünn, typische Motoneuronaxone stellten nur einen kleinen Teil der regenerierten Axone. Gruppenunterschiede fanden sich im Axon-Myelinverhältnis, das bei Kontrollen der replantierten Seiten signifikant erniedrigt war. Diese Erniedrigung war noch vorhanden, jedoch nicht mehr signifikant bei Tieren, die mit CNTF- und BDNF-behandelt wurden. Die replantierten Vorderwurzeln der CNTF+BDNF-Gruppe zeigte überwiegend eine signifikant bessere Myelinisierung als die replantierten Kontrollen. An der früheren Hinterwurzeleintrittszone am Rückenmark wurden in Tieren mit geringem Verletzungsausmaß kleine ZNS-Gewebsprotrusionen beobachtet, in denen sich myelinisierte Axone befanden. Diese Axone zeigten eine Wachstumsrichtung in die Peripherie, was auf eine Sprossung der sensorischen Rückenmarksneurone schließen lässt. Innerhalb des Spinalganglions waren Neuron- und Axondichte auf den verletzten Seiten nicht wesentlich verändert. Eine leichte Abnahme des relativen Anteils großer Neurone und Axone wurde in den verletzten Seiten der Kontrollgruppe beobachtet. Für Axone war diese Abnahme statistisch signifikant. Im Gegensatz dazu war dies in Tieren, die mit neurotrophen Faktoren behandelt wurden, nicht zu beobachten. Bei allen Tieren zeigte sich ein beträchtliches Auswachsen von Hinterwurzelaxonen aus dem Spinalganglion. Diese Axone fanden keine spontane Verbindung mit dem proximalen Rest der Wurzel, sondern waren durch Bindegewebe eingehüllt. Bei etwa der Hälfte der Tiere zeigte sich, dass einer Untergruppe dieser Axone in Richtung des Narbengewebes der replantierten Vorderwurzel gewachsen war und über Defekte in der Bindegewebshülle teilweise sogar in die Vorderwurzel einwuchsen. Ein möglicher Einfluss der applizierten neurotrophen Faktoren auf das quantitative Regenerationsergebnis scheint also in diesem Modell gering zu sein. Auf eine qualitative Verbesserung deutet die Normalisierung des Axon-Myelinverhältnisses großer regenerierter Axone bei Kombinationsbehandlung hin. Die im vorliegenden Modell beträchtliche Regenerationskapazität der Hinterwurzel scheint bisher unterschätzt worden zu sein. Das unerwartete Einwachsen von Hinterwurzelaxonen in die Vorderwurzel könnte mit einer funktionellen Beeinträchtigung der regenerierten Vorderwurzel verbunden sein. / Treatment of brachial plexus lesions is attempted by surgical replantation of avulsed nerve roots. Prerequisites for successful regeneration of motoneuron axons are 1. survival of a large number of motoneurons, 2. restoration of connectivity between avulsed nerve roots and spinal cord and 3. high quality of regenerated axons. Regeneration and survival of motoneurons can be supported by neurotrophic factors. In the present study, the influence of CNTF and BDNF on regeneration of motoneurons after C7 ventral root avulsion and replantation after 3 weeks and 6 months was analysed. Additionally, detailed morphological analyses of dorsal root ganglia (DRG), severed dorsal roots and injured dorsal horns were performed. In adult rabbits C7 dorsal roots were severed, ventral roots were avulsed and replanted ventrolaterally. CNTF, BDNF, or both was applied to the replantation site, controls were replanted without application of neurotrophic factors (n>5). After 3 weeks (n= 3 controls) and 6 months (n= 27) after avulsion and replantation semi-thin sections of ventral roots and DRGs as well as cryostat serial sections from C7 spinal cord segment were prepared. DiI-fluorescence tracing, myelin-sheath staining, modified Klüver-Barrera staining of cryostat section and touloidinblue staining of semi-thin sections served for morphological and quantitative analyses. Six months after lesion, a survival of 30% of the C7 motoneurons was found without differences between the experimental groups. Retrograde fluorescent tracing and histological analysis documented that many axons had regrown through the original ventral exit zones or had exited the spinal cord at the lateral replantation site. However, many laterally exiting axons had not grown out directly from the ventral horn through the lateral white matter but had elongated vertically before leaving the spinal cord. The mean axonal diameter was significantly higher in regenerated axons that had exited through the original ventral exit zones in comparison with axons which had grown out laterally. Application of BDNF and/or CNTF did not show any effects on the pathways of regeneration into the replanted root. Three weeks after ventral root avulsion and replantation the number of axons was rare. After six months, the number of myelinated axons increased to 45% compared to unlesioned sides. Regenerated axons were mainly of small caliber with few axons showing typical properties of motoneuron axons. In controls myelination was significantly reduced compared to the unlesioned sides. This was not observed after CNTF, BDNF and CNTF+BDNF treatment. In CNTF+BDNF treated animals myelination was significantly increased compared to replanted controls in the majority of cases. At the dorsal root entry zone, small myelinated axons extended into central tissue protrusions, in cases with well-preserved morphology. This suggested sprouting of spinal neuron processes into the central dorsal root remnant. In lesioned DRGs, the density of neurons and myelinated axons was not significantly altered, but a slight decrease in the relative frequency of large neurons and an increase of small myelinated axons was noted (significant for axons). Unexpectedly, differences in the degree of these changes were found between control and neurotrophic factor-treated animals. Central axons of DRG neurons formed dorsal root stumps of considerable length which were attached to fibrous tissue surrounding the replanted ventral root. In cases where gaps were apparent in dorsal root sheaths, a subgroup of dorsal root axons entered this fibrous tissue. Continuity of sensory axons with the spinal cord was never observed. Some axons coursed ventrally in the direction of the spinal nerve. In summary, the number of surviving motoneurons and regenerating axons appeared not to be influenced by a single- dose application of neurotrophic factors in this model. However, improvement of myelination indicated that the quality of regeneration can be increased especially by CNTF+BDNF- treatment. Moreover, the considerable capacity of dorsal root regeneration we observed in this study has possibly been underestimated previously. The unexpected ingrowth of dorsal root axons into the regenerated ventral roots could be harmful for ventral root regeneration.