Growth plate is a highly organized cartilaginous tissue found at the end of long bones and is responsible for longitudinal growth of the bones. Growth plate fracture leads to retarded growth and unequal limb length, which might have a lifelong effect on a person’s physical stature. This research is a tissue engineering approach for the treatment of growth plate injury. Insulin-like growth factor I (IGF-I), which can stimulate cartilage formation, was encapsulated within PLGA microspheres that were then used to form porous scaffolds. The release profile of the IGF-I from the PLGA scaffold showed a biphasic release pattern. In vitro studies were done by seeding rat bone marrow cells (BMCs) on the top of IGF-I encapsulated PLGA scaffolds, and the results showed an increase in cell multiplication and glycosaminoglycan content. The final in vivo studies were conducted by creating growth plate injury and implanting scaffolds in the tibiae of the New-Zealand white rabbits. Histological analysis of tissue sections showed regeneration of cartilage, albeit with disorganized structure, at the site of implantation of IGF-I encapsulated scaffolds. This work will be a significant step towards tissue engineering of growth plate cartilage.

Dunaliella, a genus of unicellular, biflagellate green algae, is one of the most studied microalgae for mass culture and is of commercial importance as a source of natural -carotene. Dunaliella species have the desirable properties of halotolerance and photoautotrophy that makes their large-scale culture simple and cheap using resources unsuitable for conventional agriculture. The ease and cost-effectiveness of culture makes Dunaliella a desirable target for increased production of natural compounds by metabolic engineering or for exploitation as biological factories for the synthesis of novel high-value compounds. However, the lack of efficient genetic transformation systems has been a major limitation in the manipulation of these microalgae. In chapter four we describe the development of a nuclear transformation system for Dunaliella tertiolecta. The gene encoding the phleomycin-binding protein from Streptoalloteichus hindustanus, was chosen as the selectable marker as this protein retains activity at high salt concentrations. To drive expression of the chosen selectable marker, two highly expressed Dunaliella tertiolecta RbcS genes and their associated 5' and 3' regulatory regions were isolated and characterised (chapter three). Dunaliella transformation cassettes containing the RbcS promoter and terminator regions flanking the ble antibiotic resistance gene were constructed. These expression cassettes were tested in Chlamydomonas reinhardtii cells and found to drive expression of the ble gene in this heterologous system. This study also demonstrated that truncation of both the D. tertiolecta RbcS1 and RbcS2 regulatory regions significantly increases the expression of the ble gene in C. reinhardtii cells. To determine if the foreign DNA could stably integrate into the Dunaliella genome, four transformation methods: microprojectile bombardment, glass bead-mediated transformation, PEG-mediated transformation and electroporation were tested and a number of parameters varied. Southern blot analysis revealed that the plasmid DNA transiently entered the Dunaliella cells following electroporation but was rapidly degraded. Following electroporation, one stably transformed Dunaliella line was recovered. This is the first demonstration of the stable transformation of this alga. Chloroplast transformation is becoming a favoured method for the production of recombinant proteins in plants, as levels of heterologous protein are often higher than those achieved by transforming the nucleus. The Dunaliella chloroplast genome has not been genetically characterised, and thus there were no existing promoter and terminator sequences or sequences of intergenic regions that could be used for vectors in transformation of the chloroplast. Therefore, this study aimed to isolate and characterise promoters of highly expressed genes and matching terminators capable of driving transgene expression, and also to characterise intergenic regions that would be suitable insertion sites for the vector construct (chapter five). The complete gene sequence of two highly expressed Dunaliella chloroplast genes psbB and rbcL including the promoter and terminator regions as well as the coding sequence of the psbA gene were cloned and sequenced. In addition, the psbA gene is useful as a selectable marker as introduced mutations confer resistance to the herbicide 3-(3,4-Dichlorophenyl)-1,1-Dimethylurea (DCMU). Two homologous transformation constructs based on mutated psbA genes were developed and tested using microprojectile bombardment. A number of parameters were tested including: the size of the gold microprojectile particle, the distance of the plates from the point of discharge, plating onto membranes or filter paper, helium pressure, addition of an osmoticum to the medium and recovery time. Although no chloroplast transformants were recovered in this study, these homologous recombination constructs should prove useful in the development of a chloroplast transformation protocol. The other major component of this study was to investigate the use of microalgae as an expression system for the production of recombinant proteins. Transformation of Chlamydomonas reinhardtii, a species related to Dunaliella, is well developed. In chapter six, this study examined the expression of two human proteins, -lactalbumin and IGF-1 in Chlamydomonas reinhardtii. Plasmids containing the C. reinhardtii RbcS2 promoter upstream of the cDNAs of these two proteins were introduced into C. reinhardtii cells using glass-bead mediated transformation. Transgenic C. reinhardtii lines were generated and shown to contain the transgenes by PCR and Southern hybridisation. RT- PCR and northern hybridisation were subsequently used to demonstrate that the transgenes were transcriptionally active. The transcripts however, could only be detected by RT-PCR indicating that the genes were transcribed at low levels. Accumulation of the -lactalbumin protein could not be demonstrated, suggesting that although the transgenes were transcribed, they were either not translated or translated at levels below the sensitivity of western blot analysis or that any protein produced was rapidly degraded. Previous studies have indicated that in microalgae codon usage is vital in translation of the foreign protein. Codon modification of the IGF-I and -lactalbumin genes should lead to higher levels of protein accumulation. This study reports the first successful stable nuclear transformation of Dunaliella tertiolecta. Therefore it is now feasible that Dunaliella can be examined as a bioreactor for the expression of recombinant proteins. In addition, two chloroplast genes (psbB and rbcL) and their corresponding promoters and terminators have been characterised and a selectable marker cassette based on the mutated psbA gene constructed.

Context and objective: Experimental models demonstrate an important role of growth hormone (GH) in retinal development. However, the interactions between GH/IGF-I axis and the neuro-vascularization of the human retina are still not clear. A model of untreated congenital isolated GH deficiency (IGHD) may clarify the action of GH on the retina. The purpose of this work was to assess the retinal neuro-vascularization in adults with congenital IGHD. Methods: In a cross sectional study, we performed fundus photographs (to assess the number of retinal vascular branching points and the optic disc and cup size), and optical coherence tomography (to assess the thickness of macula) in 25 adults IGHD subjects (13 males, 50,9 yr. [12,0]) homozygous for a null mutation (c.57+1 G>A) in the GH releasing hormone receptor gene and 28 controls (14 males, 46,4 yr. [14,7]). Results: Fisher's exact test revealed that IGHD subjects presented more reduction of vascular branching points in comparison to controls (91% vs. 53% [p=0.049]). Conversely, the percentage of moderate reduction in IGHD was higher than in control (p=0,01). The rates of individuals with increased optic disc and cup size were increased in IGHD in comparison to controls (92,9% vs. 57,1 for optic disc and 92,9% vs. 66,7% for cup [p<0.0001 in both cases]). The percentage of increased optic disc and cup in IGHD was higher than in control (p=0,005 for optic disc and p=0,028 for cup). There was no difference in fovea thickness or in any of the macula areas. Conclusions: Most IGHD individuals present moderate reduction of vascular branching points, increase of optic disc and cup size, but equal thickness of the macula. / Contexto e Objetivo: O hormônio do crescimento (GH) tem importante papel no desenvolvimento da retina, como demonstram diversos modelos experimentais. Contudo, a relação entre o eixo GH/IGF-I e a neuro-vascularização da retina humana ainda não é completamente conhecida. O modelo da deficiência isolada, congênita e vitalícia do GH (DIGH) não tratada é ideal para estudar a ação do GH nos tecidos oculares. O objetivo deste trabalho é avaliar a neuro-vascularização retiniana em indivíduos adultos com DIGH. Casuística e Métodos: Neste estudo transversal, examinamos 25 indivíduos (13 homens, idade 50,9 anos [12,0]) com DIGH devido a mutação c.57+1G>A no gene do receptor do hormônio liberador do GH e 28 controles (14 homens, idade 46,4 anos [14,7]), pareados por idade e sexo. Ambos os grupos foram submetidos a retinografia (para avaliar o número de ramificações nos vasos retinianos, o tamanho do disco óptico e da escavação) e tomografia de coerência óptica (OCT) (para avaliar a espessura da mácula). Resultados: Os indivíduos com DIGH apresentaram maior redução no número de ramificações vasculares em relação aos controles (91% vs. 53% [p=0,049]), segundo teste exato de Fisher. A porcentagem de redução moderada foi maior na DIGH que nos controles (p=0,01). A taxa de indivíduos com aumento do disco óptico e da escavação foi maior na DIGH em relação aos controles (92,9% vs. 57,1% para o disco e 92,9% vs. 66,7% para a escavação [p<0,0001 em ambos os casos]). A porcentagem de aumento do disco óptico e da escavação foi maior na DIGH (p=0,005 e p=0,028, respectivamente). Não houve diferença na espessura da mácula. Conclusões: A DIGH congênita e vitalícia não tratada provoca diminuição no número de ramificações dos vasos da retina, aumento do disco óptico e da escavação, mas não altera a espessura da mácula.

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The overall focus of this dissertation is on how chronic shear stress alters the synthesis and secretion of important regulatory molecules by endothelial cells. Our hypothesis was that inclusion of chronic pulsatile shear stress in our model would lead to changes in endothelial cell release of regulatory molecules. We distinguished between high arterial shear stresses and low venous shear stresses and used static cell cultures as reference. The first part of this research thus entailed the complete characterization of the flow dynamics in our experimental biomechanical model. Cell stretching can have a physiological effect on endothelial cells; hence we implemented a laser based optical technique for real time strain measurement of the growth fibers used in our culture system, and found that no significant strains were occurring during shear treatment. After characterization of the mechanical environment of the cells, we focused the scope of our research on metabolism of proteoglycans and insulin-like growth factor-I (IGF-I) and related IGF binding proteins (IGFBPs) in bovine aortic endothelial cells cultured under chronic pulsatile shear. We found that shear stress increased the release of proteoglycans and significantly altered proteoglycans distribution. We also found that there was an inverse relationship between the shear level treatment used to obtain the purified proteoglycans from endothelial cells and their potency in inhibiting coagulation. IGF-I release and message (IGF-I mRNA) was decreased at high shear stress compared to low shear stress. Further, the levels found under shear were significantly greater than those observed in the static cell culture model. IGFBPs released were also significantly increased by shear. This research thus establishes a link between chronic pulsatile shear stress and the metabolism of both primary (IGF-I) and secondary (IGFBPs, proteoglycans) regulators of vascular cell activity. The improved realism of our experimental biomechanical model has proved to be a valuable tool in improving the relevance of this study to vascular research. Ultimately, this research calls for further investigation in the molecular mechanisms underlying the phenomenological effects documented, which may help in understanding fundamental aspects in cardiovascular disease and its link to hemodynamics but our work is an important first step. / Ph. D.

A longitudinal study was designed to characterize developmental patterns of plasma (PL) and synovial fluid (SF) total insulin-like growth factor-I (IGF-I) concentrations, as well as their association with measurements of skeletal growth in Thoroughbred horses. Horses were randomly assigned to one of two dietary treatment groups and fed diets with either a high or low starch content to examine the effects of dietary energy source on PL and SF IGF-I. At 3, 6, 9, 12 and 15 mo of age, PL and carpal SF samples were collected for analysis of total IGF-I. Body weight gain, wither height gain and forearm length gain were calculated for the 90 day periods between SF and PL sampling. No influence of diet on PL or SF IGF-I was detected (P > 0.05). Average SF IGF-I concentrations were 30.1 ± 1.8% of that found in PL, and PL and SF IGF-I were positively correlated (r = 0.48, P = 0.0003) There was an effect of month of age on both PL and SF IGF-I concentrations (P < 0.05). There was a positive correlation between all measures of gain except forearm length gain with PL and SF IGF-I (r = 0.41 to 0.55, P < 0.05). In our second study, we evaluated the use of a liquid-nitrogen cooled mortar and pestle, motorized freezer mill and rotor-stator homogenizer for homogenization of small (<50mg) articular cartilage samples. The rotor-stator homogenizer produced quanitfiable RNA yields, and was used to evaluate three different RNA isolation protocols. Two of the protocols were commercially available RNA extraction kits, with the third a modified guanidinium isothiocyanate/acid-phenol extraction procedure. The combined average yield for all protocols was 91.9 ng RNA/mg of cartilage. All protocols yielded a sufficient quantity of quality RNA suitable for gene expression analysis. / Master of Science

Zahlreiche klinische Faktoren beeinflussen Blutserumspiegel von IGF-I (Insulin-like growth factor I) und dem zugehörigen Bindungsprotein IGFBP-3 (IGF binding protein 3). Diese sind in ihrer Gesamtheit nicht einheitlich untersucht und beschrieben. Der vorliegenden Arbeit liegt die Fragestellung zugrunde, ob zwischen neuen alters-, geschlechts- und pubertätsadjustierten Referenzbereichen für IGF-I beziehungsweise IGFBP-3 sowie BMI, Kontrazeptiva (CD, contraceptive drugs) sowie peri- und postmenopausaler Hormonersatztherapie (HRT, hormone replacement therapy) signifikante Assoziationen bestehen. Die Studienteilnehmer entstammen hauptsächlich den beiden Kohortenstudien LIFE Child und LIFE Adult. Wir untersuchten knapp 9400 Serumproben von mehr als 7000 gesunden und 1278 adipösen Probanden im Altersbereich zwischen drei Monaten und 81 Jahren. Die laborchemische Messung erfolgte mittels eines neuen Chemilumineszenz-Immunoassays. Mithilfe linearer Regressionsmodelle wurden Assoziationen zwischen IGF-I beziehungsweise IGFBP-3 und den Prädiktoren BMI, CD und HRT untersucht. Im Ergebnis weisen adipöse Kinder um bis zu 1 SDS höhere IGF-I-SDS-Werte vor, die im Alter von zirka 13 Jahren mit den Werten der normalgewichtigen Kinder konvergieren. Im Alter zwischen 20 und 40 Jahren zeigen adipöse Probanden um bis zu -0,5 SDS geringere IGF-I-SDS-Werte. Der Einfluss der Adipositas fällt für IGFBP-3 geringer ausgeprägt aus. Östrogen- und progestinbasierte CD (epCD), jedoch nicht HRT, verringern IGF-I und erhöhen IGFBP-3 signifikant (p<0.01) bei Jugendlichen (β IG F-I = −0,45; β IGFBP-3 = 0,94) und Erwachsenen (β IGF-I = –0,43, β IGFBP-3 = 1,12). Für rein progestinbasierte CD (pCD) zeigen sich dagegen signifikant positive Assoziationen zu IGF-I (β IGF-I = 0,82). Aufgrund der ermittelten Assoziationen sind BMI und CD bei der Untersuchung und Interpretation klinischer Werte für IGF-I und IGFBP-3 stets zu berücksichtigen.:Inhalt I Abkürzungsverzeichnis 1 Vorbemerkung 2 Einführung 2.1 Bedeutung der GH-IGF-Achse 2.1.1 Biochemische Merkmale und Wirkungen von GH, IGF I und IGFBP 3 2.1.2 IGF I und IGFBP 3 in Diagnostik und Therapie von Störungen der GH-IGF-Achse 2.2 Störungen der GH-IGF-Achse im Kindes- und Erwachsenenalter 2.2.1 Wachstumshormonmangel im Kindes- und Erwachsenenalter 2.2.2 Gigantismus und Akromegalie 2.3 RI für IGF I und IGFBP 3 2.3.1 Anforderungen an RI 2.3.2 Assoziationen mit BMI, Kontrazeption (CD) und Hormonersatztherapie (HRT) 2.3.3 Aktuelle Datenlage zu RI für IGF I und IGFBP 3 3 Problemstellung und abgeleitete Hypothesen 4 Publikation 4.1 Manuskript der Publikation: 4.2 Ergänzungsmaterial zur wissenschaftlichen Publikation 5 Zusammenfassung der Arbeit 6 Literaturverzeichnis II Darstellung des eigenen Beitrags III Erklärung über die eigenständige Abfassung der Arbeit V Danksagung