The developing chick retina is an extrapituitary site of growth hormone (GH) synthesis and action. GH, GH receptor (GHR) and their mRNAs are present in the neural retina when the neural cells are undergoing proliferation and differentiation during early embryogenesis. It is thus likely that GH acts as an autocrine or paracrine in this location. The present study shows that intra-vitreal injection of a chick GH (cGH) small interfering RNA (siRNA) into the eyes of early embryos [embryonic day (ED) 4] suppresses GH expression in the neural retina and increases the incidence of spontaneous retinal cell death. Our current work also demonstrates a reduction of local IGF-1 expression after retinal GH gene knockdown, suggesting that GH action in retinal cells is regulated through IGF-1 signalling. These results demonstrate that retinal GH is an autocrine/paracrine hormone that acts as a neuroprotective factor in the retina of chick embryos.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 7 uppsatser.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Sept. 15, 2009). Includes bibliographical references.

The purpose of the study is to purify human growth hormone from the fermentation broth by affinity chromatography. For this purpose, human growth hormone specific oligonucleotide aptamers are selected among an aptamer library / selected oligonucleotides were synthesized and used as ligands. Effect of pH on ligand-human growth hormone complex formation was investigated and the highest complex formation was obtained at pH= 7.0. Human growth hormone is separated from the fermentation broth with 99.8% purity and 41% overall yield. The equilibrium data obtained was described by Langmuir type isotherm where saturation constant (q0) and affinity constant (K) are calculated as 0.338 mg hGH/&micro / mol aptamer and 0.059 mg hGH/ml, respectively. Further, equilibrium data obtained using aptamer affinity column was described by Langmuir type isotherm where saturation constant (q0) and affinity constant (K) are 0.027 mg hGH/&micro / mol aptamer and 1.543 mg hGH/ml, respectively. It is possible that, selected aptamer can be used for purification of bulk amounts of recombinant human growth hormone by using aptamer affinity chromatography.

Creutzfeldt-Jakob disease (CJD) is the commonest form of human prion disease and occurs in sporadic, genetic and acquired forms. The causative agents (prions) appear to be composed entirely of a modified host protein, the prion protein, which undergoes misfolding to a disease-associated isoform closely associated with infectivity that is resistant to conventional methods of decontamination. Prions can be transmitted from one individual to another by medical and surgical procedures, resulting in iatrogenic CJD (iCJD). The commonest cause of iCJD is the inoculation of cadaveric pituitary-derived human growth hormone (hGH) to treat growth hormone deficiency in children; this form of treatment was abandoned in 1985 after the first UK case of iCJD in a hGH recipient was identified. Seventy-eight cases of iCJD have since occurred in the UK cohort of 1849 hGH recipients, including a case in 2016. This thesis describes a comprehensive tissue-based and molecular genetic analysis of the largest series (35 cases) of UK hGH-iCJD cases reported to date, including in vitro kinetic molecular modelling of genotypic factors influencing prion transmission. The results show that the polymorphism at codon 129 of the prion protein gene strongly influences the disease incubation period in hGH-iCJD (from 7.8-32.3 years in this series) and interacts with the infectious prion strain to govern the molecular and pathological characteristics of iCJD. The findings are consistent with the hypothesis that the UK hGH-iCJD epidemic resulted from transmission of the V2 human prion strain, which is found in the second most common form of sporadic CJD. The investigation also found accumulation of the amyloid beta (Aβ) protein associated with Alzheimer’s disease (AD) in the brains and cerebral blood vessels in 18/35 hGH-iCJD patients and 5/12 control patients who had been treated with hGH, but died from causes other than iCJD. In contrast, Aβ accumulation was markedly less prevalent in age-matched patients who died from sporadic CJD (1/15 cases) and variant CJD (2/33 cases). These results are consistent with the hypothesis that Aβ, which can accumulate in the pituitary gland, was present in the inoculated hGH preparations and seeded into the brains of around 50% of all hGH recipients, producing AD-like neuropathology and cerebral amyloid angiopathy (CAA). This provides further evidence of the prion-like properties of Aβ and gives insight into the potential for possible transmission of AD/CAA. It is uncertain whether any Aβ seeding within the brains of surviving patients in the UK hGH recipient cohort will ultimately result in clinical AD; however, the CAA in these patients may be complicated by intracerebral haemorrhage resulting from rupture of the blood vessels damaged by Aβ accumulation within their walls.

The growth and development of an organism requires the coordinated actions of many factors. During development individual cells undergo proliferation, migration and differentiation to form the adult organism. Two structurally related members of the glucagon superfamily, growth hormone releasing hormone (GRF) and pituitary adenylate cyclase-activating polypeptide (PACAP), are thought to modulate vertebrate development. In mammals, GRF modulates the development of pituitary somatotrophs and the release of fetal growth hormone. In contrast, PACAP appears to have a more general role during development. PACAP may be involved in the patterning of the embryonic axis and in the development of the neural tube. The objectives of my study were to isolate GRF, PACAP and their receptors from the zebrafish, characterize their expression in the developing embryo and adult embryo and examine the role of PACAP during brain development. To study the role of GRF and PACAP, I isolated a genomic clone encoding the GRF and PACAP peptides from the zebrafish genomic library and characterized its gene copy number and adult tissue expression pattern. The GRF-PACAP gene isolated from the zebrafish was comprised of five exons with the GRF peptide encoded on the fourth exon and the PACAP peptide encoded on the fifth exon. This gene structure is similar to that found in other non-mammalian vertebrates and supports the hypothesis that the gene duplication leading to the encoding of the GRF and PACAP peptides on separate genes occurred later in evolution. In addition, the zebrafish genome was found to contain only one copy of the GRF-PACAP gene. The GRF-PACAP gene was widely expressed in the adult zebrafish in tissues developmentally derived from all three germ layers, suggesting that the gene may be widely expressed in the embryo as well. To examine the functional significance of the co-expression of GRF and PACAP in zebrafish, I isolated the GRF and PACAP receptors and characterized their expression pattern. I isolated three distinct cDNAs from zebrafish encoding the GRF receptor, the PACAP specific PAC1 receptor and the shared vasoactive intestinal peptide/PACAP receptor VPAC1. In addition, four isoforms of the PAC1 receptor were isolated from zebrafish including a novel isoform found in the gill. All three receptors were widely expressed in adult zebrafish and receptors for both GRF and PACAP were found in most tissues. This indicates that GRF and PACAP may modulate each other’s function. To determine the developmental role of GRF and PACAP, I characterized the expression pattern of the GRF-PACAP gene and the GRF, PAC1 and VPAC1 receptors in the zebrafish embryo. The GRF and PAC1 receptors are the earliest to be expressed in development starting at the cleavage stage. Later, the GRF-PACAP gene and the VPAC1 receptor are first expressed at the late blastula/early gastrula stage in the zebrafish and are expressed throughout the developmental period. Strong expression of the GRF, PACAP and their receptors during mid gastrulation indicates that these peptides may be involved in modulating the formation of the embryonic axis. During the segmentation period the GRF-PACAP gene is widely expressed in the zebrafish embryo and the PAC1 receptor short and hop isoforms are differentially expressed. Therefore, PACAP may regulate cell cycle exit or cell proliferation through activation of different PAC1 receptor isoforms during the segmentation stage. In the subsequent pharyngula period, the GRF-PACAP transcript is localized mainly to the hatching gland. However, expression is seen also in tissues that undergo differentiation during this stage. Therefore, the timing of the expression of the GRF-PACAP gene indicates that it may be involved in early patteming events and promoting cell cycle exit prior to differentiation. To investigate the role of GRF and PACAP in the developing brain, I localized the expression of GRF, PACAP and the PAC1 receptor in neuroblasts derived from an embryonic day 3.5 chick. PACAP was found to stimulate the cAMP pathway in these cells, indicating that PACAP may modulate brain development. This work indicates that GRF and PACAP play an important role in vertebrate development. / Graduate

Studies have shown that growth hormone (GH) is a polypeptide with immunomodulatory properties. Herein, studies were performed to determine in vivo and in vitro effects of GH on macrophage by using cultures of resident peritoneal macrophages from swiss mice. The microscopical analyses revealed alterations in the macrophage morphology after GH treatment with 20 and 200 ng/mL for 12 and 24 hours. It was also observed that GH-treated macrophages have an increase in fibronectin and laminin deposition evaluated by indiret immunocytochemistry. By using flow cytometry, it was shown that GH-treatment (200 ng/mL) for 6 and 24 hours altered the Mac-1 and VLA-6 integrins expression on macrophage surface. Moreover, the same GH concentration, during 6 hours of treatment, was able to decreased macrophage adhesion to laminin. In the in vitro migration assays, the GHtreated cells (200 ng/mL) showed opposite effects depending on the treatment time, after 6 hours, there was a increase in migrating cells whereas after 12 hours was a decrease in cell migration. The in vitro GH treatment did not influence the phagocytic activity of macrophages but when the GH treatment was perfomed in vivo, for 7 consecutive days, the peritoneal macrophages from GH-treated mice (20 and 200 μg/kg) showed a higher porcentage of phagocytosis and also a higher phagocytic capacity than cells from control animals. Taken together, these results reinforce the literature data which show that GH can act as an macrophage activating-factor in the immune response. / Fundação de Amparo a Pesquisa do Estado de Alagoas / Estudos mostram que o hormônio do crescimento (GH) é um polipeptídio com propriedades imunomoduladoras. Assim, o objetivo desse estudo foi avaliar os efeitos in vivo e in vitro do GH sobre macrófagos, utilizando culturas de macrófagos peritoneais residentes obtidos de camundongos swiss. Inicialmente, através da análise microscópica foram observadas alterações na morfologia dos macrófagos em cultura, tratados com GH, nas concentrações de 20 e 200 ng/mL, pelos períodos de 12 e 24 horas, quando comparado às células não-tratadas. A presença de ligantes de moléculas da matriz extracelular em macrófagos foi analisada por imunocitoquímica, em que se evidenciou um aumento na deposição de fibronectina e laminina quando as células foram tratadas com GH nas concentrações de 20 e 200 ng/mL, nos tempos de 6, 12 e 24 horas. Por citofluorimetria, observou-se que o tratamento com GH (200 ng/mL), por 6 e 24 horas alterou a expressão das integrinas Mac-1 e VLA-6 na superfície dos macrófagos. Além disso, o tratamento com GH, nesta mesma concentração, pelo período de 6 horas, foi capaz de diminuir a adesão de macrófagos à laminina. No ensaio de migração in vitro, o tratamento com GH (200 ng/mL), apresentou efeitos opostos nos diferentes tempos de tratamento, aumentando o número de células migrantes após 6 horas e diminuindo o número de macrófagos migrantes após 12 horas de tratamento. Demonstrou-se ainda, que o tratamento in vitro com GH, em ambas as concentrações, não modulou a atividade fagocítica dos macrófagos, contudo macrófagos peritoneais obtidos de animais tratados com GH nas doses de 20 e 200 μg/kg, por um período de 7 dias, apresentaram uma maior porcentagem de fagocitose e uma maior capacidade fagocítica quando comparados aos macrófagos de animais do grupo controle. De uma forma geral, os resultados apresentados reforçam os dados já constantes na literatura de que o GH pode agir na resposta imune como um ativador de macrófagos.