Atualmente, a maioria dos endocrinologistas pediátricos usa uma dose fixa de GH, calculada por quilo de peso ou área de superfície corporal, para todos os pacientes. Algumas crianças com DGH, tratadas com as doses atuais, não atingem uma estatura normal e outras não atingem a estatura-alvo geneticamente prevista pela altura dos pais. Além disso, algumas crianças com DGH desenvolvem características acromegalóides após o uso por longo prazo da medicação. A existência de um marcador preciso e eficiente seria útil para a individualização da dose de rGH. Esse marcador deveria ser mensurado em um período de tempo menor que a velocidade de crescimento (VC). Neste estudo usamos as concentrações de IGF-I como esse marcador. Durante 12 meses, acompanhamos trinta crianças portadoras de DGH grave, em tratamento prévio por cerca de quatro anos, divididas em dois grupos de 15, para comparar o tratamento com a dose de rGH baseada no peso versus o tratamento com a dose de rGH ajustada pelas concentrações de IGF-I para mantê-las em uma faixa alvo (entre 0 a +2 DP). Foi possível manter concentrações de IGF-I dentro de valores predeterminados pelo ajuste da dose de rGH em 13 dos 15 pacientes, mas a VC não foi diferente entre os grupos (6,8±2,6 vs. 6,9±2,7 cm/ano; p=NS); porém, quando considerados apenas os pacientes prépúberes que mantiveram concentrações de IGF-I entre 0 e +2DP em pelo menos 75% das dosagens, sua velocidade de crescimento foi maior em comparação com as crianças que mantiveram concentrações de IGF-I abaixo de 0DP em 50% ou mais das dosagens (8,8±1,8 vs. 6,3±2,9 cm/ano; p<0,05). Em paralelo, comparamos as concentrações de IGF-I de diferentes genótipos do exon 3 do receptor de GH (GHR), nessas trinta crianças, durante o tratamento. Duas das mais comuns isoformas em seres humanos são geradas pela retenção (full length GHR - GHRfl) e exclusão do exon 3 (exon 3 deleted GHR - GHRd3). A influência desse polimorfismo na resposta ao tratamento com rGH em pacientes com DGH tem sido alvo de controvérsia. No presente estudo, apesar de receberem a mesma dose de rGH (41±10 vs. 41±8 ug/kg d; p=NS), pacientes carreando ao menos um alelo-d3 GHR, como grupo, tiveram maiores níveis de IGF-I que aqueles homozigotos para o alelo GHR full-length (0,9±0,9 vs. -0,3±1,2 DP; p<0,05) , indicando uma melhor sensibilidade ao GH para o primeiro genótipo. A VC não foi diferente entre os grupos GHRd3 vs. GHRfl (7,3±1,9 vs. 6,4±3,1cm/ano; p=NS). / Currently, most pediatric endocrinologists use a fixed rGH dose calculated according to the weight or body surface area for all patients. Some children with GHD, treated with the present doses, do not achieve normal height, and some, even reaching normal height, do not achieve the genetic target height determined by their parents\' heights. At the same time, some children with GHD develop acromegalic characteristics after long-term treatment with rGH. The existence of a specific and effective marker to individualize rGH dose would be useful to control therapy of children with GHD. This marker ideally should be obtained in a shorter time interval than the growth velocity (GV). In the present study, we measured insulin-like growth factor (IGF-I) as this marker. During one year, we followed 30 children with severe GHD, treated previously with rGH for an average of 4yrs, divided in two groups of 15, to compare weight-based versus IGF-I-based rGH dosing to reach IGF-I levels between a target range (0 and +2 SDS). It was feasible to maintain IGF-I levels in this predetermined range by adjusting the rGH dose in 13 of 15 patients, but growth velocity was not different in groups with weight-based or IGF-I based rGH dose (6.8±2.6 vs. 6.9±2.7cm/y; p=NS); however, in prepubertal children who reached predetermined target IGF-I levels in 75% of the measurements GV was higher than in those who did not (8.8±1.8 vs. 6.3±2.9 cm/y; p<0.05). In parallel, we compared IGF-I levels of different GH receptor (GHR) exon 3 genotypes in these 30 children during treatment. Two of the more common GHR isoforms in humans are generated by retention (full length GHR - GHRfl) and by exclusion of exon 3 (exon 3 deleted GHR - GHRd3). The influence of this polymorphism on the response to rGH treatment in patients with GH deficiency has been controversial. In the present study, despite receiving similar rGH doses (41±10 vs. 41±8 ug/kg d; p=NS), patients carrying at least one GHR d3-allele, as a group, had higher IGF-I levels than those homozygous for the GHR full-length allele (0.9±0.9 vs. -0.3±1.2 SDS; p<0.05), indicating a greater GH sensitivity with the former genotype. GV was not different between groups GHRd3 vs. GHRfl (7.3±1.9 vs. 6.4±3.1cm/y; p=NS).

A neuraminidase 1 (Neu1) é a enzima que regula o catabolismo de sialoglicoconjugados nos lisossomos. A deficiência da Neu1 é a base da sialidose, doença grave associada a um amplo espectro de manifestações, incluindo hipotonia e fraqueza muscular. Camundongos com deficiência de Neu1 desenvolvem degeneração muscular caracterizada principalmente por atrofia, invasão das fibras musculares por fibroblastos e expansão da matriz extracelular. A Neu1 controla a proliferação de fibroblastos de pacientes por meio da desialilação dos receptores de PDGF e IGF. Além disso, há enzimas lisossomais que são moduladas pela Neu1, tais como as catepsinas, que são capazes de degradar componentes musculares e estariam excessivamente ou erroneamente ativas (sialiladas) em decorrência da deficiência de Neu1. O objetivo deste trabalho foi identificar se o fenótipo da musculatura esquelética de camundongos Neu1-/- poderia estar associado à atividade do IGF-1R, PDGFR e/ou à sialilação de catepsina B, através da análise histológica e proteica de músculos esqueléticos e fibroblastos de camundongos Neu1+/+ e Neu1-/- tratados com inibidores de IGF1-R e PDGFR. O estudo da expressão proteica de catepsina B foi realizado nos músculos tratados com os inibidores de IGF-1R e PDGFR, e nas frações citosólica e lisossomal de fibroblastos tratados com neuraminidase exógena. Em comparação com camundongos Neu1+/+, os músculos de animais Neu1-/- apresentam menor área de fibra, peso corporal, expressão de pAkt e maior expressão de catepsina B; e os fibroblastos Neu1-/- exibem maior proliferação e expressão de pAkt. A inibição do IGF-1R em camundongos Neu1-/- aumentou a área das fibras musculares, expressão de pAKt e diminuiu a expressão de catepsina B; em relação aos fibroblastos Neu1-/-, entretanto aumentou a proliferação celular com diminuição de pAkt. A inibição do PDGFR em músculos de camundongos Neu1-/- levou ao aumento da expressão de pAkt, da área das fibras, com diminuição de pERK e catepsina L, quando comparados com os controles Neu1-/-; a mesma inibição in vitro conduziu à diminuição da expressão de pAkt, pERK e proliferação. A catepsina B encontra-se bastante ativa na fração lisossomal e o tratamento com neuraminidase foi eficaz na correção de seu peso molecular e compartimentalização lisossomal. De forma geral, o fenótipo muscular de camundongos Neu1-/- parece estar relacionado com a atividade de IGF-1R e PDGFR, e a catepsina B hipersialilada é potencialmente deletéria para o músculo esquelético / Neuraminidase 1 (Neu1) is an enzyme that regulates the catabolism of sialoglycoconjugates in lysosomes. Neu1 deficiency is the basis of sialidosis, a severe disease associated with a broad spectrum of manifestations, including hypotonia and muscle weakness. Neu1 deficient mice develop muscular degeneration characterized by atrophy, invasion of muscle fibers by fibroblasts, and expansion of the extracellular matrix. Neu1 controls the proliferation of fibroblasts from patients through the desialylation of PDGF and IGF receptors. In addition, lysosomal enzymes are modulated by Neu1, such as cathepsins, which degrade muscle components and are excessively or erroneously active (sialylated) as a result of Neu1 deficiency. The aim of this study was to identify whether skeletal muscle phenotype of Neu1-/- mice may be associated with IGF-1R, PDGFR and/or sialylation of cathepsin B, through protein and histological analysis of skeletal muscles and fibroblast from Neu1+/+ and Neu1-/- mice treated with IGF-1R and PDGFR inhibitors. The study of cathepsin B protein expression was performed in skeletal muscles treated with IGF-1R and PDGFR inhibitors, and in the cytosolic and lysosomal fractions of fibroblasts treated with exogenous neuraminidase. Compared with Neu1+/+ animals, Neu1-/- muscles showed smaller muscle fiber area, body weight, pAkt expression and higher cathepsin B expression; and Neu1-/- fibroblasts exhibited increased proliferation and expression of pAkt. The inhibition of IGF-1R Neu1-/- mice increased the area of muscle fibers, expression of pAkt and decreased expression of cathepsin B; but, considering Neu1-/- fibroblasts, there was increased cell proliferation with reduction of pAkt. The inhibition of PDGFR in muscles of Neu1-/- mice led to increased expression of pAkt, muscle fiber area, with decreased expression of pERK and cathepsin L, when compared with the Neu1-/- controls; the same inhibition in vitro led to reduced expression of pAkt, pERK and cell proliferation. Cathepsin B presented high activity in the lysosomal fraction and the treatment with neuraminidase was effective in the correction of its molecular weight and lysosomal compartmentalization. In general, the muscular phenotype of Neu1-/- mice is possibly related to IGF-1R and PDGFR activity, and oversialylated cathepsin B is potentially deleterious for the skeletal muscle

研究目的：脊髓損傷是中樞神經系統的嚴重創傷，致殘率高。脊髓損傷後的再生修復一直是當前醫學的難題。迄今為止，脊髓損傷依然缺乏一種有效地治療方法。既往研究證明，胰島素樣生長因子-1對鼠和兔脊髓損傷有保護作用，為了進一步把這些發現應用到臨床方面，我們採用與人類生理更相近的豬只作為實驗動物，構建與臨床相似的脊髓損傷動物模型，并以此為基礎，系統性研究胰島素樣生長因子-1的脊髓保護作用，評估該治療的功效。 / 研究方法：以運動誘發電位為指導，通過直接壓迫和牽拉造成脊髓損傷。18頭猪只隨機分為3組：胰島素樣生長因子-1治療組、生長激素治療組及生理鹽水對照組。脊髓損傷后1小時、24小時及48小時經鞘內注射給藥。于術後第1天、第3天及第21天收集腦脊液檢測胰島素樣生長因子-1和生長激素濃度。連續21天使用修正的 Tarlov 評分標準對動物的運動功能進行評估。第21天處死動物並取材，檢測脊髓中NeuN, GFAP, caspase-3 的活性，并通過TUNEL染色觀察細胞凋亡情況，比較各組之間有無差別。 / 研究結果：通過這種方法建立的脊髓損傷動物模型穩定可靠，各組之間無明顯差異。鞘內給藥24小時及48小時后，腦脊液中胰島素樣生長因子-1和生長激素濃度明顯升高，術後21天檢測，其濃度恢復至基礎值。胰島素樣生長因子-1治療組的運動功能的恢復優於其它各組。與生理鹽水對照組比較，胰島素樣生長因子-1治療組可以明顯提高脊髓損傷后神經元的存活數量，抑制星形膠質細胞增生，減少細胞凋亡。而生長激素治療組僅抑制星形膠質細胞增生，其它方面與生理鹽水對照組無明顯差別。 / 結論：胰島素樣生長因子-1通過提高神經元存活數量，抑制星形膠質細胞增生，以及減少細胞凋亡促進脊髓損傷的恢復。 / Objective: Spinal cord injury is a devastating condition that leads to long-term disabilities. Currently, there is no effective treatment that minimizes spinal cord damage or enhances neurological recovery. Recent studies in rats or rabbits suggested that neurologic recovery after spinal cord injury could be improved with the administration of neurotropic hormones, such as insulin-like growth factor-1 (IGF-1). In order to apply such bench-side discovery to clinical practice, we conducted a study in a higher animal model, akin to human physiology, to evaluate the effectiveness of intrathecal injections of IGF-1to improve neurological recovery in a porcine model of acute traumatic spinal cord injury. / Methods: Traumatic spinal cord injury model was produced by controlled compression and distraction of the exposed T12 segment of the spinal cord. Eighteen pigs were randomly assigned to receive intrathecal injections of either IGF-1, growth hormone or saline at 1, 24 and 48 hours after spinal cord injury. Locomotor function was assessed daily using the validated modified Tarlov’s scale for 21 days. Spinal cord segments were then harvested and the survival of neurons, reactive astrogliosis and apoptosis were determined using neuronal-specific nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays. / Results: Intrathecal injections of IGF-1 and growth hormone significantly increase the concentrations of the neurotropic hormones in the cerebrospinal fluid after injury (p < 0.01). These concentrations returned to baseline by 21 days after drug delivery. Motor deficits on the first day after injury were comparable between animals in the treatment and control groups. By the end of the third week, neurologic recovery was better in animals receiving IGF-1 treatment (p < 0.05). Immunohistological and western blot studies of the injured segments of spinal cord showed that treatment with both IGF-1 and growth hormone prevented reactive astrogliosis (p < 0.05) while only IGF-1 improved the survival of mature neurons (p < 0.05). IGF-1 also inhibited apoptosis after spinal cord injury (p < 0.05). / Conclusions: In our clinically relevant model of traumatic spinal cord injury in pigs, intrathecal injection of IGF-1 demonstrated beneficial effects on neurological and histological recovery. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Qinzhou. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 105-122). / Abstract also in Chinese. / Declaration of origination --- p.I / Abstract --- p.II / Acknowledgements --- p.VI / Table of Contents --- p.VIII / List of Tables --- p.XII / List of Figures --- p.XIII / Abbreviations --- p.XVIII / Chapter Part 1 --- Spinal Cord Injury: A Review --- p.1 / Chapter Chapter 1-1 --- Acute Spinal Cord Injury: Epidemiology, Socioeconomic Impact --- p.2 / Chapter 1.1.1 --- Epidemiology of Spinal Cord Injury --- p.2 / Chapter 1.1.2 --- Socioeconomic Impact of Acute Spinal Cord Injury --- p.5 / Chapter Chapter 1-2 --- Mechanisms of Spinal Cord Injury --- p.6 / Chapter Chapter 1-3 --- Putative Treatments for Spinal Cord Injury --- p.8 / Chapter 1.3.1 --- Methylprednisolone --- p.8 / Chapter 1.3.2 --- Stem Cell Therapy --- p.11 / Chapter 1.3.3 --- Riluzole --- p.11 / Chapter 1.3.4 --- Other Pharmacological Therapies for Spinal Cord Injury --- p.12 / Chapter Chapter 1-4 --- Insulin-like Growth Factor-1 for the Treatment of Spinal Cord Injury --- p.13 / Chapter Chapter 1-5 --- Summary --- p.17 / Chapter Part 2 --- Insulin-like Growth Factor-1 and Growth Hormone for Spinal Cord Injury --- p.18 / Chapter Chapter 2-1 --- Hypothesis and Objectives --- p.19 / Chapter Chapter 2-2 --- Establishment of Animal Models for Acute Spinal Cord Injury --- p.22 / Chapter 2.2.1 --- Introduction --- p.22 / Chapter 2.2.2 --- Experimental Animals --- p.22 / Chapter 2.2.3 --- Anesthesia --- p.23 / Chapter 2.2.4 --- Transcranial Electrical Motor Evoked Potential --- p.26 / Chapter 2.2.5 --- Surgery --- p.28 / Chapter 2.2.6 --- Statistics --- p.34 / Chapter 2.2.7 --- Results --- p.34 / Chapter 2.2.8 --- Discussion --- p.38 / Chapter Chapter 2-3 --- Optimal Stimulation Protocols for Transcranial Electrical Motor Evoked Potential. --- p.42 / Chapter 2.3.1 --- Introduction --- p.42 / Chapter 2.3.2 --- Methods --- p.42 / Chapter 2.3.2.1 --- Experimental Animals and Anesthesia --- p.42 / Chapter 2.3.2.2 --- Transcranial Electrical Motor Evoked Potential Recording --- p.44 / Chapter 2.3.2.3 --- Stimulation Protocol --- p.44 / Chapter 2.3.3 --- Analyses --- p.44 / Chapter 2.3.4 --- Results --- p.45 / Chapter 2.3.5 --- Discussion --- p.52 / Chapter Chapter 2-4 --- Evaluation of the Efficacy of Insulin-like Growth Factor-1 and Growth Hormone in a Porcine Model --- p.54 / Chapter 2.4.1 --- Introduction --- p.54 / Chapter 2.4.2 --- Materials and Methods --- p.54 / Chapter 2.4.2.1 --- Study Design --- p.54 / Chapter 2.4.2.2 --- Intrathecal Injection and Collection of Cerebrospinal Fluid --- p.58 / Chapter 2.4.2.3 --- Measurements --- p.58 / Chapter 2.4.2.3.1 --- Clinical Evaluation --- p.58 / Chapter 2.4.2.3.2 --- Biochemical Assessments --- p.58 / Chapter 2.4.2.3.3 --- Spinal Cord Section, Histological and Immunochemical Staining --- p.63 / Chapter 2.4.2.3.4 --- Western Blot --- p.69 / Chapter 2.4.3 --- Statistical Analysis and Sample Size Calculation --- p.72 / Chapter 2.4.3.1 --- General Analysis --- p.72 / Chapter 2.4.3.2 --- Sample Size --- p.72 / Chapter 2.4.4 --- Results --- p.73 / Chapter 2.4.4.1 --- Changes of TceMEP --- p.73 / Chapter 2.4.4.2 --- Motor Deficit after Spinal Cord Injury at Baseline --- p.75 / Chapter 2.4.4.3 --- Insulin-like Growth Factor-1 and Growth Hormone in Cerebrospinal Fluid --- p.77 / Chapter 2.4.4.4 --- Clinical Assessment --- p.80 / Chapter 2.4.4.5 --- Demyelination, Neuron Survival and Astrocyte Reaction --- p.85 / Chapter 2.4.4.6 --- Apoptosis --- p.89 / Chapter 2.4.5 --- Discussion --- p.93 / Chapter 2.4.5.1 --- Principal Findings --- p.93 / Chapter 2.4.5.2 --- Insulin-like Growth Factor-1 and Neuroprotection after Spinal Cord Injury --- p.93 / Chapter 2.4.5.3 --- Growth Hormone and Neuroprotection after Spinal Cord Injury --- p.95 / Chapter 2.4.5.4 --- Strengths and Limitations of Our Study --- p.96 / Chapter 2.4.5.5 --- Summary --- p.97 / Chapter Part 3 --- Summary and Future Directions --- p.99 / Chapter Chapter 3-1 --- Summary --- p.100 / Chapter Chapter 3-2 --- Future Directions --- p.103 / Chapter Part 4 --- References and appendixes --- p.104 / References --- p.105 / Appendixes --- p.123

Crianças nascidas pequenas para a idade gestacional (PIG) apresentam maior risco de permanecerem com baixa estatura na vida adulta. Os fatores de crescimento insulina-símile 1 e 2 (IGF-1 e IGF-2) são os principais fatores endócrinos determinantes do crescimento fetal. Na vida pós-natal o GH, principal hormônio promotor de crescimento, exerce a maior parte de seus efeitos por meio do IGF-1. A grande maioria das ações conhecidas do IGF-1 e IGF-2 são mediadas via receptor tirosina quinase conhecido como receptor tipo 1 de IGFs (IGF-1R). Os objetivos deste trabalho foram estudar os genes IGF1 e IGF1R em crianças nascidas pequenas para a idade gestacional que não recuperaram o crescimento na vida pós-natal. Foram selecionados 145 pacientes nascidos PIG, 72 sem catch up e 73 com catch up. Em 54 PIG sem catch up foi estudado toda a seqüência codificadora do gene IGF1 por meio de PCR e seqüenciamento direto, nos demais PIG sem catch up e nos 73 PIG com catch up foi estudado apenas o exon 6 do IGF-1 por PCR e seqüenciamento direto para avaliação de um polimorfismo encontrado nesta região. Nos pacientes que apresentavam concentração sérica de IGF-1 e IGFBP-3 acima da média para idade e sexo e seqüência do IGF1 normal (n=23) foi realizada coleta de sangue periférico com posterior separação de leucócitos mononucleares pelo gradiente de ficoll seguido por extração de RNA pelo método de Trizol® Posteriormente, a partir do RNA, sintetizamos o cDNA (DNA complementar) utilizando primers randômicos. Foi realizado PCR e seqüenciamento direto do cDNA, além de análise da expressão do IGF1R por PCR em tempo real. Nenhuma mutação foi encontrada no gene IGF1. Entretanto um locus altamente polimórfico foi encontrada na região 3\' não traduzida do exon 6 deste gene, região esta envolvida no processo de poliadenilação. A freqüência das variantes alélicas foi semelhante em PIG com e sem catch-up e em controles nascidos AIG. Analisando o fenótipo de pacientes PIG que apresentavam a variante alélica wild type ou uma das três variantes alélicas mais freqüentemente encontradas, não observamos diferenças significativas entre peso e comprimento ao nascimento, níveis de IGF-1 e crescimento na vida pós-natal. No gene IGF1R encontramos duas variantes alélicas nunca descritas previamente. A primeira variante encontrada está localizada no exon 1, em uma região de peptídeo sinal do pro IGF-1R e consiste na troca do nucleotídeo guanina pelo nucleotídeo adenina na posição 16 da região codificadora (c.16G>A), levando a troca do aminoácido glicina por arginina na posição 6 da proteína (p.G6R). A outra mutação encontrada está localizada no exon 7 onde observamos uma troca do nucleotídeo citosina por timina na posição 1531 do cDNA (c.1531 C>T), levando a uma troca de arginina por triptofano na posição 511 do IGF1R (p.R511W). Adicionalmente, foi observada uma expressão do IGF1R diminuída em 5 pacientes estudados.Concluímos que as variantes alélicas encontradas na região de poliadenilação do IGF1 não influenciam significativamente as características ao nascimento e pós-natais de crianças nascidas PIG ou a altura adulta de indivíduos normais nascidos AIG. O estudo do IGF1R identificou duas novas variantes alélicas em heterozigose no gene IGF1R e, em cinco pacientes, observamos uma expressão reduzida deste gene. Pacientes com alterações no gene IGF1R não apresentam um fenótipo característico que os diferencie de outras crianças nascidas PIG sem alterações neste gene, mostrando a importância dos estudos moleculares. / Children born small for gestational age (SGA) have a higher risk of remaining short in adulthood. The insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) are the main factors determining endocrine fetal growth. GH is the main promoter of linear growth in the postnatal life, exerting its effects mostly through the IGF-1. The vast majority of known actions of IGF-1 and IGF-2 are mediated by the insulin-like growth factor type 1 receptor (IGF-1R), a member of the tyrosine kinase receptors family. The aim of this study was to investigate IGF1 and IGF1R genes mutations in children born small for gestational age without catch up growth in postnatal life. We selected 145 patients born SGA, 72 without catch-up and 73 with catch up. The whole coding region of the IGF1 gene was sequenced in 54 patients without catchup. In the other SGA children without catch-up and in 73 SGA with catch-up, only the exon 6 of IGF1 was sequenced to assess the influence of allelic variants present in this region. In patients with normal IGF1 sequence and IGF-1 and IGFBP-3 serum levels above the mean for age and sex (n = 23) total RNA was extracted from peripheral blood lymphocytes followed by cDNA synthesis with random primers. The IGF1R cDNA was amplified using specific primers followed by direct sequencing. IGF1R expression was analyzed by real-time PCR. No mutations were found in the IGF1 gene. However a highly polymorphic sequence was identified in the upstream core polyadenylation signal (UCPAS) located in IGF1 3\' UTR at exon 6. The frequency of the identified allelic variants was similar in SGA children with and without catch-up and in controls. Furthermore, children homozygous for the wild-type allele and those carrying the allelic variants in homozygous or heterozygous state presented similar weight and length at birth, as well as serum IGF-1 levels and postnatal growth features. Two novel nonconservative allelic variants were identified in IGF1R in 23 SGA children (8.7%) in the heterozygous state. The first variant (c.16G>A) was located in the exon one, leading to a substitution of glicine by arginine in the pro-IGF-1R signaling peptide (p.G6R). The second variant was located in exon 7 (c.1531 C>T), leading to a substitution of arginine by tryptophan in the amino acid 511 of the IGF1-R (p.R511W). Moreover, a decreased IGF1R expression was observed in 5 of the 23 patients with elevated serum IGF-1 concentrations. We conclude that the UCPAS allelic variants did not significantly influence the birth and postnatal characteristics of children born SGA, neither the adult height of normal individuals born adequate for gestational age. The IGF1R study identified two novel allelic variants in two patients and a reduced expression of the IGF1R was observed in five patients. Patients with alterations in IGF1R did not have a distinctive phenotype when compared with other children born SGA without changes in this gene, indicating the importance of molecular studies.

<p>The interest for the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis and its role in the central nervous system (CNS) has grown during the past decade. GH has been associated with psychological functions as sleep, mood, general well-being and learning and memory. The present thesis is a contribution to clarify the functions and mechanisms involved in the actions of GH and IGF-1 in the CNS. A variant of the GH receptor (GHR) gene transcript lacking exon 3 (GHR3-) was cloned from ovine choroid plexus epithelial cells and tissue. The GHR3- transcript has previously only been identified in human tissue. Further, an anatomical study of the localization of GHR mRNA in the rat brain stem and spinal cord was carried out by the use of in situ hybridization. High densities of GHRs were found in areas associated with the regulation of food intake, sleep and nociception, functions known to be influenced by the GH/IGF-1 axis. The interaction with the opioid system was studied by an acute treatment with morphine. The levels of the transcripts for GHR and GHBP in the rat hippocampus and spinal cord were decreased 4 h after the injection of the opiate and restored to normal levels after 24 h. Young and aged rats injected with GH or IGF-1 showed differential gene regulation of subunits of the NMDA subtype of glutamate receptor in the hippocampus. This indicates an age-related difference in the sensitivity to GH/IGF-1 mediated effects on memory functions. Moreover, hypophysectomized rats treated with GH showed improved performance in the Morris water maze, a spatial memory task. The effect was accompanied with an increase in transcripts for NMDA receptor subunits and its associated membrane anchoring PSD-95 protein. Taken together, the results suggest that GH and/or IGF-1 play important roles in mechanisms associated with cognitive functions.</p>

The objective of this thesis was to investigate the effects of the somatotrope GH/IGF-1 axis upon the thymus. This work included two parts: 1. Translational research study: Thymus function in adult GH deficiency (AGHD) with and without GH treatment Background: Despite age-related adipose involution, T cell generation in the thymus (thymopoiesis) is maintained beyond puberty in adults. In rodents, growth hormone (GH), insulin-like growth factor-1 (IGF-1), and GH secretagogues reverse agerelated changes in thymus cytoarchitecture and increase thymopoiesis. GH administration also enhances thymic mass and function in HIV-infected patients. Until now, thymic function has not been investigated in adult GH deficiency (AGHD). The objective of this clinical study was to evaluate thymic function in AGHD, as well as the repercussion upon thymopoiesis of GH treatment for restoration of GH/IGF-1 physiological levels. Methodology/Principal Findings: Twenty-two patients with documented AGHD were enrolled in this study. The following parameters were measured: plasma IGF-1 concentrations, signal-joint T-cell receptor excision circle (sjTREC) frequency, and sj/b TREC ratio. Analyses were performed at three time points: firstly on GH treatment at maintenance dose, secondly one month after GH withdrawal, and thirdly one month after GH resumption. After 1-month interruption of GH treatment, both plasma IGF-1 concentrations and sjTREC frequency were decreased (p,0.001). Decreases in IGF-1 and sjTREC levels were correlated (r = 0.61, p,0.01). There was also a decrease in intrathymic T cell proliferation as indicated by the reduced sj/b TREC ratio (p,0.01). One month after reintroduction of GH treatment, IGF-1 concentration and sjTREC frequency regained a level equivalent to the one before GH withdrawal. The sj/b TREC ratio also increased with GH resumption, but did not return to the level measured before GH withdrawal. Conclusions: In patients with AGHD under GH treatment, GH withdrawal decreases thymic T cell output, as well as intrathymic T cell proliferation. These parameters of thymus function are completely or partially restored one month after GH resumption. These data indicate that the functional integrity of the somatotrope GH/IGF-1 axis is important for the maintenance of a normal thymus function in human adults. 2. Fundamental study: intrathymic expression of members of the GH/IGF-1 axis and effects of GH on T-cell differentiation in murine fetalthymic organ cultures (FTOC). We here address the question of expression and role of GH/IGF axis in the thymus. Methods: Using RT-qPCR, the expression profile of various components of the somatotrope GH/IGF axis was measured in different thymic cell types and during thymus embryogenesis in Balb/c mice. Effect of GH on T-cell differentiation was explored through thymic organotypic culture. Results: Transcription of Gh, Igf1, Igf2 and their related receptors predominantly occurred in thymic epithelial cells (TEC), while a low level of Gh and Igf1r transcription was also evidenced in thymic T cells (thymocytes). Gh, Ghr, Ins2, Igf1, Igf2, and Igfr1, displayed distinct expression profiles depending on the developmental stage. The protein concentration of IGF-1 and IGF-2 were in accordance with the profile of their gene expression. In fetal thymus organ cultures (FTOC) derived from Balb/c mice, treatment with exogenous GH resulted in a significant increase of double negative CD4-CD8- T cells and CD4+ T cells, with a concomitant decrease in double positive CD4+CD8+ T cells. These changes were inhibited by concomitant treatment with GH and GHR antagonist pegvisomant. However, GH treatment also induced a significant decrease in FTOC Gh, Ghr and Igf1 expression. Conclusion: These data show that the thymotropic properties of the somatotrope GH/IGF-1 axis involve an interaction between exogenous GH and GHR expressed by TEC. Since thymic IGF-1 is not increased by GH treatment, the effects of GH upon T-cell differentiation could implicate a different local growth factor or cytokine.

The interest for the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis and its role in the central nervous system (CNS) has grown during the past decade. GH has been associated with psychological functions as sleep, mood, general well-being and learning and memory. The present thesis is a contribution to clarify the functions and mechanisms involved in the actions of GH and IGF-1 in the CNS. A variant of the GH receptor (GHR) gene transcript lacking exon 3 (GHR3-) was cloned from ovine choroid plexus epithelial cells and tissue. The GHR3- transcript has previously only been identified in human tissue. Further, an anatomical study of the localization of GHR mRNA in the rat brain stem and spinal cord was carried out by the use of in situ hybridization. High densities of GHRs were found in areas associated with the regulation of food intake, sleep and nociception, functions known to be influenced by the GH/IGF-1 axis. The interaction with the opioid system was studied by an acute treatment with morphine. The levels of the transcripts for GHR and GHBP in the rat hippocampus and spinal cord were decreased 4 h after the injection of the opiate and restored to normal levels after 24 h. Young and aged rats injected with GH or IGF-1 showed differential gene regulation of subunits of the NMDA subtype of glutamate receptor in the hippocampus. This indicates an age-related difference in the sensitivity to GH/IGF-1 mediated effects on memory functions. Moreover, hypophysectomized rats treated with GH showed improved performance in the Morris water maze, a spatial memory task. The effect was accompanied with an increase in transcripts for NMDA receptor subunits and its associated membrane anchoring PSD-95 protein. Taken together, the results suggest that GH and/or IGF-1 play important roles in mechanisms associated with cognitive functions.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, with two predominant histologic subtypes: embryonal and alveolar. These histologies display distinct clinical courses, and despite refinements in dose and duration of multimodality therapy, the 5-year overall survival of patients diagnosed with metastatic RMS remains <30%. Thus, there is an urgent need to define novel targets for therapeutic intervention. Interrogation of cancer cell signal transduction pathways that regulate the pathogenic behaviours of tumor cells has been successful in defining targets in numerous tumor types. These have ultimately yielded clinically-relevant drugs that have improved the disease-free and overall survival of patients diagnosed with cancer. Work contained in this thesis describes the interrogation of several potential targets for inhibition in RMS. Interruption of RMS cell proliferation, survival and apoptosis is examined through disruption of the protein kinase integrin-linked kinase (ILK) and the nuclear receptor estrogen-receptor β. ILK, in particular, is demonstrated to have dual competing functions through the regulation of c-jun amino-terminal kinase (JNK) signaling: an oncogene in alveolar, and a tumor suppressor in embryonal RMS. These findings are recapitulated in other tumor cell lines, indicating that expression levels of JNK1 correlate with ILK function in a broad spectrum of tumor types. Furthermore, interruption of rhabdomyosarcoma cell migration as a surrogate marker of metastasis is examined through disruption of the stromal-cell derived factor 1α/chemokine (CXC)receptor 4 signaling network, as well as through cooperative interactions between ILK and the mammalian target of rapamycin. Finally, we demonstrate that the insulin-like growth factor pathway is a potential target for therapeutic inhibition, which also distinguishes tumors of embryonal and alveolar histology. These studies provide a rationale for the development of novel agents, as well as the use of established drugs targeting these pathways in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, with two predominant histologic subtypes: embryonal and alveolar. These histologies display distinct clinical courses, and despite refinements in dose and duration of multimodality therapy, the 5-year overall survival of patients diagnosed with metastatic RMS remains <30%. Thus, there is an urgent need to define novel targets for therapeutic intervention. Interrogation of cancer cell signal transduction pathways that regulate the pathogenic behaviours of tumor cells has been successful in defining targets in numerous tumor types. These have ultimately yielded clinically-relevant drugs that have improved the disease-free and overall survival of patients diagnosed with cancer. Work contained in this thesis describes the interrogation of several potential targets for inhibition in RMS. Interruption of RMS cell proliferation, survival and apoptosis is examined through disruption of the protein kinase integrin-linked kinase (ILK) and the nuclear receptor estrogen-receptor β. ILK, in particular, is demonstrated to have dual competing functions through the regulation of c-jun amino-terminal kinase (JNK) signaling: an oncogene in alveolar, and a tumor suppressor in embryonal RMS. These findings are recapitulated in other tumor cell lines, indicating that expression levels of JNK1 correlate with ILK function in a broad spectrum of tumor types. Furthermore, interruption of rhabdomyosarcoma cell migration as a surrogate marker of metastasis is examined through disruption of the stromal-cell derived factor 1α/chemokine (CXC)receptor 4 signaling network, as well as through cooperative interactions between ILK and the mammalian target of rapamycin. Finally, we demonstrate that the insulin-like growth factor pathway is a potential target for therapeutic inhibition, which also distinguishes tumors of embryonal and alveolar histology. These studies provide a rationale for the development of novel agents, as well as the use of established drugs targeting these pathways in rhabdomyosarcoma.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 199-250.