Thesis (doctoral)--Lund University, 1993. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1993. / Added t.p. with thesis statement inserted.

Proefschrift Universiteit Maastricht. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Thesis (Ph.D.)--Baylor University, 2009. / Includes bibliographical references (p. 126-143).

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<p>The RAS family is a group of small GTPases that can become constitutively activated by point mutations that are found in about 30% of all cancer patients. There are three well-characterized RAS family members: HRAS, NRAS, and KRAS, the latter of which is alternatively spliced at the C-terminus into KRAS4A and KRAS4B. The RAS proteins are all nearly identical at their N-termini and core effector binding domains, but have divergent C-terminal membrane-binding regions that impart different subcellular localization and subtle differences in signaling. Although the role of constitutively activated oncogenic RAS has been well established to play a role in cancer, recent work has suggested that wild-type RAS signaling may also be important in tumorigenesis. Wild-type RAS proteins have been shown to be activated in the presence of oncogenic KRAS. However, the consequences of this activation are context-dependent, as signaling through the wild-type RAS proteins has been shown to both suppress neoplastic growth and promote tumorigenesis under different circumstances.</p><p>I sought to investigate the role of the wild-type RAS proteins in two clinically –relevant models of cancer: pancreatic, the type of cancer most frequently associated with KRAS mutations, and lung cancer, the cancer in which KRAS mutations affect the highest number of patients. First, I tested whether a loss of wild type Hras altered tumorigenesis in a mouse model of pancreatic cancer driven by oncogenic Kras. Hras homozygous null mice (Hras-/- ) exhibited more precancerous lesions of the pancreas as well as more off-target skin papillomas compared to their wild type counterparts, indicating that Hras suppresses early Kras-driven pancreatic tumorigenesis. Loss of Hras also reduced the survival of mice engineered to develop aggressive pancreatic cancer by the additional disruption of one allele of the tumor suppressor p53 (Trp53R172H/+). However, this survival advantage was lost when both alleles of Trp53 were mutated, suggesting that wild-type HRas inhibits tumorigenesis in a p53-dependant manner. </p><p>Next, I investigated the role that wild-type Hras and Nras play in a chemical carcinogen-induced model of lung cancer. In mice treated with urethane, a carcinogen that induces Kras-mutation positive lung lesions, Hras-/ mice once again developed more tumors than wild-type mice. Interestingly, however, this effect was not observed in mice lacking wild-type Nras. Mice lacking both Hras and Nras alleles developed approximately the same number of tumors as Hras-/- mice, thus the additional loss of Nras does not appear to enhance the tumor-promoting effects of loss of Hras. In summary, signaling through wild-type Hras, but not Nras, suppresses tumorigenesis in a carcinogen-induced model of lung cancer.</p><p>The tumor-suppressive effects of wild-type Ras signaling were traced to the earliest stages of pancreatic tumorigenesis, suggesting that wild-type Ras signaling may suppress tumorigenesis as early as the time of initiation. These findings suggest that differences in expression of the wild-type Ras isoforms could potentially play a role in an individual’s predisposition for developing cancer upon oncogenic insult.</p> / Dissertation

Le diabète de type 1 (DT1) résulte de la destruction des cellules β productrices d’insuline par le système immunitaire. Cette condition représente un enjeu de santé publique majeur car, malgré les thérapies actuelles, les patients atteints développent trop souvent des complications cardio-vasculaires. Des thérapies alternatives se doivent donc d’être mises au point. Ainsi, diverses approches visent à reprogrammer/différencier (in vitro ou in vivo) différents types cellulaires pancréatiques afin de générer des cellules β (productrices d’insuline) fonctionnelles. Dans ce but, notre laboratoire a notamment montré que les cellules α (productrices de glucagon) embryonnaires peuvent être régénérées et converties en cellules β fonctionnelles par l’expression ectopique du seul gène Pax4 (un gène normalement impliqué dans la spécification embryonnaire du lignage β - (Collombat and Mansouri, 2009)). Dans la première partie de ce travail, nous démontrons que les cellules α à l’âge adulte (Al-Hasani et al., 2013) retiennent leur capacité de régénération et de conversion en cellules β, celles-ci étant fonctionnelles et capable de remplacer plusieurs fois l’ensemble des cellules β du pancréas. Cependant, cette approche transgénique serait difficile à mettre en œuvre chez l’homme. De nombreux cribles furent donc initiées dans le but de trouver des petites molécules/composés chimiques mimant les effets de Pax4. Un composé potentiel, GABA, fut ainsi identifié et caractérisé. / Type 1 diabetes (T1D) results from the destruction of insulin-producing β-cells by the immune system. This condition is a major public health issue because, despite current therapies, patients often develop cardiovascular complications. Therefore alternative therapies need to be developed. Thus, various approaches are designed to reprogram / differentiate (in vitro or in vivo) different pancreatic cell types to generate functional (insulin-producing) β-cells. To this end, our laboratory has shown that especially the embryonic α-cells (producing glucagon) can be regenerated and converted into functional β-cells by the ectopic expression of the Pax4 gene (usually a gene involved in the specification of embryonic lineage β - (Collombat and Mansouri, 2009)). In the first part of this work, we show that α-cells in adulthood (Al-Hasani et al., 2013) retain their capacity for regeneration and conversion into β-cells, the latter being functional and able to replace repeatedly all the β-cells of the pancreas. However, this transgenic approach would be difficult to implement in humans. Many screens were therefore initiated in order to find small molecules / chemical compounds that mimic the effects of Pax4. A potential compound, GABA, was identified and characterized. Our results demonstrate that treatment of WT mice with GABA results in a significant increase in the number and size of the islets (caused by insulin+ cell hyperplasia). By using lineage tracing tools, our results indicate that these "β-like" neo-generated cells are coming from glucagon+ cells.

Pancreatic ductal adenocarcinoma (PDAC) is a cancer of unmet need with a 5-year survival following diagnosis of 3% with limited surgical, radiotherapy and chemotherapy treatment options. Central to PDAC tumorigenesis is acquisition of an oncogenic Kras mutation to drive acinar-to-ductal metaplasia and progression to PDAC that is potentiated by NF-kB deregulation. However, PDAC requires the additional loss of tumour suppressors such as p53, SMAD4 or p16. ASPP family members ASPP2 and iASPP regulate both p53 and NF-kB, and are classified as a tumour suppressor and oncogene respectively. However, the precise roles of ASPP2 and iASPP in pancreatic cancer are unknown. In this thesis I demonstrate that ASPP2 suppresses metastasis and iASPP suppresses the pro-inflammatory tumour microenvironment. In a mouse model of PDAC development, ASPP2-deficiency does not alter metaplasia, PanIN progression or primary PDAC onset. However, median survival due to metastasis is significantly reduced in an ASPP2-deficient PDAC model. I demonstrate ASPP2-deficient PDAC can result in increased squamous differentiation defined histologically or via increased p63 expression. I propose ASPP2 is a key suppressor &Delta;Np63 and the squamous PDAC subtype in vivo. Conversely, iASPP is a putative oncogene and high expression in cancer associates with poor prognosis. However, in a mouse model of PDAC, loss of iASPP accelerates PDAC onset and metastasis. I demonstrate that iASPP is a functional tumour suppressor of a pro-inflammatory phenotype in response to oncogenic Kras and pancreatitis. I propose ASPP2- and iASPP-deficient mouse models of PDAC represent in vivo the squamous and immunogenic subtypes of PDAC respectively; and are relevant tools to study mechanisms of metastasis and inflammation-driven carcinogenesis.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2015. / Made available in DSpace on 2015-09-29T04:08:50Z (GMT). No. of bitstreams: 1 334525.pdf: 7690982 bytes, checksum: a566cb5bfdf076e3559a98ca79c5fc74 (MD5) Previous issue date: 2015 / A hiperglicemia crônica proveniente da diminuição da produção insulínica e do aumento da resistência tecidual à insulina é a principal causa de danos teciduais associados à diabetes e insulinoresistência, e pode ser desencadeada por fatores como menor disponibilidade de GLUT4. Compostos insulinoestimulantes e que regulam de forma rápida a homeostase da glicose e a translocação de GLUT4 para a membrana plasmática são de grande importância na terapêutica da diabetes, e nesse âmbito se encontram os triterpenos pentacíclicos (TPs) e a 1,25 (OH)2 vitamina D3 (1,25-D3). Os TPs são metabólitos vegetais amplamente disponíveis na dieta humana e sua potencial ação nutracêutica desperta atenção devido ao efeito antidiabético frequentemente relatado, porém, cientificamente pouco comprovado. Ainda, estudos sugerem que a deficiência em 1,25-D3 prejudica a secreção de insulina em ratos, podendo levar a diabetes melito tipo 2, enquanto o tratamento com esse hormônio aumentou a sensibilidade à insulina por estimular a expressão do receptor de insulina e o transporte de glicose. Contudo, os mecanismos de ação da 1,25-D3 neste aspecto ainda são pouco elucidados. Neste trabalho foi estudado o efeito antihiperglicêmico e o mecanismo de ação da 1,25-D3 e de triterpenos naturais na secreção insulínica e absorção tecidual de glicose. Para análise de curva tempo e dose resposta dos triterpenos, ratos Wistar machos após jejum (16 h) foram pré-tratados com os TPs (ácido betulínico (AB), ácido ursólico (AU), trihidroxioleanano (THO), 3ß-hidroxihopeno (BHH) ou fernenodiol) e após 30 min foi induzida a hiperglicemia para análise da tolerância à glicose. Do soro foram determinadas a glicemia, insulina, lactato desidrogenase, GLP-1 e a concentração de cálcio. Ainda nestes animais, o músculo sóleo e o fígado foram utilizados para dosagem de conteúdo total de glicogênio. Para estudos in vitro de mecanismo de ação, foram investigados os processos de captação de 14C-deoxiglicose, incorporação de 14C-timidina ao DNA, de 14C-leucina em proteínas em presença ou não de inibidores farmacológicos e expressão, síntese e translocação de transportadores GLUT4 no músculo e tecido adiposo. Em ilhotas pancreáticas isoladas de ratos foram estudados os efeitos desses compostos na secreção de vesículas contendo insulina, no influxo de 45Ca2+ e captação de 14C-deoxiglicose. Os triterpenos estudados reduziram fortemente a glicemia dos animais hiperglicêmicos, bem como aumentaram a insulina, o GLP-1 séricos e o conteúdo de glicogênio muscular e hepático, sem aumentar a atividade da lactato desidrogenase (LDH) e a concentração sérica de cálcio. Os ácidos betulínico, ursólico e trihidroxioleanano agiram estimulando a captação de glicose por uma via dependente de PI3K, contudo, diferentemente do ácido ursólico, o AB e THO não estimularam prioritariamente as vias de ação nuclear, mas estimularam a translocação de GLUT4 no período estudado. Estudos com a 1,25-D3 mostraram que esse hormônio age por mecanismo independente de PI3K, estimulou o aumento na incorporação de timidina ao DNA e o conteúdo total de GLUT4. Estudos com ilhotas pancreáticas isoladas incubadas em presença de ácido ursólico e 3ß-hidroxihopeno mostraram que estes tratamentosestimulam a secreção de insulina pelas células ß-pancreáticas. Os triterpenos 3ß-hidroxihopeno, fernenodiol e AB estimularam o influxo de 45Ca2+ em ilhotas pancreáticas por mecanismo dependente de canais de cálcio do tipo L e de potássio sensíveis a ATP e em especial, o AB agiu também através de mecanismo mediado por canais de cloreto. Estes resultados juntos mostram que todos os triterpenos testados apresentaram forte ação nas vias de sinalização voltadas para a homeostasia da glicose, estimulando à secreção de insulina em ilhotas pancreáticas, bem como o aumentaram no influxo de cálcio e/ou ativaram a captação de glicose no músculo esquelético e tecido adiposo assim como a 1,25-D3 estimulou a captação de glicose em músculo esquelético por meio de aumento de síntese e/ou translocação do GLUT4.<br> / Abstract : Chronic hyperglycemia resulting from decreased insulin production and increased tissue insulin resistance is the major cause of tissue damage associated with diabetes and insulin resistance, and can be triggered by factors such as low availability of GLUT4. Insulinogenic compounds that quickly regulates glucose homeostasis and translocation of GLUT4 to the plasma membrane are of great importance in diabetes therapy, like the pentacyclic triterpenes (TPs) and 1,25 (OH)2 vitamin D3 (1,25-D3). The TPs are plant metabolites widely available in the human diet and its potential nutraceutical action arouses attention due to the often reported antidiabetic effect, however, scientifically need proves. Also, studies suggest that 1,25-D3 deficiency impairs insulin secretion in rats, leading to type 2 diabetes, while the treatment with this hormone increased insulin sensitivity by stimulating insulin receptor expression and glucose transport. However, the mechanisms of action of 1,25-D3 in this regard are still poorly understood. In this work, was studed the antihyperglycemic effect and the mechanism of action of 1,25-D3 and natural triterpenes in insulin secretion and tissue uptake of glucose. For analysis of time and dose-response curve of the triterpenes, male Wistar rats after fasting (16 hours) were pretreated with the TPs (betulinic acid (BA), ursolic acid (UA), trihydroxyoleanane (THO), 3ß-hydroxyhopene (BHH) or fernenediol) and after 30 min was induced hyperglycemia for analysis of glucose tolerance. Were determined from serum glucose, insulin, lactate dehydrogenase, GLP-1 and calcium concentration. Also of these animals, the liver and soleus muscle were used for determination of the total glycogen content. In vitro mechanism of action studies of 14C-deoxyglucose uptake, 14C-thymidine incorporation into the DNA and 14C-leucine into protein in the presence or absence of pharmacological inhibitors and expression were investigated. Also, synthesis and translocation of GLUT4 transporters in muscle and adipose tissue were analysed. In isolated pancreatic islets of rats were studied the effects of these compounds in secretory vesicles containing insulin, in 45Ca2+ influx and uptake of 14C-deoxyglucose. The triterpenes studied reduced the blood glucose of hyperglycemic animals and stimulates the insulin increase, serum GLP-1 and the contents of muscle and liver glycogen, without increasing the activity of lactate dehydrogenase (LDH) and serum calcium concentration. The betulinic acid, ursolic and trihydroxyoleanane acted stimulating glucose uptake by a PI3K-dependent pathway, however, unlike the ursolic acid, AB and THO not primarily stimulated nuclear courses of action, but stimulated GLUT4 translocation of the study period. The 1,25-D3 showed a PI3K-independent mechanism, but stimulated the increase in thymidine incorporation to DNA and the total content of GLUT4. Studies using isolated pancreatic islets incubated in the presence of ursolic acid and 3ß-hydroxyhopene showed that these treatments stimulate insulin secretion from pancreatic ß-cells. The 3ß-hydroxyhopene, fernenediol and AB-stimulated 45Ca2+ influx in pancreatic islets was by a mechanism dependent of L-type calcium and ATP-sensitive potassium channels, andespecially, the AB act also through mechanisms mediated by chloride channels. These results together show that all triterpenes tested showed strong action in signaling pathways focused on glucose homeostasis by stimulating the secretion of insulin in pancreatic islets, as well as the increased calcium influx and/ or glucose uptake in skeletal muscle and adipose tissue as well as the 1,25-D3 stimulated glucose uptake in skeletal muscle by increasing the synthesis and GLUT4 translocation.

Diabetes and congenital hyperinsulinism (CHI) are severe diseases affecting the pancreas. Current models for testing drugs to treat these diseases are in vivo in rodents or isolated rodent islets. Differences between the human and rodent pancreas, and ethical issues, mean that in vitro human models are needed. To develop a novel in vitro model for pancreatic diseases, mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs) were derived from the pancreas of patients with CHI. MSCs from three forms of CHI were phenotypically normal for MSCs, and maintained the CHI-causing mutation. When compared to MSCs from bone marrow, the CHI pancreatic MSCs expressed pancreas-specific gene ISL1 and showed promoter hypomethylation of other pancreatic genes, including PDX1. The CHI pMSCs could be differentiated to cells resembling immature beta-cells, with some beta-cell gene expression (INS, PDX1), but no glucose responsive insulin secretion. CHI associated hypersecretion of insulin was not seen as the ATP-sensitive potassium KATP channels were not being expressed. Addition of the Wnt inhibitor DKK1 markedly enhanced differentiation via induction of neuronal genes. Alongside high insulin secretion, CHI also features increased proliferation. CHI MSCs were also hyperproliferative, and showed alterations to the cell cycle. These changes were related to p27Kip1 localisation, a known affected protein in CHI tissue, and CDK1, a novel regulator for CHI. iPSCs were also derived from focal CHI MSCs and were also phenotypically normal, but did not maintain the pancreatic hypomethylation present in MSCs. The CHI iPSCs were efficiently differentiated to definitive endoderm and PDX1 positive cells. Terminally differentiated iPSCs were endocrine, but were not mature beta-cells. In conclusion, authentic MSCs and iPSCs were derived for the first time from patients with CHI. These stem cells could be differentiated towards beta-cells, but mature glucose responsive beta-cells were not produced. MSC derived beta-like cells secreted insulin but did not have KATP channels, whereas iPSC derived beta-like cells had KATP channel gene expression but not INS. With further optimisation to resolve these, CHI stem cell derived beta-cells may be used for in vitro modelling. Further, the undifferentiated MSCs only show hyperproliferation associated with p27Kip1 and CDK1 and so can be a useful resource for modelling hyperproliferation seen in CHI.