Diss. (sammanfattning) Linköping : Linköpings universitet, 2004. / Härtill 4 uppsatser.

The Rab family of proteins are low molecular weight GTPases that have the ability to switch between GTP- (active) and GDP- (inactive) bound form, and in that sense act as molecular switches. Through distinct localization on various vesicles and organelles and by cycling through GTP/GDP bound forms, Rabs are able to recruit and activate numerous effector proteins, both spatially and temporally, and hence behave as key regulators of trafficking in both endocytic and biosynhtetic pathways. The Rab5 protein has been shown to regulate transport from plasma membrane to the early endosome as well as activate signaling pathways from the early endosome. This dissertation focused on understanding Rab5 activation via endocytosis of receptor tyrosine kinases (RTKs). First, tyrosine kinase activity of RTKs was linked to endosome fusion by demonstrating that tyrosine kinase inhibitors block endosome fusion and activation of Rab5, and a constitutively active form of Rab5 is able to rescue endosome fusion. However, depending on how much ligand is available at the cell surface, the receptor-ligand complexes can be internalized via a number of distinct pathways. Similarly, Rab5 was activated in a ligand-dependent concentration dependent manner via clathrin- and caveolin-mediated pathways, as well as a pathway independent of both. However, overexpression Rabex-5, a nucleotide exchange factor for Rab5, is able to rescue activation even when all of the pathways of EGF-receptor internalization were blocked. Next, the three naturally occurring splice variants of Rabex-5 selectively activated Rab5. Lastly, Rabex-5 inhibits differentiation of 3T3-L1 and PC12 cells through 1) degradation of signaling endosome via Rab5-dependent fusion with the early endosome, 2) and inhibition of signaling cascade via ubiquitination of Ras through the ZnF domain at the N-terminus of Rabex-5. In conclusion, these data shed light on complexity of the endosomal trafficking system where tyrosine kinase activity of the receptor is able to affect endosome fusion; how different endocytic pathways affect activation of one of the key regulators of early endocytic events; and how selective activation of Rab5 via Rabex-5 can control adipogenesis and neurogenesis.

Insulin and insulin-like growth factor 1 (IGF-1) and 2 (IGF-2) are related protein hormones with different but overlapping biological functions. All the hormones interact with a receptor within the insulin-IGF system (insulin receptor A and B, IGF-1 receptor), however with different affinity. The different interaction with individual receptors is just one of the main tools for regulation of the system that is essential for the proper functioning of the organism. Although the residues directly interacting with receptors are mainly located in A and B domains, the C and D domains probably play a role in receptor specificity. Here, we firstly focused on the impact of D domains of IGF-1 and 2 (D1 and D2 domains) and C domain of IGF- 2 (C2 domain). To probe the impact of C and D domains, we prepared insulin analogues containing a part of or an entire domain following a pattern seen in IGFs. The receptor-binding affinities of these analogues and their receptor autophosphorylation potentials were characterised. Our results revealed that the initial part of D1 domain has a detrimental effect on IR affinity that is only slightly enhanced by the rest of the D1 domain. D2 domain has rather neutral effect on IR affinity. We further showed that the addition of amino acids derived from the C2 domain to the...

has been significant progress in the development of new technologies and methodologies to characterize gene expression. The fluorescent-based real-time reverse transcription (RT) polymerase chain reaction (PCR) is an important tool used for clinical and molecular research, biotechnology and as a diagnostic test. Insulin-like growth factors (IGF-1 and IGF-2) and insulin are ubiquitously expressed and play important roles in the regulation of cell growth, differentiation and the maintenance of cell differentiation in mammals. The IGF system (IGF-1, IGF-2, IGF -1 receptor, IGF-2 receptor and six IGF-binding proteins) and insulin are consequently essential to most aspects of male and female reproduction. IGF-1 is produced in multiple tissues but predominately in the liver, from where it enters the circulation. Insulin is secreted by β-cells of the pancreas’ islets of Langerhans. Both IGF-1 and insulin polypeptides bind to specific cell surface receptors. These receptors are members of the superfamily known as tyrosine protein kinases, and are composed of two α and two β subunits linked by disulfide bonds to form an αβ–αβ heterotetramer. The α subunits include ligand binding sites, whereas the β subunits contain tyrosine kinase activity. The aim of this project was to develop real-time RT-PCR assays for quantification of equine insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor (INS-R) mRNA. The assays were developed using stallion testicular tissue samples, obtained by excisional biopsy, from three horse breeds (Friesan, Thoroughbred and Warmblood). The assays developed were efficient, sensitive and had a broad linear range of detection (seven logs for IGF-1R and six logs for INS-R). The assays worked well in our hands and were both sensitive and specific for the detection of equine IGF-1R and INS-R mRNA in a variety of equine tissues. / Dissertation (MMedVet)--University of Pretoria, 2011. / Production Animal Studies / Unrestricted

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A mutação recorrente JAK2V617F é a lesão molecular com maior impacto na fisiopatologia das neoplasias mieloproliferativas (NMP) BCR ABL1 negativas. A ausência de resposta clínica completa ao inibidor seletivo de JAK1/2, ruxolitinibe, indica a necessidade de novas abordagens terapêuticas. Dados recentes sugerem que IGF1R/IRS representa um potencial alvo de inibição para o tratamento das NMP: (i) o substrato do receptor de insulina 2 (IRS2) coopera com JAK2V617F na transformação maligna em NMP; (ii) a desregulação da via de sinalização de IGF1R induz NMP. O composto NT157 foi desenvolvido para inibir IRS1/2 e apresentou efeitos antineoplásicos em neoplasias sólidas. Os objetivos deste trabalho foram avaliar os efeitos celulares e moleculares do tratamento com o inibidor de IRS1/2, NT157, isolado e em combinação com ruxolitinibe, em NMP JAK2V617F. Células HEL e SET2 JAK2V617F foram tratadas com veículo, NT157 e/ou ruxolitinibe e submetidas à avaliação da viabilidade celular, apoptose, proliferação, clonogenicidade, ciclo celular, expressão gênica e/ou expressão/ativação proteica. Células primárias de pacientes com policitemia vera foram submetidos a tratamento com NT157 e avaliação de formação espontânea de colônias eritroides. O efeito do NT157 in vivo foi avaliado utilizando modelo de xenotransplante de células HEL em camundongos NSG. A análise estatística foi realizada através do teste ANOVA ou t de Student. Em células HEL e/ou SET2 JAK2V617F, o tratamento com NT157 promoveu redução da viabilidade, clonogenicidade e proliferação celular, aumentou a apoptose e resultou em parada do ciclo celular em G2/M (p?0,05). Exposição ao NT157 resultou em inibição da fosforilação de STAT3, STAT5 e ERK e na modulação da expressão de 23 oncogenes (CCND1, MYB e WT1) e genes supressores tumorais (CDKN1A, JUN e FOS) em células HEL (p?0,05). O tratamento combinado com ruxolitinibe não apresentou efeito potencializador, sendo que a redução da viabilidade nas condições de combinação corresponde ao efeito das monoterapias nas linhagens celulares avaliadas. Em células primárias de pacientes com policitemia vera (n=3), NT157 reduziu a formação espontânea de colônias eritroides (p?0,05). O tratamento in vivo com veículo ou NT157 na dose de 70mg/kg, 3 vezes por semana, via intraperitoneal, em modelos de xenotransplante com células HEL em camundongos NSG (n=5 para cada grupo) não apresentou efeitos antineoplásicos. Em conclusão, a inibição farmacológica de IRS1/2 apresentou efeitos antineoplásicos significativos em modelos de linhagens celulares e amostras primárias de pacientes com NMP JAK2V617F. A inibição farmacológica combinada de IRS1/2 e JAK1/2 não potencializou o efeito antineoplásico das monoterapias nos processos celulares investigados. Os resultados dos estudos in vivo em modelos de xenotransplante indicam a necessidade de estudos de farmacocinética e farmacodinâmica para o NT157. Os efeitos moleculares identificados permitiram uma melhor compreensão sobre os mecanismos de ação da droga NT157 em NMP. / The recurrent V617F mutation in JAK2 is a major contributor to the pathogenesis of BCR-ABL1 negative myeloproliferative neoplasms (MPN). Absence of complete clinical response to ruxolitinib, a JAK1/2 inhibitor, highlights the need for targeting other signaling pathways that contribute to JAK2. Recent data indicate that IGF1R/IRS is a potential target in MPN: (i) insulin receptor substrate 2 (IRS2) cooperates to malignant transformation induced by JAK2V617F, (ii) IGF1R signaling upregulation induces MNP phenotype. NT157 is a synthetic compound designed as IRS1/2 inhibitor and was able to induce anti-neoplastic effects in solid tumors. We, herein, aimed to characterize the molecular and cellular effects of NT157 treatment, combined or not with ruxolitinib, in MPN JAK2V617F. HEL and SET2 JAK2V617F cells were treated or not with vehicle, NT157 and/or ruxolitinib and submitted to evaluation of cell viability assay, apoptosis, proliferation, clonogenicity, cell cycle, gene expression and protein expression/activation. Primary cells from polycythemia vera (PV) patients (n=3) were exposed to NT157 treatment and evaluated for erythropoietin-independent colony formation. NT157 effects in vivo were evaluated in a xenograft model of leukemogenesis induced by HEL cells in NSG mice. Statistical analysis was performed using ANOVA or Student\'s t test. In MPN cell lines, NT157 treatment significantly decreased cell viability, clonogenicity and cell proliferation, increased apoptosis and cell cycle arrest in G2/M (all p<0.05). NT157 exposure resulted in inhibition of STAT3, STAT5 and ERK phosphorylation. NT157 also modulated the expression of 23 oncogenes (CCND1, MYB and WT1) and suppressor tumor genes (CDKN1A, FOS and JUN) in HEL cells (p?0.05). In both cell lines, the combined treatment, NT157 plus ruxolitinib, did not potentiate the effects of monotherapies. In primary cells from polycythemia vera patients, NT157 exposition reduced spontaneous erythroid colony formation (all p<0.05). In vivo treatment with vehicle or NT157 (70mg/kg intraperitoneal), three times a week, showed no antineoplastic effects in NSG mice transplanted with HEL cells (n = 5 for each group). In summary, the IRS1/2 pharmacological inhibitor NT157 displayed remarkable antineoplastic effects in JAK2V617F cells lines and MPN primary cells. The combined treatment of NT157 plus ruxolitinib did not present potentializing effects when compared to the monotherapy. The results of in vivo treatment using a xenograft model highlight the need for pharmacokinetic and pharmacodynamic studies for the NT157 compound. The molecular effects identified allowed a better understanding about the mechanisms of NT157 action in MPNs.

Bardet Biedl Syndrome (BBS) displays heterogeneity in the genes involved and clinical features. Mutations in 19 genes have been associated with BBS. Eight BBS proteins (BBS1, 2, 4, 5, 7, 8, 9 and 18) form the BBSome. Assembly of the BBSome is mediated by three BBS proteins (BBS6, 10, 12) in a complex with the CCT/Tric chaperonins. The BBSome is involved in formation and maintenance of primary cilia and vesicle trafficking. The clinical features of BBS include obesity, degenerative retinopathy, polydactyly, renal dysfunction, hypogonadism, and learning disability. Diabetes mellitus is commonly associated with BBS, but the mechanisms remain unknown. Our objective was to understand the molecular mechanism of BBS-associated diabetes. The role of BBS in insulin receptor (IR) signaling in Bbs4-/-mice was tested by preventing obesity using calorie restriction. These studies demonstrate the genetic defect in BBS directly contributes to the diabetes phenotype independently from the obesity phenotype. Emerging evidence implicating neuronal mechanisms in various BBS phenotypes led us to test the possibility that loss of Bbs1 in the central nervous system (CNS) disrupts glucose homeostasis. We found that deletion of the Bbs1 gene throughout the CNS or in specific hypothalamic neurons leads to hyperglycemia, glucose intolerance and insulin resistance. Our data demonstrate the critical role of neuronal Bbs1 in the regulation of glucose in an insulin-independent manner. Finally, the IR was found to interact with BBS proteins. The loss of BBSome proteins leads to a specific reduction in the amount of IR at the cell surface. The results demonstrate that BBSome proteins are required to maintain adequate levels of IR at the cell surface. The role of BBS proteins in transporting IR has not been previously described. Loss of the BBSome appears to be a novel mechanism of insulin resistance.

Social insects have complex forms of social organization. Molecular mechanisms involved in the regulation of their reproduction are not fully understood. This dissertation investigated the vitellogenin receptor (VgR), short neuropeptide F (sNPF) receptor, and two insulin receptors (InRs) in the red imported fire ant Solenopsis invicta, focusing on their possible roles in the regulation of queen reproduction. Knowledge of these receptors may provide novel ways to manipulate either reproductive castes or overall reproductive outcome, diminishing the fire ant impact as invasive pest. Fire ant virgin queens have more abundant VgR (SiVgR) transcripts than newly-mated queens, but limited egg formation. To elucidate whether queen maturation involved changes in SiVgR expression, we investigated both virgin and mated queens. In both queens, immunofluorescence analysis of ovaries revealed differential SiVgR localization in early and late stage oocytes; however, mated queens showed higher SiVgR expression than virgin queens. In virgin queens, the SiVgR signal was first observed at the oocyte membrane beginning at day 12 post-emergence, coinciding with the maturation period required before a mating flight. SiVgR silencing in virgins through RNA interference abolished egg formation, demonstrating that SiVgR is involved in queen ovarian development pre-mating. The sNPF and insulin signaling pathways have been implicated in the regulation of food intake and body size, and these peptides also play a gonadotropic role in the ovaries of some insect species. To elucidate the sites of action of the sNPF peptide(s), the sNPF receptor tissue expression and cellular localization were analyzed in the queen brain, subesophageal ganglion (SEG), and ovaries by immunofluorescence. Results suggest that the sNPF signaling cascade may be involved in diverse functions, and the sNPF peptide(s) may act in the brain and SEG as neurotransmitter(s) or neuromodulator(s), and in the ovaries as neurohormone(s). In addition, to elucidate the role of insulin signaling pathway in the fire ant, two putative InRs were cloned. Transcriptional expression analyses show that the receptor abundance was negatively correlated with body size and nutrition status in fire ant immatures. In queens, the expression of InRs in different queen tissues correlates with tissue requirements for queen reproductive physiology and behaviors.

Die Mitglieder der Insulinrezeptorfamilie spielen eine wichtige Rolle in der Funktion von Zellen. Die Hauptangriffspunkte liegen hierbei im Bereich der Glucosehomöostase, sowie weiterhin bei der Proteinbiosynthese, dem Fettstoffwechsel, Elektrolyttransport, Zellwachstum, Differenzierung und Apoptose. Die beiden Hauptvertreter der Insulinrezeptorfamilie, Insulinrezeptor (InsR) und Insulin-like Growth Factor 1 Rezeptor (IGF1R) sind am besten untersucht und viele ihrer Funktionen aufgeklärt. Ein weiteres Mitglied der Familie stellt hier noch eine Ausnahme dar, der Insulin receptor-related receptor (IRR). Obwohl er hohe Sequenzhomologien zum InsR und IGF1R aufweist und in Untersuchungen gezeigt werden konnte, dass er die Möglichkeit besitzt wichtige Punkte der Insulinsignalkaskade zu aktivieren, bleibt seine Funktion unverstanden. Auch ein Ligand wurde bisher nicht identifiziert. Untersuchungen an aktivierbaren IRR-Rezeptorkonstrukten zeigten einen möglichen Einfluss auf Differenzierung, Proliferation und Apoptose von Zellen. Der IRR wird gewebespezifisch exprimiert und v.a. in neuronalen Zellen und β-Zellen des Pankreas nachgewiesen. In der vorliegenden Doktorarbeit wurde der mögliche Einfluss eines stimulierbaren IRR-Rezeptorkonstruktes auf die Proliferation und Apoptose von β-Zellen untersucht. Weiterhin wurde ermittelt, in wie fern dabei zwei Hauptsignalwege der Insulinrezeptorkaskade, der AKT/PKB und der MAPK/ERK Signalweg, aktiviert werden. Hierzu wurde ein bereits beschriebenes Colony stimulating factor 1 receptor/Insulin receptor-related receptor- (CSF1R/IRR-) Konstrukt (Dandekar et al.1998) in INS-1E Zellen überexprimiert und anschließend mit Macrophage colony stimulating factor (MCSF), dem Liganden des CSF1R, stimuliert. Dieses CSF1R/IRR-Konstrukt besteht aus dem extrazellulären Teil des CSF1R, der Transmembrandomäne des InsR und dem intrazellulären Teil des IRR. Es zeigte sich, dass das Konstrukt zu einer transienten Aktivierung des ERK-Signalweges fähig ist, ein Einfluss auf den AKT-Weg allerdings ausblieb. Ferner konnte kein Einfluss auf Proliferation oder Apoptose gezeigt werden. Dies lässt vermuten, dass die mögliche Funktion über alternative Wege verwirklicht wird, wie z.Bsp. eine Hybridrezeptorbildung zwischen IRR und IGF1R. Die angefertigte Arbeit liefert somit einen weiteren Beitrag zum Verständnis der Rolle des IRR in der Zelle.