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Seawater-transferred rainbow trout (Oncorhynchus mykiss) has become an attractive aquaculture product in recent years. Industrial interest is mainly due to its resistance to infectious pancreatic necrosis and its adaptability to brackish water, which allows farming in otherwise unexploited locations. However, most practices for the aquaculture of this species have been imported from Atlantic salmon (Salmo salar) without evidence data supporting their suitability for the species. These include protocols to induce their preadaptation to seawater (smoltification). As a result, following seawater transfer, large numbers of fish die (around 10%) or become growth-stunted (GS; 10-60%). Therefore, species-specific rearing protocols and seawater-readiness biomarkers are needed. In the present PhD thesis, the effects of different photoperiod and temperature protocols for rearing in freshwater were assessed on the development of smoltification traits and subsequent seawater performance. This was achieved by using an array of molecular tools to measure osmoregulation-, growth- and haematopoiesis-related genes, proteins and hormones. Moreover, the discovery of potential seawater-readiness biomarkers and the study of smoltification, seawater adaptation and GS fish development were performed using several mass spectrometry proteomic and lipidomic approaches. Results suggest that winter light signals are inadvisable for the species, while all tested summer signals produced similar good results. Moreover, increased temperature protocols not only failed to improve smoltification and growth but potentially compromised the immune system of the fish. Overall, continuous light seems an advisable light regime, irrespective of temperature. Moreover, new promising potential biomarkers for seawater-readiness were identified using proteomics, while also suggesting a previously unknown role of these proteins in smoltification and seawater adaptation. Finally, GS development was shown to be related to low insulin-like growth factor 1 levels following seawater transfer. Moreover, other related factors to the phenotype were higher stress levels, possibly caused by bullying by bigger fish, and hepatic anomalies related to oxidative stress.

Since Choukroun introduced the use of platelet-rich fibrin (PRF) for oral surgical procedures in 2000, PRF has been researched as autologous biologic material to aid in accelerated healing, regulation of inflammation and surgical site integrity. Autologous fibrin glue (AFG) was introduced by Sohn in 2010 to fabricate a growth-factor-enriched bone graft matrix, also called sticky glue, for bone augmentation. Currently, PRF and AFG are widely used in maxillofacial surgeries to improve healing and predictability of procedures. Various studies evaluated the release kinetics of specific growth factors (PDGF-BB, TGFβ-1, VEGF and IGF-1) from PRF. However, an evaluation of an extensive array of available growth factors in PRF and AFG has not been conducted. This study evaluated the presence of 41 human growth factors within PRF and AFG and their change over time over a 3-hour period, replicating the potential handling time associated with usage of PRF in a clinical setting. 3 healthy volunteers were recruited from the UCSF Division of Periodontology. Venous blood from each volunteer was collected in three 10ml glass tube and one 10ml plastic tube and centrifuged at 2700rpm for 13 minutes to obtain 3 tubes of PRF and 1 tube of AFG. Each sample was separated into three equal parts and analyzed using a human growth factor antibody array kit. Samples were evaluated at 0, 1.5 and 3 hours after collection. The array data revealed significant peak level concentrations for several growth factors at 1.5 hour and the presence of high concentrations of IGFBP and MCSF R growth factors. The latter growth factors have not been previously reported in studies of PRF and AFG.

Cancer is the abnormal growth of cells. This growth can kill by disrupting the normal function of its organ of residence or through long-range pathophysiological interactions with distant tissues. Despite the morbidity and mortality associated with cancer, the mechanisms underlying these lethal interactions have remained elusive.We have established a Drosophila tumor model to explore the poorly-understood mechanisms of tumor-host interactions. Using a classic technique, we transplanted larval tumors into adult flies and investigated the effects imposed on host tissues. We find that only 5 days post-transplantation, malignant fly tumors induce robust wasting of muscle, adipose and gonadal tissues. Interestingly, these wasting phenotypes recapitulate key characteristics of the enigmatic cancer cachexia suffered by about half of human cancer patients. We have identified the Insulin Growth Factor Binding Protein (IGFBP) homolog ImpL2 as key mediator of these cachexia-like phenotypes. This factor is secreted specifically by malignant tumors and is both necessary and sufficient for tissue wasting. Consistent with its role as an insulin antagonist, tumor-secreted ImpL2 interrupts systemic insulin signaling to induce insulin resistance, resulting in the cachexia-like wasting of peripheral host tissues.Our work demonstrates that this Drosophila model lends itself to the dissection of complex tumor-host interactions, such as cachexia. We have also used this model to investigate other important features of tumor-host interactions, including metastasis, bloating, immune response and lethality. The genetic manipulability of this system has facilitated the identification of a critical factor mediating the observed cachexia-like phenotypes and can now be used to explore other contributors to the tumor-induced systemic wasting or the molecular mechanisms underlying other important tumor-host interactions.

The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGFR) are membrane bound receptors belonging to the IR superfamily. IR, stimulated by insulin, conducts signals resulting in energy homeostasis. Defect insulin secretion leads to type I diabetes mellitus; insufficient insulin signaling, usually due to insulin receptor resistance, results in type II diabetes mellitus. While, IGFR, bound to type 1 insulin-like growth factor (IGF-I), is responsible for the regulation of mitogenic and differentiating effects on most cell types. Excessive IGF-I-IGFR signaling are commonly found in biopsies from patients with cancer and in cultured immortalized cells. Although, structural similarities are found in between the ligands, insulin and IGFs, as well as their receptors, the IR and IGFR, the receptors bind insulin and IGFs with different affinities. Our goal is to identify and characterize determinants on the receptors responsible for ligand specificity, which allows us to understand the structure-function relationships of the IR and IGFR, their mechanisms of specific activation, which will ultimately lead to the design of novel IR agonists for diabetes therapy and IGFR antagonists for cancer therapy.In our investigations, we used chimeric IRs and IGFRs generated by site specific mutagenesis based on structural information from sequence alignments and crystallographic conformation comparison between the IRs and IGFRs. The ligand binding affinities were characterized by competition binding assays using radio-active isotope labeled insulin and IGF-I analogues. Several mutants based on the IR with deletion or charge reversal in the module 6 and the exon 11 displayed significant changes in binding affinity for IGF-I while retaining the affinity for insulin, indicating the importance of the module 6 and exon 11 in determining ligand specificity. From our data, we found that the major ligand specificity determinants reside in the regions with major structural difference and are complementary to the characteristics of their ligands, suggesting the structure-function relationship plays a core role in protein-protein interactions. Based on this principle and our findings, a chimeric IR with substitutions and deletions of twenty-five residues from the module 6 and exon 11 was generated and showed similar insulin affinity as the IR and similar IGF-I affinity as the IGFR, and thus, no specificity against either ligand.

Insulin-like growth factor-binding protein (IGFBP)-5 is a secreted protein that binds to IGFs and modulates IGF actions. IGFBP-5 is also found in the nuclei of mammalian cells and has transactivation activity. The transactivation domain of IGFBP-5 was mapped to its N-terminal region. Sequence analysis revealed several amino acids in the IGFBP-5 N-domain that are not present in IGFBP-1. The activities of mutants in which these residues were changed to the corresponding IGFBP-1 sequence were determined. Mutations that changed acidic residues to neutral residues (e.g. E8A, D11S, E12A, E30S/P31A, E43L, and E52A) or a polar to a basic residue (e.g. Q56R) significantly reduced transactivation activity. These results suggest that IGFBP-5 may function as a transcriptional regulator in the nucleus. Previously, we used short double-stranded RNA (siRNA) to trigger RNA interference of IGFBP-5 in U2 osteosarcoma (U2OS) cells. Knock-down of IGFBP-5 resulted in significant increase in the number of TUNEL-positive cells. I tried to rescue this apoptotic phenotype by introducing nuclear localization mutations and ligand binding mutations into the siRNA-resistant IGFBP-5. The results showed that the nuclear localization mutant was able to rescue the apoptotic phenotype; however, the ligand binding mutant failed to rescue the apoptotic phenotype. These results indicate that IGFBP-5 promotes U2OS cell survival in an IGF-dependent but nuclear localization independent manner. We also investigated nuclear functions of IGFBP-5. IGFBP-5 overexpression reduced body length in the zebrafish embryos. This action of IGFBP-5 is due to its nuclear presence and its transcriptional activity and is independent of IGF binding. Using several cell biological and biochemical approaches, we showed that the nuclear IGFBP-5 is derived from the secretion pathway via endoplasmic reticulum-cytosol retro-translocation. We also found that PDI facilitates and ERp72 inhibits the IGFBP-5 retro-translocation. Injection of ERp72 mRNA suppressed the IGFBP-5 overexpression phenotype in zebrafish embryos. These results suggest that nuclear IGFBP-5 may exert cell autonomous functions in zebrafish embryonic development. Taken together, the evidences show that several acidic residues are important for IGFBP-5 transactivation activity, IGFBP-5 promotes U2OS cell survival in an IGF-dependent manner, and nuclear IGFBP-5 is derived from the secretion pathway and may exert IGF-independent functions.