The JNK signal transduction pathway mediates a broad, complex biological process in response to inflammatory cytokines and environmental stress. These responses include cell survival and apoptosis, proliferation, tumorigenesis and the immune response. The divergent cellular responses caused by the JNK signal transduction pathway are often regulated by spatial and cell type contexts, as well as the interaction with other cellular processes. The discovery of additional components of the JNK signal transduction pathway are critical to elucidate the stress response mechanisms in cells. This thesis first discusses the cloning and characterization of two novel members of the JNK signal transduction pathway. JIP1 and JMP1 were initially identified from a murine embryo library through a yeast Two-Hybrid screen to identify novel JNK interacting proteins. Full length cDNAs of both genes were cloned and analyzed. JIP1 represents the first member of the JIP group of JNK scaffold proteins which were characterized. The JNK binding domain (JBD) of JIP1 matches the D-domain consensus of other JNK binding proteins, and it demonstrates JNK binding both in vitro and in vivo. This JNK binding was demonstrated to inhibit JNK signal transduction and over-expression of JIP1 inhibits the JNK mediated pre-B cell transformation by bcr-abl. Over-expressed JIP1 also sequesters JNK in the cytoplasm, which may be a mechanism of the inhibition of JNK signaling. A new, high-resolution digital imaging microscopy technique using deconvolution demonstrated the absence of JNK1 in the nucleus of co-transfected JIP1 and JNK1 cells. The other protein discussed in this thesis is JMP1, a novel JNK binding, microtubule co-localized protein. There is a JBD in the JMP1 carboxyl end and a consensus D-domain within this region. The JMP1 JBD demonstrates an increased association with phospho-JNK from UV irradiated cells compared to un-irradiated cells in vivo. JMP1 also has 12 WD-repeat motifs in its amino terminal end which are required for microtubule co-localization. JMP1 demonstrates a cell cycle specific localization at the mitotic spindle poles. This co-localization is dependent on intact microtubules and the amino-terminal WD-repeats are required for this localization. JMP1 mRNA is highly expressed in testis tissues. Immunocytochemistry on murine testis sections using an affinity purified anti-JMP1 antibody demonstrates JMP1 protein in the lumenal compartment of the seminiferous tubules. JMP1 protein is expressed in primary and secondary spermatocytes, cells which are actively undergoing meiosis. The results obtained from the localization of JMP1 in meiotic spermatocytes led to an investigation of the roles of JNK signal transduction in the testis. The testis is an active region of cellular proliferation, apoptosis and differentiation, which make it an appealing model for studying JNK signal transduction. However, the roles JNK signaling have in the testis are poorly understood. I investigated the reproduction capability of Jnk3-/- male mice and discovered older Jnk3-/- males had a reduced capacity to impregnate females compared to younger animals and age-matched wild type controls. The testis morphology and sperm motility of these animals were similar to wild-type animals, and there was no alteration of apoptosis in the testis. The final section of this thesis involves the study of this breeding defect and investigating for cellular defects that might account for this age-related Jnk3-/- phenotype.

Maternally supplied mRNAs encode for necessary developmental regulators that pattern early embryos in many species until zygotic transcription is activated. In Caenorhabditis elegans, post-transcriptional regulatory mechanisms guide early development during embryogenesis. Maternal transcripts remain in a translationally silenced state until fertilization. A suite of RNA-binding proteins (RBP’s) regulate these maternally supplied mRNAs during oogenesis, the oocyte-to-embryo transition, and early embryogenesis. Identifying the target specificity of these RNA-binding proteins will reveal their contribution to patterning of the embryo. We are studying post-transcriptional regulation of maternal mRNAs during oocyte maturation, which is an essential part of meiosis that prepares oocytes for fertilization. Although the physiological events taking place during oocyte maturation have been well studied, the molecular mechanisms that regulate oocyte maturation are not well understood. OMA-1 and OMA-2 are essential CCCH-type tandem zinc finger (TZF) RBP’s that function redundantly during oocyte maturation. This dissertation shows that I defined the RNA-binding specificity of OMA-1, and demonstrated that OMA-1/2 are required to repress the expression of 3ʹUTR reporters in developing oocytes. The recovered sequences from in vitro selection demonstrated that OMA-1 binds UAA and UAU repeats in a cooperative fashion. Interestingly, OMA-1 binds with high affinity to a conserved region of the glp-1 3ʹUTR that is rich in UAA and UAU repeats. Multiple RNA-binding proteins regulate translation of GLP-1 protein, a homolog of Notch receptor. In addition to previously identified RBP’s, we showed that OMA-1 and OMA-2 repress glp-1 reporter expression in C. elegans oocytes. Mapping the OMA-1 dependent regulatory sites in the glp-1 mRNA and characterizing the interplay between OMA-1 and other factors will help reveal how multiple regulatory signals coordinate the transition from oocyte to embryo but the abundance of OMA-1 binding motifs within the glp-1 3ʹUTR makes it infeasible to identify sites with a functional consequence. I therefore first developed a strategy that allowed us to generate transgenic strains efficiently using a library adaptation of MosSCI transgenesis in combination with rapid RNAi screening to identify RBP-mRNA interactions with a functional consequence. This allowed me to identify five novel mRNA targets of OMA-1 with an in vivo regulatory connection. In conclusion, the findings in this dissertation provide new insights into OMA-1 mediated mRNA regulation and provide new tools for C. elegans transgenesis. Development of library MosSCI will advance functional mapping of OMA-1 dependent regulatory sites in the target mRNAs. Extending this strategy to map functional interactions between mRNA targets and RNAbinding proteins in will help reveal how multiple regulatory binding events coordinate complex cellular events such as oocyte to embryo transition and cell-fate specification.

How do embryos develop with such poise from a single zygote to multiple cells with different identities, and yet survive? At the four-cell stage of the C. elegans embryo, only the blastomere EMS adopts the endo-mesoderm identity. This fate requires SKN-1, the master regulator of endoderm and mesoderm differentiation. However, in the absence of the RNA binding protein POS-1, EMS fails to fulfill its fate despite the presence of SKN-1. pos-1(-) embryos die gutless. Conversely, the RNA binding protein MEX-5 prevents ectoderm blastomeres from adopting the endo-mesoderm identity by repressing SKN-1. mex-5(-) embryos die with excess muscle at the expense of skin and neurons. Through forward and reverse genetics, I found that genes gld-3/Bicaudal C, cytoplasmic adenylase gld-2, cye-1/Cyclin E, glp-1/Notch and the novel gene neg-1 are suppressors that restore gut development despite the absence of pos-1. Both POS-1 and MEX-5 bind the 3’UTR of neg-1 mRNA and its poly(A) tail requires GLD-3/2 for elongation. Moreover, neg-1 requires MEX-5 for its expression in anterior ectoderm blastomeres and is repressed in EMS by POS-1. Most neg-1(-) embryos die with defects in anterior ectoderm development where the mesoderm transcription factor pha-4 becomes ectopically expressed. This lethality is reduced by the concomitant loss of med- 1, a key mesoderm-promoting transcription factor. Thus the endo-mesoderm identity of EMS is determined by the presence of SKN- 1 and the POS-1 repression of neg-1, whose expression is promoted by MEX-5. Together they promote the anterior ectoderm identity by repressing mesoderm differentiation. Such checks and balances ensure the vital plurality of cellular identity without the lethal tyranny of a single fate.

Drosophila is host to the steroid hormone ecdysone, which regulates development and immune functions using a common group of transcription factors. Developmentally-induced ecdysone pulses activate the expression of the EcR, BR-C, HR46, Eip74EF, Eip75B, Eip78C, and Eip93F, which assume control of hundreds of other genes involved in the transition from larva to pupa stage. Many of the transcription factors are related to mammalian nuclear hormone receptors by homology. In addition to these transcription factors, the ecdysoneregulated GATA factors SRP and PNR are required for the proper expression of the peptidoglycan sensor PGRP-LC, which belongs to a conserved class of proteins in innate immunity. Although the transcriptional network has been elucidated in development, it is unclear why ecdysone control of PGRP-LC gene activity involves these nine transcription factors and how ecdysone is regulated in the context of an infection in vivo. An ecdysone-activated enhancer was located upstream of the PGRP-LC locus using a reporter plasmid. Female flies that lacked the enhancer had reduced PGRP-LC expression, but survived infection. Male flies did not experience these changes. Therefore, PGRP-LC enhancer appears to be a female-specific cis-regulatory element. The lack of survival phenotype could be caused by using an improper injection site. Bioinformatics software was used to identify putative individual and overlapping binding sites for some transcription factors. Site-directed mutations of the motifs reduced PGRP-LC promoter activity without abolishing the signal. These results suggest that the transcription factors assemble at multiple locations on the PGRP-LC enhancer and form strong protein-protein bonds. Septic injury led to elevated ecdysone in whole flies, which could be a neuroendocrine response to stress similar to the mammalian system. Steroid hormone regulation of immune receptors is a common theme in humans and flies, and these results could advance our understanding of the transcriptional regulation of related genes and gender differences observed in innate immune responses at the transcriptional level.

Indiana University-Purdue University Indianapolis (IUPUI) / Sterol Regulatory Element Binding Protein-1 (SREBP-1) is a conserved transcription factor of the basic helix-loop-helix leucine zipper family (bHLH-Zip) that primarily regulates glycolytic and lipogenic enzymes such as L-pyruvate kinase, acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, and mitochondrial glycerol-3-phosphate acyltransferase 1. SREBP-1c activity is higher in the liver of human obese patients, as well as ob/ob and db/db mouse models of obesity and type 2 diabetes, underscoring the role of this transcription factor as a contributor to hepatic steatosis and insulin resistance. Nonetheless, SREBP-1 deficient ob/ob mice, do not display improved glycemia despite a significant decrease in hepatic lipid accumulation, suggesting that SREBP-1 might play a role at regulating carbohydrate metabolism. By silencing SREBP-1 in the liver of normal and type 2 diabetes db/db mice, we showed that indeed, SREBP-1 is needed for appropriate regulation of glycogen synthesis and gluconeogenesis enzyme gene expression. Depleting SREBP-1 activity more than 90%, resulted in a significant loss of glycogen deposition and increased expression of Pck1 and G6pc. Hence, the benefits of reducing de novo lipogenesis in db/db mice were offset by the negative impact on gluconeogenesis and glycogen synthesis. Some studies had also indicated that SREBP-1 regulates the insulin signaling pathway, through regulation of IRS2 and a subunit of the PI3K complex, p55g. To gain insight on the consequences of silencing SREBP-1 on insulin sensitivity, we analyzed the insulin signaling and mTOR pathways, as both are interconnected through feedback mechanisms. These studies suggest that SREBP-1 regulates S6K1, a downstream effector of mTORC1, and a key molecule to activate the synthesis of protein. Furthermore, these analyses revealed that depletion of SREBP-1 leads to reduced insulin sensitivity. Overall, our data indicates that SREBP-1 regulates pathways important for the fed state, including lipogenesis, glycogen and protein synthesis, while inhibiting gluconeogenesis. Therefore, SREBP-1 coordinates multiple aspects of the anabolic response in response to nutrient abundance. These results are in agreement with emerging studies showing that SREBP-1 regulates a complex network of genes to coordinate metabolic responses needed for cell survival and growth, including fatty acid metabolism; phagocytosis and membrane biosynthesis; insulin signaling; and cell proliferation.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Cell membranes have remarkable properties both at the microscopic level and the molecular level. The current research describes the use of physisorbed polymer-grafted lipids in model membranes to investigate some of these properties on both of these length scales. On the microscopic scale, plasma membranes can be thought of as heterogenous thin films. Cell membranes adhered to elastic substrates are capable of sensing substrate/film mismatches and modulating their membrane stiffness to more closely match the substrate. Membrane/substrate mismatch can be modeled by constructing lipopolymer-enriched lipid monolayers with different bending stiffnesses and physisorbing them to rigid substrates which causes buckling. This report describes the use of atomic force microscopy and epimicroscopy to characterize these buckled structures and to illustrate the use of the buckled structures as diffusion barriers in lipid bilayers. In addition, a series of monolayers with varying bending stiffnesses and thicknesses are constructed on rigid substrates to analyze changes in buckling patterns and relate the experimental results to thin film buckling theory. On the molecular scale, plasma membranes can also be thought of as heterogeneous mixtures of lipids where the specific lipid environment is a crucial factor affecting membrane protein function. Unfortunately, heterogeneities involving cholesterol, labeled lipid rafts, are small and transient in live cells. To address this difficulty, the present work describes a model platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains into which transmembrane proteins are incorporated (αvβ3, and α5β1integrins). This flexible platform enables the use of confocal fluorescence fluctuation spectroscopy to quantitatively probe the effect of cholesterol concentrations and the binding of native ligands (vitronectin and fibronectin for αvβ3, and α5β1) on protein oligomerization state and on domain-specific protein sequestration. In particular, the report shows significant ligand-induced integrin sequestration with a low level of dimerization. Cholesterol concentration increases rate of dimerization, but only moderately. Ligand addition does not affect rate of dimerization in either system. The combined results strongly suggest that ligands induce changes to integrin conformation and/or dynamics without inducing changes in integrin oligomerization state, and in fact these ligand-induce conformational changes impact protein-lipid interactions.

[ES] Los inhibidores de la tirosina quinasa (TKIs) son fármacos anticancerígenos que actúan atacando selectivamente a las células cancerosas, inhibiendo su crecimiento descontrolado y, por tanto, la consiguiente aparición de tumores. Sin embargo, muchos TKIs presentan un cromóforo en su estructura que les permite interaccionar con luz solar y, consecuentemente, pueden generar reacciones de fotosensibilización. A pesar de ello, existen pocos estudios sobre el mecanismo de reacción asociado a estos eventos. En este sentido, conocer la fotorreactividad de un fármaco en medio biológico es clave para entender dichos mecanismos y correlacionar el comportamiento fotofísico del fármaco con el daño fotobiológico que genera. La espectroscopía de fluorescencia y de absorción transitoria son técnicas altamente sensibles que permiten estudiar las especies transitorias generadas tras la absorción de luz por parte de un fármaco, las cuales podrían ser las responsables de las reacciones de fotosensibilización inducidas por éste. Habiendo estudiado recientemente la foto(geno)toxicidad asociada al TKI gefitinib (GFT) y a sus principales metabolitos fenólicos (GFT-M1 y GFT-M2), se ha llevado a cabo un estudio completo de la fotorreactividad de éstos tanto en disolución orgánica, utilizando disolventes de distintas polaridades, como en medio biológico, utilizando las proteínas transportadoras de origen humano albúmina sérica (HSA) y ¿1-glicoproteína ácida (HAG). Para ello, se han utilizado las técnicas espectroscópicas de absorción, fluorescencia y absorción transitoria, entre otras. Adicionalmente, se han llevado a cabo estudios teóricos de modelización molecular para investigar la unión ligando@proteína, así como las interacciones que tienen lugar en el complejo supramolecular tras la absorción de luz UV-A. Con este trabajo, se ha conseguido establecer una buena correlación entre el comportamiento fotofísico observado para gefitinib y sus dos metabolitos fenólicos con su potencial fototóxico. / [CA] Els inhibidors de la tirosina quinasa (TKIs) són fàrmacs anticancerígens que actuen atacant selectivament a les cèl·lules canceroses, inhibint el seu creixement descontrolat i, per tant, la consegüent aparició de tumors. No obstant això, molts TKIs presenten un cromòfor en la seua estructura que els permet interaccionar amb llum solar i, conseqüentment, poden generar reaccions de fotosensibilització. Malgrat això, existixen pocs estudis sobre el mecanisme de reacció associat a estos esdeveniments. En este sentit, conéixer la fotorreactivitat d'un fàrmac al mig biològic és clau per a entendre estos mecanismes i correlacionar el comportament fotofísic del fàrmac amb el dany fotobiològic que genera. La espectroscopia de fluorescència i d'absorció transitòria són tècniques altament sensibles que permeten estudiar les espècies transitòries generades després de l'absorció de llum per part d'un fàrmac, les quals podrien ser les responsables de les reaccions de fotosensibilització induïdes per aquest. Havent estudiat recentment la foto(geno)toxicitat associada al TKI gefitinib (GFT) i d'els seus principals metabòlits fenòlics (GFT-M1 i GFT-M2), s'ha dut a terme un estudi complet de la fotorreactivitat d'estos tant en dissolució orgànica, utilitzant dissolvents de diferents polaritats, com al mig biològic, utilitzant les proteïnes transportadores d'origen humà albúmina sèrica (HSA) i ¿1-glicoproteïna àcida (HAG). Per a això, s'han utilitzat les tècniques espectroscòpiques d'absorció, fluorescència i absorció transitòria, entre altres. Addicionalment, s'han dut a terme estudis teòrics de modelització molecular per a investigar la unió lligant@proteína, així com les interaccions que tenen lloc en el complex supramolecular després de l'absorció de llum UV-A. Amb este treball, s'ha aconseguit establir una bona correlació entre el comportament fotofísic observat per a gefitinib i els seu dos metabòlits fenòlics amb el seu potencial fototòxic. / [EN] Tyrosine kinase inhibitors (TKIs) are anticancer drugs that target cancer cells, inhibiting their uncontrolled growth and thus the subsequent development of tumours. However, many TKIs contain a chromophore in their structure that allows them to interact with sunlight and, consequently, to induce photosensitivity reactions. Despite this, there are few studies on the reaction mechanism associated with these events. In this regard, a better understanding on the photoreactivity of a drug in a biological environment is key to correlate the photophysical behaviour of the drug with the photoinduced damage it can induce to biomolecules. Fluorescence and transient absorption spectroscopies are very sensitive techniques that allow the study of the transient species generated after drug excitation, which could be responsible for drug-induced photosensitivity reactions. Having recently studied the photo(geno)toxicity associated with the TKI gefitinib (GFT) and its main phenolic metabolites (GFT-M1 and GFT-M2), a complete study of their photoreactivity has been carried out both in organic medium, using solvents of different polarities, and in a biological environment, using transport proteins such as human serum albumin (HSA) and human ¿1-acid glycoprotein (HAG). For this purpose, absorption, fluorescence and transient absorption techniques, among others, have been used. In addition, molecular dynamics simulation studies have been carried out to investigate the ligand@protein binding, as well as the interactions arising in the supramolecular complex after absorption of UV-A light. Therefore, a good correlation between the photophysical behaviour of gefitinib and its two phenolic metabolites and their phototoxic potential has been established in this research work. / Tamarit Mayo, L. (2024). Fotorreactividad de gefitinib y sus metabolitos fenólicos en disolución y en proteínas transportadoras [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/204145

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 4 uppsatser. Även tryckt utgåva.

Over the last decade, the innate immune system has been the subject of extensive research. Often overlooked by the robustness and specificity of the adaptive immune system, the innate immune system is proving to be just as complex. The identification of several families of pattern recognition receptors (PRRs) has revealed an ancient yet multifaceted system of proteins that are responsible for initiating host defense. A wide array of pathogens, from virus to bacteria, is detected using this assortment of receptors. One such family, the Toll-like receptors (TLRs), has been at the forefront of this research. To date, 10 TLRs have been described in the human genome. Activation of TLRs leads to the induction of immune-related genes that ultimately control the response of the host. However, the signaling pathways emanating from activated TLRs and other PRRs are not fully understood. In particular, the pathway leading to the activation of interferon regulatory factor 3 (IRF3), a transcription factor crucial for the induction of type I interferon, remains undefined. IRF3 activation occurs as the consequence of viral infection and through the activation of TLRs 3 and 4 by dsRNA and lipopolysaccharide (LPS), respectively. The focus of this research is to describe components of the IRF3 activation pathway, partly through the analysis of TLR signal transduction. IRF3 normally resides in the cytoplasm of cells. Upon infection with certain viruses and bacteria, IRF3 is activated though phosphorylation at its C-terminus. Phosphorylated IRF3 homodimerizes and associates with co-activators CBP-p300. After translocating to the nucleus, the activate IRF3 complex induces the activation of type 1 interferon and interferon related genes. Little is known about the pathways that lead to the activation of IRF3, especially the kinases involved. In this study we report that the non-canonical IкB kinase homologues, IкB kinase epsilon (IKKε) and TANK-binding kinase-1 (TBK1), which were previously implicated in NF-кB activation, are also essential components of the IRF3 signaling pathway. In particular, mouse embryonic fibroblasts from TBK1 deficient mice fail to activate IRF3 in response to both viral infection and stimulation with LPS or poly (IC), a dsRNA analog. Thus, both IKKε and TBK1 play a critical role in innate immunity and host defense. In addition to viral infection, IRF3 activation also occurs via the activation of TLR3 and 4. TLRs signal through a subfamily of Toll-IL-1-Resistance (TIR) domain containing adapter molecules. One such adapter, MyD88, is crucial for all TLRs, with the exception of TLR3. MyD88 participates in a signal transduction pathway culminating in the activation of the transcription factor NF-кB. Studies from MyD88-deficient mice reveal that both TLR3 and 4 still are capable of activating NF-кB, although with slightly delayed kinetics. Another aspect of the MyD88-independent signal transduction pathway is the activation of IRF3. A second TIR domain containing adapter molecule called Mal/Tirap was discovered and originally thought to mediate the MyD88-independent pathway. However, Mal-deficient mice were found to be defective in both TLR2 and 4 mediated NF-кB activation. We hypothesized that other TIR domain containing adapters could mediate this MyD88-independent pathway of TLR3 and 4 leading to the activation of IRF3. Two additional TIR adapters were discovered, TRIF and TRAM. TRIF was shown to mediate TLR3 signal transduction. In this study, we report that both TRIF and TRAM mediate the activation of the MyD88-independent pathway in response to LPS/TLR4 activation. Unlike any of the other known TIR domain containing adapters, TRAM appears to be restricted to the LPS/TLR4 activation pathway while TRIF plays a role in both TLR3 and TLR4 pathways leading to IRF3 target gene expression. Our studies revealed that TRAM could be acting upstream of TRIF in the LPS/TLR4 pathway. To this end, we sought to determine the localization of TRAM within the cell. We found that TRAM localizes to the plasma membrane. TRAM localization is the result of myristoylation since mutation of the predicted myristoylation site (G2A) resulted in the re-distribution of TRAM from the membrane into the cytoplasm. Reconstitution of TRAM-deficient macrophages with TRAM G2A is unable to rescue LPS/TLR4 signal transduction. Thus, myristoylation and membrane association of TRAM are critical for LPS/TLR4 signal transduction. The data generated in this dissertation extends our understanding of the signaling pathways of the innate immune system. Indeed, the molecules and pathways described herein could prove to be beneficial targets for ameliorating symptoms of disease, both autoimmune and pathogen-associated. Finally, the research described here will spur further insight into the complex signaling pathways of a once ignored arm of the immune system.