Regulation and Function of Stress-Activated Protein Kinase Signal Transduction Pathways: A Dissertation

The c-Jun NH2-terminal kinase (JNK) group and the p38 group of mitogen-activated protein kinases (MAPK) are stress-activated protein kinases that regulate cell proliferation, differentiation, development, and apoptosis. These protein kinases are involved in a signal transduction cascade that includes a MAP kinase (MAPK), a MAP kinase kinase (MAP2K), and a MAP kinase kinase kinase (MAP3K). MAPK are phosphorylated and activated by the MAP2K, which are phosphorylated and activated by various MAP3K. The work presented in this dissertation focuses on understanding the regulation and function of the JNK and p38 MAPK pathways. Two different strategies were utilized. First, I used molecular and biochemical techniques to examine how MAP2K and MAP3K mediate signaling specificity and to define their role in the MAPK pathway. Second, I used gene targeted disruption studies to determine the in vivo role ofMAP2K and MAP3K in MAPK activation. I specifically used these approaches to examine: (1) docking interactions between p38 MAPK and MAP2K [MKK3 and MKK6 (Chapter II)]; (2) the differential activation of p38 MAPK by MAP2K [MKK3, MKK4, and MKK6 (Chapter III)]; and (3) the selective involvement of the mixed lineage kinase (MLK) group of MAP3K in JNK and p38 MAPK activation (Chapter IV and Appendix). In addition, I analyzed the role of the MKK3 and MKK6 MAP2K in cell proliferation and the role of the MLK MAP3K in adipocyte differentiation (Chapter III and Chapter IV). Together, these data provide insight into the regulation and function of the stress-activated MAPK signal transduction pathways

Compositionally Complex Titanium Niobium Oxynitride Materials for Solar-Driven Photochemistry

The focus of this thesis is to examine co-incorporation of cationic and anionic dopants simultaneously as a strategy for increasing visible light absorption in TiO2. Co-incorporation has been well-studied theoretically as a viable mechanism for introducing low-energy transitions into the stable TiO2 host lattice; the history and fundamental motivation for co-incorporation is examined here in detail. However, experimental preparations of co-doped and co-incorporated materials remain limited, and a general synthetic method that establishes rigorous control over both cationic and anionic dopant stoichiometry has yet to emerge. In this work, TiO2 co-incorporated with the charge-compensating pair Nb5+/N3- to form titanium niobium oxynitride (TiNbON) is prepared by three synthetic routes and its photochemical properties investigated. First, TiNbON with 25% Nb is prepared by a traditional hydrolytic sol-gel method followed by high-temperature ammonolysis. The resultant material is modified with 1 wt % of RuO2 and evaluated as a photochemical water oxidation catalyst in a solution of NaIO3 sacrificial oxidant. Under 6 times the intensity of solar illumination (6 suns), TiNbON-25 produces oxygen via water oxidation at the rate of ~100 μmol h-1 g-1. Water oxidation occurs regardless of excitation wavelength, though diminished proportionally to the material absorptivity at each wavelength. The rate constant exhibits a zero-order dependence on iodate. Finally, oxygen evolution experiments in 18O-labeled water produce primarily 36O2, suggesting that the dominant pathway for oxygen evolution is the coupling of two water molecules. Secondly, we undertake a new synthetic preparation for TiNbON materials by adapting the urea-glass synthesis for metal nitrides. The reaction produces micron-sized particles of mixed-metal titanium niobium nitride for a variety of niobium contents. These materials are then oxidized to form photoactive anatase/rutile TiNbON that absorbs visible light of λ ≤ 550 nm. Contrary to previous results, the optimized material contains 8% Nb of total metals and degrades methylene blue with a first-order Langmuir-Hinshelwood rate constant of 0.704 h-1 under 5 suns illumination (0.595 h-1 when restricted to λ ≥ 400 nm). Full compositional analysis of TiNbON-5 reveals an empirical formula of Ti0.92Nb0.08O1.97N0.03. A further refinement of the urea-glass synthesis for TiNbON is presented in which the alkaline-earth cation Ca2+ is added to the synthesis to slow the rate of ammonia release during the initial heating step. TiNbON-5 prepared by this method exhibits superior visible light absorption to 600 nm. Tauc analyses of optical spectra suggest a direct band gap. TiNbON-5 produced by this route exhibits a superior first-order Langmuir-Hinshelwood rate constant of 1.785 h-1 under 5 suns solar irradiation. It is hypothesized that the material’s improved properties are due to increased nitrogen content. Finally, a preparation for layered transition metal tungstate/tungsten oxide (MWO4/WO3) photoanodes for photoelectrochemical water oxidation is presented. This strategy seeks to improve on the fundamental shortcomings of the well-studied photoanode material WO3, namely its instability and propensity to participate in side reactions, by adding a more stable and chemoselective interface layer. The interface layers chosen for this study are CuWO4 and Cu0.95Ni0.05WO4. The photoelectrochemical reactivity of MWO4/WO3 electrodes does not change appreciably with respect to bare WO3. Furthermore, we introduce α-thujone, a water-soluble organic molecule with the potential to report on radical chemistry near an electrode surface. Experiments including α-thujone suggest decreased radical prevalence near the surface of MWO4/WO3 compared to WO3.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138737/1/jbrancho_1.pd

Dels jardins als boscos: invasió reeixida del Pitòspor del Japó (Pittosporum toriba, Pittosporaceae) al Delta del Llobregat (nord-est de la península Ibèrica)

Les invasions vegetals constitueixen una amenaça per a la biodiversitat mundial, essent especialment difícil suprimir els seus impactes ecològics i socioeconòmics un cop una espècie sestableix a una localitat determinada. El coneixement previ sobre el potencial de les plantes al·lòctones per esdevenir invasores és essencial per dissenyar polítiques de gestió adequades. Aquí presentem un cas destudi duna planta al·lòctona el pitòspor del Japó, Pittosporum tobira que ha establert grans poblacions en boscos duna zona humida costanera de Catalunya. El nostre treball ha geolocalitzat més de 1000 individus en hàbitats naturals i seminaturals. Lespècie sha convertit en un arbust molt estès al sotabosc estudiat. Discutim les propietats paisatgístiques que probablement van promoure una invasió tan exitosa i les possibilitats de gestió. El potencial invasiu de plantes com el pitòspor del Japó shauria de reconsiderar atès que espècies ornamentals com la tractada estan modificant les comunitats de flora autòctones.Plant invasions are recognized as a threat to the worlds biodiversity, being particularly hard to supress its ecological and socio-economic impacts once an species establishes viable populations in a given locality. Prior knowledge about the potential of allochthonous plants to become invasive is essential to design adequate management policies. Here we present a study-case where an allochthonous plant the Japanese Mock Orange, Pittosporum toriba has established large populations in forests of a coastal wetland of Catalonia. Our work has geolocated more than 1000 individuals in natural and semi natural habitats. The species has become a widespread shrub in the understory of studied woods. We discuss the landscape properties that likely promoted such a successful invasion and the management possibilities. The invasive potential of plants as the Japanese Mock Orange should be reconsidered while such ornamentals are changing autochthonous flora communities

Activation of p38 Mitogen-Activated Protein Kinase Promotes Epidermal Growth Factor Receptor Internalization

Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions

The Mixed-Lineage Kinase DLK Is a Key Regulator of 3T3-L1 Adipocyte Differentiation

The mixed-lineage kinase (MLK) family member DLK has been proposed to serve as a regulator of differentiation in various cell types; however, its role in adipogenesis has not been investigated. In this study, we used the 3T3-L1 preadipocyte cell line as a model to examine the function of DLK in adipocyte differentiation.Immunoblot analyses and kinase assays performed on 3T3-L1 cells showed that the expression and activity of DLK substantially increase as differentiation occurs. Interestingly, DLK appears crucial for differentiation since its depletion by RNA interference impairs lipid accumulation as well as expression of the master regulators of adipogenesis C/EBPalpha and PPARgamma2 at both the mRNA and protein levels. In contrast, neither the expression nor the DNA binding activity of C/EBPbeta, an activator for C/EBPalpha and PPARgamma, is affected by DLK loss.Taken together, these results suggest that DLK is important for expression of mature adipocyte markers and that its action most likely takes place via regulation of C/EBPbeta transcriptional activity and/or initiation of C/EBPalpha and PPARgamma2 gene transcription

Compound A, a Dissociated Glucocorticoid Receptor Modulator, Inhibits T-bet (Th1) and Induces GATA-3 (Th2) Activity in Immune Cells

Background: Compound A (CpdA) is a dissociating non-steroidal glucocorticoid receptor (GR) ligand which has antiinflammatory properties exerted by down-modulating proinflammatory gene expression. By favouring GR monomer formation, CpdA does not enhance glucocorticoid (GC) response element-driven gene expression, resulting in a reduced side effect profile as compared to GCs. Considering the importance of Th1/Th2 balance in the final outcome of immune and inflammatory responses, we analyzed how selective GR modulation differentially regulates the activity of T-bet and GATA-3, master drivers of Th1 and Th2 differentiation, respectively. Results: Using Western analysis and reporter gene assays, we show in murine T cells that, similar to GCs, CpdA inhibits T-bet activity via a transrepressive mechanism. Different from GCs, CpdA induces GATA-3 activity by p38 MAPK-induction of GATA-3 phosphorylation and nuclear translocation. CpdA effects are reversed by the GR antagonist RU38486, proving the involvement of GR in these actions. ELISA assays demonstrate that modulation of T-bet and GATA-3 impacts on cytokine production shown by a decrease in IFN-c and an increase in IL-5 production, respectively. Conclusions: Taken together, through their effect favoring Th2 over Th1 responses, particular dissociated GR ligands, fo

Usefulness of PCR-based assays to assess drug efficacy in Chagas disease chemotherapy: value and limitations

One major goal of research on Chagas disease is the development of effective chemotherapy to eliminate the infection from individuals who have not yet developed cardiac and/or digestive disease manifestations. Cure evaluation is the more complex aspect of its treatment, often leading to diverse and controversial results. The absence of reliable methods or a diagnostic gold standard to assess etiologic treatment efficacy still constitutes a major challenge. In an effort to develop more sensitive tools, polymerase chain reaction (PCR)-based assays were introduced to detect low amounts of Trypanosoma cruzi DNA in blood samples from chagasic patients, thus improving the diagnosis and follow-up evaluation after chemotherapy. In this article, I review the main problems concerning drug efficacy and criteria used for cure estimation in treated chagasic patients, and the work conducted by different groups on developing PCR methodologies to monitor treatment outcome of congenital infections as well as recent and late chronic T. cruzi infections

Cancer Genomics Identifies Regulatory Gene Networks Associated with the Transition from Dysplasia to Advanced Lung Adenocarcinomas Induced by c-Raf-1

Background: Lung cancer is a leading cause of cancer morbidity. To improve an understanding of molecular causes of disease a transgenic mouse model was investigated where targeted expression of the serine threonine kinase c-Raf to respiratory epithelium induced initialy dysplasia and subsequently adenocarcinomas. This enables dissection of genetic events associated with precancerous and cancerous lesions. Methodology/Principal Findings: By laser microdissection cancer cell populations were harvested and subjected to whole genome expression analyses. Overall 473 and 541 genes were significantly regulated, when cancer versus transgenic and non-transgenic cells were compared, giving rise to three distinct and one common regulatory gene network. At advanced stages of tumor growth predominately repression of gene expression was observed, but genes previously shown to be upregulated in dysplasia were also up-regulated in solid tumors. Regulation of developmental programs as well as epithelial mesenchymal and mesenchymal endothelial transition was a hall mark of adenocarcinomas. Additionaly, genes coding for cell adhesion, i.e. the integrins and the tight and gap junction proteins were repressed, whereas ligands for receptor tyrosine kinase such as epi- and amphiregulin were up-regulated. Notably, Vegfr- 2 and its ligand Vegfd, as well as Notch and Wnt signalling cascades were regulated as were glycosylases that influence cellular recognition. Other regulated signalling molecules included guanine exchange factors that play a role in an activation of the MAP kinases while several tumor suppressors i.e. Mcc, Hey1, Fat3, Armcx1 and Reck were significantly repressed. Finally, probable molecular switches forcing dysplastic cells into malignantly transformed cells could be identified. Conclusions/Significance: This study provides insight into molecular pertubations allowing dysplasia to progress further to adenocarcinoma induced by exaggerted c-Raf kinase activity