The goal of this model is to create and to implement qualitative model of the signal track of olfactory sensory neuron, including the feedback with a focus on diffusion of substances that allows to conduct more simulations for the better understanding of dynamics of the signal track. This model is expected to be used for the simulation of influencing during the activation of two receptors in firstly defined distance. Model was created and therefore implemented in a programming language Python with the use of library STEPS. Then I have conducted sensitivity analysis by a method Morris OAT on the model, together with an optimization with the usage of change of individual parameters with a great importance on the output of the model. Model is conducting good and biologically comparable results when there are from 10 to 100 active receptors at the beginning of the track. Unfortunately with a lower numbers, the results are not valid and therefore not to be used for the simulation of influencing of two activated receptors. Despite this is a main benefit of the work the model of signal transduction for the whole signal track with an included feedback and emphasis on diffusion. Another benefit is a set of scripts for the sensitivity analysis by a method Morris OAT and optimization.

Membrane adaptor proteins are characterized by the lack of enzymatic activity and the presence of various interaction sites for other proteins and cellular membranes. They typically function as scaffolds connecting receptors or other adaptors with proximal signaling molecules at cellular membranes. Their overall effects on signaling can be activating or inhibiting depending on the nature of the effector molecules they recruit. SCIMP is one of the membrane adaptors discussed in this thesis. It is expressed in antigen- presenting cells and it has been previously shown to enhance MHCII signaling in B cells. This thesis covers the analysis of SCIMP functions beyond B cells and describes the first analysis of SCIMP deficient mice. Although the results of this analysis did not show any alterations in immune cell populations, the novel function of SCIMP in dendritic cell signaling downstream of DECTIN- 1 was uncovered. DECTIN-1 is a pattern recognition receptor involved in antifungal immunity. The data presented in this thesis describe the role of SCIMP in sustaining DECTIN-1 signaling over relatively long periods of time and the contribution of SCIMP signaling to maintaining prolonged production of pro-inflammatory cytokines. PSTPIP2 is another interesting adaptor discussed in this thesis. It is...

Cancer is the second leading cause of death in the United States and is expected to become the primary cause of disease-related death within the next decade. There are significant country-to-country variations in cancer incidence, which suggests that nutrition and dietary factors are important to the carcinogenesis process. An increased risk of cancer is associated with obesity and a high body mass index demonstrating that nutrition has a central role in the promotion of cancer. Healthy eating habits protect against cancer, while unhealthy eating habits increase the risk of cancer. Mediterranean societies have a lower risk for many cancers than those of northern Europe and the Americas. Mediterranean diets consist of a high consumption of fruits, vegetables, grains, beans, nuts, and seeds, with olive oil as an important source of monounsaturated fat. These foods are rich in lipid soluble antioxidants such as vitamin E. Vitamin E may prevent cancer by decreasing the formation of mutagens arising from the oxidation of lipids, decreasing oxidative stress in the epithelial cells as well as modulating molecular mechanisms that influence cell death, cell cycle, and transcriptional events. Vitamin E is a major fat-soluble antioxidant and it occurs naturally as eight compounds (alpha-, beta gamma-, and delta-tocopherol or alpha-, beta-, gamma-, and delta-tocotrienol). Since the recognition of vitamin E in 1922 as an essential nutrient for reproduction, alphatocopherol has been considered the major form of vitamin E. It has the highest concentration in the plasma and has been studied more in epidemiological and clinical studies than any other form of vitamin E. Recent data suggests that other isoforms of vitamin E may be important in the control of cancer. These isoforms of vitamin E have varying anti-carcinogenic potencies. Data indicate that gamma-tocopherol may be a more effective anti-cancer agent than alpha-tocopherol. Our laboratories and others have demonstrated that tocotrienols are even more effective than tocopherols at inhibiting cell proliferation in cancer cells. Differences in apoptotic induction among the various vitamin E isoforms are reflective of different avenues of apoptotic signaling and may be tissue specific. Dietary fat has been linked to an increase in a number of cancers including colon, prostate, and breast cancer. Vitamin E modulates a number of molecular mechanisms involved in fat metabolism. These include: the peroxisome proliferator activator receptor (PPAR), arachidonic acid metabolism, de novo sphingolipid metabolism, and cholesterol metabolism. Vitamin E family members have demonstrated the potential to activate pathways involved in cell proliferation, differentiation, apoptosis, and cell cycle. This chapter reviews data that identify the molecular targets of vitamin E action against the development of cancer.

Microglia are the resident innate immune cells that are critical for innate and adaptive immune responses within the CNS. They recognize and are activated by pathogen-associated molecular patterns (PAMPs) present on the surface of pathogens. β-glucans, the major PAMP present within fungal cell walls, are recognized by Dectin-1, which mediates numerous intracellular events invoked by β-glucans in various immune cells. Previously, we showed that Dectin-1 mediates phagocytosis of β-glucan and subsequent superoxide production in microglia. Here, we report that the guanine nucleotide exchange factor Vav1 as well as phosphoinositide-3 kinase (PI3K) are downstream mediators of what is now recognized as the Dectin-1 signaling pathway. Both Vav1 and PI3K are activated upon stimulation of microglia with β-glucans, and the two proteins are required for phagocytosis of the glucan particles and for subsequent superoxide production. We also show that Vav1 functions upstream of PI3K and is required for activation of PI3K. Together, our results provide an important insight into the mechanistic aspects of microglial activation in response to β-glucans.

In this study, we evaluated whether blocking myeloid differentiation factor-88 (MyD88) could decrease cardiac myocyte apoptosis following pressure overload. Adenovirus expressing dominant negative MyD88 (Ad5-dnMyD88) or Ad5-green fluorescent protein (GFP) (Ad5-GFP) was transfected into rat hearts (n = 8/group) immediately followed by aortic banding for 3 wk. One group of rats (n = 8) was subjected to aortic banding for 3 wk without transfection. Sham surgical operation (n = 8) served as control. The ratios of heart weight to body weight (HW/BW) and heart weight to tibia length (HW/TL) were calculated. Cardiomyocyte size was examined by FITC-labeled wheat germ agglutinin staining of membranes. Cardiac myocyte apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and myocardial interstitial fibrosis was examined by Masson's Trichrome staining. Aortic banding significantly increased the HW/BW by 41.0% (0.44 ± 0.013 vs. 0.31 ± 0.008), HW/TL by 47.2% (42.7 ± 1.30 vs. 29.0 ± 0.69), cardiac myocyte size by 49.6%, and cardiac myocyte apoptosis by 11.5%, and myocardial fibrosis and decreased cardiac function compared with sham controls. Transfection of Ad5-dnMyD88 significantly reduced the HW/BW by 18.2% (0.36 ± 0.006 vs. 0.44 ± 0.013) and HW/TL by 22.3% (33.2 ± 0.95 vs. 42.7 ± 1.30) and decreased cardiomyocyte size by 56.8%, cardiac myocyte apoptosis by 76.2%, as well as fibrosis, and improved cardiac function compared with aortic-banded group. Our results suggest that MyD88 is an important component in the Toll-like receptor-4-mediated nuclear factor-κB activation pathway that contributes to the development of cardiac hypertrophy. Blockade of MyD88 significantly reduced cardiac hypertrophy, cardiac myocyte apoptosis, and improved cardiac function in vivo.

A role for the PI3K/Akt/mTOR pathway in cardiac hypertrophy has been well documented. We reported that NFκB activation is needed for cardiac hypertrophy in vivo. To investigate whether both NFκB activation and PI3K/Akt/mTOR signaling participate in the development of cardiac hypertrophy, two models of cardiac hypertrophy, namely, induction in caAkt-transgenic mice and by aortic banding in mice, were employed. Rapamycin (2 mg/kg/daily), an inhibitor of the mammalian target of rapamycin, and the antioxidant pyrrolidine dithiocarbamate (PDTC; 120 mg/kg/daily), which can inhibit NFκB activation, were administered to caAkt mice at 8 weeks of age for 2 weeks. Both rapamycin and PDTC were also administered to the mice immediately after aortic banding for 2 weeks. Administration of either rapamycin or PDTC separately or together to caAkt mice reduced the ratio of heart weight/body weight by 21.54, 32.68, and 42.07% compared with untreated caAkt mice. PDTC administration significantly reduced cardiac NFκB activation by 46.67% and rapamycin significantly decreased the levels of p70S6K by 34.20% compared with untreated caAkt mice. Similar results were observed in aortic-banding-induced cardiac hypertrophy in mice. Our results suggest that both NFκB activation and the PI3K/Akt signaling pathway participate in the development of cardiac hypertrophy in vivo.

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Much research has been conducted and significant progress has been made in understanding how signals are transmitted in response to extracellular stimuli. Continued research is necessary to elucidate how specificity in signaling from the cell surface is achieved and to understand how disruption of such signaling pathways leads to disease. The question of specificity is important since many of the same proteins can be activated by different stimuli in a variety of signaling pathways. One means of achieving specificity would be for different cell types to express different sets of proteins that interact with "common" proteins such as GRB2, Shc, and Ras. There were two goals to my dissertation research. The first goal was to study disease which results from perturbation of the EGF receptor. The second was to explore the possibility that signals generated in response to EGF or other stimuli might be transmitted through MAP kinase proteins other than ERKs 1 and 2. Chapter II of this thesis centers around further analyzing the disease potential of erbB in chickens. I have conducted oncogenicity tests with an erbB oncogene that contains no mutations, only the characteristic N-terminal truncation. My studies have revealed that truncation of the EGF receptor (erbB) can induce tumorigenesis of the kidney as well as the erythroblastosis originally associated with erbB. Previous studies have only found kidney tumors in association with mutated forms of the erbB oncogene. Furthermore, I have demonstrated that mutational removal of a negative regulatory serine phosphorylation site increases the number of wing web tumors caused by the erbB oncogene. Chapter III describes the identification of the Ser/Thr protein kinase p56 KKIAMRE and characterization of p56 KKIAMRE and p42 KKIALRE. These kinases have homology to the MAP kinase group and contain the conserved Thr-Xaa-Tyr dual phosphorylation site. I have demonstrated that both kinases can be activated by treatment of cells with EGF. Interestingly, phosphorylation of Thr and Tyr in the Thr-Xaa-Tyr motif is not necessary for EGF stimulated activity.

Signal transduction by G-proteins (a heterotrimer membrane protein composed of an α, β, and γ subunit) requires that the α-subunit undergoes a transition from a GDP-bound inactive state to an activated GTP-bound state. The exchange of GDP for GTP leads to a conformational change in the α-subunit that results in the loss of affinity for the βγ subunits. We predicted that appropriate genetic manipulation of key regions of the α-subunit could result in the induction of the active conformation that would mimic at least in part the activated GTP-bound state. We have demonstrated that the substitution of the 38 amino acid residue carboxyl termimus of Gαs with the last 36 amino acid residues of Gαi2 resulted in a chimeric Gα-subunit (C4) that exhibits a constitutively active Gαs-like activity. Similarly, the substitution of the amino terminal 61 amino acid residues of Gαs with the first 54 residues of Gαi2 also resulted in a chimeric Gα-subunit that is persistently active (Gs like). We have also generated point mutations in the Gαs subunit that are comparable to the activating mutations in the ras protein. Our results suggest that point mutations in the signature sequence of the A (Val 49) and C (Thr 225) homologous regions that are implicated in regulating the GTPase activity of the molecule also resulted in the activation of the subunit. The present study has identified four key regions of the α-subunit that are critical for the activity and regulation of the Gs protein.

The Insulin Receptor Substrate (IRS) proteins IRS-1 and IRS-2 are cytoplasmic adaptor proteins that organize and propagate intracellular signaling downstream of specific growth factor receptors, including the Insulin and Insulin-Like Growth Factor-1 Receptors (IR and IGF-1R, respectively). Despite sharing a high level of homology and the ability to stimulate Phosphotidylinositol-3-Kinase (PI3K) and Mitogen-Activated Protein Kinase (MAPK) signaling, IRS-1 and IRS-2 play distinct roles in mammary tumor progression. Specifically, IRS-1 promotes growth and proliferation, whereas IRS- 2 promotes motility, invasion, survival, aerobic glycolyis, and metastasis. To further understand the differences between IRS-1 and IRS-2, I investigated the mechanistic basis of IRS-2-mediated PI3K activation. I identified tyrosines in IRS-2 that mediate its recruitment and activation of PI3K in response to insulin and IGF-1 stimulation. Using a PI3K-binding deficient IRS-2 mutant, I demonstrated that IRS-2-dependent PI3K signaling promotes aerobic glycolysis through its ability to selectively regulate the phosphorylation of the Akt effector Glycogen Synthase Kinase-3β (Gsk-3β). I also performed a rigorous comparison of IRS-1 and IRS-2 signal transduction and their ability to regulate functions associated with tumor progression. These studies required the generation of a novel model system where IRS-1 and IRS-2 function could be compared in a genetically identical background. Using this model, I confirmed a role for IRS-1 in growth regulation and IRS-2 in tumor cell invasion, as well as expanded the understanding of differential IRS protein function by showing that IRS-2 more vi effectively promotes Akt activation. The model system I have established can be used for further characterization of IRS-1 and IRS-2-specific functions.