Solid tumors can hijack many of the same programs used in neurogenesis to enhance tumor growth and metastasis, thereby generating a plethora of neurogenesis-related molecules including semaphorins Among them, we have identified Semaphorin7A (SEMA7A) in breast cancer We first used to the DA-3 mammary tumor model to determine the effect of tumor-derived SEMA7A on immune cells We found that tumor-derived SEMA7A can modulate the production of proangiogenic chemokines CXCL2/MIP-2 and CXCL 1, and prometastatic MMP-9 in macrophages We next aimed to determine the expression and function of SEMA7A in mammary tumor cells We found that SEMA7A is highly expressed in both metastatic human and murine breast cancer cells We show that both TGF-β and hypoxia elicits the production of SEMA 7 A in mammary cells SEMA7 A shRNA silencing in 4T1 cells resulted in decreased mesenchymal markers MMP-3, MMP-13, Vimentin and TGF-β) SEMA7A silenced cells show increased stiffness with reduced migratory and proliferative potential In vivo, SEMA7A silenced 4T1 tumor bearing mice showed decreased tumor growth and metastasis Genetic ablation of host-derived SEMA7A synergized to further decrease the growth and metastasis of 4T1 cells Our findings suggest novel functional roles for SEMA7A in breast cancer and that SEMA7A could be a novel therapeutic target to limit tumor growth and metastasis / Includes bibliography / Dissertation (PhD)--Florida Atlantic University, 2016 / FAU Electronic Theses and Dissertations Collection

Hedgehog (Hh) est un morphogène secrété qui contrôle la croissance et la différentiation cellulaire chez les métazoaires. La dérégulation de son activité entraine des maladies développementales et de nombreux cancers chez l’adulte. Chez la drosophile, la transduction du signal Hh est initiée par la fixation de Hh sur son récepteur Patched (Ptc), conduisant à la stabilisation de la protéine membranaire Smoothened (Smo) et à l’activation du complexe de transduction composé de 5 protéines : les kinases Fused (Fu), PKA, GprK2, la kinésine Costal 2 (Cos2), et le facteur de transcription Cubitus Interruptus (Ci). Ma thèse a porté sur l’étude de la régulation et des interactions moléculaires entre les composants du complexe de transduction. Par des approches complémentaires, j’ai montré qu’en absence d’Hh, les protéines PKA et Fu interagissent du côté C-terminal de Ci, alors que la présence d’Hh induit leur relocalisation vers le domaine N-terminal de Ci. J’ai pu prouver que l’élément déclencheur de ce remaniement protéique est Smo. En présence d’Hh, Smo s’incorpore dans le complexe de transduction, conduisant à l’activation et au déplacement de Fu vers la région N-terminale de Ci. Ce remaniement entraine la phosphorylation et l’activation de Ci. Ma thèse révèle l’importance des changements de conformation au sein du complexe de transduction de la voie Hh. Le mécanisme de transduction étant conservé entre invertébrés et invertébrés, mon doctorat apporte des éléments de recherche pour mieux comprendre le fonctionnement normal et pathologique des cellules. / Hedgehog (Hh) is a secreted morphogen that controls growth and differentiation in both vertebrates and invertebrates. The dysregulation of its activity leads to severe developmental defects, and the onset of cancer in adults. In Drosophila, the Hh signal transduction is initiated by the binding of Hh to its receptor Patched (Ptc). This induces the stabilization of the transmembrane protein Smoothened (Smo) and the subsenquent activation of a transduction complex consisting of 5 proteins: the kinases Fused (Fu), PKA and Gprk2, the kinesin Costal2 (Cos2), and the transcription factor of the pathway Cubitus Interruptus (Ci). The aim of my thesis was to study the regulation and molecular interactions between the different components of the transduction complex. Thanks to complementary techniques, I have shown that in absence of Hh the proteins Fu and PKA interact in C-terminal part of Ci, whereas on the presence of Hh induces their relocalization toward the N-terminal domain of Ci. I have proved that the trigger element of this moving is Smo. In presence of Hh, Smo goes into transduction complex, allowing the activation and the moving of Fu toward N-terminal domain of Ci. This relocalization is responsible of Ci phosphorylation and activation. My thesis reveals the importance of conformational changes inside the transduction complex of Hh pathway. As the mechanism of transduction is conserved between species, my PhD provides research elements in order to better understand the normal and abnormal functioning of cells.

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.