Regulation of intracellular pH is responsible for many cellular processes, such as metabolism, cell cycle progression, and apoptosis. Many chemotherapeutic agents work by inducing target cells to undergo apoptosis, a cell death process still poorly understood. Previous studies demonstrated that a rise in intracellular pH activated apoptotic proteins leading to cytochrome C release. This "apoptotic alkalinization" occurred upon activation of the plasma membrane protein, sodium hydrogen exchanger-1 (NHE1), whose activity is regulated by the stress kinase p38 MAPK. In previous studies, upon cytokine withdrawal from cytokine-dependent lymphocytes induced the activity of the p38 MAP kinase which then phosphorylated the C-terminus of NHE1. To identify the p38 MAPK phosphorylation sites on NHE1, in vitro p38 MAP kinase assays coupled to deletion analysis of NHE1 and mass spectrometry, identified four possible p38 MAPK phosphorylation sites. To establish that NHE1 causes apoptotic alkalinization and determine whether the identified phosphorylation sites on NHE1 are functionally significant, we used PCR site directed mutagenesis to mutate T717, S722, S725, and S728 on the C-terminus of NHE1. Stable NHE1 deficient cell lines, expressing wild type (WT) NHE or the four mutated sites (F4MUTNHE), were assessed for apoptotic alkalinization using the pH-sensitive fluorescent protein, destabilized YFP. Our results show that NHE1 is required for apoptotic alkalinization, since expression of WT NHE restored alkalinization in an NHE deficient cell line, and that this process requires the phosphorylation of the p38 MAPK target sites, since mutation of all four sites prevented the apoptotic alkalinization response.

Mitogen-activated protein kinases (MAPKs) are a family of highly conserved serine/threonine protein kinases which link upstream receptors to their downstream targets. These targets can be localized in the cytoplasm or the nucleus. Pathogens produce pathogen-associated molecular patterns (PAMPs) that are known to trigger the activation of MAPK cascades. In plants, MAPK signaling cascades regulate development and cellular processes such as stress responses, immunity, and apoptosis by means of the phosphorylation of specific targets. Phosphoproteomics analysis of PAMP-induced Arabidopsis plants led to the identification of several putative MAPK targets. USP1 (Universal Stress Protein A) (At1g11360) and WLIM2A (At2g39900) are two potential phosphorylation targets of MAPKs, and are the focus of this thesis. So far, little is known about their role in plant immunity. CRISPR-Cas9 generated knockout usp1 mutant lines enhanced resistance to infection by Pst DC3000, usp1 mutant showed a reduced level of apoplast reactive oxygen species accumulation and upregulation of defense marker genes such as WRKY29 and FRK1. Transcriptome analyses revealed that immune hormone signaling genes such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are differentially regulated. These hormones are responsible for primary defense responses against biotrophic and necrotrophic pathogens. Although the physiological role of USP1 has been established, the biochemical and molecular functions are unknown. We discovered a new role for USP1, demonstrating that it functions as a molecular chaperone and is involved in thermal priming. Overall, these data show that phosphoprotein USP1 plays an important role in orchestrating plant immunity. CRISPR-Cas9 generated knockout wlim2a mutant showed susceptibility to infection by Pst DC3000. wlim2a mutants showed a reduced level of apoplast reactive oxygen species accumulation and upregulation of defense marker genes such as WRKY29 and FRK1. Transcriptome analyses revealed that immune hormone signaling genes such as salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are differentially regulated in wlim2a mutants. These hormones are responsible for primary defense responses against biotrophic and necrotrophic pathogens. wlim2a mutants show enhanced fungal infection by Botrytis cinerea. Overall, the data shows that WLIM2A phosphorylation is important during plant immunity.

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Mammary cancer is one of the leading causes of death in both the human and companion animal population. There are many histological and pathophysiological similarities between human and feline mammary cancer, therefore investigating the molecular pathways of disease progression in one species may provide useful information for both. The EGFR/MAPK signal transduction pathway is upregulated in many human breast tumors, and both EGFR and MAPK have been implicated as independent prognostic indicators for decreased survival times in human breast cancer patients. We report here that active MAPK levels can be detected in both peripheral white blood cells (WBC) and mammary adipose tissue in cats. Adipose tissue levels of active MAPK were similar to those observed in peripheral WBC, suggesting that WBC MAPK might serve as a useful biomarker in the diagnosis or follow-up treatment of disease. PUFA have been reported to influence breast cancer risk in humans, and may modulate the EGFR/MAPK pathway through a variety of mechanisms. Dietary PUFA n-6-to-n-3 ratio in cats was reflected in mammary adipose tissue and resulted in altered active MAPK levels in both adipose tissue and peripheral white blood cells, suggesting that PUFA may have similar effects on the feline and human MAPK pathway. In human breast tumor cell line studies, rather than having opposing effects, as was hypothesized, it was demonstrated that n-6 and n-3 PUFA exerted similar effects on EGFR+ breast tumor cell proliferation and activity of the EGFR/MAPK pathway. Slightly more than twice the concentration of n-3 PUFA was needed to elicit the same response as n-6 in cells. These results indicate n-6 and n-3 PUFA can modulate proliferation of EGFR+ tumor cells similarly and may be exerting their effects, in part, through the EGFR/MAPK pathway. Decreasing total PUFA intake, while increasing the n-3-to-n-6 PUFA ratio, may be practical as preventative or adjuvant therapy for breast cancer. / Ph. D.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

No / Cryptolepis sanguinolenta and its bioactive alkaloid, cryptolepine have shown anti-inflammatory activity. However, the underlying mechanism of anti-inflammatory action in neuronal cells has not been investigated. In the present study we evaluated an extract of C. sanguinolenta (CSE) and cryptolepine (CAS) on neuroinflammation induced with IL-1β in SK-N-SH neuroblastoma cells. We then attempted to elucidate the mechanisms underlying the anti-neuroinflammatory effects of CAS in SK-N-SH cells. Cells were stimulated with 10 U/ml of IL-1β in the presence or absence of different concentrations of CSE (25–200 μg/ml) and CAS (2.5–20 μM). After 24 h incubation, culture media were collected to measure the production of PGE2 and the pro-inflammatory cytokines (TNFα and IL-6). Protein and gene expressions of cyclooxygenase (COX-2) and microsomal prostaglandin synthase-1 (mPGES-1) were studied by immunoblotting and qPCR, respectively. CSE produced significant (p < 0.05) inhibition of TNFα, IL-6 and PGE2 production in SK-N-SH cells. Studies on CAS showed significant and dose-dependent inhibition of TNFα, IL-6 and PGE2 production in IL-1β-stimulated cells without affecting viability. Pre-treatment with CAS (10 and 20 μM) was also found to inhibit IL-1β-induced protein and gene expressions of COX-2 and mPGES-1. Further studies to determine the mechanism of action of CAS showed inhibition of NF-κBp65 nuclear translocation, but not IκB phosphorylation. At 10 and 20 μM, CAS inhibited IL-1β-induced phosphorylation of p38 MAPK. Studies on the downstream substrate of p38, MAPK-activated protein kinase 2 (MAPKAPK2) showed that CAS produced significant (p < 0.05) and dose dependent inhibition of MAPKAPK2 phosphorylation in IL-1β-stimulated SK-N-SH cells. This study clearly shows that cryptolepine (CAS) inhibits neuroinflammation through mechanisms involving inhibition of COX-2 and mPGES-1. It is suggested that these actions are probably mediated through NF-κB and p38 signalling.