Introduction: Cardiometabolic syndrome is considered a chronic low-grade inflammatory condition that affects various organs and tissues. Individuals with type 2 diabetes mellitus (T2DM) and obesity are at an increased risk for developing the cardiometabolic syndrome and have greater rates of cardiovascular disease (CVD). These conditions are also associated with increased systemic and local inflammation and greater expression of pro-inflammatory markers such as monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor-α (TNF-α), and interleukin 1β (IL-1β) in many tissues. The heart is adversely affected by the inflammation and metabolic changes induced by diabetes and obesity. Nuclear transcription factor kappa B (NF-κB) activity is known to be related to inflammation and cytokine production. However, there is limited information on whether NF-κB signaling and inflammation play a role in early cardiac pathogenesis related to obesity and diabetes and whether lifestyle changes known to prevent or treat these diseases are effective in the heart. Purpose: Therefore, the purpose of this study was to compare the effect of exercise (EX) and caloric restriction (CR) to alter NF-κB signaling, inflammation, and markers of cardiac dysfunction in the heart of 20-week old Otsuka Long Evans Tokushima (OLETF) rats. Methods: Hearts of male 20 week old OLETF rats from a previous study (Crissey et al., 2014) were collected for gene expression (RT-PCR), NF-κB activity, and markers of inflammation and immune cell infiltration. Results: There were no significant differences detected in markers of cardiac dysfunction including, α-MHC, β-MHC, ANP, BNP, COL1, COL3 (all p>0.05). Second, 1-way ANOVA showed that there was trend for an overall effect of group (p=0.07) on NF-κB activation where CR tended to be greater compared to SED and WR (p=0.06). Finally, there were no significant differences between groups in inflammatory and immune cell markers; CD4, F4/80, CD68, IL-1β, MCP-1, TGFB1, and TNF-α (all p>0.05). Conclusion: This study shows that at 20 weeks, a time when OLETF animals exhibit characteristics of the metabolic syndrome such as hypertension, mild obesity, and increased insulin resistance, EX and CR do not reduce markers of cardiac dysfunction and inflammation, potentially because inflammation does not influence the heart at this early time period in the development of the disease. Further, the trend of greater NF-κB activity in CR compared to EX and SED, needs further exploration.

Nuclear factor kappa B (NFκB) is an inflammatory transcription factor that plays an important role in the host immune response to infection. The potential for chlamydiae to activate NFκB has been an area of interest, however most work has focused on chlamydiae impacting human health. Given that inflammation characteristic of chlamydial infection may be associated with severe disease outcomes or contribute to poor overall fitness in farmed animals, we evaluated the ability of porcine chlamydiae to induce NFκB activation in vitro. C. pecorum infection induced both NFκB nuclear translocation and activation at 2 hours post infection (hpi), an effect strongly enhanced by suppression of host de novo protein synthesis. C. suis and C. trachomatis showed less capacity for NFκB activation compared to C. pecorum, suggesting a species-specific variation in NFκB activation. At 24 hpi, C. pecorum induced significant NFκB activation, an effect not abolished by penicillin (beta lactam)-induced chlamydial stress. C. pecorum-dependent secretion of interleukin 6 was also detected in the culture supernatant of infected cells at 24 hpi, and this effect, too, was unchanged by penicillin-induced chlamydial stress. Taken together, these results suggest that NFκB participates in the early inflammatory response to C. pecorum and that stressed chlamydiae can promote inflammation.

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Cancer is one of the major causes of morbidity in the world. Although the overall survival of cancer has been significantly improved by chemotherapy in the last three decades, the success of cancer chemotherapy is still severely limited by the lack of selectivity of anti-cancer drugs to malignant cells leading to dose-limiting toxicity and the resistance of cancer cells to the conventional anti-cancer drugs. Gene-directed enzyme prodrug therapy (GDEPT) was designed to direct the anti-cancer drugs to specifically target the cancer cells by using cancer specific promoter to drive the expression of enzyme which can convert prodrug into anti-cancer drug specifically in cancer cells. However, this strategy is hindered by the lack of strong cancer specific promoters to specifically express drug-converting enzymes in cancer cells. In consequence, there is not enough anti-cancer drug activated inside the cancer cells. The first part of this study was to employ NF-κB binding sites as a novel enhancer system to improve the promoter activity of carcinoembryonic antigen (CEA) and human telomerase reverse transcriptase (hTERT) for GDEPT. In this system, the basal CEA promoter sequences were placed downstream of the 4 or 8 NF-κB DNA binding sites linked in tandem (κB4 or κB8). The system was designed to serve two particular purposes: to exploit the high levels of intratumoural NF-kB expression and keep the relative tumour specificity of the CEA and hTERT promoters. The results demonstrated that κB enhancer systems increased the transcriptional activity of CEA and hTERT promoter without compromising its cancer specificity. The fidelity of the κB4-CEA enhancer-promoter system was therefore improved by the increased transcriptional contrast between the cancer and normal cells. Moreover, in comparison with CEA promoter alone, κB-CEA enhancer-promoter system expressed human thymidine phosphorylase (TP) protein at significantly higher levels which were comparable to those expressed by CMV promoter. The κBCEA- TP system transfected cells demonstrated significantly higher sensitivity to 5'-Deoxy-5-Fluorouridine (5'-DFUR), a prodrug of 5-fluorouracil (5-FU). The second part of this study was involved in using NF-κB inhibitor as a chemosensitizer to sentizise the anti-cancer drug-induced chemoresistance cells to anti-cancer drugs. The results derived from this study manifested that the anti-alcoholism drug, Disulfiram (DS), and anti-inflammatory drug, triptolide (PG490), markedly enhanced the cytotoxicity of several conventional anti-cancer drugs in colon, lung and breast cancer cell lines. PG490 induced caspase-dependent cell death accompanied by a significant decrease in Bcl-2 levels. PG490 induced the expression of p53 and down-regulated p21 expression. This study indicated that some clinically used non-cancerchemotherapeutic drugs may be developed as chemosensitizers for cancer chemotherapy

Sodium butyrate (NaBt) is a four-carbon short chain fatty acid, produced naturally in colon as the end product of the bacterial anaerobic metabolism on dietary fibers. It was previously shown that NaBt can induce differentiation and may inhibit proliferation. The objective of this study was to investigate the effect of NaBt-induced differentation on inflammatory pathways in HT29 colon cancer cells. For this purpose, first, cells were treated with varying concentrations of NaBt from 1-5 mM and amount required to induce differentiation was determined as 3 mM. To understand the effect of NaBt on inflammation, the NF-kappaB pathway (p50 and p65) was investigated. Immunofluorescent staining showed increased nuclear translocation of p50 subunit with no remarkable change in subcellular localization of p65 / moreover a synergistic effect was observed when cells were co-treated with NaBt and an NF-kappaB repressor, Bay 11-7085 / implying the formation of repressive p50 homodimers in the nucleus. Our preliminary chromatin immunoprecipitation results showed that p65 recruitment v to the promoters of ICAM-1 was reduced, whereas p50 recruitment was increased. However, analysis of NF-kappaB target genes showed that cells treated with 3 mM NaBt have higher expression of the cytokines IL1-&beta / and TNF-&alpha / , adhesion molecules ICAM-1 and VCAM-1 but not COX-2. These results suggest that NaBt-induced differentiation could cause the emergence of an inflammatory signal in HT29 cells as an anti-tumor mechanism, independent from the NFkappaB activity. This work will be important in understanding the role of SCFAs in the colon microenvironment and may provide alternative therapeutic options in colorectal cancer.

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Septic shock is a major cause of deaths resulting from uncontrolled inflammation and circulatory failure. Recent studies suggest that the vascular isoform of ATP-sensitive K+ (KATP) channels is an important contributor to septic susceptibility. To understand the molecular mechanisms for channel regulation during sepsis, we performed studies in isolated endothelium-denuded mesenteric rings. Lipopolysaccharides (LPS) induced vascular relaxation and hyporeactivity to phenylephrine. The LPS-treated aortic smooth muscle cells displayed hyperpolarization and augmentation of KATP channel activity. Both were due to an up-regulation of Kir6.1 and SUR2B surface expression. The up-regulation relied on transcriptional and translational mechanisms, in which nuclear factor-¦ÊB (NF-¦ÊB) and Protein kinase A (PKA) played a critical role. Oxidative stress occurs during sepsis and may act as another regulatory mechanism affecting KATP channel activity and vascular contractility. We found that micromolar concentrations of H2O2 impaired the pinacidil-induced vasodilation. The effect attributed to the suppression of KATP channel activity, which can be fully produced by reactivity oxidants. Unlike the Kir6.1/SUR2B channel, the Kir6.2/SUR2B channel was insensitive to 1mM H2O2, indicating that the modulation sites are located in Kir6.1. Site-directed mutational analysis showed that three cysteine residues located in N-terminus and the core region of Kir6.1 were likely to mediate the redox-dependent channel modulation. Arginine vasopressin (AVP) is a vasoconstrictor that is successfully applied to manage sepsis. However, the downstream target of AVP is uncertain. Our studies show that AVP-induced vasoconstriction depended on V1a receptor, Protein kinase C (PKC) and KATP channel. Additionally, AVP decreased Kir6.1/SUR2B channel activity through V1a receptor. The inhibitory effect was caused by a suppression of the channel open state probability. The channel inhibition was mediated by phosphorylation of the channel protein by PKC. The widespread involvement of the vascular KATP channel in vascular responses to endotoxemia strongly suggests that the temporospatial control of channel activity may constitute an important intervention to vascular tone, blood pressure and organ-tissue perfusion in septic shock. Such a control appears feasible by targeting several modulatory mechanisms of intracellular signaling, Kir6.1/SUR2B expression, redox state and channel protein phosphorylation as demonstrated in this dissertation.

ABSTRACT Renal cell carcinoma (RCC), the most common type of kidney cancer, is a highly metastatic disease. Late stage metastatic RCC is essentially incurable and lethal. The average survival of patients, following metastatic RCC, is about 4 months and only 10% of patients survive for one year. None of the currently available chemotherapy, radiotherapy, hormonal or biological therapies have a significant impact on the progression of the disease. Novel chemotherapeutics are urgently required for the treatment of this deadly disease. The mechanisms that pose the greatest challenges to chemotherapeutics are resistance of tumour cells to apoptosis, tumour angiogenesis and multi-drug resistance. Resistance to apoptosis may be mediated by the up-regulation of anti-apoptotic proteins, especially Bcl-2 and Bcl-XL, and/or by the down- regulation of pro-apoptotic proteins, particularly Bax. Angiogenesis is pivotal for tumour growth and metastasis. Of all identified pro-angiogenic molecules, vascular endothelial growth factor (VEGF) is considered to be a key molecule. Drug resistance is thought to be mediated by the up-regulation of multi-drug resistance molecules such as MDR-1 and MRP-1. Up-regulation of Bcl-2 also confers drug resistance to cancer cells. The main hypothesis of this thesis was that treatment targets of metastatic RCCs are likely to multifactorial and that inhibition of molecules that regulate the processes of apoptosis, angiogenesis and multidrug resistance are likely to be better targets than those that regulate only one of these processes. In this regard, the transcription factor nuclear factor kappaB (NF-kB) meets the criterion, regulating the apoptotic, angiogenic and multi-drug resistance pathways of cancer cells. Its inhibition appeared to be an attractive strategy for the treatment of metastatic RCC. Many studies have demonstrated an association between the over-expression NF-kB and RCC. Thus, the major aim of this thesis was to explore the anti-cancer effect of pyrrolidine dithiocarbamate (PDTC), a potent NF-kB inhibitor on human metastatic RCC cell lines. The thesis is divided into seven Chapters. In Chapter 1, the literature on RCC, NF-B and the role of NF-kB in RCC development and progression are reviewed. The rationale for the inhibition of NF-kB as a potential anti-RCC strategy using PDTC is established. During the course of this research, the use of PDTC as an anti-cancer agent has risen to prominence. Chapter 2 describes the materials and methods used in the project. In Chapter 3, the expression of NF-kB in human kidney and the RCC cell lines, ACHN and SN12K1, was established. The proof of hypothesis that NF-kB inhibition using PDTC is an effective anti-cancer strategy was demonstrated. PDTC was selectively toxic to the RCC cell lines, but not to normal human kidney cells. PDTC induced apoptosis and inhibited proliferation of the RCC cells. PDTC also inhibited NF-kB, its upstream regulatory molecules such as the inhibitory protein family of the IkBs, and the kinase IKK complex. PDTC also inhibited anti-apoptotic Bcl-2 and Bcl-XL, but not pro-apoptotic Bax. Chapter 4 demonstrated the in vitro and ex vivo anti-angiogenic and anti-metastatic effects of PDTC. Protein microarrays for angiogenic factors produced controversial results. PDTC inhibited epidermal growth factor (EGF) produced in endothelial cells. VEGF had neutral effect on angiogenesis under the experimental conditions used. In the RCC cell lines, several pro-angiogenic molecules were modulated. Interestingly, the pro-angiogenic molecule interleukin (IL)-8 was up-regulated in both RCC cell lines. The monocyte chemoattractant protein-1 (MCP-1) was decreased in ACHN cells, but increased in SN12K1 cells. The implications of these controversial findings are discussed. Chapter 5 demonstrated the ability of PDTC to overcome drug resistance in a synergism with cisplatin. Individual non-toxic concentrations of PDTC and cisplatin, when combined, induced significant toxicity of RCC cell lines. The synergistic effect was not mediated by the inhibition of NF-kB, but rather through the inhibition of transcriptional activation of NF-kB. Bcl-2 rather than MDR-1 or the regulatory protein MRP-1 may be important in overcoming drug resistance in RCC. Chapter 6 showed the anti-cancer effect of PDTC in an animal model of RCC. PDTC significantly decreased the growth of RCC implanted in the kidney of severe combined immunodeficiency (SCID) mice. PDTC inhibited NF-kB and was not toxic to normal cells. The expression of Bcl-2, Bcl-XL and Bax were contradictory to the in vitro findings and a theory about the spread of RCC based on these findings is discussed. In Chapter 7, the findings are summarised. A case for PDTC as a potential therapeutic agent for RCC is established. Under the experimental conditions used, PDTC was demonstrated to be an effective anti-RCC agent by targeting the three most important characteristics of RCC that pose the greatest challenges to chemotherapeutics: resistance of tumour cells to apoptosis, tumour angiogenesis and multi-drug resistance. PDTC was selectively toxic to RCC, but not to normal renal cells. Thus PDTC appears to be a promising anti-cancer agent. This is supported by the current increase in interest, and in the number of publications, on the use of PDTC in several cancers. Some future directions are also discussed in this Chapter. These include, but are not limited to, an investigation of what is protecting normal cells from the toxicity of PDTC, the creation of an Australian database on RCC, and the characterisation of RCC based on NF-kB expression.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.