Chronic inflammation in the brain results in the development of several CNS diseases, including Alzheimer’s and Parkinson’s diseases, multiple sclerosis, and tumors. IL-1, a pro-inflammatory cytokine released by activated microglia and astrocytes, instigates the expression of factors promoting the progression of these CNS disorders, including cytokines, chemokines, and components of matrix remodeling systems, such as the plasminogen activator system. IL-1 also increases the mRNA expression and activity of SphK, the enzyme that phosphorylates Sph to form S1P, a bio-active sphingolipid. This thesis demonstrates that IL-1 and S1P enhance the mRNA and protein expression of PAI-1 and uPAR, two key components of the plasminogen activator system, in glioblastoma cells. The S1P-induced mRNA expression of PAI-1 and uPAR is mediated by the S1P2 receptor, and requires Rho-kinase and MEK1. However, IL-1 regulation of PAI-1 and uPAR mRNA expression is independent of SphK, and thus S1P. IL-1- and S1P-induced mRNA expression of PAI-1 and uPAR results in the increased in vitro invasion of glioblastoma cells. Since significant amounts of IL-1 are secreted from gliomas, and it increases the production of S1P via inciting the activity and mRNA expression of SphK, we propose a mechanism by which S1P and IL-1 influence the invasion of glioblastoma cells by increasing the mRNA and protein expression of uPAR and PAI-1. IL-1 and S1P also influence the mRNA expression of chemokines implicated in the development and progression of multiple sclerosis, namely IP-10 and RANTES, in primary human astrocytes. IP-10 and RANTES attract T cells, which are the major pathological cause of multiple sclerosis. This thesis demonstrates a novel mechanism by which S1P significantly inhibits the IL-1-induced mRNA expression of these chemokines. The mechanism by which S1P reduces IL-1-induced IP-10 and RANTES mRNA expression involves the prolonged hyperphosphorylation of TAK1, as well as the inhibition of IL-1-stimulated IFN beta production and the phosphorylation of STAT1 and STAT2. In summary, this dissertation describes the mechanisms by which S1P and IL-1 control the mRNA expression of two chemokines associated with multiple sclerosis, and the components of the plasminogen activator system, which are critical for the invasion of glioblastoma cells; thus, indicating future therapeutic targets for destructive CNS disorders.

FTY720 (aussi connu sous le nom de Fingolimod ou Gilenya) agit sur les récepteurs sphingosine-1-phosphate (S1P) et induit la suppression du système immunitaire (immunosuppression). Cette molécule est reconnue pour avoir une activité contre plusieurs cellules cancéreuses. Cette activité est indépendante de l’action sur les récepteurs S1P et on attribue plutôt la mort (apoptose) des cellules cancéreuse à la capacité que possède la molécule à réduire le transport des nutriments dans la cellule. Toutefois, malgré ses nombreux avantages, FTY720 ne peut pas être utilisé afin de traiter des humains puisque l’activation secondaire des récepteurs S1P1 et S1P3 mènent à une diminution du rythme cardiaque (bradycardie) chez les patients. Notre groupe s’est donc concentré sur la synthèse d’analogues qui potentiellement n’activeraient pas le récepteur S1P tout en gardant une activité biologique contre plusieurs cellules cancéreuses. Malgré le fait que nos analogues agissent également sur la diminution du transport des nutriments dans les cellules, nous ne connaissons pas le mécanisme d’action par lequel ceux-ci agissent. Au passage, le projet de recherche ci-présenté nous aura par ailleurs permis de développer une grande variété de sondes photo-actives dans l’espoir d’isoler une ou plusieurs protéines qui seraient impliquées dans le mécanisme d’action. / FTY720 (also known as Fingolimod, or Gilenya) functions as an immunosuppressant due to its effect on sphingosine-1-phosphate receptors. FTY720 also exhibits anticancer activity in a number of cancer cell lines. This result is independent of its effects on S1P receptors and due instead, to its ability to induce nutrient transporter down-regulation. However, FTY 720 cannot be used in human patients due to bradycardia that occurs secondary to the activation of S1P1 and S1P3 receptors. Our group focused on the design and synthesis of constrained FTY720 analogues that do not activate S1P receptors and also have the potential to be safely used in humans due to their potent antiproliferative activity against selected cancer cell lines. Although we know that our analogues, like FTY 720, act as nutrient transporter down-regulators, the proteins involved in the process are not known. We are developing a wide variety of Photo-Cross-Linking Probes to hopefully isolate one or more proteins that may be involved in one of the pathways.

Cholangiocarcinoma (CCA) is a fatal primary malignancy that is formed in the bile ducts. Cancer-associated myofibroblasts play a crucial role in CCA proliferation and invasion. Furthermore, there is a growing interest in the role of the exosome in the interaction between the cancer-associated myofibroblasts and cholangiocarcinoma which lead to CCA growth. However how cholangiocarcinoma-derived exosome affect the cancer-associated myofibroblasts in the tumor microenvironment remain unknown. In this study, we examined whether exosome produced by cholangiocarcinoma could involve in the prompt of CCA cells growth by regulation of myofibroblast. We found that cholangiocarcinoma-derived exosome could prompt elevated α-smooth muscle actin and stromal cell-derived factor one expression that induces myofibroblast proliferation. We then demonstrated that cholangiocarcinoma-derived exosome upregulated periostin expression that plays an important role in cancer metastasis. In 3D organotypic rat CCA coculture model, TCA and S1P considerably increase the growth of CCA cell. Conclusion: cholangiocarcinoma-derived exosome trigger cancer-associated myofibroblasts proliferation in the tumor microenvironment that leads to prompt CCA growth.

Sphingolipids modulate multiple cellular functions, including steroid hormone biosynthesis. Sphingosine is an antagonist ligand for the nuclear receptor steroidogenic factor 1 (SF-1), which is the primary transcriptional regulator of most steroidogenic genes. Furthermore, sphingosine-dependent repression of SF-1 function is dependent on the expression of acid ceramidase (ASAH1), an enzyme that forms sphingosine. Based on these data, I hypothesized that ACTH/cAMP signaling regulates ASAH1 function at both transcriptional and post-transcriptional levels. In addition, because SF-1 is predominantly a nuclear protein, I postulated that ASAH1 modulates SF-1 function and, therefore, steroidogenic gene expression by controlling the nuclear concentrations of SPH. To test these hypotheses, I first examined the effect of chronic ACTH/cAMP signaling on the transcription of the ASAH1 gene. Next, the functional significance of ASAH1 expression in adrenocortical cells was probed by generating an ASAH1-knockdown cell line. I subsequently characterized the role of ASAH1 as a transcriptional nuclear receptor coregulator. Finally, I defined the role of sphingosine-1-phosphate, a bi-product of ASAH1 activity, in the acute phase of cortisol biosynthesis. Using a variety of experimental approaches, I identified cAMP response element binding protein as an essential transcriptional activator of the ASAH1 gene. Analysis of adrenocortical cells lacking ASAH1 revealed that ASAH1 is a global regulator of steroidogenic capacity. Furthermore, I identified ASAH1 as a nuclear protein and defined the molecular determinants of the interaction between ASAH1 and SF-1. Collectively, this body of work establishes the integral role of ASAH1 in the regulation of ACTH-dependent adrenocortical cortisol biosynthesis.

Type 2 diabetes is a disease characterized by the inability of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normoglycemia. Increased levels of saturated fatty acids such as palmitate are believed to contribute to β-cell failure and the development of the disease. In the present thesis, mechanisms behind palmitate-induced β-cell apoptosis were explored. Palmitate augmented insulin secretion after short exposure to the fatty acid, but attenuated the secretory response after longer exposure. Elevated levels of palmitate increased endoplasmic reticulum (ER) stress and induced apoptosis. When insulin secretion was inhibited by diazoxide, palmitate-induced ER stress and apoptosis were reduced. In comparison to palmitate, the mono-unsaturated fatty acid oleate increased neither ER stress nor apoptosis. Furthermore, shuttling of fatty acids into triglycerides and β-oxidation was favored in cells exposed to oleate compared to palmitate. When the levels of stearoyl-CoA desaturase 1 (SCD1), the enzyme responsible for conversion of saturated to mono-unsaturated fatty acids, were reduced, up-regulation of ER chaperones and components of the proteasome was observed. Cells with reduced levels of SCD1 showed increased sensitivity to palmitate, as exposure to the fatty acid increased levels of ER stress and apoptosis. Palmitate-induced apoptosis of the β-cell has been linked to alterations in sphingolipid metabolism. In cells with reduced levels of sphingosine kinase (SphK) 2, palmitate failed to induce apoptosis, and ER stress was reduced. Furthermore, SphK2 was required for the palmitate-induced activation of c-Jun N-terminal kinase (JNK). In contrast, knockdown of SphK1 sensitized the cell to palmitate-induced apoptosis independently of ER stress. In summary, palmitate induces β-cell apoptosis, which is partly dependent on the induction of ER stress. The mechanisms investigated support the notion that increased protein load on the ER, low degree of triglyceride formation and β-oxidation, and perturbations in sphingolipid metabolism contribute to palmitate-induced apoptosis in insulin-producing β-cells.

In this thesis a methodology to synthesize anti-β-hydroxy-α-amino esters possessing olefins has been investigated. The developed procedures originate from two already established procedures in which α-amido-β-keto esters, which do not contain olefins, has been stereoselectively reduced to the corresponding anti-β-hydroxy-α-amino alcohols via asymmetric transfer hydrogenation coupled with dynamic kinetic resolution. Both established methods, one solvent free and one emulsion procedure, have been investigated on the expanded substrate scoop. Four different α-amido-β-keto ester containing olefins were tested and it was found that the ketones were reduced to desired anti-β-hydroxy-α-amino esters in both procedures, but also side products were formed where the olefins were reduced. The ratio of the different products was dependent on the structure of the starting α-amido-β-keto ester, ligand used on the catalyst and reaction conditions such as number of equivalents of base and reaction temperature. The diastereoselectivity for the desired products was in favor of the anti stereoisomer, however, the dr was worse than in the established procedures. The usefulness of this methodology was then demonstrated by a formal total synthesis of Sphingosine.

Binding of the Rev protein with Rev Response Element (RRE) RNA present in singly- and unspliced mRNA transcripts is necessary for the replication of HIV-1. This interaction transports the mRNA transcripts from the nucleus to the cytoplasm for translation of the necessary structural and enzymatic proteins for the newly budding virus as well as for providing its genetic material. Given the high rate of mutation in HIV-1, the highly conserved and pertinent RRE RNA is of high interest for pharmaceutical intervention. Consequently, a branched peptide library containing unnatural amino acids was developed to target RRE RNA with the goal of increasing stability, potency, selectivity, and in vivo activity for RRE RNA. An unnatural amino acid branched peptide library (46,656 sequences) was synthesized and screened against RRE IIB and several hits in the sub-micromolar regime were found. A number of hits demonstrated selectivity in the presence of other RNAs in addition to two hits, 4A5 and 4B3, significantly inhibiting HIV-1 growth in vitro. These peptides inhibited HIV-1 replication in a concentration dependent manner and were demonstrated to be non-toxic. Further analysis of 4A5 and 4B3 via footprinting and SHAPE-MaP experiments determined that these peptides blocked binding of Rev through binding at the primary and secondary binding sites of RRE RNA. Sphingosine 1-phosphate (S1P) is a signaling molecule that plays a role in various biological processes including immunity, neurogenesis, and angiogenesis. The role S1P plays is largely determined by its location, in which Spinster homolog 2 (spns2) and mfsd2b are the two known transporters. The two transporters exist in different cell types and cellular localizations, with spns2-produced S1P being responsible for trafficking of lymphocytes. As such, spns2 has become of interest for therapeutic targeting in autoimmune and inflammatory diseases. To validate spns2 as a target in pharmaceutical intervention, a series of spns2 inhibitors were developed. A screening of a library of inhibitors found that compound SLP7120922 demonstrated inhibition of spns2 transport activity. The design, synthesis, and biological evaluation of inhibitors based on SLP7120922 is described. Modifications to the lipophilic tail region were performed with one compound 4.40f discovered to be potent, minimally toxic, and active in vivo. A series of modifications to the head region were then conducted that evaluated linear head derivatives with alkyl-, amide-, and amino acid-based groups. A number of compounds are reported that demonstrate good in vitro activity and minimal toxicity with two compounds, 4.48b and 4.52c, showing favorable in vivo activity in mice. / Doctor of Philosophy / Human immunodeficiency virus (HIV-1) has a high rate of mutation, which commonly leads to the need for many types of medications throughout the lifetime of a patient. In order to design a therapeutic that the virus has a low chance of growing resistance to, a target needs to be chosen with a low mutation rate. One such target is the Rev Response Element (RRE) RNA and it is necessary for the virus to replicate. A protein named Rev binds to RRE RNA in order for RRE to carry out its pertinent function. To block this function we have chosen branched peptides to target the RNA. Peptides are made of the same building blocks of proteins, but are much shorter than proteins. The peptides described here are made up of modified building blocks, called unnatural amino acids. This work describes the generation of an unnatural amino acid branched peptide library and how it was screened in order to find branched peptides that bind RRE RNA. Many peptides were found to bind RRE RNA but two in particular, 4A5 and 4B3, were the best binders that inhibited HIV-1 growth. The remainder of the work describes how these peptides bind to RRE RNA, while demonstrating that they are non-toxic and bind HIV-1 in a concentration dependent manner. A transporter protein termed Spinster homolog 2 (spns2) transports a signaling molecule known as sphingosine 1-phosphate (S1P). For our immune system to function properly, spns2 has to transport S1P to the appropriate places to signal to immune cells. Unfortunately, this is a problem in autoimmune and inflammatory diseases, such as multiple sclerosis, due to these diseases having an overactive immune system. A potential way to treat these diseases would be by inhibiting spns2. This work describes the design, synthesis, and biological evaluation of spns2 inhibitors. Many compounds were found to inhibit spns2 to a degree, but three compounds, in particular, show potent and effective inhibition in mice.

Cancer progression and physiological changes within the cells are accompanied by alterations in the biophysical properties. Therefore, the cell biophysical properties can serve as promising markers for cancer detection and physiological activities. To aid in the investigation of the biophysical markers of cells, a microfluidic chip has been developed which consists of a constriction channel and embedded microelectrodes. Single-cell impedance magnitudes at four frequencies and entry and travel times are measured simultaneously during their transit through the constriction channel. This microchip provides a high-throughput, label-free, automated assay to define biophysical signatures of malignant cells and monitor the therapeutic efficacy of drugs. Here, we monitored the dynamic cellular biophysical markers in response to sphingosine kinase inhibitors (SphKIs), and compared the effectiveness of drug delivery using Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with SphKIs versus conventional delivery. Cells treated with SphKIs showed significantly higher impedance magnitudes at all four frequencies. The bioelectrical parameters extracted using a model also revealed that the highly aggressive breast cells treated with SphKIs shifted electrically towards that of a less malignant phenotype; SphKI-treated cells exhibited an increase in cell-channel interface resistance and a significant decrease in specific membrane capacitance. Furthermore, SphKI-treated cells became slightly more deformable as measured by a decrease in their channel entry and travel times. We observed no significant difference in the bioelectrical changes produced by SphKI delivered conventionally or with NPs. However, NPs-packaged delivery of SphKI decreased the cell deformability. In summary, the results showed that while the bioelectrical properties of the cells were dominantly affected by SphKIs, the biomechanical properties were mainly changed by the NPs. / Master of Science

Doctor of Philosophy / In healthy individuals, the autoimmune system is the body's natural defense against foreign materials and organisms. The main tools utilized for this defense mechanism are immune cells. However, in patients suffering from autoimmune diseases, the autoimmune system is overactive resulting in its attack on healthy cells, which leads to reduced or eliminated function of the targeted organs. To suppress these overreactive immune responses, pharmaceutical intervention is needed. An integral part of autoimmune response is the lipid sphingosine-1-phosphate (S1P). Interactions of S1P with its response-inducing receptors prompts the release of immune cells, lymphocytes in particular, from lymph tissue to migrate and participate in the invoked immune response. The pharmaceutical industry has produced five FDA approved drugs that disrupt this S1P-receptor interaction by blocking the receptor to reduce the autoimmune response in patients suffering from autoimmune diseases such as multiple sclerosis and ulcerative colitis. However, these treatments had adverse side effects on the cardiovascular system due to the presence of S1P receptors in the heart. Due to this, there is attraction to target a different node of the S1P signaling pathway to avoid these side effects while still suppressing the immune response. A node that is a viable target for therapeutic target that has recently become the focus of medicinal chemistry campaigns is the transporter protein spinster homolog 2 (Spns2). This protein is responsible for the transport of S1P from intracellular space to extracellular space to interact with its receptors and induce the immune response. Recently, our group has developed several effective inhibitors of Spns2. In this dissertation, several improvements of previously reported inhibitors are revealed. The pinnacle of this work is the development of 4.22v that is optimized to have drug-like properties for testing in mice. Administration of 4.22v to mice resulted in reduced circulating lymphocytes and without showing signs of toxicity following chronic dosing for 14 days. These results suggest that 4.22v is a potential drug candidate and is currently undergoing further biological evaluation.

The potential antitumor activity of mixed CB1/CB2 cannabinoid receptor agonists, such as the aminoalkylindole WIN55,212-2 (WIN2), has been extensively studied, but little information is available as to their potential interaction with conventional cancer therapies, such as ionizing radiation (IR). In the present work, we investigated the effects of WIN2 on the antiproliferative effects of radiation in human (MCF-7 and MDA-MB-231) and murine (4T1) breast cancer cells, as well as an immortalized human breast epithelial cell line (MCF-10A). WIN2 or radiation alone inhibited breast tumor growth, while the combination of WIN2 and radiation was more effective than either agent alone in breast cancer cells. WIN2 showed lower potency in MCF-10A cells than MCF-7 cells, but was still able to augment the effects of radiation at higher doses. The stereoisomer of WIN2, WIN55,212-3 (WIN3) failed to inhibit growth or potentiate the growth-inhibitory effects of radiation, indicating stereospecificity in all cell lines tested. The combination of WIN2 and IR was examined in vivo but the results were inconclusive. Interestingly, while other aminoalkylindoles, pravadoline and JWH-015, enhanced the antiproliferative effects of radiation, this was not the case for other synthetic cannabinoids (i.e., nabilone, CP55,940 and methanandamide) or phytocannabinoids (i.e., ∆9-tetrahydrocannabinol and cannabidiol). The antiproliferative actions of WIN2 were not ameliorated by CB1, CB2, TRPV1, or PPAR receptor antagonists, suggesting the possibility of a novel site of action. Studies utilizing sphingosine-1-phosphate (S1P) agonists and estradiol suggest that WIN2 interferes with S1P signaling in cell proliferation, but agonist stimulated [³⁵S]GTPγS binding assays show that this antagonism is not occurring at the level of S1P receptors. In addition, WIN2 did not alter radiation-induced DNA damage or the rate of DNA repair based on γH2AX staining. Treatment with WIN2 and radiation promoted both autophagy and senescence, but not apoptosis or necrosis. Time course studies combined with senescence and cell death data suggest that radiation-induced senescence, while WIN2 induced classical growth arrest and the WIN2/IR combination produced parallel mechanisms of both senescent growth arrest and classical growth arrest. Taken together, these findings raise the possibility that aminoalkylindole compounds targeting a novel site of action represents a potential strategy to augment the effectiveness of radiation treatment in breast cancer.