Rheumatoid arthritis (RA) is a debilitating chronic inflammatory disease causing bone and cartilage degradation. Macrophages are known to play a role in RA pathology by producing pro-inflammatory cytokines and chemokines, which activates immune cells, drives inflammation and facilitates the degradation of bone and cartilage. Alterations in epigenetic mechanisms, processes that regulate gene expression, have been implicated in the regulation of pro-inflammatory cytokines in RA. Therefore, the aim of this thesis was to determine specific epigenetic variation between RA patient blood and synovial fluid (SF)-derived macrophages (SF MLS). Granulocyte and macrophages colony stimulating factor (GM-CSF) was used to differentiate healthy donor and RA patient blood monocytes into macrophages. Lipopolysaccharide (LPS) was used to stimulate blood and SF-derived macrophages to initiate inflammatory cytokine production. A library of small molecule inhibitors was used to identify key epigenetic regulators of pro-inflammatory cytokine production. Bromodomain and extra-terminal (BET) protein inhibitors (JQ1, I-BET151, PFI-1) were the only class of inhibitor to show consistent down regulation of pro-inflammatory cytokines in both healthy and RA patient-derived macrophages. However, only JQ1 was shown to reduce TNFα production significantly in SF MLS. Transcriptional profiling of RA patient SF MLS indicated a preference for a pro-inflammatory phenotype, and a resistance to steroids (a trait found in 30% of RA patients); SF MLS production of chemokines and cytokines were not downregulated by glucocorticoids in comparison to corresponding blood-derived macrophages. However, JQ1 treatment successfully suppressed these genes. In addition, silencing of BRD4 in blood-derived macrophages from healthy donors reduced pro-inflammatory cytokine production. Chromatin immunoprecipitation studies showed BRD4 was localised to pro-inflammatory promoter regions upon LPS stimulation and displaced in the presence of JQ1. These studies identified BET proteins BRD2, 3 and 4, as essential epigenetic regulators of pro-inflammatory cytokine and chemokine production in both healthy donors and RA patient macrophages. Furthermore, the observation that BET inhibitors can regulate genes that are steroid resistant in RA patient SF MLS, highlights their therapeutic potential in RA.

INTRODUCTION: For many years, stress and anxiety disorders have taken a heavy toll on the American population. Affecting approximately 40 million individuals over the age of 18, the discovery of treatment options is very important. Ever since the 1950s, a wide variety of compounds have been discovered and proven to have antagonistic properties for such disorders. For the last three decades, however, researchers have focused on a specific peptide that was discovered in 1981 by Dr. Wylie Vale and his colleagues at the Salk Institute in San Diego, California, corticotropin releasing factor (CRF). CRF is a 41 amino acid peptide that has been shown to play a very important role in an organism's endocrine response to stress through the activation of the hypothalamic–pituitary–adrenal (HPA) axis. Ever since its discovery, the identification and characterization of the CRF receptors and family members have allowed for the development of novel peptide and non–peptide antagonists. Unfortunately, these compounds have been unsuccessful in the progression to later stage clinical trials that could lead to promising therapeutics. There are two receptor subtypes for this family of peptides known as CRFR1 and CRFR2. While there have been many compounds identified that can block CRFR1, currently, there are no known selective non–peptide antagonists for the CRFR2 subtype. As the two receptor subtypes share 70% sequence identity, close observation of the functional properties of antagonist ligands for CRFR1 may lead to the development of such ligands for CRFR2. METHODS: In our current study, we focused on two residues in transmembrane domains (TMD) 3 (His199) and 5 (Met276) of CRFR1 that have proven to be important for the function of the highly selective small molecule antagonist antalarmin. In order to further prove the importance of these sites, we have mutated the two corresponding amino acids in CRFR2β to those of CRFR1: V215H in TMD 3 and V292M in TMD 5. In addition, we mutated a third amino acid residue, M293I, in order to avoid the positioning of two adjacent methionine amino acids. With this mutant construct, CRE–luciferase and cyclic AMP radioimmunoassay methodologies were used to observe the function of antalarmin on CRFR1, the mutant and wild type CRFR2β. The accumulation of cAMP was measured intracellularly following stimulation by the CRF receptor peptide agonists sauvagine, isolated from frog, and urocortin 1, isolated from rat. RESULTS: For the initial CRE–luciferase functional assay, we used the CRF receptor agonist sauvagine on our mutant CRFR2β to indirectly measure the accumulation of intracellular cAMP through the enzyme luciferase. In the presence or absence of the antagonist antalarmin, there were no significant changes on the function of the mutant CRFR2β. On the other hand, when directly measuring the accumulation of intracellular cAMP via radioimmunoassay, antalarmin successfully showed a functional inhibitory effect on the mutant CRFR2β receptor. As expected, Ucn1 stimulation of CRFR1 in the presence of antalarmin indicated a decrease in the EC50 for the peptide agonist, and thus an inhibitory effect by antalarmin. Compared to CRFR1, we observed a similar effect for Ucn1 stimulation of the mutant CRFR2β receptor in the presence of antalarmin. While the presence or absence of antalarmin did not have a significant inhibitory effect on the wild type CRFR2β, it can be concluded that the mutant CRFR2β receptor possessed similar properties to the CRFR1 receptor with respect to antalarmin antagonist activity. CONCLUSION: In our study, we were able to further support the importance of the two amino acid residues in TMD 3 and 5 of CRFR1 for the function of small molecule antagonists. In addition, we were able to show that antalarmin, a small molecule antagonist known to be highly selective for CRFR1, can have a functional inhibitory effect on the mutant CRFR2β. The progressive study of these discrete differences between the two CRF receptor subtypes may enable the discovery of novel selective non–peptide CRFR2β receptor antagonists.

Mesenchymal stem cells are the stem cells that replace the bone, fat and cartilage tissues of the human body. In addition, these cells can form muscles, ligaments and neurons. This wide multipotency has made mesenchymal stem cells of particular interest in the fields of tissue engineering and regenerative medicine. Furthermore, mesenchymal stem cells can modulate the immune system by reducing factors that increase inflammation and immune recognition. This immune recognition suppression has resulted in their application as part of bone marrow transplantation in the prevention of 'graft versus host‘ disease. There are hundreds of on-going clinical trials using these cells for the treatment of autoimmune diseases such as type I diabetes, arthritis and multiple sclerosis. The increasing importance of these cells has brought in to focus the culture methods used to for their expansion and manipulation. Currently, animal derived components are used as surfaces for their growth and as components in the culture media. This exposes these cells to animal pathogens and antigens that can be passed to the recipients of these cells. In the first part of this thesis, polymer microarrays were employed to identify alternatives to the biological surfaces currently used for mesenchymal stem cell culture. This platform allowed hundreds of polyacrylates/acrylamides and polyurethanes to be simultaneously scrutinised to identify surfaces that could support their growth and maintain their stem cell characteristics. Identified polymer surfaces were monitored in long-term culture (10 passages) and were shown to retain the cell phenotype and capacity to differentiate, thus providing chemically defined substrates for long-term mesenchymal stem cell culture. In the second part of this thesis, a 'smart‘ polymer microarray of hydrophilic cross-linked polymers (hydrogels) were used to remove another key biological component of culture, trypsin. These 'smart‘ hydrogels modulated their properties depending on the temperature. Hydrogels that could trigger mesenchymal stem cell release after a reduction in temperature were identified. A unique passaging system using a modest temperature reduction for 1h was developed as a passaging method. Cells were maintained and monitored for 10 passages using this novel enzyme free passaging method. Analysis of the mesenchymal stem cell phenotype and differentiation capacity revealed this method superior than conventional culturing methods. In the final part of this thesis, a 'knowledge-based‘ small molecule library was designed, which could potentially yield small molecules to manipulate/enhance the mesenchymal stem cell state without the use of biological components. The key protein pathways that control the stem cell state were examine with the bioinformatics tool GeneGo was used to identify compounds that affected these pathways, resulting in selection of 200 small molecules. The effect of the small molecules on the mesenchymal phenotype was examined and 5 small molecules were identified that enhanced the phenotype of these cells. The anti-inflammatory properties associated with the hit compounds led to the investigation of their effects on key surface proteins associated with the immune-modulatory state of the cells. In this preliminary study, two of the small molecules, estriol and spermine, increased the expression of a key mesenchymal stem cell marker STRO-1 and down regulated ICAM-1, a critical component of the immune modulation capacity of this cell type.

abstract: Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that represents the ancient fusion of two major thiol-disulfide oxidoreductase gene families: thioredoxin and ERV. QSOX1 was first linked with cancer after being identified as overexpressed in pancreatic ductal adenocarcinoma (but not in adjacent normal ductal epithelia, infiltrating lymphocytes, or chronic pancreatitis). QSOX1 overexpression has been confirmed in a number of other histological tumor types, such as breast, lung, kidney, prostate, and others. Expression of QSOX1 supports a proliferative and invasive phenotype in tumor cells, and its enzymatic activity is critical for promoting an invasive phenotype. An in vivo tumor growth study utilizing the pancreatic tumor cell line MIAPaCa-2 containing a QSOX1-silencing shRNA construct revealed that QSOX1 expression supports a proliferative phenotype. These preliminary studies suggest that suppressing the enzymatic activity of QSOX1 could represent a novel therapeutic strategy to inhibit proliferation and invasion of malignant neoplasms. The goal of this research was to identify and characterize biologically active small molecule inhibitors for QSOX1. Chemical inhibition of QSOX1 enzymatic activity was hypothesized to reduce growth and invasion of tumor cells. Recombinant QSOX1 was screened against libraries of small molecules using an enzymatic activity assay to identify potential QSOX1 inhibitors. Two lead QSOX1 inhibitors were confirmed, 2-phenyl-1, 2-benzisoselenazol-3-one (ebselen), and 3-methoxy-n-[4-(1 pyrrolidinyl)phenyl]benzamide. The biological activity of these compounds is consistent with QSOX1 knockdown in tumor cell lines, reducing growth and invasion in vitro. Treatment of tumor cells with these compounds also resulted in specific ECM defects, a phenotype associated with QSOX1 knockdown. Additionally, these compounds were shown to be active in pancreatic and renal cancer xenografts, reducing tumor growth with daily treatment. For ebselen, the molecular mechanism of inhibition was determined using a combination of biochemical and mass spectrometric techniques. The results obtained in these studies provide proof-of-principle that targeting QSOX1 enzymatic activity with chemical compounds represents a novel potential therapeutic avenue worthy of further investigation in cancer. Additionally, the utility of these small molecules as chemical probes will yield future insight into the general biology of QSOX1, including the identification of novel substrates of QSOX1. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

abstract: Acute Myeloid Leukemia (AML) is a disease that occurs when genomic changes alter expression of key genes in myeloid blood cells. These changes cause them to resume an undifferentiated state, proliferate, and maintain growth throughout the body. AML is commonly treated with chemotherapy, but recent efforts to reduce therapy toxicity have focused on drugs that specifically target and inhibit protein products of the cancer’s aberrantly expressed genes. This method has proved difficult for some proteins because of structural challenges or mutations that confer resistance to therapy. One potential method of targeted therapy that circumvents these issues is the use of small molecules that stabilize DNA secondary structures called G-quadruplexes. G-quadruplexes are present in the promoter region of many potential oncogenes and have regulatory roles in their transcription. This study analyzes the therapeutic potential of the compound GQC-05 in AML. This compound was shown in vitro to bind and stabilize the regulatory G-quadruplex in the MYC oncogene, which is commonly misregulated in AML. Through qPCR and western blot analysis, a GQC-05 mediated downregulation of MYC mRNA and protein was observed in AML cell lines with high MYC expression. In addition, GQC-05 is able to reduce cell viability through induction of apoptosis in sensitive AML cell lines. Concurrent treatment of AML cell lines with GQC-05 and the MYC inhibitor (+)JQ1 showed an antagonistic effect, indicating potential competition in the silencing of MYC. However, GQC-05 is not able to reduce MYC expression significantly enough to induce apoptosis in less sensitive AML cell lines. This resistance may be due to the cells’ lack of dependence on other potential GQC-05 targets that may help upregulate MYC or stabilize its protein product. Three such genes identified by RNA-seq analysis of GQC-05 treated cells are NOTCH1, PIM1, and RHOU. These results indicate that the use of small molecules to target the MYC promoter G-quadruplex is a viable potential therapy for AML. They also support a novel mechanism for targeting other potentially key genetic drivers in AML and lay the groundwork for advances in treatment of other cancers driven by G-quadruplex regulated oncogenes. / Dissertation/Thesis / Masters Thesis Molecular and Cellular Biology 2017

abstract: Surface plasmon resonance (SPR) has emerged as a popular technique for elucidating subtle signals from biological events in a label-free, high throughput environment. The efficacy of conventional SPR sensors, whose signals are mass-sensitive, diminishes rapidly with the size of the observed target molecules. The following work advances the current SPR sensor paradigm for the purpose of small molecule detection. The detection limits of two orthogonal components of SPR measurement are targeted: speed and sensitivity. In the context of this report, speed refers to the dynamic range of measured kinetic rate constants, while sensitivity refers to the target molecule mass limitation of conventional SPR measurement. A simple device for high-speed microfluidic delivery of liquid samples to a sensor surface is presented to address the temporal limitations of conventional SPR measurement. The time scale of buffer/sample switching is on the order of milliseconds, thereby minimizing the opportunity for sample plug dispersion. The high rates of mass transport to and from the central microfluidic sensing region allow for SPR-based kinetic analysis of binding events with dissociation rate constants (kd) up to 130 s-1. The required sample volume is only 1 μL, allowing for minimal sample consumption during high-speed kinetic binding measurement. Charge-based detection of small molecules is demonstrated by plasmonic-based electrochemical impedance microscopy (P-EIM). The dependence of surface plasmon resonance (SPR) on surface charge density is used to detect small molecules (60-120 Da) printed on a dextran-modified sensor surface. The SPR response to an applied ac potential is a function of the surface charge density. This optical signal is comprised of a dc and an ac component, and is measured with high spatial resolution. The amplitude and phase of local surface impedance is provided by the ac component. The phase signal of the small molecules is a function of their charge status, which is manipulated by the pH of a solution. This technique is used to detect and distinguish small molecules based on their charge status, thereby circumventing the mass limitation (~100 Da) of conventional SPR measurement. / Dissertation/Thesis / Ph.D. Electrical Engineering 2013

The use of oncolytic viruses (OVs) to selectively destroy cancer cells is poised to make a major impact in the clinic and potentially revolutionize cancer therapy. Pre-clinical and clinical studies have shown that OV therapy is safe, well-tolerated and effective in a broad range of cancers. Still, resistance due to tumour heterogeneity highlights areas for improvement in OV based therapeutics. Combining OVs and small molecules is a promising strategy to selectively enhance OV-mediated anti-tumour effects. To this end, we have previously identified the synthetic compound Viral Sensitizer 1 (VSe1) that enhances the spread of oncolytic vesicular stomatitis virus (VSVΔ51) in resistant cancer cell lines up to 1000-fold, resulting in synergistic cell killing and improved efficacy in vitro and in vivo. The electrophilic nature of VSe1 prompted us to investigate the scaffold to identify active analogs with more favourable physiochemical properties and explore structure-activity relationships (SAR). In vitro assays and a rational approach in the design of VSe1 analogs allowed us to identify functional groups that can be modified without hampering activity. Lead compounds created in this study based on a pyrrole scaffold increase OV growth up to 2000-fold in vitro and demonstrate remarkable selectivity for cancer cells over normal tissue ex vivo and in vivo. Compared to the parental VSe1, these small molecules also possess enhanced stability with reduced electrophilicity and are well-tolerated in animals, leading to reduced tumour burden and prolonged survival in vivo when used in combination with VSVΔ51. It was known from previous studies that VSe1 suppresses the type I interferon response generated by cancer cells to defend against viral infection. In this study, further investigation revealed that VSe1 and its analogs inhibit the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), resulting in dampened transcriptional expression and secretion of IFN-β and interferon stimulated genes, thereby increasing viral replication and spread. While these findings further elucidated the effect these compounds have on the innate antiviral response, the molecular mechanisms leading to NFκB inhibition remained unclear. We used the newly generated VSe1 analogs to perform ligand-based affinity capture studies leading to the identification of glutathione-s-transferases as interacting proteins, catalytically inhibited by VSe1 and to a lesser extent by its pyrrole analogs. Further inquiry revealed that VSe1 and its analogs cause an imbalance in cellular glutathione homeostasis and increase oxidative stress, which is associated with inhibition of the nuclear translocation of NFκB. However, further studies are required to assess whether these phenomena are directly or indirectly linked. Overall, this study highlights a novel approach to improving OV therapy by using a previously uncharacterized class of compounds that ultimately alter the innate cellular antiviral response through inhibition of NFκB.

Uma das várias formas da hemoglobina do peixe Leporinus Frederici (piava) não apresenta efeito Borh (variação da afinidade ao O2 com o pH). Purificação, caracterização e experimentos de cristalização foram conduzidos visando a determinação da estrutura através de difração de raios X, embora sem resultados positivos. O espectro óptico desta forma particular de hemoglobina foi medido no intervalo de 300-700 nm e subseqüentemente simulado, interpretado e comparado com o espectro da humana. Em outra área do trabalho experimental, três pequenas estruturas moleculares foram determinadas: uma é um intermediário na síntese de alcalóides, com um esqueleto sarpagina; outra é um dipeptídeo complexado com Cu2+; e a terceira é um complexo de picrato com Ce3+. As intensidades das reflexões foram medidas com um difratômetro automático de quatro ciclos CAD-4. As estruturas foram resolvidas por Patterson ou Métodos Diretos e foram refinadas por método de mínimo quadrado. Cetona, C19H21N3O, é um intermediário chave no caminho de reação para síntese de indoloquinolisidinas, pertence ao sistema, P21/C, a=12.200(7), b=16,795(2), c=16,655(l)&#197, &#946=104,18(3)&#176, Z=8, Dc= 1,234 gcm-3, V=3308(3)&#1973. As duas moléculas independentes são aproximadamente relacionadas por um centro de inversão, a principal diferença sendo relativa às configurações dos grupos nitril e metil. As moléculas enantioméricas estão mantidas por ligação de hidrogênio através do N(3)-O(1\') e N(3\')-O(1). A junção N(1)-C(6) é trans e o grupo CH2CN é axial. L-(triptofil)-L-glicinato-cobre(II). C13H13CuN3O3, um composto modelo para conseguir informações para interpretar os dados disponíveis para proteínas azuis, é ortorrômbico, P212121, a=8,284(6), b=9,345(2). c=16.503(2)&#197, Z=4, Dc=1.678 gcm-3, V=1277(2)&#1973. O íon Cu2+ é coordenado por um oxigênio e dois nitrogênios de um dipeptídeo e com um oxigênio de um ligante simetricamente relacionado. A estrutura polimérica resultante está alinhada com o eixo b e tem uma estabilidade maior devido a uma ligação de hidrogênio entre o oxigênio carbonil de um dipeptídeo e o nitrogênio do triptofano do ligante vizinho. A coordenação é essencialmente quadrado planar. O complexo de picrato com Ce3+, CeO33N9C18H30, foi analisado dentro de um grande programa de pesquisa para estudar a química de coordenação dos lantanóides. Duas formas cristalinas são estudadas: uma é monoclínica, P21/n, a=7,799(2), b=26,925(2), c=17,465(2)&#197, &#946=98,93(3)&#176 , Z=4, Dc= 1,908 gcm-3, V=3623(2)޵ e a outra é monoc1ínica, C2/c, a=40,225(5), b=8,08(4), c=24,35l(9), &#946=111,46(2), Z=8, Dc= 1,893 gcm-3, V=7300(8)&#1973. A primeira é relativamente instável sobre a incidência de raios-X e embora a medida das intensidades apresentasse erros sistemáticos significantes, a estrutura pode ser resolvida. O número de coordenação dos dois complexos é 9 e os poliedros de coordenação são intermediários entre antiprisma quadrado monoencapuzado e prisma trigonal triencapuzado / One of the various forms of hemoglobin of the fish Leporinus Frederici (piava) does not present any Bohr effect (variation of the affinity to O2 with pH). Purification, characterization and crystallization experiments were conducted, aimed at the structure determination through X-ray diffraction, although with no positive results. The optical spectrum of this particular hemoglobin form was measured in the range 300-700 nm and subsequently simulated, interpreted and compared with the human hemoglobin spectra. In another area of experimental work, three small molecules structures were determinate: one is an intermediate in the synthesis of alkaloids, with a sarpagine backbone; another is a dipeptide complexes with Cu2+; and a third one is a complex of picrate with Ce3+. The intensities of the reflections were measured with an automatic four-circle difractometer CAD-4. The structures were solved by Patterson or Directs Methods, and were refined by the least squares methods. Ketone, C19H21N3O, is a key intermediate in the reaction pathway for synthesis of indoloquisidines, belongs to the monoclinic system, P21/c, a=12.200(7), b=16,795(2), c=16,655(l)&#197, &#946=104,18(3)&#176, Z=8, Dc= 1,234 gcm-3, V=3308(3)&#1973. The two independent molecule are approximately related by an inversion center, the main difference being the relative configurations of the nitril and methyl groups. The enantiomeric molecules are hydrogen bonded through N(3)-O(1\') and N(3\')-O(1). The junction N(l)-C(6) is trans and the group CH2CN is axial. L-(tryptophyl)-L-glycinate-copper(II), C13H13CuN3O3, a model compound to get information to interpret spectroscopic data available for blues proteins, is orthorhombic, P212121, a=8,284(6), b=9,345(2). c=16.503(2)&#197, Z=4, Dc=1.678 gcm-3, V=1277(2)&#1973. The Cu-ion is coordinated by one oxygen and two nitrogen atoms of the one dípeptide and with an oxygen of a symmetrically related ligand. The resulting polymeric structure is aligned with the b-axis and is further stabilized by an H-bond between the carbonyl-oxygen of the one dipeptide and the tryptophan side-chain nitrogen of the neighboring ligand. The coordination is essentially square-planar. The complex of picrate with Ce3+, CeO33N9C18H30, was analyzed within a broader research program to study the chemistry of coordination of the lantanoids. Two crystalline forms are studied: one is monoclinic, P21/n, a=7,799(2), b=26,925(2), c=17,465(2)&#197, &#946=98,93(3)&#176 , Z=4, Dc= 1,908 gcm-3, V=3623(2)޵ and the other is monoclinic, C2/c, a=40,225(5), b=8,08(4), c=24,35l(9), &#946=111,46(2), Z=8, Dc= 1,893 gcm-3, V=7300(8)&#1973. The former is relatively unstable under the X-rays and although the measured intensities presented significant systematic errors, the structure could be solved. The coordination number of the two complexes is 9 and the coordination polyedra are intermediate between mono-coupled square antiprism and tri-coupled trigonal prism

No description available.

One of the largest problems in current medicine is the shortage of organs for transplant due to technological limitations in the storage of organs for any length of time. A possible solution to this problem would involve cryopreservation. However, current cryopreservatives such as sucrose or DMSO have concerning cytotoxic issues that limit their possible applications. A major cause of cryoinjury is the uncontrolled recrystallization of inter and intra-cellular ice crystals that occurs during the thawing process leading to mechanical damage and dehydration. The Ben lab has thus been interested in the design of compounds that are capable of inhibiting this process but do not possess other undesirable properties found in the native compounds. These synthetic analogues have been shown to increase cellular viability post-thaw. A series of mixed α/β glycopeptides are prepared and analyzed for antifreeze properties. The results of this study imply that it is not the gross conformation of the glycopeptide that is responsible for activity, but rather that intramolecular relationships may be responsible for disrupting the reorganization of ice. A technique was devised for the incorporation of triazoles into the analogues to investigate the importance of the linker and to greatly simplify the synthesis of a library of glycoconjugates. It was found that the IRI activity of glycopeptides is very sensitive to the distance between carbohydrate and peptide backbone. The electron density at the anomeric oxygen is an important parameter with respect to intramolecular networks. A series of substituted galactosides is presented that modify the electronics of the anomeric oxygen. The results demonstrate that decreasing electron density at this position appears to improve IRI activity in a predictable manner. To better understand the remarkable IRI activity of a key analogue, it was systematically truncated. This study led to the serendipitous discovery of a series of very highly IRI active analogues that do not contain a peptide backbone. These compounds represent the first non-glycopeptides that can show very significant IRI activity even at very low concentrations. The final portion of the thesis reports the efforts towards the preparation of a carbasugar analogue of AFGP-8.