Tyrosine kinases regulate fundamental signal pathways in cells including cell proliferation, motility, and differentiation. The kinase activity is tightly controlled in normal cells but is usually excessive activated in cancers. Several tyrosine kinase inhibitors are used in cancer therapies nowadays. Our novel tyrosine kinase inhibitor, 1J-309, is a multiple kinase inhibitor that targets several receptors including vascular endothelial growth factor receptors (VEGFRs). We find 1J-309 dramatically reduces cell proliferation of VEGFR3+/VEGF-C+ A549 human lung cancer cells by decreasing the expression of CDK1 and cyclin B1 following growth arrest at G2/M phase. After long term drug treatment, 1J-309 causes cell death. Moreover, 1J-309 represses CDK1 expression at early stage but it does not change CDK1 RNA expression and protein stability. Additionally, 1J-309 significantly decreases the migration ability of A549 cells. 1J-309 also reduces gelatin-related invasion potency. The AKT and p38 MAPK activity are significantly repressed by 1J-309 and it dramatically drives the expression of tumor suppressor, p53, at low-dose treatment. Our results demonstrate that 1J-309 significantly attenuates cell proliferation by inducing G2/M growth arrest, reduces the invasion and migration potency, and promotes a dramatic increase of p53 in A549 cells.

No description available.

Tolerance to ethanol is an endophenotype of alcoholism, allowing the study of a complex psychiatric condition using animal models. To identify new genes involved in the acquisition of tolerance, I designed an automated and high-throughput tolerance assay and screened a collection of deficiency mutants for the inability to develop tolerance. The screen yielded several “regions of interest” where more than one overlapping deficiency failed to develop tolerance. One of these regions comprised nine genes, and testing the expression levels of each gene revealed that diazepam binding inhibitor (Dbi) showed grossly increased expression in the deficiency mutant compared to wild type. Another mutant stock, with a P-element transposon inserted downstream of the Dbi gene, both failed to develop tolerance and showed further increased expression of Dbi. There are two insulator binding sites flanking Dbi, and the P-element transposon also contains insulator binding sites. Based on these results, it was hypothesized that an insulator complex kept Dbi expression low in wild type flies and that disrupting the insulator complex allowed aberrantly high expression of Dbi in the mutants. Furthermore, we assumed that induction of Dbi blocked tolerance by making the mutants resistant prior to the first sedation. A UAS-DBI transgene was constructed to over-express Dbi. Induction of the UAS-DBI with a heat shock gal4 driver induced resistance to ethanol sedation; a similar response was observed in the parental control, but the effect was smaller. Although driving UAS-DBI with the neural elav-gal4 driver did not block tolerance, the experimental stock was resistant to ethanol sedation compared to the parental controls, indicating that increased Dbi expression produced “pre-tolerance.” To confirm the theory that insulator disruption was responsible for the increase in Dbi and the resulting no-tolerance phenotype, the P-element in the second mutant was mobilized by introducing a transposase source. These offspring lines were analyzed using qualitative PCR to determine whether the transposon excised precisely, left a portion of the transposon behind, or removed some of the flanking region. A precise excision mutant was identified, but this mutation did not rescue tolerance as predicted. This result might indicate that genetic background was the cause of the no-tolerance phenotype, or it might indicate that the excision was not exactly precise and removed the native insulator binding site, causing the insulator complex to remain disrupted. / text

Over one-third of the world’s population is at risk for infection with dengue virus (DENV), a mosquito-borne virus that can cause a severe febrile disease. There are no specific treatments available for dengue infection, and much remains unknown about how DENV interaction with the host cell leads to a successful infection. This dissertation examines DENV entry using small molecule inhibitors and mutagenesis of the envelope (E) protein, the major protein on the viral surface. This work grew from our initial observation that small molecule GNF-2 is capable of lowering DENV yield when present at two separate points during DENV infection. Treatment of infected cells with GNF-2 post-entry significantly lowered DENV yield, most likely due to GNF-2’s documented activity against Abl kinase. However, we also observed that treatment of virus inocula with GNF-2 prior to cellular infection significantly lowered DENV yield. We discovered that GNF-2 bound directly to the dengue virion and co-localized with DENV envelope protein shortly after cellular infection. Using GNF-2 as a scaffold, we performed a structure-activity relationship study and identified twenty-one compounds that have similar or increased potency as GNF-2 when pre-incubated with virus. Using a subset of compounds from this study, we demonstrated that they block completion of DENV fusion in vitro, suggesting that the compounds inhibit DENV entry by preventing the completion of viral fusion inside cellular endosomes. In experiments complementing the mechanism of action studies, we selected for inhibitor-resistant virus by passaging virus in the presence of small molecules. We identified a single point mutation in the envelope protein located in the domain I/II interface that enhanced viral entry and conferred resistance to virus particles against select compoundsin a single-cycle reporter virus system. Further examination of this E protein “hinge region” found that mutations in this area may affect both release and entry of reporter virus particles. The work presented in this dissertation may inform the design of future small molecule inhibitors of DENV as well as increase our understanding of how point mutations in the DENV E protein can influence viral entry and other steps of the viral life cycle.

Many common cancers, including breast, prostate and lung, have a predilection to metastasize to the bone, bringing not only bone destruction but severe pain. Although novel chemotherapeutic agents have increased life expectancy, patients are experiencing higher incidences of fracture, pain and drug-induced side effects; furthermore, recent findings suggest that patients are severely under-treated for their cancer pain. Strong analgesics, namely opiates, are the first-line therapy in alleviating cancer-related pain despite severe side effects including enhanced bone destruction with sustained administration. Bone resorption is primarily treated with bisphosphonates, which can bring highly undesirable side-effects including nephrotoxicity and osteonecrosis of the jaw. Thus novel therapeutics are needed to treat the pain of metastatic cancer patients. Animal models of cancer-induced bone pain (CIBP) have revealed that the neurochemistry of cancer has distinctive features from other chronic pain states. These include factors released from the cancer cells, tumor activated macrophages and increased osteoclast degredation of bone within the bone microenvironment, all acting to sensitize free nerve endings.One possibility of inhibiting cancer-mediated pain inducing factors includes agonism of the Cannabinoid 2 receptor agonists. Cannabinoid CB2 receptor-specific agonists have been shown to reduce bone loss and stimulate bone formation in a model of osteoporosis. CB2 agonists produce analgesia in both inflammatory and neuropathic pain models. Notably, mixed CB1/CB2 agonists also demonstrate a reduction in ErbB2-driven breast cancer progression. Osteolytic sarcoma within the femur produced spontaneous and touch evoked behavioral signs of pain within the tumor-bearing limb. The systemic administration of AM1241 both acutely and for 7 days significantly attenuated spontaneous and evoked pain in the inoculated limb. Sustained AM1241 significantly reduced bone loss and decreased the incidence of cancer-induced bone fractures. In addition, CB2 agonists significantly reduce breast cancer-induced bone pain, bone loss and breast cancer proliferation in part via cytokine/chemokine suppression. Studies utilized the spontaneously-occurring syngenic murine mammary cell line (66.1) implanted and sealed into the femur intramedullary space. Measurements were made of spontaneous pain, bone loss and cancer proliferation. The central and systemic administration of the CB2 agonist JWH015 for seven days significantly attenuates pain. Pharmacological characterization with cannabinoid 1 and 2 antagonists demonstrates that the effects JWH015 on pain were mediated by the CB2 receptor. We and others have found that bone induced cancer pain increases the expression of GFAP and Iba1 in the lumbar spinal cord which are markers of astrocytes and microglia respectively, compared to control animals. After administration of JWH015 (i.t), the release of spinal pro-inflammatory cytokines, IL-6 and TNFá, are reduced suggesting that modulation of glial cytokines may be one mechanism by which CB2 agonists can attenuate pain centrally. On the other hand, systemic administration of JWH015 reduces cancer-induced elevation of cytokines in the tumor microenvironment, suggesting a mechanism by which CB2 agonist is attenuating pain peripherally. Additionally, systemic administration improves bone modification, as demonstrated via micro-computed tomography and bone serum markers while decreasing femoral tumor burden. In vitro, JWH015 reduced cancer cell proliferation and other inflammatory mediators shown to promote pain, bone loss and proliferation. These results suggest CB2 agonists as a novel treatment for breast cancer-induced bone pain, where disease modifications include a reduction in bone loss, suppression of cancer growth, attenuation of severe bone-pain and increased survival without the major side effects of current therapeutic options. Another future therapeutic option for metastatic bone cancer pain may include cathepsin inhibitors. Cysteine cathepsins (B, C, F, H, K, L, O, L2/V, W, X/Z) are highly expressed in many human cancers and have been associated with poor patient prognosis. In the RIP1-Tag2 transgenic model of pancreatic cancer, mice treated with VBY-825, a reversible inhibitor of cathepsins S, B, V, L, K showed a significant reduction in tumor incidence and growth. Here we demonstrate the cathepsin inhibitor VBY-825 reduces cancer-induced pain behaviors. Additionally, tumor bearing animals treated with VBY-825 demonstrate a reduction in bone resorption, possibly mediated through a reduction in osteoclast activity. These results indicate that a cathepsin inhibitor targeting multiple cathepsins, such as VBY-825, could be a novel therapeutic for bone metastases.Part of the failure to palliate cancer pain is due to a poor understanding of the etiology of cancer pain. Preclinical studies have just begun to scratch the surface on how such cancers may interact with the bone microenvironment to result in pain and bone loss. Further studies are desperately needed at both the preclinical and clinical level to determine the unique molecular profile of cancer pain that may lead to the development of superior therapeutics for CIBP. The studies presented herein provide preclinical evidence that warrant the investigation of these compounds in the clinic as treatment for cancer-induced bone pain.

Pastaraisiais metais vyksta labai daug elgsenos endokrininės kontrolės tyrimų. Mūsų darbe mes tyrėme galimą estrogenų vaidmenį vištų patelių lytinės elgsenos difirenciacijai. Eksperimentiniai gyvūnai 8 embrioninio vystymosi dieną buvo aveikti aromatazės inhibitoriumi, fadrozoliu, tokiu būdu blokuojant estradiolio gamybą. Kontroliniams gyvūnams buvo suleistas fizioliginis tirpalas. Suaugusių vištų elgsena buvo stebima dvejuose skirtinguose lytinės elgsenos testuose: su suaugusiu intaktiniu (nepaveiktu) gaidžiu ir su intaktine (kontroline) višta. Testuojant su gaidžiu fadrozoliu paveiktos vištos rodė mažesnį pasiruošimą kopuliuoti su gaidžiu, turėjo polinkį dominuoti ir vengti gaidį. Testuojant su kontroline višta, eksperimentiniai paukščiai rodė vyrišką elgseną - sparno rėžimą, bandymus kopuliuoti ir giedojimą. Kelios fadrozoliu paveiktos vištos rodė pilną kopuliacinės elgsenos seką, tame tarpe kloakų kontakto judesius. Mūsų tyrimai palaiko hipotezę, kad estrogenai yra pagrindinis faktorius, lemiantis naminių vištų lytinės elgsenos diferenciaciją. / In recent years much scientific reseach is devoted to the endocrine control of behaviour. In our work we studied the possible role of estrogens in differentiation of sexual behaviour in female chickens. Eksperimental animals were treated on day 8 of embrionic development with an aromataze inhibitor, fadrozole, thus blocking the oestradiol production. Control animals received injections of vehicle (saline). In adulthood behaviour of hens was observed in two different sexual behaviour tests: with mature non-treated (intact) cocks and with non-treated (control) hens. When tested with a cock, fadrozole-treated hens showed reduced readiness to copulate with a cock, tended to dominate and to avoid a cock. When tested with a control hen, experimental birds displayed male-type behaviour – waltzing, mount attempts and crowing,. Some of fadrozole-treated hens showed a full sequence of copulatory behaviour, including cloacal contact movement. Our study supports the hypothesis that oestrogens play a major role in differentiation of sexual behaviour in the domestic chickens.

No description available.

Atherosclerotic cardiovascular disease is the leading cause of death of adults in North America. Diabetes is a classical risk factor for atherosclerotic cardiovascular disease. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of fibrinolysis. Elevated levels of PAI-1, oxidized low-density lipoprotein (oxLDL) and glycated LDL (glyLDL) were detected in patients with diabetes. Increased oxidative stress is associated with diabetic cardiovascular complications. Previous studies in our laboratory demonstrated that oxLDL or glyLDL increased the production of PAI-1 or reactive oxygen species (ROS) in vascular endothelial cells (EC). This study was undertaken to investigate transmembrane signaling mechanisms involved in oxLDL or glyLDL-induced upregulation of PAI-1 in cultured vascular EC. Further, we examined the mechanism for oxLDL or glyLDL-induced oxidative stress in EC. The results of the present studies demonstrated novel transmembrane signaling pathway for oxLDL-induced PAI-1 production in vascular EC. We demonstrated that lectin-like oxLDL receptor-1, H-Ras, a small G-protein and Raf-1/ERK-1/2 mediate oxLDL-induced PAI-1 expression in cultured EC. GlyLDL may activate EC via a distinct transmembrane signaling pathway. The results of the present study demonstrated that receptor for advanced glycation end products, NADPH oxidase and H-Ras/Raf-1 are implicated in the upregulation of heat shock factor-1 or PAI-1 in vascular EC under diabetes-associated metabolic stress. We investigated the effects of oxLDL or glyLDL on mitochondrial function in EC. Treatment with oxLDL or glyLDL significantly impaired the activities of electron transport chain (ETC) enzymes and also increased mitochondria-associated ROS in EC. The findings suggest that oxLDL or glyLDL attenuated activity of ETC and increased ROS generation in EC, which potentially contributes to oxidative stress in vasculature. In conclusion, diabetes-associated lipoproteins may upregulate stress response mediators and PAI-1 production via distinct transmembrane signaling pathways. OxLDL or glyLDL may increase ROS production via NOX activation and the impairment of mitochondrial ETC enzyme activity in EC. The understanding and identification of the regulatory mechanisms involved in diabetes-associated lipoprotein-induced signaling may help pharmacological design for the management of diabetic cardiovascular complications.

<p>The incessant increase of antibiotic resistance among Gram-negative pathogens is a serious threat to public health worldwide. A lack of new antimicrobial agents, particularly those against multidrug-resistant Gram-negative bacteria further aggravates the situation, highlighting an urgent need for development of effective antibiotics to treat multidrug-resistant Gram-negative infections. Past efforts to improve existing classes of antimicrobial agents against drug-resistant Gram-negative bacteria have suffered from established (intrinsic or acquired) resistance mechanisms. Consequently, the essential LpxC enzyme in the lipid A biosynthesis, which has never been exploited by existing antibiotics, has emerged as a promising antibiotic target for developing novel therapeutics against multidrug-resistant Gram-negative pathogens. </p><p>In Chapter I, I survey the medically significant Gram-negative pathogens, the molecular basis of different resistance mechanisms and highlight the benefits of novel antibiotics targeting LpxC. In Chapter II, I discuss a structure-based strategy to optimize lead compounds for LpxC inhibition, revealing diacetylene-based compounds that potently inhibit a wide range of LpxC enzymes. The elastic diacetylene scaffold of the inhibitors overcomes the resistance mechanism caused by sequence and conformational heterogeneity in the LpxC substrate-binding passage that is largely defined by Insert II of LpxC. In Chapter III, I describe the structural basis of inhibitor specificity of first-generation LpxC inhibitors, including L-161,240 and BB-78485 and show that bulky moieties of early inhibitors create potential clashes with the &#61538;a-&#61538;b loop of Insert I of non-susceptible LpxC species such as P. aeruginosa LpxC, while these moieties are tolerated by E. coli LpxC containing long and flexible Insert I regions. These studies reveal large, inherent conformational variation of distinct LpxC enzymes, providing a molecular explanation for the limited efficacy of existing compounds and a rationale to exploit more flexible scaffolds for further optimization of LpxC-targeting antibiotics to treat a wide range of Gram-negative infections. </p><p>In Chapters IV and V, a fragment-based screening and structure-guided ligand optimization approach is presented, which has resulted in the discovery of a difluoro biphenyl diacetylene hydroxamate compound LPC-058 with superior activity in antibacterial spectrum and potency over all existing LpxC inhibitors. In Chapter VI, I describe our efforts to improve the cellular efficacy of LPC-058 by reducing its interaction with plasma proteins, such as human serum albumin (HSA). The binding mode of LPC-058 was captured in the crystal structure of HSA/LPC-058 complex. The acquired structural information facilitated the development of the dimethyl amine substituted compound LPC-088 that displays significantly improved cellular potency in presence of HSA.</p> / Dissertation

Pluripotent stem cells (PSCs) can self-renew indefinitely in culture while maintaining their capacity to differentiate into any cell type of an organism, thus offering novel sources for drug screening, in vitro disease modelling, and cell replacement therapies. However, due to their sensitive nature, many PSC lines are still cultured using undefined components such as serum or serum-derived components, on either feeder cells or complex protein mixes such as Matrigel or gelatine. In order to fully realize the potential of these cells we need controlled, completely defined and xeno-free culturing conditions that maintain growth and survival of homogenous, non-differentiated colonies. This thesis focuses on the in vitro maintenance of both mouse and human PSCs, analysing the media and substrate requirements of these cells and linking them to the intracellular signalling pathways involved in the maintenance of pluripotency and self-renewal. Benchmarking of commercially available culture methods for PSCs has been performed, evaluating their capacity to maintain pluripotency and growth of undifferentiated PSCs over several passages and reporting new characteristics, like the tendency of mouse PSCs to grow as floating spheres in 2i medium, a novel media formulation that uses two inhibitors to hinder differentiation capacity and subsequently induce pure, undifferentiated cultures. The major finding in this thesis is the identification of Inter-α-Inhibitor (IαI) as a protein able to activate the previously described signal-transduction pathway Yes/YAP/TEAD in mouse PSCs and to induce transcription of the well-known stem cell transcription factors Nanog and Oct3/4. IαI is a serum protein found in high concentration in human serum that had been traditionally described as an extracellular matrix remodelling protein. For the first time, we describe IαI to have signalling capacity on PSCs. Moreover, IαI is demonstrated to induce attachment, growth and long-term survival of undifferentiated mouse and human PSCs when added to serum-free, chemically defined media. IαI is the first molecule described to date to induce attachment of human PSCs on uncoated, standard tissue-culture treated plastic, just by supplementation as a soluble molecule at the seeding step. Following this discovery, we evaluate a novel culture method using the completely defined, serum-free E8 medium supplemented with IαI (E8:IαI) for long-term propagation of four different human PSC lines and discover that IαI can indeed support long-term culture with maintained pluripotency, differentiation capacity, growth rate and genetic stability. Moreover, in contrast to the control culture method using a commercially available surface coating, IαI supplementation can support single cell passaging of human PSCs, and adapt feeder-dependent cultured human PSCs to E8:IαI with high efficiency. A mouse PSC line is also grown for over 20 passages in IαI with retained pluripotency, differentiation capacity and genetic stability. IαI is inexpensive to produce and derived from human plasma, and could therefore be produced in compliance with Good Manufacturing Practices. Ultimately, our group aims to develop and test large-scale, completely defined, xeno-free culturing methods for PSCs, suitable for pharmacological and medical applications.