Submitted by Michele Mologni (mologni@unoeste.br) on 2018-11-30T12:45:47Z No. of bitstreams: 1 Marina Platzeck Chaves.pdf: 891914 bytes, checksum: 43d527b64ad46e92f25496042406c5e9 (MD5) / Made available in DSpace on 2018-11-30T12:45:47Z (GMT). No. of bitstreams: 1 Marina Platzeck Chaves.pdf: 891914 bytes, checksum: 43d527b64ad46e92f25496042406c5e9 (MD5) Previous issue date: 2018-05-30 / Steroids and gonadotrophins are essential for the regulation of antral follicular development and the late stages of preantral development. Although the luteinizing hormone receptor (LHR) has been detected in the preantral follicles of rats, rabbits, and pigs, the expression of this receptor in bovine fetal ovary has not been demonstrated. The present study aimed to quantify the expression of the LHR and the mRNA abundance of the genes LHR binding protein (LRBP), STAR, HSD3B1, CYP17A1, and CYP19A1 during the development of bovine fetal ovary. In addition, we aimed to identify and quantify the expression of bta-miR-222 (a regulatory microRNA of the LHCGR gene). In summary, LHR expression was observed in the preantral follicle in bovine fetal ovary, from oogonias to primordial, primary and secondary stages, and the mRNA abundance was lower on day 150 than day 60. However, the mRNA abundance of LRBP followed the opposite pattern. The LHR protein was detected in oogonia, primordial, primary, and secondary follicles. Moreover, both oocytes and granulosa cells showed positive immunostaining for LHR. Similar to LRBP, the abundance of bta-miR-222 was higher on day 150 than day 60 or 90 of gestation. With regard to the gene expression of steroidogenic enzymes; only the mRNA abundance of STAR was higher on day 150 than on day 60. In conclusion, these results suggested the involvement of LHCGR/LRBP regulation with mechanisms related to the development of preantral follicles, especially during the establishment of secondary follicles. Furthermore, the present data reinforced that the reduced expression of LHR mRNA in bovine fetal ovaries on day 150 was related to the higher expression of LRBP and bta-miR-222. / Esteroides e gonadotrofinas são essenciais para a regulação do desenvolvimento folicular antral e os estágios finais do desenvolvimento pré-antral. Embora o receptor do hormônio luteinizante (LHR) tenha sido detectado nos folículos pré-antrais de ratos, coelhos e porcos, a expressão deste receptor no ovário fetal bovino não foi demonstrada. O presente estudo teve como objetivo quantificar a expressão do LHR e a abundância de mRNA da proteína de ligação LHR (LRBP), STAR, HSD3B1, CYP17A1 e CYP19A1 durante o desenvolvimento do ovário fetal bovino. Além disso, objetivamos identificar e quantificar a expressão de bta-miR-222 (microRNA regulador do gene LHCGR). Em resumo, a expressão de LHR foi observada no folículo pré-antral no ovário fetal de bovino e a abundância de mRNA foi menor no dia 150 do que no dia 60. No entanto, a abundância de mRNA da LRBP seguiu o padrão oposto. Semelhante a LRBP, a abundância de bta-miR-222 foi maior no dia 150 do que no dia 60 ou 90. Com relação à expressão gênica de enzimas esteroidogênicas; apenas a abundância de mRNA de STAR foi maior no dia 150 do que no dia 60. A proteína LHR foi detectada em oogônia, folículos primordiais, primários e secundários. Além disso, ambos os oócitos e células da granulosa apresentaram imunolocalização positiva para LHR. Em conclusão, estes resultados sugeriram o envolvimento da regulação do LHCGR / LBPB com mecanismos relacionados ao desenvolvimento de folículos pré-antrais, especialmente durante o estabelecimento de folículos secundários. Além disso, os presentes dados reforçaram que a expressão reduzida de mRNA de LHR em ovários fetais bovinos no dia 150 estava relacionada à maior expressão de LRBP e bta-miR-222.

The statin family of hydroxymethylglutaryl coenzyme A reductase (HMGCR) inhibitors, used to control hypercholesterolemia, triggers apoptosis of various human tumour cells. HMGCR is the rate-limiting enzyme of the mevalonate (MVA) pathway, a fundamental metabolic pathway required for the generation of a number of biochemical end-products including cholesterol and isoprenoids, but the contribution of the MVA pathway to human cancer remains largely unexplored. Furthermore, as only a subset of tumour cells has been shown to be highly responsive to statins, the identification of appropriate subsets of patients will be required to successfully advance these agents as anticancer therapeutics. To this end, there were two major aims to this work: 1) Elucidate a molecular rationale for the observed therapeutic index of statin-induced apoptosis in normal and tumour cells; 2) Identify molecular determinants of sensitivity for statin-induced apoptosis in human tumour cells. To address the first aim we demonstrated that dysregulation of the MVA pathway, achieved by ectopic expression of either full length HMGCR (HMGCR-FL) or its novel splice variant lacking exon 13 (HMGCR-D13), increases transformation. Ectopic HMGCR promotes growth of transformed and non-transformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice. We also show that high mRNA levels of HMGCR and four out of five other MVA pathway genes correlate with poor prognosis in primary breast cancer, suggesting the MVA pathway may play a role in the etiology of human cancers. To address the second aim, we show that dysregulation of the MVA pathway is a key determinant of sensitivity to statin-induced apoptosis in multiple myeloma. In a panel of 17 distinct myeloma cell lines, half were sensitive to statin-induced apoptosis and the remainder were insensitive. Interestingly, in sensitive cells, the classic feedback response to statin exposure is lost, a feature we demonstrated could distinguish a subset of statin-sensitive primary myeloma cells. We further illustrated that statins are highly effective and well tolerated in an orthotopic model of myeloma using cells harboring a dysregulated MVA pathway. Taken together, this work provides a molecular rationale and determinants of sensitivity for statin-induced apoptosis of human tumour cells.

Mycobacterium tuberculosis, the causative pathogen of tuberculosis, currently infects one-third of the world’s population, resulting in two million deaths annually. This clearly shows that tuberculosis is one of the most serious diseases of our times. The often unpleasant side effects from the current drugs, combined with the difficulty of ensuring patient compliance, and the emergence of drug-resistant and multidrug-resistant strains, makes the need for new and better drugs urgent. In this thesis, all the steps, from cloning, purification, crystallization, to activity determination, and structure determination are presented for two different M. tuberculosis enzymes. The structures, which were modeled from X-ray crystallographic data, provide the framework for structure-based drug design. Here, new potential inhibitors can be tailor-made based on the specific interactions in the enzyme’s active site. The bacteria have two different peptidyl-prolyl cis-trans isomerases that catalyze the isomerization of peptide bonds preceding proline residues, a process of high importance for correct folding. Here we present the structure of peptidyl-prolyl cis-trans isomerase A, an enzyme present inside the bacteria, and distinguish it from the B form of the enzyme, which is membrane bound, placing its active site outside the bacteria. The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step within the non-mevalonate pathway, which leads to the production of isopentenyl diphosphate. This compound is the precursor of various isoprenoids, vital to all living organisms. In humans, isopentenyl diphosphate is produced via a different pathway, indicating that all the enzymes within the non-mevalonate pathway may be suitable drug targets in M. tuberculosis. Several structures of both wild type and mutant 1-deoxy-D-xylulose-5-phosphate reductoisomerase in complex with different substrates, and also with the known inhibitor fosmidomycin, provide valuable information not only to the field of drug design, but also, in this case, into the catalysis.

Overall survival of the metastatic forms of epithelial derived cancers, especially head and neck squamous cell carcinomas (HNSCC), has not significantly improved even with the application of aggressive combined modality approaches incorporating radiation and chemotherapy. Cumulative evidence implicates the epidermal growth factor receptor (EGFR) as an important therapeutic target in HNSCC. We have previously demonstrated that the combination of lovastatin, a potent inhibitor of the mevalonate pathway, with EGFR tyrosine kinase inhibitors induced robust synergistic cytotoxicity. However, the use of high dose statins in our clinical trial was associated with significant toxicities including higher than anticipated rate of muscle pathologies. Our goal was to uncover novel downstream targets of the mevalonate pathway that may enhance the efficacy or limit toxicities of this novel combination therapeutic approach. In this study we have demonstrated that GGTI-298, an inhibitor of protein geranylgeranylation, through its ability to disrupt the actin cytoskeleton, inhibits EGFR dimerization and cellular trafficking. This novel mechanism targeting the EGFR has clinical implications as GGTI-298 in combination with tarceva, a clinically relevant EGFR inhibitor, showed enhanced cytotoxicity and inhibitory effects on EGFR activation and its downstream signaling.

The statin family of hydroxymethylglutaryl coenzyme A reductase (HMGCR) inhibitors, used to control hypercholesterolemia, triggers apoptosis of various human tumour cells. HMGCR is the rate-limiting enzyme of the mevalonate (MVA) pathway, a fundamental metabolic pathway required for the generation of a number of biochemical end-products including cholesterol and isoprenoids, but the contribution of the MVA pathway to human cancer remains largely unexplored. Furthermore, as only a subset of tumour cells has been shown to be highly responsive to statins, the identification of appropriate subsets of patients will be required to successfully advance these agents as anticancer therapeutics. To this end, there were two major aims to this work: 1) Elucidate a molecular rationale for the observed therapeutic index of statin-induced apoptosis in normal and tumour cells; 2) Identify molecular determinants of sensitivity for statin-induced apoptosis in human tumour cells. To address the first aim we demonstrated that dysregulation of the MVA pathway, achieved by ectopic expression of either full length HMGCR (HMGCR-FL) or its novel splice variant lacking exon 13 (HMGCR-D13), increases transformation. Ectopic HMGCR promotes growth of transformed and non-transformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice. We also show that high mRNA levels of HMGCR and four out of five other MVA pathway genes correlate with poor prognosis in primary breast cancer, suggesting the MVA pathway may play a role in the etiology of human cancers. To address the second aim, we show that dysregulation of the MVA pathway is a key determinant of sensitivity to statin-induced apoptosis in multiple myeloma. In a panel of 17 distinct myeloma cell lines, half were sensitive to statin-induced apoptosis and the remainder were insensitive. Interestingly, in sensitive cells, the classic feedback response to statin exposure is lost, a feature we demonstrated could distinguish a subset of statin-sensitive primary myeloma cells. We further illustrated that statins are highly effective and well tolerated in an orthotopic model of myeloma using cells harboring a dysregulated MVA pathway. Taken together, this work provides a molecular rationale and determinants of sensitivity for statin-induced apoptosis of human tumour cells.

This dissertation is dedicated to the research and investigation of novel enzymes and the methods used to study them, with physiological roles ranging from isoprenoid biosynthesis to neurotransmitter production. Using a combination of bioinformatics, recombinant cloning, enzymology, and proteomics, we have contributed to the understanding and exploration of several human illnesses, including malaria, cancer, and endocrine dysfunction. Our first project involved studying the enzymes responsible for N-acylarylalkylamide biosynthesis in Bombyx mori. Very little is known how these potent signaling molecules are produced in vivo, however, one possible pathway is the direct conjugation of an acyl-CoA to a corresponding arylalkylamide by the enzyme arylalkylamine N-acyltransferase (AANAT). In insects, this enzyme is responsible for controlling melanism, the inactivation of biogenic amines, the sclerotization of the insect cuticle, photoperiodism, and the penultimate intermediate in the production of melatonin. We studied a pair of AANAT enzymes from B. mori: Bm-AANAT and Bm-AANAT3. The former was found to catalyze the direct formation of long-chain acylarylalkylamines (only the second enzyme discovered to do such chemistry), while the latter exhibited potent inactivation of several amines through acetylation. We conducted a full kinetic characterization of Bm-AANAT3, including double-reciprocal plots, pH-rate profiling, dead-end inhibition, and the construction of mutants to elucidate catalytically-relevant amino acids. Our hope is that new insights and discoveries on these enzymatic pathways in model organisms will yield novel molecular targets for human health and disease. We then developed an innovative, microwave-assisted synthesis of a binding-based probe capable of enriching proteins that bind adenine ribose derivatives (AdoRs). We employed this probe in activity-based protein profiling studies to qualitatively assess the AdoR-binding proteome in three bacterial cell lines from the genus Bacillus. This proof of concept experiment demonstrated a unique subset of proteins distinctive to each species, and confirmed the efficacy of the probe tagging and subsequent enrichment. This technology can be used in clinical applications for the detection and identification of cancerous biomarkers. Finally, we successfully truncated and recombinantly-expressed 1-deoxy-D-xylulose-5-phosphate synthase (DXS) from both P. vivax and P. falciparum. We elucidated the order of substrate binding for both of these TPP-dependent enzymes using steady-state kinetic analyses, dead-end inhibition, and intrinsic tryptophan fluorescence titrations. Both enzymes adhere to a random sequential mechanism with respect to binding of both substrates: pyruvate and D-glyceraldehyde-3-phosphate. These findings are in contrast to other TPP-dependent enzymes, which exhibit classical ordered and/or ping-pong kinetic mechanisms. A better understanding of the kinetic mechanism for these two Plasmodial enzymes could aid in the development of novel DXS-specific inhibitors that might prove useful in treatment of malaria.

Overall survival of the metastatic forms of epithelial derived cancers, especially head and neck squamous cell carcinomas (HNSCC), has not significantly improved even with the application of aggressive combined modality approaches incorporating radiation and chemotherapy. Cumulative evidence implicates the epidermal growth factor receptor (EGFR) as an important therapeutic target in HNSCC. We have previously demonstrated that the combination of lovastatin, a potent inhibitor of the mevalonate pathway, with EGFR tyrosine kinase inhibitors induced robust synergistic cytotoxicity. However, the use of high dose statins in our clinical trial was associated with significant toxicities including higher than anticipated rate of muscle pathologies. Our goal was to uncover novel downstream targets of the mevalonate pathway that may enhance the efficacy or limit toxicities of this novel combination therapeutic approach. In this study we have demonstrated that GGTI-298, an inhibitor of protein geranylgeranylation, through its ability to disrupt the actin cytoskeleton, inhibits EGFR dimerization and cellular trafficking. This novel mechanism targeting the EGFR has clinical implications as GGTI-298 in combination with tarceva, a clinically relevant EGFR inhibitor, showed enhanced cytotoxicity and inhibitory effects on EGFR activation and its downstream signaling.

No description available.

The thesis describes over-expression, purification and crystallization of three proteins from Staphylococcus aureus (S. aureus). S. aureus is an important human pathogen and methicillin-resistant S. aureus (MRSA) is a serious problem in hospitals nowadays. The crystal structure of 3-Methyladenine DNA glycosylase I (TAG) was determined by single-wavelength anomalous diffraction (SAD) method. TAG is responsible for DNA repair and is an essential gene for both MRSA and methicilin-susceptible S. aureus (MSSA). The structure was also determined in complex with 3-methyladenine (3-MeA) and was solved using molecular replacement (MR) method. An assay was carried out and the molecular basis of discrimination between 3-MeA and adenosine was determined. The native crystal structure of fructose 1-phosphate kinase (PFK) from S. aureus was determined to 2.30 Å and solved using molecular replacement method. PFK is an essential enzyme involved in the central metabolism of MRSA. Despite extensive efforts no co-complex was determined, although crystals were obtained they diffracted poorly. An assay which can be used to test for inhibitors has been developed. Mevalonate Kinase (MK) is another essential enzyme in MRSA and is a key drug target in the mevalonate pathway. Native data diffracting to 2.2 Å was collected. The structure was solved using multiple isomorphorus replacement (MIR) method. A citrate molecule was bound at the MK active site, arising from the crystallization condition. The citrate molecule indicates how substrate might bind. The protein was kinetically characterized. A thermodynamic analysis using fluorescence-based method was carried out on each protein to investigate binding interactions of potential fragments and thus a drug design starting point.

Breast cancer is the second leading cause of death due to cancer in women. A number of effective therapeutic strategies have been implemented in clinics to cope with the disease yet recurrent disease and toxicity reduce their effectiveness. Hence, there is a need to identify and develop more effective therapies with reduced toxic side effects to improve overall survival rates. This dissertation investigates the mechanisms of action of two natural forms of vitamin E and a cholesterol lowering drug, simvastatin, as a therapeutic strategy in human breast cancer cells. Vitamin E in nature consists of eight distinct forms which are fat soluble small lipids. Until recently, vitamin E was known as a potent antioxidant but emerging work suggests they may be resourceful agents in managing a number of chronic diseases including cancer. Anticancer properties of vitamin E have been identified to be limited to the γ- and δ- forms of both tocopherols and tocotrienols. Gamma-tocopherol ([gamma]T) and gamma-tocotrienol ([gamma]T3) have both already been identified to induce death receptor 5 (DR5) mediated apoptosis in breast cancer cells. Studies here show that similar to [gamma]T3, [gamma]T induced DR5 activation is mediated by c-Jun N-terminal kinase/C/EBP homologous protein (JNK/CHOP) proapoptotic axis which in part contributed to [gamma]T mediated dowregulation of c-FLIP, Bcl-2 and Survivin. Also, both agents activate de novo ceramide synthesis pathway which induces JNK/CHOP/DR5 proapoptotic axis and downregulates antiapoptotic factors FLICE inhibitory protein (c-FLIP), B-cell lymphoma 2 (Bcl-2) and Survivin leading to apoptosis. Simvastatin (SVA) has been identified to display pleiotropic effects including anticancer effects but mechanisms responsible for these actions have yet to be fully understood. In this dissertation, it was observed that simvastatin induced apoptosis in human breast cancer cells via activation of JNK/CHOP/DR5 proapoptotic axis and down regulation of antiapoptotic factors c-FLIP and Survivin which are in part dependent on JNK/CHOP/DR5 axis. The anticancer effects mediated by simvastatin can be reversed by exogenously added mevalonate and geranylgeranyl pyrophosphate (GGPP), implicating the blockage of mevalonate as a key event. Furthermore, work has been done to understand the factors responsible for drug resistance and identify therapeutic strategies to counteract the same. It was observed that development of drug resistance was associated with an increase in the percentage of tumor initiating cells (TICs) in both tamoxifen and Adriamycin resistant cells compared to their parental counterparts which was accompanied by an increase in phosphorylated form of Signal transducer and activator of transcription 3 (Stat3) proteins as well as its downstream mediators c-Myc, cyclin D1, Bcl-xL and Survivin. Inhibition of Stat3 demonstrated that Stat3 and its downstream mediators play an important role in regulation of TICs in drug resistant breast cancer. Moreover, SVA, [gamma]T3 and combination of SVA+[gamma]T3 has been observed to target TICs in drug resistant human breast cancer cells and downregulate Stat3 as well as its downstream mediators making it an attractive agent to overcome drug resistance. From the data presented here, the mechanisms responsible for the anticancer actions of [gamma]T, [gamma]T3 and SVA have been better understood, providing the necessary rationale to test these agents by themselves or in combination in pre-clinical models. / text