Infection by the hepatitis C virus (HCV) leads to inflammation of the liver, i.e. hepatitis. The acute infection often progresses to a chronic phase during which the liver function is gradually impaired. Approximately 20% of these chronic cases develop liver cirrhosis, with an ensuing increased risk of liver cancer. Global estimates of the total number of chronic cases range from 123–170 million. Yet, neither specific anti-HCV drugs nor vaccines are available. When drugs become available for daily clinical use, rapid development of drug-resistant strains is expected, making resistance an important issue. One of the most studied targets for specific anti-HCV drugs is the NS3 protease. The main objectives of the work presented in this thesis were to design and synthesise peptidomimetic inhibitors of this enzyme, and to establish the structure–activity relationships (SARs) regarding the inhibition of the wild type as well as of the known resistant variants A156T and D168V. Substituted prolines are common P2 residues in HCV NS3 protease inhibitors. To decrease the peptide character of the inhibitors, the non-coded phenylglycine was evaluated as a proline replacement in combination with known and novel P3 and P1 residues and P2 substituents. The results confirmed that phenylglycine is a promising P2 scaffold, with a possible π-stacking interaction with histidine 57 of the active site. However, to benefit from its full potential, additional optimisation is required. A 2(1H)-pyrazinone-based scaffold was introduced as P3 residue. Utilising the scope of the method developed for the pyrazinone scaffold synthesis, the phenylglycine side-chain was transferred to the scaffold. In combination with an aromatic P1 building-block, this design yielded achiral, peptidomimetic inhibitors, three times more potent than the tripeptide lead. The SARs for the inhibition of the resistant variants A156T and D168V were investigated for compounds based on either P2 proline or phenyl­glycine. It was concluded that the vulnerability of the inhibitors to alterations in the enzyme depends on the P2 and the P1 residue, not only on the P2 as previously suggested. These results provide important information for the design of a new generation of inhibitors with improved properties.

HIV-1 protease (PR) inhibitors (PIs) are important anti-HIV drugs for the treatment of AIDS and have shown great success in reducing mortality and prolonging the life of HIV-infected individuals. However, the rapid development of drug resistance is one of the major factors causing the reduced effectiveness of PIs. Consequently, various drug resistant mutants of HIV-1 PR have been extensively studied to gain insight into the mechanisms of drug resistance. In this study, the crystal structures, dimer stabilities, and kinetics data have been analyzed for wild type PR and over 10 resistant mutants including PRL24I, PRI32V, PRM46L, PRG48V, PRI50V, PRF53L, PRI54V, PRI54M, PRG73S and PRL90M. These mutations lie in varied structural regions of PR: adjacent to the active site, in the inhibitor binding site, the flap or at protein surface. The enzymatic activity and inhibition were altered in mutant PR to various degrees. Crystal structures of the mutants complexed with a substrate analog inhibitor or drugs indinavir, saquinavir and darunavir were determined at resolutions of 0.84 – 1.50 Å. Each mutant revealed distinct structural changes, which are usually located at the mutated residue, the flap and inhibitor binding sites. Moreover, darunavir was shown to bind to PR at a new site on the flap surface in PRI32V and PRM46L. The existence of this additional inhibitor binding site may explain the high effectiveness of darunavir on drug resistant mutants. Moreover, the unliganded structure PRF53L had a wider separation at the tips of the flaps than in unliganded wild type PR. The absence of flap interactions in PRF53L suggests a novel mechanism for drug resistance. Therefore, this study enhanced our understanding of the role of individual residues in the development of drug resistance and the structural basis of drug resistance mechanisms. Atomic resolution crystal structures are valuable for the design of more potent protease inhibitors to overcome the drug resistance problem.

Cellular differentiation is a complex but fascinating process in all multicellular organisms. Differentiation can involve changes in numerous aspects of cell physiology; size, shape, polarity, metabolic activity, responsiveness to signals, and changes in gene expression profiles. These changes form the basis for differentiation to occur. The human hemochorial placenta is an intricate apposition of fetal and maternal tissues that is strategically juxtaposed at the interface, with its widespread ‘villous’ or tree-like projections, directly in contact with maternal blood. It is therefore, ideally suited to perform life-sustaining functions such as exchange of nutrients, respiratory gases and metabolic wastes, with the maternal supply. It also plays a central role in the maintenance of the immunologically privileged status of the fetal semi-allograft. Placental development is directed towards the establishment of a continuous nutrient supply to the developing fetus. This requires efficient access of maternal blood to a transporting surface, the multinucleate syncytiotrophoblast layer. This is made possible by the rapid proliferation and ensuing invasion of mononuclear trophoblasts into the maternal uterus and remodeling of the spiral arteries therein. It is interesting to note that in early pregnancy, it is the placenta that first engages its active growth and proliferation and only then, permits the logarithmic growth phase of the embryo. As a developing organ, the placenta undergoes constant tissue remodeling, which is characterized by the functional loss of trophoblast cells by apoptosis. Most of these changes occur at the trophoblast layer of the placental villous that is composed of two cell types: cytotrophoblasts (CT) and syncytiotrophoblasts (ST). The mononuclear cytotrophoblast cells, which are located between the syncytiotrophoblast layer and its basement membrane, proliferate and fuse during trophoblast differentiation to form the overlying multinucleated syncytium. CT are highly proliferating and invasive cells, in contrast to the ST which are non proliferative less invasive and functionally very active. Syncytiotrophoblast cells form the continuous, uninterrupted, multinucleated, epithelium-like surface of the placental villous that separates maternal blood from the villous interior. ST performs a crucial role in feto-maternal exchanges and serves as an endocrine tissue by its ability to synthesize and secrete a variety of hormones such as GnRH, chorionic gonadotrophin (CG), placental lactogen (PL) and steroid hormones involved in the homeostasis during pregnancy. Thus, differentiation of CT into ST serves as an ideal model to study cellular differentiation as morphologically and functionally these cells exhibit highly contrasting features. The molecular basis of cytotrophoblast differentiation has been studied using primary cultures of human trophoblast cells as a model system. Highly purified preparations of mononucleated cytotrophoblast cells can be isolated from preterm and term placental tissue by enzymatic dispersion. The isolated cells from term placental tissue aggregate spontaneously in culture and fuse to form a multinucleated syncytiotrophoblast which synthesizes and secretes placental lactogen (hPL), chorionic gonadotropin (hCG) and other syncytiotrophoblast-specific protein and steroid hormones . These in vitro changes, which recapitulate important activities accomplished by normal cytotrophoblast cells during in vivo maturation, implicate a critical relationship between the differentiation of cytotrophoblast cells into syncytiotrophoblast cells. Though primary cell culture is an ideal model to study these changes, it comes inherently associated with various problems like health risk of handling human tissues, time involved, variability in each placental samples depending on health status of the subject and quite often lack of history of the subject which makes the results from these experiments difficult to reproduce and assess. One way to overcome this is the cell culture model which is a reproducible experimental system and permits the direct observation of time-dependent processes and their experimental manipulation. BeWo cells, the cells which we have used in our study, were derived from human gestational choriocarcinoma. These cells are the highly invasive malignant counterparts of the normal human trophoblast wherein, the limited capacity for cell proliferation is far exceeded. However, they still retain important features of their normal counterpart, like the potential of hormone production and induced differentiation. Differentiation of CT to ST is precisely controlled by different agents such as transcription factors, hormones, growth factors, cytokines and oxygen levels. BeWo cells have been used by other investigators as well as by us and it has been shown that these cells can be induced to differentiate with the agents mentioned above and terminally differentiate into cells which express typical characteristics of the normal differentiating trophoblast; like morphological transition from cytotrophoblast to syncytiotrophoblast-like cells, increased production of protein and steroid hormones (hCG, hPL, estrogens, progesterone); increased activity of cellular alkaline phosphatase and arrested cell proliferation. Since these cells can be triggered by external agents to differentiate, they serve as a useful model for the study of changes that occur during differentiation. Using primary cells and various cell lines including BeWo cells, various groups have attempted to study trophoblast differentiation and the regulators that control this process. The results of such study have only come out with a list of genes or proteins which might be having a role in this process and no functional correlation has been drawn so far from these studies. The members of the syncytin protein family, ADAM (a disintegrin and metalloprotease) proteins may well be some of the main players in the process of trophoblast fusion; some of the requisites of trophoblast fusion being redistribution of phosphatidylserine to the outer leaflet of the plasma membrane and activity of certain intracellular proteases. Clearly, further studies on trophoblast differentiation are needed to answer the question of the precise identity of regulatory proteins and role of these proteins during differentiation. The present study is aimed at gaining insights into the process of trophoblast differentiation and the molecular events which occur during this process. Our aim is also to study the regulated process of differentiation using BeWo cell model and identify the differentially expressed genes and relate the known function of these gene products to changes seen during differentiation process. We have employed the Differential Display Reverse Transcriptase Polymerase Chain Reaction (DD-RTPCR) and Microarray analysis to monitor the changes in gene expression. In CHAPTER 1, a brief account of morphological, biochemical and physiological changes which occur during placentation and trophoblast differentiation is discussed. Various aspects of placental function are discussed in brief, with special reference to the many unique abilities of trophoblast cells that contribute to a successful pregnancy. Detailed accounts of molecular mechanism of cellular differentiation, the models used in these studies and the advantages and drawbacks have been highlighted. The results of the previous studies from our laboratory using different model system and the outcome of the study are also outlined in this chapter. The advantages and disadvantages of the primary cell lines and the ease of handling of continuous cell culture model, BeWo is also presented in this chapter. The aim and objective of our study is to understand the molecular mechanisms underlying the trophoblast differentiation and the literature available is reviewed in the light of the objective and the aims and scope of the present study. The details regarding the materials used and the techniques employed during the entire study are outlined in CHAPTER 2-‘Materials and Methods’. The conditions for culture of BeWo human choriocarcinoma cell line are described and details of procedures employed for the validation of BeWo cells as a model system for monitoring the process of cellular differentiation are mentioned in this chapter. The details of the procedures employed for isolation of RNA, Reverse Transcriptase Polymerase Chain Reaction (RTPCR), Differential Display RT-PCR (DD-RT-PCR), Microarray analysis, Northern Blot analysis and Western Blot analysis are also described. The principle of the MTT assay used for verifying the viability of cells following various treatments is provided along with the working protocol. This chapter also includes protocols of the in vivo studies in rat, the methods employed for rat uterine mince cultures and isolation of rat uterine epithelial cells and dose and duration of the various treatments with steroid hormones and their inhibitors, treatment with protein kinase inhibitors in cell culture system are also described. In addition, this chapter also describes the procedures for transfection of hTERT, silencing of SLPI gene using SiRNA approach, gelatin zymography, MAP Kinase assay, FACS, cloning and expression of SLPI protein and procedure employed for raising antibodies to SLPI in rabbit. Finally, details of statistical tests employed fro anlaysis of data are presented. The results obtained in the present study are presented in 4 chapters(Chapters 3-6), CHAPTER 3 describes the characterization and validation of model system employed- BeWo cells to study human trophoblastic differentiation. BeWo cells under normal culture conditions resemble cytotrophoblasts like cells and when treated with various effectors of differentiation can be induced ot differentiate into syncytiotrophoblasts. We used 10 µM Forskolin to induce differentiation in BeWo cells. Forskollin is known to induce characteristic changes associated with human trophoblast differentiation in these cells. Incubation of BeWo cultures in the presence of 10 µM Forskolin resulted in dramatic morphological biochemical changes intheir cytotrophoblast-like phenotype. Mononuclear cells were seen to fuse to form multinucleate syncytial structures over a period of 72-96 hours in culture. This process was also associated with an increased production of β-hCG, Endoglin and hTERT thereby validating this model system for study of human trophoblastic differentiation. Analysis of cell cycle genes in this system established the arrest of proliferation thus further validating the system. The viability of these cells, during the entire period of culture, was verified using the MTT assay. This chapter discusses the importance of in vitro cell culture systems in the study of human placental development, and also addresses the suitability of these model systems for the study of human trophoblast proliferation and differentiation. One of the important finding of our earlier studies was that arrest of proliferation was a prerequisite for trophoblast differentiation to occur. This conclusion was based on the fact that telomerase expression which is a hallmark of all proliferating cells was down regulated in BeWo cells by 48h as assessed by TRAP (Telomere Repeat Amplification Protocol) assay or RT-PCR analysis for hTERT which is the catalytic subunit of telomerase. Telomerase activity was undetectable by about 96th by which time syncytium formation is normally completed after the addition of differentiation inducing agents like Forskolin, TGF β etc. Although the telomeric holo enzyme consists of many components the subunits which are critical for enzyme action are hTERT and hTR; hTR; hTR which is the RNA component of telomerase is ubiquitously expressed in most cell types including telomerase negative cells such as differentiated somatic cells. Since the BeWo cells can be induced to differentiate into multinucleated ST by addition of Forskolin and periodically the aged ST are eliminated by apoptosis. It is very well documented that the life span of ST is very limited and the ST have to be replaced by the freshly formed ST out of fusion of CT. Considering this, it was of interest to test whether differentiation can be prevented or delayed by extending the expression of telomerase activity. This would further validate our system that one of the requisites for cells to differentiate is down regulation of hTERT in BeWo cells. This was achieved by transfection of BeWo cells with hTERT expression vector. The results of the study clearly established that we were able to over express hTERT in BeWo cells; we also noticed an increase in the proliferation of BeWo cells as assessed by BrdU incorporation. In agreement with this observation is the fact that, in contrast to the empty vector transfected cells, in hTERT transfected group, the cell density appeared to be clearly more at 72 h. That the decrease in the hTERT expression in the control (empty vector transfected) is not due to cell death was established by MTT assay, which indicated that there was no difference in the viability between control and hTERT transfected cells. Further more, results of analysis for a variety of cell proliferation and differentiation markers by RT-PCR and Western blot analysis clearly supports the conclusion that hTERT over expression delays syncytium formation. Although reports are available on the differential expression of genes during differentiation of CT to ST with both primary cell lines as well as BeWo cell line, relatively less is known about the functional importance of differentially expressed genes. In CHAPTER 4, results of our studies to profile the differentially expressed genes during Forskolin induced differentiation in BeWo cells by two approaches DD-RTPCR and microarray analysis and relate the known functions of these genes to changes that occur during the differentiation of CT to ST are presented. We identified several genes that had robust change during differentiation by DD RTPCR and the differential expression of ten transcripts was confirmed by Northern blot analysis. The genes which we identified were SLPI, Elongation factor-1 alpha -1, Prolyl hydroxylase beta, LIMO-4 etc. These genes were either shown to have a role during differentiation of cells or have functional role in the syncytiotrophoblasts. Secretory Leucocyte Protease Inhibitor was one of the differentially expressed transcripts which were significantly up regulated during Forskolin induced differentiation of BeWo cells. SLPI which is a 12 KDa protein reported to exhibit a variety of activities which include inhibition of proteases and elastase, in addition to antibacterial and anti inflammatory activities. It was chosen for our further studies because of its multifunctional role in placenta and also during implantation. Micro array analysis revealed the up-regulation of hCG, hCS, and Endoglin thus validating the experimental system. Several candidate genes that could influence trophoblast differentiation, cell adhesion and cellular proliferation were identified. Genes involved in cellular proliferation include cyclin M3, replication factor 3, signal-induced proliferation-associated gene 1, osteonectin, clusterin, etc clearly indicating a growth-arrested phenotype for the differentiating BeWo cells. Trophoblastic differentiation associated genes included adipose differentiation-related protein, GADD45A, PPAR binding protein, galectin 3, tubulins, collagen, stathmin, etc. The p53 tumor suppressor protein plays a major role in cellular response to DNA damage and other genomic aberrations. Activation of p53 can lead to either cell cycle arrest or DNA repair or apoptosis. Although we did not observe any change in the p53 mRNA levels, the total protein level as well the phosphorylation status of p53 was up regulated upon differentiation. We confirmed the down regulation of Cyclin D1, D2 and PCNA in differentiated cells and up regulation of CDK inhibitors, P27kip1, P21cip1which are p53 induced genes by RT-PCR and Western blot analysis. Phosphorylation of ser 20 leads to reduced interaction of p53 with its negative regulator MDM2. MDM2 inhibits the accumulation of p53 by targeting it for ubiquitination and proteosomal degradation. Analysis for the phosphorylated status of p53 revealed that specifically the ser 20 phosphorylated p53, was increased upon differentiation. Phosphorylation of ser-392 has been reported to influence the growth repressor function, DNA binding and transcriptional activation of p53 and in agreement with this, western blot analysis revealed an increase in the ser-392 phosphorylated p53. These results suggest that p53, a nuclear protein regulating several genes involved in proliferation and differentiation is playing a pivotal role in growth arrest during trophoblast differentiation. We also noticed that several components of the apoptotic cascade are differentially expressed in cytotrophoblast and the syncytiotrophoblasts layer, and these changes appear to be associated with the stage of apoptosis. Apoptosis is involved in the removal of aging syncytiotrophoblasts and it also promotes cytotrophoblast fusion and formation of the syncytial layer. We found that various apoptosis related genes are up regulated and anti apoptotic genes suppressed following differentiation in our micro array analysis. We identified the involvement of p53 in this process also and chapter 4 deals with this aspect. Genes which regulate the invasive behaviour of trophoblasts which include MMP2, cathepsin K, cystatin N, SLPI and cysterine-rich angiogenic inducer 61, etc. were found to be up regulated following differentiation in our micro array analysis, which establishes these differences in gene expression reflects the physiological changes that occur during placentation. The Co-ordinated regulation of ptoteases and protease inhibitors I (for example SLPI, cystatin B and MMP2) suggests that these genes play an important role in the regulation trophoblast invasion at the uterine-placenta interface in vivo. Our studies revealed that one of the transcripts,namely, SLPI(Secretory leukocyte protease inhibitor) was robustly up regulated as assessed in DDRT-PCR, micro-array, Northern blot and RT-PCR analysis. Considering its importance in implantation, placentation and maintenance of pregnancy several aspects of this multifunctional protein were studied in detail and the results are presented in CHAPTER 5. Studies on the regulation of this transcript in Be-Wo cells revealed that SLPI mRNA is regulated by progesterone in Be-Wo cells. The up regulation of SLPI mRNA by progesterone was specifically inhibited by Progesterone receptor antagonist, RU 486 and estradiol 17β did not have any effect on the expression of SLPI mRNA expression in BeWo cells. The absence of regulation of SLPI by estradiol in BeWo cells was also established by the fact that simultaneous addition of progesterone and aromatase inhibitor, fadrazole did not block the increase in SLPI expression. Interestingly in vivo and in vitro studies using rat uterine minces and rat epithelial cells revealed that SLPI mRNA is regulated by Estradiol 17β and the effect is specifically inhibited by estrogen receptor antagonists such as ICI 182780, Tamoxifen, and Centchorman. Promoter analysis of rat and human SLPI revealed the absence of a consensus progesterone responsive element (PRE) in human and estrogen responsive element (ERE) in rat, suggesting the possibility of a non-genomic action of progesterone or estrogen in the induction of SLPI mRNA. This was confirmed by the observation that induction of SLPI mRNA could be effectively blocked by the addition of Staurosporine, an inhibitor of protein kinase C along with progesterone and estrogen to either BeWo cells or rat uterine epithelial cells. These results suggest that the non-genomic action of steroid hormones may be involved in the induction of SLPI. In the present study, we have also identified the intracellular signaling pathway that regulates SLPI gene expression by using various protein kinase inhibitors. We have also shown that activation of MAP kinase pathway upon progesterone treatment and the involvement of protein kinases in this activation, permitting us to conclude the non genomic action of progesterone in induction of SLPI mRNA in BeWo cells. The results of these studies are presented in detail in Chapter 5. The observation that SLPI expression is markedly increased during differentiation and differentially regulated by progesterone and estradiol, and induction by non genomic pathway prompted us to undertake studies to investigate its role during differentiation. This was accomplished by using SiRNA to silence the expression of SLPI in Forskolin induced differentiating BeWo cells and the results of this study are presented in CHAPTER 6. Different concentrations and combinations of oligos were used to silence the SLPI gene and we found that effective knockdown (>80%) was achieved with SiRNA concentrations ranging from 5-25nM. A combination of oligos also increased the knockdown from 50% to 90% as assessed by RT-PCR and western blot analysis for mRNA and protein levels of SLPI respectively. We found that inhibition of SLPI expression by SiRNA also inhibited the morphological differentiation of BeWo cells. Functionally this was reflected, by increase in the protease activity as assessed by gelatin zymography. It should be noted that SLPI is a protease inhibitor; it inhibits a variety of proteases, including proteases from neutrophils, pancreatic acinar cells and mast cells and SLPI present in the syncytiotrophoblast may have a crucial role in controlling protease activity associated with invasiveness and differentiation. Inhibition of differentiation by silencing the expression of SLPI provides an opportunity to monitor the changes in gene expression where in a single gene has been silenced in contrast to the model employed in chapter 4. We carried out microarray analysis using control (Forskolin treated) and SLPI silenced (Forskolin treated) samples. The results revealed that proliferation and differentiation, apoptosis and inflammatory pathways genes are affected due to SLPI silencing and the results of this study are presented in CHAPTER 7. We confirmed the changes in gene expression by semi quantitative RT-PCR analysis of the some important genes in each pathway. A comparison of the results obtained with that of our earlier microarray analysis which is described in chapter 4 revealed that the changes in levels of expression of the genes involved in cell proliferation, differentiation, apoptosis and inflammation were completely reversed after silencing the expression of SLPI. We have presented in chapter 5 the importance of MAP kinase pathway in Forskolin induced differentiation and the activation of this pathway when SLPI expression is increased following progesterone treatment. Interestingly after silencing the expression of SLPI we found that MAP kinase pathway is affected. It was observed that silencing of SLPI expression resulted in inhibition of activation of MAP kinase as assessed by the phosphorylation status by ELISA and no activation of MAP kinase was observed in SLPI silenced Forskolin treated cells. CHAPTER 8 provides a general discussion of the results obtained in the present study in the light of current understanding the type of genes involved, changes during human trophoblastic proliferation and differentiation and the key players during this process. This chapter also brings out the importance of SLPI during trophoblastic differentiation, placentation, implantation and its regulation by steroid hormones. The highlights and salient features of the present study are summarized in this chapter. In CONCLUSION, the present investigation has led to the identification of specific genes involved in trophoblast differentiation, human placental growth and development. Also evident from this study is the usefulness of the trophoblastic cell culture system for the study of cellular differentiation. We have attempted to relate the gene expression changes to physiological changes that occur during placentation, implantation and pregnancy. Many of the regulatory events that we have described during human trophoblastic differentiation, may not only be restricted to these cells, but may represent common principles/features of cellular differentiation in general. Loss of differentiation is a wide-spread feature of tumor progression, and frequently accompanies aggressive neoplastic behavior. Our studies provide unequivocal evidence to support cellular differentiation as a natural barrier to malignant transformation. Most importantly we have shown that silencing of a single gene can disrupt this differentiation process and the importance of SLPI during differentiation process perse.

Submitted by Raniere Barreto (raniere.barros@ufvjm.edu.br) on 2018-04-12T16:59:34Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) lara_franca_colares.pdf: 2530593 bytes, checksum: f6b37bbb6a95d44536fc10c983a32afa (MD5) / Rejected by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br), reason: Verificar: "Ara?jo e Silva" e alterar, deixando Silva. Inserir as keywords adicionado campos devidos. UFVJM n?o ? ag?ncia financiadora. on 2018-04-20T14:32:10Z (GMT) / Submitted by Raniere Barreto (raniere.barros@ufvjm.edu.br) on 2018-05-15T17:49:49Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) lara_franca_colares.pdf: 2530593 bytes, checksum: f6b37bbb6a95d44536fc10c983a32afa (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2018-05-16T11:27:14Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) lara_franca_colares.pdf: 2530593 bytes, checksum: f6b37bbb6a95d44536fc10c983a32afa (MD5) / Made available in DSpace on 2018-05-16T11:27:14Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) lara_franca_colares.pdf: 2530593 bytes, checksum: f6b37bbb6a95d44536fc10c983a32afa (MD5) Previous issue date: 2016 / Plantas medicinais s?o comumente usadas por comunidades tradicionais, principalmente em regi?es com menor desenvolvimento humano. Algumas esp?cies vegetais possuem entre seus componentes subst?ncias denominadas inibidores de proteases. Os inibidores de proteases se destacam na prote??o de fluidos e tecidos contra sua degrada??o por prote?lise e poss?veis falhas na degrada??o de prote?nas de meia-vida que podem interferir de forma dr?stica nas fun??es celulares. Diante do exposto, esse estudo objetivou identificar e caracterizar inibidores de proteases em cinco esp?cies vegetais nativas do Cerrado e da Mata Atl?ntica. As esp?cies de Punica granatum L. (Rom?), Plantago major L. (Tansagem), Ocimum gratissimum L. (Alfavaca), Anadenanthera colubrina Vellozo (Angico) e Stryphnodendron adstringens Mart. Coville (Barbatim?o) foram selecionados nas cidades de Pot?, Ladainha, Atal?ia, Te?filo Otoni e Ara?ua?, devido ao seu uso tradicional como anti-inflamat?rio. A sequencia gen?mica de inibidores de proteases foi pesquisada para essas esp?cies vegetais no GenBank, mas nenhuma sequencia foi descrita para as esp?cies selecionadas. As amostras provenientes dos procedimentos de extra??o foram submetidas ?s quantifica??o de prote?nas e a presen?a de inibidores de proteases foi detectada por eletroforese em gel de poliacrilamida 12% SDS-PAGE. Somente os extratos das sementes de Punica granatum e das folhas do Anadenanthera colubrina tiveram detec??o satisfat?ria de inibidores de proteases e foram submetidos ? an?lise por cromatografia l?quida de alta efici?ncia em sistema de HPLC. Este trabalho demonstra pela primeira vez a detec??o e extra??o de inibidores de proteases em folhas de Anadenanthera colubrina e sementes de Punica granatum. / Disserta??o (Mestrado Profissional) ? Programa de P?s-Gradua??o em Tecnologia, Sa?de e Sociedade, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / Traditional communities, especially in regions with lower human development, commonly use medicinal plants. Some plant species have among their components substances called protease inhibitors. Protease inhibitors act protecting fluids and tissues from degradation by proteolysis and possible failures in the degradation of half-life proteins that can drastically interfere with cellular functions. This study aimed to identify and characterize protease inhibitors in five native plant species of Cerrado and Atlantic Forest. The species of Punica granatum L. (Rom?), Plantago major L. (Tansagem), Ocimum gratissimum L. (Alfavaca), Anadenanthera colubrina Vellozo (Angico) and Stryphnodendron adstringens Mart. Coville (Barbatim?o) were selected in the cities of Pot?, Ladainha, Atal?ia, Te?filo Otoni and Ara?ua? due to their traditional use. The genomic sequence of protease inhibitors was screened for these plant species in GenBank, but no sequence was described for the selected species. Samples from the extraction procedures were subjected to protein quantification and the presence of protease inhibitors was detected by 12% SDS-PAGE polyacrylamide gel electrophoresis. Only the extracts of the seeds of Punica granatum and of the leaves of Anadenanthera colubrina had satisfactory detection of proteases inhibitors and were submitted to the analysis by high performance liquid chromatography system. This work demonstrates for the first time the detection and extraction of protease inhibitors in leaves of Anadenanthera colubrina and seeds of Punica granatum.

Orientador: Prof. Dr. Sergio Daishi Sasaki / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2017. / O Feijão Caupi (Vigna unguiculata (L.) Walp) é uma leguminosa com importante representatividade econômica e nutricional, especialmente no Brasil. Inibidores de serino proteases, como a tripsina, já foram descritos na espécie, assim como em outras plantas. No entanto, nesta espécie, ainda não foram identificados inibidores que apresentem atividade sobre a elastase de neutrófilos humana (HNE), protease envolvida em muitos processos patológicos, como na instalação e progressão da doença pulmonar obstrutiva crônica (DPOC). Nesse estudo, purificamos um inibidor a partir do extrato protéico de Vigna unguiculata que apresenta atividade sobre HNE. Inicialmente, foi realizado o processo de extração alcalina de proteínas, seguido de três passos cromatográficos distintos, utilizando as colunas Hitrap-Q (Troca-iônica), Source15RPC (Fase-Reversa) e ACE18 (Fase-Reversa). Essas etapas foram acompanhadas por testes de atividade inibitória, utilizando os substratos fluorogênicos Meo-Suc-Ala-Ala-Pro-Val-MCA (Elastase) e Z-Phe-Arg-MCA (Tripsina), além de ensaios da quantificação de concentração total de proteínas. Para determinar a massa do inibidor, foram utilizadas as técnicas de espectrometria de massa por MALDI-TOF e SDS-PAGE, o inibidor apresenta massa molecular de 10,99 KDa. O Ki para HNE foi determinado no valor de 9 pM. O inibidor não apresentou atividade inibitória sobre tripsina e trombina, porém foi observada atividade sobre subtilisina e quimotripsina. Estes dados indicam que o inibidor purificado trata-se de uma molécula ainda não caracterizada, devido às suas atividades inibitórias o nomeamos de Vigna unguiculata Elastase Inhibitor (VuEI). / The cowpea (Vigna unguiculata (L.) Walp) is a legume of important economic and nutritional representativeness, especially in Brazil. Serine protease inhibitors, such as trypsin, have been described in many species, as well as in other plants. In this specie an inhibitor with activity on human neutrophil elastase (HNE) has not yet been identified. This protease is involved in many pathological processes, such as the onset and progression of chronic obstructive pulmonary disease (COPD). We purified and characterized an inhibitor from the protein extract of Vigna unguiculata presenting activity towards HNE. Firstly, we performed the alkaline extraction procedure for proteins followed by three different chromatographic steps using Hitrap Q (ion exchange), Source15RPC (Reversed-Phase) and ACE18 (Reversed Phase) columns. These steps were followed by the inhibitory activity tests using fluorogenic substrates, MeO-Suc-Ala-Ala-Pro-Val-MCA (elastase) and Z-Phe-Arg-MCA (trypsin), and quantitation assays of protein concentration. To determinate the size of the molecule, we used MALDI-TOF mass spectrometry and SDS-PAGE. The molecular mass of the inhibitor was 10,99 kDa. The dissociation constant (Ki) toward HNE was 9 pM. HNE inhibitor showed no inhibitory activities toward trypsin and thrombin. However, the inhibitor presented activity toward subtilisin and chymotrypsin. These datas indicate that this molecule is a novel inhibitor to HNE and we named it Vigna unguiculata Elastase Inhibitor (VuEI).

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第23106号 / 医博第4733号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 松田 道行, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

CELLULAR MECHANISMS BY WHICH ALCOHOL PROMOTES HIV PROTEASE INHIBITOR-INDUCED HEPATOTOXICITY Michael Hinton, B.S. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2019 Major Director: Huiping Zhou Professor, Department of Microbiology and Immunology The development of highly-active-antiretroviral therapy(HAART) has allowed management of HIV and extended the lives of those infected. Alcohol abuse, which is very common in HIV-1 infected patients, is one of the most important co-morbid risk factors for liver injury and has been associated with the occurrence of serious metabolic syndrome and subsequent discontinuation of HAART in HIV patients. We have identified endoplasmic reticulum (ER) stress-induced proapoptotic factor CCAAT-element-binding protein homologous protein (CHOP) as an important mechanism underlying HIV PI-induced inflammation and hepatic lipotoxicity. However, little is known about the mechanistic pathways by which alcohol promotes HIV PI-induced hepatic lipotoxicity. The aim of this study was to determine if inhibition of CHOP expression prevents alcohol- and HIV PI-induced apoptosis and dysregulation of lipid metabolism. We demonstrated that co-administration of alcohol and HIV PIs induced unfolded protein response (UPR) activation, ER stress, and CHOP upregulation in rodent hepatocytes. Both alcohol and HIV PI-induced lipid accumulation and apoptosis were significantly reduced in CHOP-/- hepatocytes. Also, CHOP-/- hepatocytes treated with alcohol and HIV PIs showed inflammation.. Activation of the ER stress-induced proapoptotic factor CHOP is a key cellular mechanism underlying alcohol and HIV PI-induced hepatotoxicity. CHOP expression is key for alcohol and HIV PI-induced dysregulation of key genes involved in lipid metabolism in hepatocytes. Limitations of the study include the usage of global CHOP-/- in lieu of tissue-specific conditional knockout mouse models, nonobservance of the effects of alcohol and HIV PIs on extra-hepatic tissues, and incomplete investigation of the interplay of hepatocytes and resident macrophages.

Many diseases display a multitude of relevant factors that contribute to the persistence of the disease and difficulty treating it. The multifactorial characteristics of some diseases lead to the requirement of combination of treatments in order to restore health. The latter may necessitate the mixing of treatments, medications, and therapeutics to first halt the disease, then assist the human body in returning itself to a state of normality. For example, chronic wounds exhibit this multifactor characteristic in which there exist many factors that lead to the body’s inability to properly heal in a timely manner. This presents a further threat to the body, such as exposure to infection and long-term pain. In this example, it is important to look at the ultimate cause of a chronic wound, which may be due to presence of other diseases impairing the body’s ability to properly heal. This may include diabetes, initial antibiotic-resistant infection, autoimmune disorders, and poor vasculature. Furthermore, the mentioned causes for chronic wounds may have associations with one another in a single case of a chronic wound. Treating each interrelated cause with drug combinations may run the risk of adverse side effects or further complications due to mixing drugs in a systemic method. The goal of this study is to develop a point-specific, protein-based therapy that incorporates a single-protein molecule with multifunctional characteristics based on what we know about chronic wounds and infections, as a proof of concept of multifunctional proteins. Multifunctionality of a single therapeutic molecule is desirable because it may eliminate the unknowns of how differing individual chemical or protein therapies may interact when simply mixed. In addition, examples of peptides, such as antimicrobial peptides, are known to have synergy, and creating a single protein platform that consists of two synergistic peptides could be of value in the making of a protein with greater activity by guaranteeing that the synergistic peptides are local to one another. Furthermore, broad spectrum activity can be obtained by combining two differing peptides. This proof of concept was accomplished by targeting two proteinases that are upregulated in chronic wounds: Matrix Metalloproteinase-2 (MMP-2) and Neutrophil Elastase. Recombinant DNA techniques were used to create a fusion protein that incorporates an inhibitor of MMP-2, which is a β-Amyloid Precursor Protein-derived Inhibitory Peptide (APP-IP), and PMP-D2, an inhibitor of Neutrophil Elastase. PMP-D2 was joined to the N-terminus of an Elastin-like peptide, while the APP-IP was joined to the C-terminus of the same Elastin-like peptide. Elastin-like peptides (ELPs) are commonly used as a backbone for recombinant protein production as their distinct thermoresponsive characteristics provide adequate protein purification using an inverse transition cycling [3]. In addition, ELPs can serve as point-specific drug delivery platforms with a transition temperature (Tt) near that of normal body temperature causing low diffusivity [3]. Therefore, when ELPs are applied to a site at their Tt, they will aggregate, which provides diffusional limitations of the protein in the application site, and may decrease the reapplication rate needed for a therapeutic, as well as eliminate adverse side effects by retaining the protein to the specific application site. From this dual fusion, the final resulting protein is PMP-D2٠ELP٠APP-IP. This protein was tested for its inhibitory activity of both MMP-2 and Neutrophil Elastase. It was hypothesized that the fusion protein, PMP-D2٠ELP٠APP-IP, would inhibit MMP-2 just as effectively as APP-IP·ELP unaccompanied by PMP-D2, as well as effectively inhibit Neutrophil Elastase to the same degree as PMP-D2·ELP unaccompanied by APP-IP. Furthermore, an additional dually fused ELP fusion protein was currently made with two synergistic antimicrobial peptides fused to each end of the ELP. The two antimicrobial peptides used were human-derived LL37 and insect-derived Cecropin A. This novel fusion peptide contains synergistic increase in antibacterial activity in which preliminary data suggests.

O tratamento anti-HIV conhecido como HAART (Highly Active Antiretroviral Therapy) se tornou comum por volta de 1996, e utiliza 3 ou 4 diferentes medicamentos em combinação - geralmente dois inibidores de transcriptase reversa + 1 ou 2 inibidores de protease. A introdução desse tipo de tratamento trouxe um grande impacto na morbidade e mortalidade de indivíduos infectados pelo HIV. Os inibidores de protease (PIs) são uma boa alternativa às falhas terapêuticas observadas com o uso dos inibidores de transcriptase reversa, no entanto também são associados a vários efeitos tóxicos, como desconforto abdominal, vômito, diarréia, dor de cabeça, tontura, lipodistrofia, hipercolesterolemia, hipertrigliceridemia e hiperglicemia. Em função da existência de efeitos adversos e da condição do ritonavir como protótipo desse tipo de medicação, nosso objetivo é avaliar o efeito desse medicamento sobre os epitélios de revestimento e glandular relacionados à cavidade bucal, de forma a identificar a possibilidade da existência de complicações bucais relacionadas ao uso de inibidores de protease. Ratos albinos (Wistar) foram tratados com Ritonavir (10mg/Kg) por períodos de 4 e 8 semanas. Foram avaliadas as taxas séricas de triglicérides e colesterol (total, HDL, LDL, VLDL). Ao final dos períodos de tratamentos propostos, os animais foram sacrificados, e as peças utilizadas no estudo foram colhidas, (sangue, pele, língua, palatos e glândulas salivares). O sangue coletado foi imediatamente centrifugado sendo o plasma foi utilizado para avaliação das lipoproteínas. Os tecidos colhidos foram fixados, descalcificados quando necessário, processados para inclusão em parafina, cortados com 6µm de espessura, montados em slides e corados com hematoxilina e eosina, para avaliação histopatológica, morfométrica e estereológica. Os dados colhidos foram apresentados em valores médios, e as diferenças analisadas por testes estatísticos adequados para a comparação entre as amostras. Nossos resultados mostram pequenas variações nas características morfológicas de epitélios de revestimento e glandulares, variações essas que poderiam deixar esses tecidos mais propensos a sofrer alterações significativas com traumas ou injúrias, comuns nos tecidos bucais. Embora observadas com pequeno grau de expressão, essas variações, parecem ser progressivas, ou seja, mais expressivas com o uso continuado do medicamento. Mais estudos devem ser realizados, principalmente voltados para avaliações histoquímicas, bioquímicas e moleculares, no entanto nosso estudo é um alerta inicial para a avaliação dos tecidos bucais de pacientes que utilizam inibidores de protease. / HAART had a dramatic impact on the HIV infection, however, protease inhibitor exhibit significant drug-drug interactions, and side effects. There are only few data on effects of protease inhibitors on oral tissues. We propose to observe experimental effects of ritonavir on oral epithelial tissues, covering and glandular. Wistar rats received Ritonavir twice a week for 4-8 weeks. Controls received no treatment. At the time for sacrifice, plasma were collected for evaluation of triglycerides, total cholesterol, HDL, LDL and VLDL. Were also collected skin, tongue, palate and glandular tissues Lipoproteins were evaluated and histological examination of skin, mucosal epithelium on tongue, palate and salivar submandibular glandula were made under light microscope. Morphometric methods (cariometry and stereology) were used. Data were statistically analysed by Kruskal Wallis test for multiple samples, since our data were considered not-normal. P[U] 0.05 were considered statistically significant. Our results show that protease inhibitor may be associated with small alterations in epithelial tissues, significant mostly when on longer times using the medication. The complete significance of this data has to be better understood, and other studies has to be done to define these points.

Die Cathepsine B, H und L sind lysosomale Enzyme, die zur Gruppe der Cysteinproteasen gehören. Erhöhte Aktivitäten dieser Proteasen fand man in Gewebeproben verschiedener Tumore, vermutlich Hinweis auf eine Beteiligung an Invasion und Metastasierung. Unter physiologischen Bedingungen werden die Cathepsine von endogenen Inhibitoren kontrolliert, eine Verminderung dieser Cysteinprotease-Inhibitoren (CPI) würde die proteolytische Dysbalance verstärken und zur Ausbreitung des malignen Prozesses beitragen. Für das Prostatakarzinom gab es bisher keine Untersuchungen. Die Aktivität der Cathepsine wurde mit Hilfe spezifischer Substrate bestimmt, die zur Bildung fluorogener Produkte führten. Zur Bestimmung der inhibitorischen Aktivität der CPI wurden die Proben nach einer Hitzeaktivierung gegen reines Cathepsin B getestet. Untersucht wurden Gewebeproben verschiedener Patientengruppen, Primärzellkulturen, die aus normalem und maligne veränderten Prostatagewebe angezüchtet wurden und vergleichend dazu die drei immortalisierten Zellinien LNCaP, PC3 und DU145. Die Ergebnisse der Gewebeproben zeigten höhere Aktivitäten der Cathepsine B und L im nichterkrankten Gewebe, nicht wie erwartet im Tumorgewebe der Prostata. Hingegen waren bei den Primärzellkulturen alle drei Cathepsine und der Quotient Cathepsine/CPI in den Tumorproben erhöht. Die immortalisierten Zellinien zeigten die gleiche Verteilung bei allen Cathepsinen, DU145 mit der höchsten Aktivität, gefolgt von LNCaP und PC3. Anhand der Ergebnisse schlußfolgern wir, daß die Untersuchung von Gewebe-proben der Prostata hinsichtlich der Beteiligung der Cathepsine am Tumor-geschehen keine eindeutigen Erkenntnisse erbringt. Dies ist vermutlich auf die Heterogenität des Gewebes zurückzuführen, das nicht nur epitheliale, sondern auch stromale Zellen enthält. Die aus dem Gewebe angezüchteten Primär-zellkulturen scheinen ein genaueres Bild des Verhältnisses von Cathepsinen und den Inhibitoren zu geben und sind unserer Meinung nach den Bestimmungen in Gewebeproben vorzuziehen. / The cathepsins B, H and L (CB, CH, CL) are lysosomal proteolytic enzymes belonging to the cysteine protease family. Elevated cathepsin levels and decreased concentration of their endogenous inhibitors have been demonstrated in a variety of tumors, suggesting a contribution to invasion and metastasis. The situation for prostate cancer was so far unknown. Using fluorimetric assays, catalytic activities of the cathepsins B, H, L were measured in prostatic tissue samples obtained from different groups of patients, in primary cell cultures established from human prostate and in the immortalized cell lines LNCaP, PC3 and DU145. Inhibitory activities of cysteine protease inhibitors (CPI) were tested against purified cathepsin B. Comparing matched pairs of normal and cancerous tissue samples from the prostate, significantly decreased levels of CB and CL were found in malignant samples. In contrast, primary cell cultures from malignant tissue showed elevated levels of all three cathepsins and increased ratios of cathepsins to CPI when compared to cell cultures from non-malignant prostate. The permanent cell lines showed a similiar distribution of cathepsin levels, DU145 with the highest activity, followed by LNCaP and PC3. These results suggest that elevated cathepsin activities and increased ratios of cathepsins to CPI in malignant cell cultures compared to non-malignant samples may be an indication for a cellular proteolytic imbalance in prostatic cancer cells. Regarding different results, determinations in primary cell cultures should be preferred to tissue samples.