Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that establishes a life-long latent infection of B cells. It is usually asymptomatic in healthy individuals; however, individuals with compromised immunity often develop EBV-induced lymphoma. EBV also encodes potential oncogenes that can contribute to tumorigenesis. Therefore, vaccine and immunotherapeutic strategies targeting EBV are desirable. Recent studies have shown that infusion of EBV-specific CD8+T cells can elicit remission of lymphomas arising after administration of immunosuppressive drugs during transplantation, suggesting an important role for T cells in the prevention of EBV-induced malignancy. A better understanding of the cellular immune components involved in the control of EBV will aid in the development of methods to prevent infection and/or treat EBV-associated disease. While EBV infection is usually acquired asymptomatically during childhood, primary infection of adolescents and young adults can result in an illness termed acute infectious mononucleosis (AIM). Because of the characteristic symptoms of the illness, individuals with AIM can be readily identified and diagnosed with acute EBV infection. Thus, primary CD4+ and CD8+ T cell responses against the virus can be evaluated. It has been previously found that there is a marked expansion of lytic EBV protein-specific CD8+ T cells early during AIM, with delayed detection of lower frequencies of latent EBV protein-specific CD8+ T cells. The magnitude and specificity of CD4+T cell responses during AIM has been less well characterized. This thesis dissertation presents data from both functional assays and direct staining experiments documenting the timing, magnitude, and antigen-specificity of CD4+ T cells over the course of primary EBV infection. Lytic and latent protein-specific CD4+ T cells were readily detected by intracellular IFN-γ production at presentation with AIM and declined rapidly thereafter. Blood EBV load was also quantitated and found to decrease over time following AIM. By contrast, CD8+T cell IFN-y responses remained high for several weeks following presentation with AIM. Direct staining of lytic epitope-specific CD4+ T cells during AIM revealed high frequencies of virus-specific cells with low proliferative and IFN-γ-producing potential. Blood EBV load in these patients was persistently high through 6 wk following AIM. These data suggest a relationship between high EBV load during acute infection and impaired EBV-specific CD4+ T cell responses, which are compatible with impaired CD4+ T cell responses reported during high viremia associated with other viral infections. This may represent a mechanism by which persistent viruses, such as EBV, are able to establish a life-long infection in their hosts.

Cells have evolved different pathways in order to tolerate damage produced by different cytotoxic agents. Each agent reacts differently with DNA causing formation of different types of adducts, each eliciting the SOS stress response to induce different cellular repair pathways. One such type of substrate generated by cytotoxic agents is the DNA double strand break (DSB). The main pathway to repair such damage in the cell is through a process of recombination. In this thesis, I specifically examined the anti-cancer therapeutic agent cisplatin, which forms single- and double-strand breaks in DNA, and methylating agents, which are proposed to also be capable of forming such breaks. Neither type of agent can directly form these breaks; however, they leave a signature type of damage lesion which is recognized by different repair processes. The mismatch repair (MMR) status of a mammalian cell or an Escherichia coli dam mutant relates directly to the sensitivity of the cells to the agents mentioned above. As the dam gene product plays an important role in this pathway and in other processes in the cell, when mutated, dam cells are more sensitive to methylating agents and cisplatin than wildtype. A combination of dam and either mutS or mutL restores resistance to the same agents to wild type levels. Therefore, mismatch repair sensitizes dam bacteria to these agents. The rationale for this comes from examining the viability of dam mutants, as dammutants are only viable because they are highly recombinogenic. The presence of MMR-induced nicks or gaps results in the formation of DSBs that require recombination to restore genomic integrity. Mismatch repair proteins inhibit recombination between homeologous DNA. Homeologous recombination (recombination between non-identical, but similar, DNA sequences) is only possible when the MMR proteins, MutS and MutL, are absent. It is postulated that this is because MutS recognizes the homeologous DNA and subsequently slows down or aborts recombination completely. The double mutant, dam mutS/L shows wild type levels of sensitivity to cisplatin because mismatch repair is no longer recognizing the adducts and recombinational repair is allowed to continue. Human cells behave in an analogous fashion to the bacterial dam mutant, showing sensitivity to cisplatin and methylating agents. When an additional mutation in a mismatch repair gene is present, the cells become as resistant as wild type. Therefore, the E. coli dammutant is a useful model system to study this mechanism of drug resistance. DNA containing cisplatin adducts or lesions resulting from methylation are substrates for other types of repair processes such as nucleotide excision repair and base excision repair; however they have also been implicated as substrates for MMR and recombinational repair. The goal of the work in this thesis was two-fold. The first was to identify the gene products and mechanism necessary for repair of cisplatin damage by recombination. The second was to examine the mechanism of cisplatin toxicity, and specifically how MMR proficiency aids in the cytotoxicity of this drug by preventing recombination. Using the duplicated inactive lac operon recombination assay, we were able to determine the requirements for spontaneous and cisplatin-induced recombination, the RecBCD and RecFOR pathways. We were also able to further postulate that the cisplatin- induced signature damage recognized by recombination was the double strand break, likely formed from fork stalling and regression or a subsequent collapse during DNA synthesis, thus requiring these pathways for repair. This observation led to the experiments involving examination of the mechanism of cisplatin toxicity and where MMR could inhibit specific steps of recombination with DNA containing cisplatin lesions. Low levels of cisplatin lesions slowed the rate of RecA-mediated strand transfer in vitro, likely due to its ability to form a large bend in the DNA. MutS bound to cisplatin lesions in the DNA during heteroduplex formation in the RecA strand exchange step of recombination, inhibiting branch migration, and aborting the reaction. In order for MutS to inhibit recombination with cisplatin lesions, the results in the work in Chapter IV, show that binding to the lesion requires the C-terminus of MutS to be present, possibly due to a requirement for tetramerization of the protein, a domain contained in the C-terminus of MutS. This antirecombination function is different than the mutation avoidance function of MutS, as binding of mismatches requires only dimers. This differential sensitivity for cisplatin versus a mismatch was further exemplified in Chapter V, the experiments with dna mutants, where the greatest difference in sensitivity was observed for a dnaE mutant (catalytic subunit of polIII), which was as sensitive to cisplatin as a dam mutant, but fairly resistant to treatment with MNNG. This is indicative of the potency of a cisplatin adduct to block polymerase progression, versus a mismatch which poses little problem to synthesis. Recombination is invoked to repair DSBs caused by the cisplatin lesions through the RecBCD and FOR pathways after fork regression or collapse. A main conclusion from these studies is that a cisplatin lesion is processed differently than a mismatch. The mechanism of how a cisplatin lesion is processed, forming the DSB which invokes recombinational repair is still unclear and continues to be investigated.

The "RNA world" hypothesis makes two predictions that RNA should have been able both to catalyze RNA replication and to direct protein synthesis. The evolution of RNA-catalyzed protein synthesis should be critical in the transition from the RNA world to the modem biological systems. Peptide bond formation is a fundamental step in modem protein biosynthesis. Although many evidence suggests that the ribosome is a ribozyme, peptide bond formation has not been achieved with ribosomal RNAs only. The goal of this thesis is to investigate whether RNA could catalyze peptide bond formation and how RNA catalyzes peptide bond formation. Two systems have been employed to approach these questions, the ribozyme system and the ribosome system. Ribozymes have been isolated by in vitro selection that can catalyze peptide bond formation using the aminoacyl-adenylate as the substrate. The isolation of such peptide-synthesizing ribozymes suggests that RNA of antiquity might have directed protein synthesis and bolsters the "RNA world" hypothesis. In the other approach, a novel assay has been established to probe the ribosomal peptidyltransferase reaction in the presence of intact ribosome, ribosomal subunit, or ribosomal RNA alone. Several aspects of the peptidyltransfer reaction have been examined in both systems including metal ion requirement, pH dependence and substrate specificity. The coherence between the two systems is discussed and their potential applications are explored. Although the ribozyme system might not be a reminiscence of the ribosome catalysis, it is still unique in other studies. The newly established assay for ribosomal peptidyltransferase reaction provides a good system to investigate the mechanism of ribosomal reaction and may have potential application in drug screening to search for the specific peptidyltransferase inhibitors.

p53, known as the “guardian of the genome”, is the most well-characterized tumor suppressor gene. The central role of p53 is to prevent genome instability. p53 is the central node in an incredibly elaborate genome defense network for receiving various input stress signals and controlling diverse cellular responses. The final output of this network is determined not only by the p53 protein itself, but also by other p53 cooperating proteins. p300 and CBP (CREB-Binding Protein) act as multifunctional regulators of p53 via acetylase and ubiquitin ligase activities. Prior work in vitro has shown that the N-terminal 595 aa of p300 encode both generic ubiquitin ligase (E3) and p53-directed E4 functions. Analysis of p300 or CBP-deficient cells revealed that both coactivators were required for endogenous p53 polyubiquitination and the normally rapid turnover of p53 in unstressed cells. Unexpectedly, p300/CBP ubiquitin ligase activities were absent in nuclear extracts and exclusively cytoplasmic. In the nucleus, CBP and p300 exhibited differential regulation of p53 gene target expression, C-terminal acetylation, and biologic response after DNA damage. p300 activated, and CBP repressed, PUMA expression, correlating with activating acetylation of p53 C-terminal lysines by p300, and a repressive acetylation of p53 lysine-320 induced by CBP. Consistent with their gene expression effects, CBP deficiency augmented, and p300 deficiency blocked, apoptosis after doxorubicin treatment. Subcellular compartmentalization of p300/CBP’s ubiquitination and transcription activities reconciles seemingly opposed functions—cytoplasmic p300/CBP E4 activities ubiquitinate and destabilize p53, while nuclear p300/CBP direct p53 acetylation, target gene activation, and biological outcome after genotoxic stress. p53 is a prominent tumor suppressor gene and it is mutated in more than 50% of human tumors. Reactivation of endogenous p53 is one therapeutic avenue to stop cancer cell growth. In this thesis, we have identified S5as a critical regulator of p53 degradation and activity. S5a is a non-ATPase subunit in the 19S regulatory particle of the 26S proteasome. Our preliminary data indicates that S5a is required for p53 instability and is a negative regulator of p53 tranactivation. As a negative regulator of p53, S5a may therefore also represent a new target for cancer drug development against tumors that specifically maintain wild type p53.

<p>Sukcinimidi su jedinjenja koja pokazuju vi&scaron;estruke farmakolo&scaron;ke efekte uključujući i antiproliferativnu aktivnost, zahvaljujući prisustvu farmakofore sa dva hidrofobna regiona i dva regiona bogata elektronima. Savremeni dizajn lekova ima za cilj da se modifikacijama u strukturi (promena vrste, položaja i orijentacije supstituenata) i in silico računarskim metodama predvide i optimizuju farmakokinetske osobine i bezbednosni profil kandidata za lek. U ranoj fazi razvoja lekova se koriste postojeće baze podataka o molekulskim, farmakokinetskim i toksikolo&scaron;kim parametrima već ispitanih jedinjenja i pomoću matematičkih modela i algoritama predviđaju se osobine novih molekula, elimini&scaron;u se neodgovarajući kandidati i postiže se u&scaron;teda u vremenu i materijalnim sredstvima. Da se ispitaju fizičko-hemijske karakteristike 11 novosintetisanih metil-etil-N-aril-sukcinimida na osnovu strukture, primenom različitih softverskih paketa; da se na osnovu strukture odrede farmakokinetski i toksikolo&scaron;ki parametri, primenom različitih softverskih paketa; da se ispita retenciono pona&scaron;anje, odnosno odrede retencione konstante za svako jedinjenje primenom visokoefikasne hromatografije na tankom sloju (HP-TLC) i ispita mogućnost primene retencionih konstanti kao mere lipofilnosti ispitivanih jedinjenja; da se ispita antiproliferativna aktivnost na odabranim kulturama ćelija karcinoma i na zdravim ćelijama fibroblasta pluća; da se analizom molekulskog dokinga ustanovi vezivanje za estrogene receptore. Ispitano je retenciono pona&scaron;anje 11 novosintetisanih derivata sukcinimida primenom visokoefikasne hromatografije na tankom sloju (HP-TLC) obrnute faze uz primenu dvokomponentne sme&scaron;e vode i organskog rastvarača (metanola, acetonitrila ili acetona), sa odgovarajućim zapreminskim udelom organskog rastvarača kao mobilne faze. Iz razvijenih hromatograma su izračunate retencione konstante RM0 i S. Logaritam podeonog koeficijenta (logP) određen je in silico, kori&scaron;ćenjem različitih računarskih programa. In silico su određene fizičko-hemijske karakteristike, farmakokinetski parametri, toksikolo&scaron;ki parametri, akvatična toksičnosti i afinitet vezivanja za estrogene receptore. Izračunate su vrednosti afiniteta za 4 vrste receptora (G-protein spregnuti receptori, jonski kanali, inhibitori kinaza, nuklearni receptori). Antiproliferativna aktivnost ispitivanih derivata sukcinimida određena je primenom kolorimetrijskog testa sa tetrazolijum solima (MTT testa) na komercijalnim kulturama ćelija (MRC-5, A549, HeLa, MDA-MB-231, MCF-7, HT-29) i izračunate su IC50 vrednosti. Urađena je i doking analiza sukcinimida prema ERA (estrogen receptor alfa) i ERB (estrogen receptor beta) i dobijene su vrednosti energije formiranja kompleksa sa posmatranim receptorima (MolDock Score). Statistički najznačajnije linearne korelacije dobijene su između eksperimentalno određenih hromatografskih parametara (RM0 i S) i in silico parametara lipofilnosti MlogP i ClogP. Ispitivanjem uticaja promene RM0 i S na farmakokinetske karakteristike dobijeni su rezultati koji pokazuju paraboličnu zavisnost konstante apsorpcije (Ka) i procenta vezivanja za proteine plazme (PPB) od posmatranih retencionih konstanti, dok je zavisnost sa volumenom distribucije (Vd) i sposobno&scaron;ću prolaska kroz krvno-moždanu barijeru (logBBB) bila linearnog tipa. Toksičnost ispitivanih jedinjenja, procenjena na osnovu in silico dobijenih LD50 vrednosti, nije bila vi&scaron;a od toksičnosti već registrovanih lekova sa strukturom sukcinimida, i dala je parabolične zavisnosti u odnosu na RM0 i S vrednosti. Eksperimentalno nijedno od ispitivanih jedinjenja nije pokazalo aktivnost u odnosu na zdrave fibroblaste pluća. Najznačajniju antiproliferativnu aktivnost (najniže IC50) su pokazala jedinjenja 6 i 7 u odnosu na ćelije linije MCF-7 i jedinjenje 11 u odnosu na A549 ćelijsku liniju. Doking analiza je pokazala niže energije formiranja kompleksa sa ERA, u odnosu na ERB. Eksperimentalno određeni parametri RM0 i S se mogu koristiti kao alternativne i pouzdane mere lipofilnosti analiziranih sukcinimida. Ispitivana jedinjenja pokazuju povoljne fizičko-hemijske karakteristike, predviđene in silico metodama i povoljne farmakokinetske karakteristike: male vrednosti konstante apsorpcije, umeren volumen distribucije, povoljan afinitet vezivanja za proteine plazme, favorizovan prolazak kroz krvno-moždanu barijeru za lipofilnija jedinjenja. Procenjuje se da sva ispitivana jedinjenja, izuzev derivata sa &ndash;CN supstituentom, imaju zahtevani nizak stepen toksičnosti. Po antiproliferativnoj aktivnosti u odnosu na ćelije ER-zavisnog karcinoma dojke (MCF-7) izdvajaju se jedinjenja sa metil i nitro supstituentom u para položaju. Na osnovu malih energija formiranja kompleksa sa ERA, koji su eksprimirani na ćelijama MCF-7 linije, pretpostavlja se da bi mehanizam njihovog delovanja delimično mogao biti obja&scaron;njen uticajem na ERA, ali su potrebna dodatna istraživanja na tom polju.</p> / <p>Succinimides have exhibited various pharmaceutical effects including antiproliferative activity due to an important structural fragment (a pharmacophore) presented in form of two hydrophobic regions and two electron-rich centers. Current development of new drugs involves modifications in structure (type, position and orientation of substituents) and usage of in silico computational programs to predict and optimize pharmacokinetic and safety profile of drug candidates. In early phase of drug development, databases regarding the molecular, pharmacokinetic and toxicological parameters of already tested compounds are used, mathematical models and algorithms are applied for predicting the properties of new molecules and inadequate candidates are eliminated saving time and resources. Determination of physico-chemical properties of the analyzed methyl-ethyl-N-phenilsuccinimide derivatives by software packages; virtual pharmacokinetic and toxicology screening; investigation of retention behavior of the compounds by the reversed-phase HPTLC analysis and calculation of retention constants and their correlation with lipophilicity; in vitro evaluation of antiproliferative activity toward five carcinoma cell lines and normal fetal lung cell line; molecular behavior study on target estrogen receptors by molecular docking and correlation of antiproliferative activity toward ER+ breast carcinoma cell lines and in silico estrogen receptor affinity binding. Retention behavior of 11 newly synthesized succinimide derivatives was determined by reversed phase high performance thin layer chromatography (RP HPTLC) with the application of two-component mixtures water - organic solvent (methanol, acetonitrile or acetone) with adequate volume fractions of the organic modifier. After chromatographic development RM0 and S parameters were calculated. The logarithm of partition coefficient, logP for the analyzed compounds were calculated by different softwares. Physico-chemical properties, pharmacokinetic and toxicological parameters, aquatic toxicity and relative affinity to estrogen receptors were predicted in silico. The affinity toward 4 types of receptors (G-proteine coupled receptors, ion channels, kinase inhibitors, nuclear receptors) were calculated as well. Standard MTT assay was applied to evaluate cytotoxic activities of the analyzed succinimides after cells were exposed. Antiproliferative activity were investigated toward commercial MRC-5, A549, HeLa, MDA-MB-231, MCF-7, HT-29 cell lines and IC50 values were calculated for each compound. MolDock Score that represents energy of binding to estrogen alfa and estrogen beta receptors was determined by molecular docking. Statistically significant linear correlations were determined between the chromatographic retention constants (RM0 and S) and calculated logP, and the best two were obtained in correlation of retention constants with MlogP and ClogP. The examination of RM0 and S influence on pharmacokinetics indicated parabolic dependence of the absorption constant (Ka) and plasma protein binding predictor (PPB) from the observed constants while the volume of distribution (Vd) and the ability to cross the brain blood barrier (logBBB) had linear association with the retention parameters. The toxicity of the analysed compounds evaluated in silico as LD50 on rodents was lower in comparison with the drugs with succinimide structure that are on the market and had parabolic correlation with the RM0 and S values. The experiments indicated that none of the compounds examined had cytotoxic activity toward the healthy lung fibroblast cells. The results of the in vitro assay shown that none of the investigated compounds demonstrated antiproliferative activity toward fetal lung cells. The most potent antiproliferative agents were compounds 6 and 7 toward MCF-7 cell line, and compound 11 toward A549 cell line. Molecular docking shown lower energy for binding to ERA in comparison to ERB.</p>

The paper looks at parents' experiences related to their children's preparations for attending an eight-year gymnázium (academic secondary school), with the goal of identifying and understanding how families experience this preparatory period and what preparatory strategies they choose; or rather, how they approach the preparatory exams, what preparations the family undergoes, what emotions they experience, and how they assess this experience. The theoretical part of this theoretical and empirical paper briefly defines some of the basic terminology necessary for understanding the paper's theoretical foundations and presents the legislative background of the state admissions exams. The third chapter of the theoretical part looks at several realized studies that have focused on the subject of multi- year gymnázia and parents' views on this phenomenon. The various chapters of the paper's empirical section present the author's qualitative methodology, describe the study's approach, and show the results of a qualitative study of parents and children who underwent this experience in the 2017/18 and 2018/19 school years. Their experiences with and perception of this process were determined via semi- structured interviews with both parents and children. The study also made use of interpretative phenomenological...

A novel aryl hydrocarbon receptor nuclear translocator (ARNT)-interacting peptide (Ainpl) was characterized from human liver cDNA library using phage display. Ainpl suppresses hypoxia inducible factor-1a (HIF-1α) signaling pathway through an ARNTdependent manner. HIF-1α is known to be overexpressed in more than 90% of solid tumors, and the inhibition of HIF-1α is proved as an effective approach to suppress tumor growth. ARNT, as the obligatory heterodimeric partner of HIF-1α for downstream gene activation, was used as a bait to screen for Ainpl. Ainpl specifically interacts with the helix-loop-helix (HLH) subdomain of ARNT, but not with HIF-1α. GFP-Ainpl is localized in both cytoplasm and nucleus, and suppresses HIF-1α signaling by two mechanisms: (1) cytoplasmic GFP-Ainp 1 retains ARNT in the cell cytoplasm and (2) nuclear GFP-Ainpl inhibits HIF-1α/ARNT heterodimerization. The suppression of Ainpl on HIF-1α signaling was reversed by introducing ARNT into the cells using transient transfection. We further utilized HIV TAT protein transduction domain to deliver 6His-TAT-Ainpl into three different cancer cell lines (Hep3B, HeLa, MCF-7), and found that 6His-TAT-Ainpl co-localizes with ARNT in the cell nucleus. 6His-TATAinpl can be detected inside the cells after 30 min of transduction, and can reach the maximum level at 2 h. 6His-TAT-Ainp 1 remained detectable in the cells up to 96 h and had a half life of 24 h after transduction. In addition, 6His-TAT-Ainp 1 suppresses HIF-1α downstream genes at both message and protein levels in a dose-dependent manner. Taken together, molecules that target the HIF-1α and ARNT interface can be developed as viable drugs to suppress HIF-1α signaling.

Breast cancer, osteoporosis, and coronary heart disease (CHD) are three major threats to women’s health. Postmenopausal women with breast cancer are also at high risk for osteoporosis and CHD. Adjuvant tamoxifen therapy is not only an effective treatment for breast cancer, but has been shown to have a beneficial effect on bone and the cardiovascular system. Although tamoxifen has been convincingly demonstrated to be able to preserve bone mineral density (BMD), an unexpected increase of risk of fractures in patients treated with tamoxifen has been reported. The findings of the association of tamoxifen and CHD from previous studies were either borderline or inconsistent. To clarify the discrepancy between BMD and fractures and test the potential beneficial effect of tamoxifen on CHD, I conducted a series of retrospective studies in postmenopausal women with breast cancer who participated in the Cancer Surveillance in HMO Administrative Data (IMPACT study) or the Study of Osteoporotic Fractures (SOF). In patients who participated in the IMPACT study, I demonstrated that the association of tamoxifen and fracture incidence varied at different skeletal sites. Although the association of tamoxifen and fractures in the spine (HR=0.40, 95% CI: 0.09-1.85), wrist (HR=2.49, 95% CI: 0.88-7.06), and total body (HR=0.87, 95% CI: 0.49-1.55) was inconclusive, tamoxifen was associated with an apparent reduction of the risk of hip fracture (HR=0.41, 95% CI: 0.17-1.03, p=0.0565). Importantly, the pattern of observed association of tamoxifen with the risks of fractures among postmenopausal women with breast cancer is consistent with its widely reported preserving effect on bone mineral density. Using SOF data, I found that the association between BMD and fractures in women with breast cancer varied at different skeletal sites, and type of BMD measured. Non-specific BMD was not associated with hip fracture (HR=1.12; 95% CI: 0.78, 1.59). Site-specific BMD was more likely linked with hip fracture (HR=1.43, 95% CI: 0.99, 2.08) while change in BMD did not predict hip fracture (HR=1.05; 95% CI: 0.63, 1.72). The association of spine morphometric fracture with either non-specific or spine-specific BMD was similar (OR=1.40; 95% CI: 1.04, 1.90; OR=1.35, 95% CI: 0.99, 1.85, respectively). Overall, the association of BMD and fracture in elderly women with breast cancer is weak. Only site-specific BMD appears to have a consistently modest association with fractures in the corresponding skeletal sites. In the IMPACT study population, compared to patients without tamoxifen, the overall incidence of CHD in tamoxifen-treated patients was lower (adjusted HR=0.60, 95% CI: 0.40-0.88). For each year of tamoxifen use, there was a statistically significant decrease in the risk of CHD (HR=0.90, 95% CI: 0.82-0.98). Further analyses categorized by length of tamoxifen use showed that an apparent association with a decreased CHD risk was found in patients who received tamoxifen for two to five years (HR=0.54, 95% CI: 0.33-0.86). No association was detected after the discontinuation of tamoxifen therapy. In summary, I detected a possible benefit associated with tamoxifen on fractures in the hip, the most common fracture site. I also found that BMD did not predict osteoporotic fractures well in postmenopausal women with breast cancer. In addition, I demonstrated that tamoxifen was associated with a reduced risk of CHD in postmenopausal women with breast cancer in a dose-dependent manner. An apparent benefit was found in those patients who received tamoxifen therapy for at least two years.

In the 30 years since the Center for Disease Control's Morbidity and Mortality Weekly Report published the first mention of what later was determined to be AIDS (Acquired immunodeficiency syndrome) and HIV (Human immunodeficiency virus) recognized as the causative pathogen, much has been done to understand this disease’s pathogenesis, development of drugs and emergence of drug resistance under selective drug therapy. Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, have helped stabilize the HIV prevalence at extraordinarily high levels. Despite the recent stabilization of this global epidemic, its dimensions remain staggering with estimated (33-36 million) people living with HIV-AIDS in 2007 alone. This is because the available drugs against AIDS provide treatment for infected individuals, but HIV evolves rapidly under drug pressure and develops resistant strains, rendering the therapy ineffective. Therefore, a better understanding underlying the molecular mechanisms of viral infection and evolution is required to tackle drug resistance and develop improved drugs and treatment regimens. HIV-1 protease is an important target for developing anti-HIV drugs. However, resistant mutations rapidly emerge within the active site of the protease and greatly reduce its affinity for the protease inhibitors. Frequently, these active site drug resistant mutations co-occur with secondary/ non-active site/ associated or compensatory mutations distal to the active site. The role of these accessory mutations is often suggested to be in maintaining viral fitness and stability of protease. Many of the non-active site drug resistant mutations are clustered in the hydrophobic core in each monomer of the protease. Molecular dynamic simulation studies suggest that the hydrophobic core residues facilitate the conformational changes that occur in protease upon ligand binding. There is a complex interdependence and interplay between the inherent adaptability, drug resistant mutations and substrate recognition by the protease. Protease is inherently dynamic and has wide substrate specificity. The PI (protease inhibitor) resistant mutations, perhaps, modulate this dynamics and bring about changes in molecular recognition, such that, in resistant proteases, the substrates are recognized specifically over the PIs for the same binding site. In this thesis research, I have investigated these three complementary phenomena in concert. Chapter II examines the importance of hydrophobic core dynamics in modulating protease function. The hydrophobic core in the WT protease is intrinsically flexible and undergoes conformational changes required for protease to bind its substrates. This study investigated if dynamics is important for protease function by engineering restricted vs. flexible hydrophobic core region in each monomer of the protease, using disulfide chemistry. Under oxidizing conditions, disulfide bond established cross-link at the interface of putative moving domains in each monomer, thereby, restricting motion in this region. Upon reduction of the disulfide bond, the constraining influence was reversed and flexibility returned to near WT. The disulfide cross-linked protease showed significant loss of function when tested in functional cleavage assay. Two protease variants (G16C/L38C) and (R14C/E65C) were engineered and examined for changes in structure and enzymatic activity under oxidizing and reducing conditions. (R14C/E65C) was engineered as an internal control variant, such that cysteines were engineered between putative non-moving domains. Structurally, both the variants were very similar with no structural perturbations under oxidizing or reducing conditions. While significant loss in function was observed for (G16C/L38C) only under oxidizing conditions, (R14C/E65C) did not show any loss of function under oxidizing or reduced conditions, as expected. Successful regain of function for cross-linked (G16C/L38C) was obtained upon reversible reduction of the disulfide bond. Taken together, these data demonstrate that the hydrophobic core dynamics modulates protease function and support the hypothesis that the distal drug resistant mutations, possibly causing drug resistance by modulating hydrophobic core dynamics via long range structural perturbations. Since protease recognizes and cleaves more than 10 substrates at different rates, our further interest is to investigate if there is a differential loss of activity for some specific substrates over the others, and whether the order of polypeptide cleavage is somehow affected by restricted core mobility. In order to better answer these questions it is essential to understand: what determines the substrate binding specificity in protease? A two-pronged approach was applied to address this question as described in chapter III and IV respectively. In chapter III, I investigated the determinants of substrate specificity in HIV-1 protease by using computational positive design and engineered specificity-designed asymmetric protease (Pr3, A28S/D30F/G48R) that would preferentially bind to one of its natural substrates, RT-RH over two other substrates, p2-NC and CA-p2, respectively. The designed protease was expressed, purified and analyzed for changes in structure and function relative to WT. Kinetic studies on Pr3 showed that the specificity of Pr3 for RT-RH was increased significantly compared to the wild-type (WT), as predicted by the positive design. ITC (Isothermal Titration Calorimetry) studies confirmed the kinetic data on RT-RH. Crystal structural of substrate complexes of WT protease and Pr3 variant with RT-RH, CA-p2 and p2-NC were further obtained and analyzed. The structural analysis, however, only partially confirmed to the positive design due to the inherent structural pliability of the protease. Overall, this study supports the positive computational design approach as an invaluable tool in facilitating our understanding of complex proteins such as HIV 1 protease and also proposes the integration of internal protein flexibility in the design algorithms to make the in-silico designs more robust and dependable. Chapter IV probed the substrate specificity determining factors in HIV-1protease system by focusing on the substrate sequences. Previous studies have demonstrated that three N-terminal residues immediate to the scissile bond (P1-P3) are important in determining recognition specificity. This work investigated the structural basis of substrate binding to the protease. Catalytically active WT protease was crystallized with decameric polypeptides corresponding to five of the natural cleavage sites of protease. The structural analyses of these complexes revealed distinct P side product bound in all the structures, demonstrating the higher binding affinity of N terminal substrate for protease. This thesis research successfully establishes that intrinsic hydrophobic core flexibility modulates function in HIV-1 protease and proposes a potential mechanism to explain the role of non-active site mutations in conferring drug resistance in protease. Additionally, the work on specificity designed and N terminal product bound protease complexes advances our understanding of substrate recognition in HIV protease.