This study investigates endocytosis and ER export signals of the yeast α-factor receptor and the role that receptor oligomerization plays in these processes. The α-factor receptor contains signal sequences in the cytoplasmic C-terminal domain that are essential for ligand-mediated endocytosis. In an endocytosis complementation assay, I found that oligomeric complexes of the receptor undergo ligand-mediated endocytosis when the α-factor binding site and the endocytosis signal sequences are located in different receptors. Both in vitro and in vivo assays strongly suggested that ligand-induced conformational changes in one Ste2 subunit do not affect neighboring subunits. Therefore, the recognition of endocytosis signal sequence and the recognition of the ligand-induced conformational change are likely to be two independent events, where the signal sequence plays only a passive role in the ligand-induced endocytosis. Four amino acid substitutions (C59R, H94P, S141P and S145P) in TM domains I, II and III were identified that resulted in the accumulation of truncated receptors in the ER but did not block ER export of full-length receptors. The two DXE motifs in the C-terminal tail were required for export of the mutant receptors from the ER; however DXE was not essential for proper cell surface expression of wild-type receptors apparently because the receptors contain redundant ER export signals. An assay for oligomerization of receptors in the ER was developed based on the ability of truncated mutant receptors to exit the ER. The four substitutions (C59R, H94P, S141P and S145P) that caused DXE-dependent ER export failed to form homo-oligomers, suggesting that the DXE motifs and receptor oligomerization serve as independent ER export signals. Consistent with this view, two of the substitutions (S141P and S145P), when coexpressed, with wild-type receptors, formed hetero-oligomers that exited the ER. Finally, the full-length oligomer-defective mutant Ste2-S141P was sensitive to α-factor, suggesting that receptor monomers that reach the cell surface are able to activate the heterotrimeric G protein. The potential roles that TM1, 2 and 3 play in receptor oligomerization are discussed.

Cytokinesis is the cytoplasmic division of one cell into two independent daughter cells. Precise regulation of cytokinesis during cell cycle is essential for healthy and rapid multiplication of any organism. Schizosaccharomyces pombe has emerged as an excellent model system to study eukaryotic cell division regulation. This rod shaped organism grows by bipolar elongation in interphase when its actin cytoskeleton is concentrated at the cell ends (poles). However, growth stops in mitosis and the actin cytoskeleton is rearranged to facilitate assembly of the contractile actomyosin ring at the cell middle. Although several studies have focused on the separate processes of growth and division, it was unclear how cells regulate the cytoskeletal remodeling during the transition between the different stages of the cell cycle. The Septation Initiation Network (SIN) is a signaling cascade essential for fission yeast cytokinesis (Balasubramanian et al., 1998; Mishra et al., 2004) and the MOR (morphogenesis) signaling pathway is essential for interphase bipolar growth (Kanai et al, 2005). Interestingly, inactivation of the SIN not only failed to maintain the cytokinetic apparatus at the cell middle but also caused the redistribution of the cytoskeletal elements like actin to the cell ends that led to bipolar cell elongation similar to cells in interphase (Mishra et al., 2004). These results suggested that SIN signaling inhibits interphase bipolar growth, but it was not clear if the SIN had a direct role in inhibition of interphase growth during mitosis and this question was the major focus of this thesis. The results presented in Chapter II show a novel cross-pathway interaction between the SIN and the MOR in the fission yeast. Our results in Chapter III suggest that some of the MOR pathway components might be important for coordination between nuclear and cytoplasmic divisions in mitosis, revealing novel roles of the pathway. In a separate study (Chapter IV) we sought to identify additional regulators of the SIN and cytokinesis through a suppressor screen and found that the nucleolar rDNA transcription machinery inhibits cytokinesis in fission yeast.

The proper maintenance of the pathways governing cell growth is critical to ensure cell survival and DNA fidelity. Much of our understanding of how the cell cycle is regulated comes from studies examining the relationship between DNA viruses and the mechanisms of cell proliferation control. There are numerous examples demonstrating that viruses can alter the host cell environment to their advantage. In particular, the small DNA tumor viruses, which include adenovirus, simian-virus 40 (SV-40), and human papillomavirus (HPV), can modulate the host cell cycle to facilitate viral DNA replication. Due to the fact that these viruses infect quiescent, non-cycling cells and lack the necessary enzymes and resources to replicate their DNA (e.g. DNA polymerase), the small DNA tumor viruses must activate the host cell replication machinery in order to expedite viral DNA replication. The capacity of these viruses to perturb normal cell proliferation control is dependent upon their oncogene products, which target p53 and members of the Retinoblastoma (RB) family of proteins and inactivate their respective functions. By targeting these key cell cycle regulatory proteins, the small DNA tumor viruses induce the infected host cells to enter S-phase and activate the components involved with host cell DNA synthesis thereby generating an environment that is conducive to viral DNA replication. In contrast, the larger, nuclear-replicating DNA viruses such as those from the family Herpesviridae, do not share the same stringent requirement as the small DNA viruses to induce the infected host cell to enter S-phase. The herpesviruses encode many of the components to stimulate nucleotide biosynthesis and the necessary factors to facilitate virus DNA replication including a viral DNA polymerase and other accessory factors. Additionally, many herpesviruses encode gene products that arrest the host cell cycle, in most instances, prior to the G1/S transition point. Inducing cells to growth arrest appears to be a prerequisite for the replication of most herpesviruses. However, in addition to encoding factors that inhibit the cell cycle, many herpesviruses encode proteins that can promote cell cycle progression in a manner similar to the small DNA tumor virus oncoproteins. By targeting members of the RB family and p53 protein, the herpesvirus proteins induce S-phase and activate S-phase associated factors that playa role in DNA replication. In this manner, the herpesviruses may promote an environment that is favorable for DNA replication. Consistent with the other herpesviruses, human cytomegalovirus (HCMV)induces human fibroblasts to growth arrest. However, in other cell types, virus infection causes cells to enter S-phase. In addition, HCMV replication requires several cellular factors that are present only during S-phase. Furthermore, HCMV induces the activation of S-phase-associated events as well as the increased expression of numerous S-phase genes following infection. HCMV encodes two immediate early (IE) gene products, IE1-72 and IE2-86, which can interact with members of the RB family of proteins. Additionally, the IE2-86 protein can bind to and inhibit p53 protein function. Given the functional resemblance between the HCMV IE proteins and the oncoproteins of the small DNA tumor viruses, we hypothesized that expression of the HCMV IE proteins could modulate cell cycle control. Specifically, we determined that expression of either IE1-72 or IE2-86 can induce quiescent cells to enter S-phase and delay cell cycle exit following serum withdrawal. Moreover, IE2-86 mediates this effect in the presence or absence of p53, whereas IE1-72 fails to do so in p53-expressing cells. Furthermore, both IE1-72 and IE2-86 induce p53 protein accumulation that is nuclear localized. Because IE1-72 fails to promote S-phase entry in cells expressing p53 and induces p53 protein levels, the mechanism by which IE1-72 alters p53 levels was examined. IE1-72 elevates p53 protein levels by inducing both p19ARF protein and an ATM-dependent phosphorylation of p53 at Ser15. IE1-72 also promotes p53 nuclear accumulation by abrogating p53 nuclear shuttling. As consequence of this IE1-72-mediated increase in p53 levels, p21 protein is induced leading to a p21-dependent growth arrest in cells expressing IE1-72. These findings demonstrate that the HCMV IE proteins can alter cell proliferation control and provide further support to the notion that HCMV, through the expression of its IE proteins, induces S-phase and factors associated with S-phase while blocking cell DNA synthesis, to possibly generate an environment that is suitable for viral DNA replication.

Osteopetrosis is a metabolic bone disease in mammals characterized by a generalized skeletal sclerosis caused by reduced bone resorption. This reduced bone resorption is manifested in afflicted animals by abnormal bone shape, reduced or absent marrow cavities, extramedullary hemopoiesis, abnormal mineral homeostasis and absent or delayed tooth eruption. The available osteopetrotic animal mutations have been a constant source of fruitful investigations concerning the systemic regulation of osteoclastogenesis and bone metabolism. Tooth eruption, on the other hand, is a localized manifestation of the timely activation of bone resorption and bone formation on opposite sides of an erupting tooth. Its rate-limiting step is the speed of bone resorption to form the eruption pathway. In this dissertation, we used two osteopetrotic rat mutations, toothless (tl) and microphthalmia blanc (mib), to investigate the abnormal development of osteoclasts and tooth eruption in mutant rats with an emphasis on the role of systemic and local factors. The significant contributions to this work are listed below. 1. In the toothless rat, a mutation lacking erupted dentition due to severely reduced bone resorption, colony-stimulating factor-1 (CSF-1) promoted tooth eruption but this was delayed compared to normal rats. Eruption was accompanied by changes in the populations of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear cells in the dental follicle and TRAP+ osteoclasts on adjacent alveolar bone surfaces. These cell populations were dramatically increased in treated mutants compared to untreated tl rats, but the timing of their appearance was delayed compared to normal littermates. This lag in the appearance of osteoclasts and their precursors corresponded to the delay in eruption of first molars in treated tl rats. 2. CSF-1 also accelerated the eruption of molars in normal rats. CSF-1 increased the number of TRAP+ mononuclear cells in the dental follicle and TRAP+ osteoclasts on adjacent alveolar bone surfaces, but had no effect on the timing of their appearance in normal rats. 3. Our data revealed a differential effect on tooth eruption of the growth factors CSF-1 and epidermal growth factor (EGF). CSF-1 accelerated eruption of molars in normal rats, but had no effect on incisor eruption. On the other hand, EGF accelerated incisor eruption; but did not affect molar eruption in normal rats. 4. We have described the mechanism for the transient, mild form of osteopetrosis inherited by mib rats. Mutant animals possess a typical sclerosis at birth, which diminished--but was not resolved--during the first postnatal month. These characteristics are caused by early reductions in osteoclast number and function which improve to normal levels by 4 weeks. Osteoclast numbers were severely reduced in mib rats between birth and 2 weeks, but improved to near normal levels by 4 weeks. Neonatal abnormalities in osteoclast function included reduced staining for the functional enzymes TRAP and TrATPase, decreased levels of mRNA for both TrATPase and CAll, and inability to form a well-developed ruffled border. None of these defects were apparent after the first postnatal month. 5. Finally, we have shown that the dental abnormalities caused by the mild, transient form of osteopetrosis in mib rats are limited to incisor defects and delayed eruption of all teeth. Histologic and radiographic examination of mutant incisors revealed that, contrary to the situation in normal rats, the apex of the incisors of mib rats failed to extend past the first molar region to the third molar. The incisor apex of newborn mib rats was misshaped due to ankylosis of incisor matrices with alveolar bone. This ankylosis was temporary, being resolved by the third postnatal day. The delayed eruption of incisors in mib rats and abnormal shape and occlusion of these teeth in older animals is a consequence of the temporary ankylosis in newborn rats.

Avian leukosis virus (ALV)-induced osteopetrosis is a proliferative disorder of the bone affecting the growth and differentiation of osteoblasts. Osteopetrosis is a polyclonal disease in which cells of the bone contain, on average, multiple viral DNA copies. Osteopetrotic bone is also characterized by the accumulation of unintegrated viral DNA, suggesting an atypical life cycle of the virus in the infected osteoblasts. To better understand virus-host interactions in the induction of osteopetrosis by ALVs, infected chick osteoblast cultures and osteopetrotic bone were examined for aspects of the virus life cycle and effects of infection on osteoblast function. Levels of infection and virus expression were compared in cultured osteoblasts and osteopetrotic bone. Osteopetrotic bone contained higher levels of viral DNA and correspondingly higher levels of viral proteins than infected osteoblast cultures, suggesting a higher viral load in the diseased bone. A significant level of mature Gag protein was present in the bone, suggesting the accumulation of mature virus particles in the diseased bone. It is possible that the accumulation of virus could facilitate the high levels of infection observed in the diseased bone. The mechanism by which unintegrated viral DNA persisted in osteopetrotic bone was investigated by examining the susceptibility of infected osteoblasts to superinfection. The results indicated that, in culture, infected osteoblasts were able to establish interference to superinfection. This suggests that the persistence of unintegrated viral DNA in osteopetrotic bone may not result from the continuing infection of productively infected osteoblasts. The effect of virus infection on osteoblast function was examined in the diseased bone and in osteoblast cultures. In infected chickens, osteoblast activity, as evidenced by the expression of osteoblast phenotypic markers, was increased only in chickens developing severe osteopetrosis. In culture, virus infection had no apparent effect on either the proliferation or differentiation of osteoblasts. This indicates that infection was itself not sufficient to perturb osteoblast function. Furthermore, it suggested that additional components of the bone may be required for ALV infection to induce the abnormal activity of osteoblasts observed in osteopetrosis.

Chromatin remodeling is crucial to transcriptional regulation in vivo and a number of protein complexes capable of altering genomic architecture in the budding yeast Saccaromyces cerevisiaehave been identified. Among these, the SWI/SNF complex, a 2 MDa, eleven subunit protein assembly, has been the most extensively characterized. The SWI/SNF complex is required for the proper expression of a number of genes in yeast, although it is completely dispensable for the expression of others. Likewise, some, but not all, transcriptional activator proteins require SWI/SNF activity in order to function in vivo. The goal of this thesis work was to identify those components of the transcription process which dictate this dependence on SWI/SNF activity. Using the well characterized UASGALsystem, we have determined that one of these components is the nucleosome state of activator binding sites within a promoter. We find that while SWI/SNF activity is not required for the GAL4 activator to bind to and activate transcription from nucleosome-free binding sites, the complex is required for GAL4 to bind and function at low affinity, nucleosomal binding sites in vivo. The SWI/SNF -dependence of these nucleosomal binding sites can be overcome by 1) replacing the low affinity sites with higher affinity, consensus GAL4 binding sequences, or 2) placing the low affinity sites into a nucleosme-free region. These results provide the first in vivo evidence that the SWI/SNF complex can regulate gene expression by modulating the DNA binding of a transcriptional activator protein. To determine whether specific components of the GAL4 protein are necessary in order for the SWI/SNF complex to modulate binding to nucleosomal sites in our model system, we tested the SWI/SNF-dependent DNA binding of various derivative GAL4 proteins. We find that a functional activation domain is not required for SWI/SNF to modulate GAL4 binding in vivo. Interestingly, like the full length protein, GAL4 derivatives in which the activation domain has been mutated are able to partially occupy nucleosomal sites in the absence of SWI/SNF (binding in the absence of SWI/SNF is at least forty percent lower than in the presence of SWI/SNF), indicating the activation domain is also not required for SWI/SNF-independent DNA binding. These results support a model in which the SWI/SNF-dependence of a gene reflects the nucleosomal context of its important regulatory sequences, e.g. binding sites for transcriptional regulatory proteins. Although nucleosomal promoter regions have been correlated with SWI/SNF-dependence in the past, there has of yet been no gene at which nucleosome location has correlated with a specific genetic function. In the final part of this thesis work, we initiated a search for an endogenous SWI/SNF-dependent gene for which the nucleosome state of activator binding sites could be determined.

Synapse development and injury-induced reorganization in the nervous system have been extensively characterized morphologically, although, relatively little is known regarding the molecular and biochemical events that underlie these processes. In an attempt to better understand, at the molecular level, the role of the expression of synaptic proteins during synapse establishment and regeneration, this dissertation examines the dynamics of expression of the neuron-specific gene synapsin I during development and in response to selective lesions of the rat central nervous system. Synapsin I is the best characterized member of a family of nerve-terminal specific phosphoproteins implicated in the regulation of neurotransmitter release. During development, the expression of synapsin I correlates temporally and topographically with synapse formation, and recent physiological studies by Lu et al., (1992) have suggested that synapsin I may participate in the functional maturation of synapses. To better understand the temporal relationship between synapsin I gene expression and particular cellular events during development, we have used in situhybridization histochemistry to localize synapsin I mRNA in the rat central and peripheral nervous systems throughout embryonic and postnatal development, and into the adult period. During development, from the earliest embryonic time point examined (E12), the expression of the synapsin I gene was detectable in both the rat central and peripheral nervous systems. While, in general, levels of synapsin I mRNAs were high in utero, synapsin I cDNA probes revealed specific patterns of hybridization in different regions of the embryonic nervous system. To precisely determine the temporal onset of expression of the synapsin I gene during neuronal development, we examined in detail the appearance of synapsin I mRNA during the well characterized postnatal development of the cerebellum and hippocampus. In both regions, the onset of synapsin I gene expression correlated with the period of stem cell commitment to terminal differentiation. In a second phase, in accord with prior analyses, synapsin I gene expression increases to a maximum for a given neuronal population during synapse formation. In the adult rat brain, our data demonstrates a widespread yet regionally variable pattern of expression of synapsin I mRNA similar to that seen at earlier time points, with noteworthy exceptions. The greatest abundance of synapsin I mRNA was found in the pyramidal neurons of the CA3 and CA4 fields of the hippocampus, and in the mitral and internal granular cell layers of the olfactory bulb. Other areas abundant in synapsin I mRNA were the layer n neurons of the piriform and entorhinal cortices, the granule cell neurons of the dentate gyrus, the pyramidal neurons of hippocampal fields CA1 and CA2, and the cells of the parasubiculum. In general, the pattern of expression of synapsin I mRNA paralleled those encoding other synaptic terminal-specific proteins, such as synaptophysin, VAMP-2, and SNAP-25. Then, to determine specifically how synapsin I mRNA levels are related to levels of synapsin I protein in the adult rat brain, we employed in situhybridization histochemistry and immunohistochemistry to examine in detail the local distribution of both synapsin I mRNA and protein in the hippocampus. In short, these data revealed differential levels of expression of synapsin I mRNA and protein within defined synaptic circuits of the rat hippocampus. Based on these data we hypothesized that locally high levels of synapsin I mRNA in neuronal somata may reflect the ability of the nervous system to respond to select enviromental stimuli and/or injury by producing longterm changes in synaptic circuitry. To test this hypothesis and to better understand the regulation and putative role of synapsin I gene expression in the development of functional synapses in the central nervous system, we first examined the developmental pattern of expression of the synapsin I gene; in dentate granule neurons of the dentate gyrus and their accompaning mossy fibers during the main period of synaptogenic differentiation in the rat hippocampus. The results of these studies indicate a significant difference between the temporal expression of synapsin I mRNA in dentate granule cell somata and the appearence of protein in their mossy fiber terminals during the posmatal development of these neurons. Next, to investigate the regulation and putative role of synapsin I gene expression during the restoration of synaptic contacts in the central nervous system, we examined the expression of the synapsin I mRNA and protein following lesions of hippocampal circuitry. These studies show marked changes in the pattern and intensity of synapsin I immunoreactivity in the dendritic fields of dentate granule cell neurons following perforant pathway transection. In contrast, changes in synapsin I mRNA expression in target neurons, and in those neurons responsible for the reinnervation of this region of the hippocampus, were not found to accompany new synapse formation. On a molecular level, both developmental and lesion data suggest that the expression of the synapsin I gene is tightly regulated in the central nervous system, and that considerable changes in synapsin I protein may occur in neurons without concommitant changes in the levels of its mRNA. From a functional standpoint, our results suggest that the appearance of detectable levels of synapsin I protein in developing and sprouting synapses does not reflect simply synaptogenesis, but coincides with the acquisition of function by those central synapses.

Transcriptional regulation of cell cycle controlled genes is fundamental to cell division in eukaryotes and a broad spectrum of physiological processes directly related to cell proliferation. Expression of the cell cycle dependent human H4, H3 and H1 histone genes is coordinately regulated at both the transcriptional and posttranscriptional levels. We have systematically analyzed the protein/DNA interactions of the immediate 5'regions of three prototypical cell cycle controlled histone genes, designated H4-F0108, H3-ST519 and H1-FNC16, to define components of the cellular mechanisms mediating transcriptional regulation. Multiple biochemically distinct protein/DNA interactions were characterized for each of these genes, and the binding sites of several promoter-specific nuclear DNA binding activities were delineated at single nucleotide resolution using a variety of techniques. These findings were integrated with results obtained by others and revealed that the in vitro factor binding sites in H4, H3 and H1 histone promoters coincide with genomic protein/DNA interaction sites defined in vivofor the H4-F0108 and H3-STS19 genes, and with evolutionarily conserved cis-acting sequences shown to affect the efficiency of histone gene transcription. Specifically, we have defined binding sites for Sp1, ATF, CP1/NF-Y, HiNF-D, HiNF-M, HiNF-P and HMG-I related factors. Based on sequence-similarities and cross-competition experiments, we postulate that most of these protein/DNA interaction elements are associated with more than one class of histone genes. Thus, the protein/DNA interactions characterized in this study may represent components of a cellular mechanism that couples transcription rates of the various histone gene classes. Regulation of the protein/DNA interactions involved in transcriptional control of these H4, H3 and H1 histone genes was investigated in a spectrum of cell types using several distinct in vitro cell culture models for the onset of differentiation and quiescence, as well as cell cycle progression. Moreover, we studied control of histone gene associated DNA binding activities during hepatic development from fetus to adult in transgenic mice reflecting the onset of differentiation and quiescence in vivo. We show that the H4 histone promoter protein/DNA interaction mediated by factor HiNF-D is selectivelymodulated, and directly at the level of DNA binding activity, during the entry into, progress through and exit from the cell cycle in normal diploid cells, as well as during hepatic development. The regulation of this protein/DNA interaction occurs in parallel with analogous interactions occurring in H3 and H1 histone genes. Moreover, these proliferation-specific protein/DNA interactions are collectively deregulated during the cell cycle in four distinct cell types displaying properties of the transformed phenotype. Hence, the cellular competency to coordinately transcribe distinct classes of histone genes during the cell cycle may be mediated by the intricate interplay of constitutively expressed general transcription factors and temporally regulated, cell growth controlled nuclear factors interacting specifically with cell cycle dependent histone genes. Finally, we show that HiNF-D is represented by two electrophoretically distinct species. The ratio of these forms of HiNF-D fluctuates dramatically during the cell cycle of normal diploid cells, but remains relatively constant in tumor cells. Total HiNF-D binding activity embodied by both HiNF-D species is negatively influenced in vitro by incubation with exogenous phosphatase activity. These observations provide a first indication for the hypothesis that HiNF-D may exist in distinct post-translationally modified forms that are subject to a stringent cell growth control mechanism involving protein kinases and phosphatases. Such a cellular post-translational modification mechanism, which directly impinges on (or activates) the DNA binding activity of a key factor controlling histone genes, would provide a highly efficient means by which to influence the rate of transcription in rapid response to intra-cellular requirements for histone mRNA and extra-cellular cues signalling the onset and cessation of cell proliferation.

HIV-1 protease is a very important drug target for AIDS therapy. Nine protease inhibitors have been proved by FDA and used in AIDS treatment. Due to the high replication rate and the lack of fidelity of the HIV-1 reverse transcriptase, HIV-1 virus developed various drug-resistant variants. Although experimental methods such as crystallography and isothermal titration calorimetry provide structural and thermodynamic data on drug-resistant variants, they are unable to discern the mechanism by which the mutations confer resistance to inhibitors. Understanding the drug-resistance mechanism is crucial for developing new inhibitors more tolerant to the drug-resistant mutations. Computational methods such as free energy calculations and molecular dynamic simulations can provide insights to the drug resistance mechanism at an atomic level. In this thesis, I have focused on the elucidation of the energetic and dynamics of key drug-resistant variants of HIV-1 protease. Two multi-drug resistant variants, in comparison with wild-type HIV-1 protease were used for the comparisons: Flap+ (L10I, G48V, I54V, and V82A) which contains a combination of flap and active site mutations and ACT (V82T, I84V) that only contains active site mutations. In Chapter II, I applied free energy simulations and decomposition methods to study the differential mechanism of resistance to the two variants, Flap+ and ACT, to the recently FDA-approved protease inhibitor darunavir (DRV). In this study, the absolute and relative binding free energies of DRV with wild-type protease and the two protease variants were calculated with MM-PB/GBSA and thermodynamic integration methods, respectively. And the predicted results are in good agreement with the ITC experimental results. Free energy decomposition elucidates the mutations alter not only its own interaction with DRV but also other residues by changing the geometry of binding pocket. And the VdW interactions between the bis-THF group of DRV is predominant even in the drug-resistant variants. At the end of this chapter, I offer suggestions on developing new inhibitors that are based on DRV but might be less susceptible to drug-resistant mutations. In Chapter III, 20-ns MD simulations of the apo wildtype protease and the apo drug-resistant protease variant Flap+ are analyzed and compared. In these studies, these mutations have been found to decrease the protease flexibility in the apo form but increase the mobility when the protease is binding with inhibitor. In Chapter IV, more details of the free energy simulation and decomposition are discussed. NMR relaxation experiments were set up as a control for the MD simulation study of the dynamics of the Flap+ variant. The difficulty of finishing the NMR experiment is discussed and the solution and some preliminary results are shown. In summary, the scope of this thesis was to use computational methods to study drug-resistant protease variants’ thermodynamic and dynamic properties to illuminate the mechanism of protease drug resistance. This knowledge will contribute to rational design of new protease inhibitors which bind more tightly to the protease and hinder the development of drug-resistant mutations.

The development of methods and techniques is the driving force of scientific research. In this work, we described two large-scale screens in studying transcriptional activation and tumor suppression. In Part I, we studied transcriptional activation mechanisms by deriving and characterizing activation defective mutants. Promoter-specific transcriptional activators stimulate transcription through direct interactions with one or more components of the transcription machinery, termed the “target.” The identification of direct in vivo targets of activators has been a major challenge. We perform a large-scale genetic screen to derive and characterize tra1 alleles that are selectively defective for interaction with Gal4 in vivo. Utilizing these mutants, we demonstrated that Tra is an essential target for Gal4 activation, Gal4 and Tra1 bind cooperatively at the promoter and the Gal4–Tra1 interaction occurs predominantly on the promoter. In addition, we demonstrated that the Gal4-interaction site on Tra1 is highly selective. In Part II, we described a functional genomics approach to discover new tumor suppressor genes. A goal of contemporary cancer research is to identify the genes responsible for neoplastic transformation. Cells that are immortalized but non-tumorigenic were stably transduced with pools of short hairpin RNAs (shRNAs) and tested for their ability to form tumors in mice. ShRNAs in any resulting tumors were identified by sequencing to reveal candidate TSGs, which were then validated both experimentally and clinically by analysis of human tumor samples. Using this approach, we identified and validated 33 candidate TSGs. We found that most candidate TSGs were down-regulated in >70% of human lung squamous cell carcinoma (hLSCC) samples, and 17 candidate TSGs negatively regulate FGFR signalling pathway, and their ectopic expression inhibited growth of hLSCC xenografts. Furthermore, we suggest that by examining at the expression level of TSGs in lung cancer patients, we can predict their drug responsiveness to FGFR inhibitors. In conclusion, we have identified many new lung squamous cell cancer TSGs, using an experimental strategy that can be broadly applied to find TSGs in other tumor types.