Breast cancer is the most frequent tumor type among women. Heightened susceptibility of the breast to tumor development has been associated with early menarche, nulliparity, exposures to ionizing radiation, and family history, but the underlying molecular mechanisms are poorly understood. Unfortunately, the etiology of breast cancer is complex and is complicated by the fact that it is a heterogeneous disease. The p53 tumor suppressor gene was altered in a large proportion of these spontaneous breast tumors implicating its involvement in the progression of breast cancer development. The aim of this dissertation was to determine the regulation of p53 in the normal mammary gland and whether it is involved in suppressing the development of mammary tumors. To evaluate the effect of p53 on mammary tumor formation, the first component of this work involved the characterization of BALB/c- p53-deficient mice. BALB/c-p53+/− and p53−/− mice were examined for tumor spectrum and mammary abnormalities. Mammary transplants were performed to evaluate the role of p53 in tumor suppression in the mammary gland. This work demonstrated that p53 is critical in suppressing mammary tumorigenesis in the mammary gland as BALB/c mice deficient in p53 readily develop mammary carcinomas. The second element of this project examined the expression, localization and activity of p53 in normal mammary tissues. Since the mammary gland is a tissue that is sensitive and responsive to local and systemic hormones, the last chapter of this dissertation focused on the hormonal effects on p53 activity. Results from these experiments demonstrated that p53 was expressed at high levels localized to the cytoplasm of the ductal epithelium of the quiescent mammary gland. P53 was not responsive to radiation-induced DNA damage suggesting its function is compromised in the nulliparous mammary gland. Further experiments demonstrated that the functional state of wild type p53 in the mammary epithelium could be regulated by hormonal stimuli.

Homologous recombination is generally considered a major mechanism by which cells repair many types of DNA lesions and damaged replication forks. However, if this process is left unchecked, cells often show a hyper-recombination (hyper-rec) phenotype, and are susceptible to large deletions, duplications, or inversions of important genetic information. This dissertation describes two projects aimed at examining molecular mechanisms by which cells regulate homologous recombination. The first shows several 3'-5' exonucleases prevent RecA-GFP loading by destroying potential substrates. It is shown that two genetic pathways exist: one consisting of ExoIII and another comprised of ExoVII, ExoIX, ExoX, and ExoXI. ExoI acts upstream of both of these pathways. Although xthA cells have an increase in DSBs and recB-dependent loading of RecA-GFP, they are viable with a recB mutation and do not display a large increase in SOS expression. The increase in RecA-GFP is also independent of base excision repair (BER). These experiments uncovered that DNA in a population of wild type cells undergoes DSBs and is often repaired in a RecA-independent manner after processing by ExoI and ExoIII. The second project shows the helicase, UvrD limits the number and intensities of RecA-GFP foci. This activity is due to the ability of UvrD to remove RecA from DNA where it is loaded in a RecF pathway-dependent manner. This activity requires ATP binding by UvrD, suggesting that helicase/translocase activity is important for RecA-removal. The hyper-helicase mutation, uvrD303 confers UV sensitivity to cells. Epistasis analyses showed uvrD303 is defective in the recA pathway of UV repair and not in nucleotide excision repair (NER). Surprisingly, UvrD303 does not directly remove RecA after UV, as new RecA-GFP foci appear like in wild type cells. UvrD303 does, however, slightly inhibit SOS induction, and constitutively activating the SOS response restores UV resistance to these cells in a way that is independent of recA overexpression. Furthermore, uvrD303 was capable of suppressing the constitutive SOS phenotype of recA730. These experiments suggested that UvrD303 antagonizes the ability of RecA filaments to induce the SOS response, rendering cells UV sensitive.

The budding yeast (S. cerevisiae) centromere component, Cse4p is an evolutionarily conserved histone H3-like protein, with homologues identified in fission yeast, worm, fly and human. All histone H3-like proteins have C-terminal histone fold domains (HFD) that are highly similar to the HFD of H3, but carry very different N-termini with unknown functions. The Cse4p N-terminus contains 135 residues, with a large portion of charged amino acids and a high concentration of serines within the first 22 residues. Based on the current model that suggests that Cse4p replaces H3 in a specialized centromeric nucleosome, the Cse4p N-terminus would extend out from the putative Cse4p-nucleosome and may play a variety of roles in centromere function. To elucidate the function of the Cse4p N-terminus, we conducted two comprehensive and systematic mutagenesis studies involving alanine scanning and sequence deletions, and we defined a 33-amino acid domain that is essential for cell viability and chromosome segregation. This essential N-terminal domain (END) has functions distinct from that of the HFD as demonstrated by interallelic complementation between cse4 END and HFD mutant alleles and heterodimer formation of END-HFD mutant proteins. Mutating all the potential posttranslational sites in the END indicates that the END function does not require posttranslational phosphorylation or acetylation. Genetic studies involving dosage suppression, synthetic lethality and two-hybrid analysis reveal that the END interacts with the Ctf19p/Mcm21p/Okp1p kinetochore complex. These results are consistent with the current Cse4p-nucleosome model. Although Cse4p has an HFD resembling that of H3, unlike H3, Cse4p exclusively localizes at the centromere. An important question is whether the N-terminus of Cse4p is responsible for the specific centromere targeting of the protein. Lethal Cse4p proteins lacking regions of the N-terminus can localize to the centromere in the presence or absence of wildtype Cse4p as determined by chromatin immunoprecipitation. In contrast, some lethal Cse4p HFD mutant proteins as well as chimeric proteins consisting of the Cse4p N-terminus fused to the HFD of either H3 or the Cse4p human homologue, CENP-A, fail to localize to the centromere. We conclude that the N-terminus of Cse4p is not required for centromere targeting of the protein and that the Cse4p HFD is necessary and sufficient to confer centromere localization.

Actinomycetes have been used with enormous success in industrial processes; however, little is known about biofilm development by these filamentous microbes, and the presence of community development on insoluble cellulosic substrates such as cellulose. Cellulose is the most abundant biopolymer and renewable energy source on Earth, and its decomposition, which is carried out almost exclusively by microorganisms, is a key step in the cycling of carbon in the biosphere. It has long been known that cellulolytic bacteria may adhere to their insoluble substrate as it is degraded, although surprisingly little is known about microbial growth, colonization and community development on insoluble cellulosic substrates and non-nutritive surfaces. Previous investigations indicated that two Gram-positive cellulolytic soil bacteria, Cellulomonas uda, a facultative aerobe, and Clostridium phytofermentans , an obligate anaerobe, specifically adhered to nutritive surfaces forming a biofilm, but cells did not colonize non-nutritive surfaces. In this study is hypothesized that biofilm formation is a general strategy used by microbes in the degradation of insoluble substrates, and that it may serve as a means for microbes to secure a nutrient and persist in their environments. The objective of this study was to characterize biofilms produced by Thermobifida fusca, a Gram-positive cellulolytic actinomycete isolated from compost that rapidly degrades cellulose by means of a well-characterized extracellular cellulase system, and is a causative agent of Farmers Lung, the most common type of hypersensitivity pneumonitis. T. fusca was cultured with dialysis tubing as a nutritive surface for biofilm formation, and by using non-nutritive surfaces such as glass, plastic, metal and Teflon. Dialysis tubing was colonized by T. fusca aleuriospores but not by mycelial pellets. Surface-attached growth, examined by confocal scanning laser and scanning electron microscopy revealed structures resembling biofilms with cells embedded in fibrous material suggestive of an exopolymeric (EPS) matrix. T. fusca cells possessed higher hydrophobicity than C. uda and C. phytofermentans cells implicating higher capacity to bind to surfaces. DNase1 inhibited biofilm formation when assayed on microtiter plates suggesting a role for extracellular DNA in T. fusca biofilm formation. Concanavalin-A bound to the EPS material of biofilms and mycelial pellets, indicating alpha-linked D-mannosyl and/or alpha-linked D-glucosyl residues. The carbohydrate content of biofilms and mycelial pellets increased during growth. T. fusca biofilm formation is reduced when lack or excess of nutrients such as; iron, nitrogen and salt. Robust biofilms were developed between pHs 7 and 9, whereas minimum biofilms were produced at pH 3 and 11. Cellulose degradation rate and celE (endoglucanase E5) expression was similar for T. fusca biofilms and mycelial pellets. Also, results of this study indicate that in the life cycle of this actinomycete, cellulose is specifically colonized by aleuriospores, which germinate and degrade cellulose, ultimately developing into biofilms encased in a carbohydrate-containing EPS matrix, a hallmark of biofilm production.

The development and maintenance of multicellular organisms depends fundamentally on cell division, a series of events largely mediated by the mitotic spindle. Errors in spindle formation and/or function are often associated with severe consequences, most notably cancer. In order to elucidate the cause of such errors and the potential for therapeutic intervention, it is imperative to attain a clear understanding of how cell division normally operates. In this regard, this dissertation focuses on the activity of two microtubule-based motor proteins, Eg5 and dynein, prior to and immediately following nuclear envelope breakdown during mitosis. I show that prophase microtubules are remarkably more dynamic than their metaphase counterparts, moving both toward and away from centrosomes across a wide distribution of rates. Inhibition of Eg5, dynein and Kif2a revealed that a subset of this motion is consistent with microtubule flux, a well-established phenomenon temporally limited to metaphase and anaphase spindles by the preceding literature. My data indicates that flux is operational throughout all of mitosis, possibly functioning at early stages to collect centrosomal components. Immediately following prophase, cells begin assembling bipolar spindles. While the establishment of spindle bipolarity fails in the physical or functional absence of Eg5, I show that co-inhibition of dynein restores a cell's ability to organize microtubules into a bipolar structure. Despite inhibition of both Eg5 and dynein, these spindles are morphologically and functionally equivalent to controls. Together, these data suggest that Eg5 and dynein share an antagonistic relationship and that a balance of forces, rather than a definitive set of players, is important for spindle assembly and function. To determine how Eg5- and dynein-mediated forces functionally coordinate to bring about antagonism during spindle assembly, I utilize a nocodazole washout assay. I show, via in vivo imaging and in silico modeling, that spindle collapse in the absence of functional Eg5 requires dynein activity and an initial intercentrosomal distance of less than 5.5μm. These data are consistent with a model in which dynein antagonizes Eg5 by crosslinking and sliding antiparallel microtubules, a novel role for dynein within the framework of spindle assembly.

Chloroplast biogenesis relies on the import of thousands of nuclear encoded proteins into the organelle and proper sorting to their sub-organellar compartment. The majority of nucleus-encoded chloroplast proteins are synthesized in the cytoplasm and imported into the organelle via the Toc-Tic translocation systems of the chloroplast envelope. In many cases, these proteins are further targeted to subcompartments of the organelle (e.g. the thylakoid membrane and lumen or inner envelope membrane) by additional targeting systems that function downstream of the import apparatus. The inner envelope membrane (IEM) plays key roles in controlling metabolite transport between the organelle and cytoplasm, and is the major site of lipid and membrane biogenesis within the organelle. In contrast to the protein import and thylakoid targeting systems, our knowledge of the pathways and molecular mechanisms of protein targeting and integration at the IEM are very limited. Previous reports have led to the conclusion that IEM proteins are transferred to the IEM during protein import via a stop-transfer mechanism. Recent studies have shown that at least two components of the Tic machinery (AtTic40 and AtTic110) are completely imported into the stroma and then re-inserted into the IEM in a post-import mechanism. This led me to investigate the mechanisms and pathways involved in the integration of chloroplast IEM proteins in more detail. I selected candidates (AtTic40 for post-import and IEP37 for stop-transfer) that are predicted to have only one membrane-spanning helix and adopt the same IEM topology to facilitate my analysis. My studies confirm the existence of both stop-transfer and post-import mechanisms of IEM protein targeting. Furthermore, I conclude that the IEP37 transmembrane domain (TMD) is a stop-transfer signal and is able of diverting AtTic40 to this pathway in the absence of AtTic40 IEM targeting information. Moreover, the IEP37 TMD also functions as a topology determinant. I also show that the AtTic40 targeting signals are context dependent, with evidence that in the absence of specific information in the appropriate context, the AtTic40 TMD behaves as a stop-transfer signal. This is an indication that the stop-transfer pathway is the default mechanism of protein insertion in the IEM.

The functional aspects of specific mutations, R30K, S291G, and I392T, found associated with AChE gene of OP- and carbamate-resistant Colorado potato beetle (CPB) were determined using recombinant AChEs. From the previous studies, azinphosmethyl resistant CPB strain (AZ-R) possesses S291G and R30K mutations and their AChE was 16-fold less sensitive to azinphosmethyl than that of insecticide-susceptible (SS) strain, in terms of biomolecular rate constant (ki). A carbamate-resistant strain (BERTS) showed high resistance to carbofuran (12-fold), but was relatively less resistant to azinphosmethyl (1.35-fold) than SS CPB. The AZ-R strain was 2.6-fold more resistant to carbofuran and 8-fold more resistant to azinphosmethyl than SS CPB, respectively, in a discriminating dose bioassay. A substrain of BERTS, BERTS-R, possessing only the S291 G mutation, elicited high resistance to carbofuran and moderate resistance to azinphosmethyl. The BERTS-S substrain, possessing S291G and I392T mutations, was susceptible to both azinphosmethyl and carbofuran. In this study, enzymatic properties of altered AChEs from CPB were examined using recombinant AChEs obtained from baculovirus expression system. The S291G mutation increased hydrolysis activity of larger substrates (e.g. BTC) and increased sensitivity to inhibition by larger inhibitors (e.g. paraoxon, DFP, and N-propyl carbofuran) in the altered recombinant AChEs. The R30K mutation caused further increasement of hydrolysis activity of larger substrates and the sensitivity to inhibition by larger inhibitors in combination with the S291G mutation. The 1392T mutation compensated the effect of S291G. Thus, the altered recombinant AChE with both S291G and I392T mutations elicited a substrate specificity and inhibitory properties more similar to the susceptible form of ACNE without mutations. Two rapid molecular diagnostic systems were successfully developed for monitoring of OP and pyrethroid resistances by determining resistant allele frequencies. These techniques were used to genotyping pyrethroid resistance in 16 field populations of CPB by detection of the kdr mutation. The predicted resistant levels of populations by genotyping were closely correlated to the esfenvalerate bioassay results by the rank-correlation coefficient of a non-parametric rank correlation test for independence. These two DNA based diagnostic procedures are reliable, accurate and affordable techniques for monitoring field population of CPB for resistance.

Small GTP-binding proteins of the Rac family are important signaling switches that regulate plant growth and development due to their ability to shuttle between the inactive GDP-bound and the active GTP-bound form. The ratio between the two forms is tightly regulated in the cell. The Arabidopsis genome encodes eleven Rac proteins designated Arac1-11. Based on the C-terminus, Aracs can be divided into typeI and typeII. TypeI Aracs associate with the membrane by prenylation while typeII Aracs associate with the membrane by palmitoylation. Differences in the effector-binding region and the C-terminal hypervariable region place Arac7 in a separate phylogenetic group than the other typeII Aracs. The work described in this dissertation examines the expression patterns, functions and regulation of the Arac7 protein and provides evidence that supports a distinct role for Arac7 within type II Aracs. Arac7 expression is shown to repress auxin-induced gene expression and to enhance ABA-stimulated gene expression in Arabidopsis. Plants overexpressing Arac7 are less responsive to auxin but show increased responses to ABA while the opposite is observed in plants with decreased levels of Arac7 mRNA. Arac7 expression is high in the lateral root primordia and can be traced back to the first pericicle divisions that give rise to these primordia. This, together with the increased number of lateral roots of plants overexpressing Arac7, is consistent with a role in lateral root formation for this small GTPase. Interestingly, transcription from the Arac7 promoter is stimulated by auxin but repressed by ABA. Together with the observed functions on auxin and ABA signaling, the regulated expression of Arac7 provides a feedback mechanism to ensure that cells maintain sensitivity to signals. This work also shows that Arac7 is constitutively associated with the plasma membrane where it partitions into detergent-resistant membrane domains (DRMs). Moreover, at the plasma membrane, the majority of Arac7 partitions into high molecular weight complexes. These properties contrast with those observed for the typeI Arac5 which is distributed between the cytosol and non-DRMs regions of the plasma membrane and exists predominantly in low molecular weight forms. Together, these observations suggest different functions and regulatory mechanisms for typeI and typeII Aracs.

Wnt ligands are a family of highly conserved glycoproteins that act as morphogens to regulate development in many organisms. Drosophila Wnt-1 (Wingless) is involved in directing cell fate decisions and pattern formation during differentiation. Wnt signaling are of high interest of many developmental biologists due to their important functions, yet little is known about how these ligands function on a biochemical level. Previously it was found that Porcupine, an ER-membrane-bound acyltransferase is required for Wingless secretion. But it is unclear how a secreted morphogen requires an acyltransferase to function. Studies reported here demonstrated that Wingless undergoes a hydrophobic modification, in which a lipid moiety containing a palmitate group is covalently attached to the polypeptide through an ester linkage. And it partitions with the specialized detergent insoluble lipid raft microdomains in the plasma membrane. Porcupine is required for the modification and the raft targeting of Wingless. Blocking Wingless modification with a specific inhibitor results in the loss of rafts-association as well as loss of protein secretion. Disrupting raft microstructures by cholesterol depletion reagents also impaired Wingless secretion, indicating that the ligand secretion is dependent on its specific association with the plasma membrane. This work provided the first insight on the function of Porcupine and the important biochemical evidence on the role of specialized membrane microdomains in Wnt signaling.

Notch is crucial for multiple stages of T cell development, including the CD4+CD8+ double positive (DP) to CD8 + single positive (SP) transition, but regulation of Notch activation is not well understood. In this thesis, I explored the potential of p53, endocytosis, and Cbl-b to regulate Notch activation. p53 regulates Presenilin1 (PS1) expression, and PS1 cleaves Notch, releasing its intracellular domain (NIC), leading to the expression of downstream targets, e.g. the HES1 gene. One aim of this thesis was to determine if p53 regulates Notch activity during T cell development. I found that Notch1 expression and activation were negatively regulated by p53 in several thymoma lines. Additionally, NIC was elevated in Trp53 −/− thymocytes as compared to Trp53 +/+ thymocytes. To determine if elevated Notch1 activation in Trp53−/− thymocytes had an effect on T cell development, CD4 and CD8 expression were analyzed. The CD4+ SP:CD8+ SP T cell ratio was decreased in Trp53 −/− splenocytes and thymocytes. This alteration in T cell development correlated with the increased Notch1 activation observed in the absence of p53. These data indicate that p53 negatively regulates Notch1 activation during T cell development. Skewing of T cell development toward CD8 + SP T cells in Trp53−/− mice is reminiscent of the phenotype of NIC-overexpressing mice. Thus, I suggest that p53 plays a role in T cell development, in part by regulating Notch1 activation. In the second aim of my thesis I present preliminary data showing that endocytosis does not appear to be involved in mammalian Notch activation although there is evidence in Drosophila for a positive endocytic role in Notch activation. The ability of Cbl-b to regulate Notch activation was the final aim of this thesis. Cbl-b, like Notch, has been shown to play a role in regulating the T cell signaling threshold. With this aim, I wanted to address the possibility of Cbl-b regulating T cell signaling via regulating Notch activation. Due to technical difficulties I was only able to obtain preliminary data suggesting that Cbl-b does positively regulate Notch activation in peripheral T cells. In this dissertation I have shown that Notch activation is regulated by controlling the expression of cellular components needed for cleavage, not just by encountering ligand on neighboring cells.