The aryl hydrocarbon receptor (AhR) is a ligand-activated bHLH-PAS protein that binds its partner, the aryl hydrocarbon receptor nuclear translocator (Arnt), in the nucleus to initiate the expression of proteins involved with detoxification. Published work suggests cross-talk between both proteins and cellular pathways involving the transcription factors, HIF -1 , ER, and NFKB, whose activity is typically upregulated in cancer. This thesis focuses on using a truncated form of AhR, AhR CΔ553, which is thought to act as a dominant-negative to sequester Arnt from its other binding partners. To test this hypothesis, we transfected HeLa cells with AhR CΔ553 fused to pEGFP or a vector under a tetracycline-inducible promoter. Stable cell lines expressing pEGFP-AhR CΔ553 have been generated and confirmed to have nuclear localization. We were also interested in confirming endogenous localization patterns of AhR and Arnt to study the role of p23 in the nuclear translocation of AhR. While we were successful in showing AhR translocating to the nucleus in treated MCF-7 cells, we couldn't clearly see nuclear AhR in Hepalclc7 cells, the cell line with knockdown levels of p23. To compare DNA damage generated in Jm·kat and Hepalclc7 cells, we looked for reactive oxygen species (ROS) production and quantified DNA damage after exposure to benzo[a]pyrene (B[a]P) and some of its derivatives. Hepalclc7 cells were prone to a wide variety of DNA damage, but Jurkat cells did not appear to undergo damage specifically through ROS production. Finally, we wanted to confirm apoptosis in HeLa cells after being cocultured with Trichomonas vaginalis. The G3 lab strain was more aggressive than Tl , but Parp, and apoptotic marker, was not observed in HeLa cells, suggesting that experimental conditions need to be further optimized.

Indiana University-Purdue University Indianapolis (IUPUI) / Human papillomaviruses (HPVs) are one of the most common causes of sexually transmitted disease in the world. HPVs are divided into high-risk (HR) or low-risk (LR) types based on their oncogenic potential. HPVs 16 and 18 are considered HR types and can cause cervical cancer. HPVs 6 and 11 are classified as LR and are associated with condyloma acuminata (genital warts). Viral proteins of both HR and LR HPVs must be able to facilitate a replication competent environment. The E7 proteins of LR and HR HPVs are responsible for maintenance of S-phase activity in infected cells. HR E7 proteins target all pRb family members (pRb, p107 and p130) for degradation. LR E7 does not target pRb or p107 for degradation, but does target p130 for degradation. Immunohistochemistry experiments on HPV 6 infected patient biopsies of condyloma acuminata showed that detection of p130 was decreased in the presence of the whole HPV 6 genome. Further, the effect of HR HPV 16 E7 and LR HPV 6 E7 on p130 intracellular localization and half-life was examined. Experiments were performed using human foreskin keratinocytes transduced with HPV 6 E7, HPV 16 E7 or parental vector. Nuclear/cytoplasmic fractionation and immunofluorescence showed that, in contrast to control and HPV 6 E7-expressing cells, a greater amount of p130 was present in the cytoplasm in the viii presence of HPV 16 E7. The half-life of p130, relative to control cells, was decreased in the cytoplasm in the presence of HPV 6 E7 or HPV 16 E7, but only decreased by HPV 6 E7 in the nucleus. Inhibition of proteasomal degradation extended the half-life of p130, regardless of intracellular localization. Experiments were also conducted to detect E7-binding partners. Cyclin C and cullin 5 were identified as proteins capable of binding to both HPV 6 E7 and HPV 16 E7. Preliminary experiments showed that decreasing protein levels of p600, a binding partner of both HPV 6 E7 and HPV 16 E7, by RNA interference might affect p130 stability. Elucidating the mechanisms of p130 degradation may identify potential targets for preventing degradation of p130 and allowing restoration of cell cycle control.

This study investigated the effects of ionic strength, pH, gum concentration, and protein type on protein - xanthan gum interactions. Commercial soy sauce and tamari sauce as well as model systems of soy protein isolate and whey protein concentrate were the sources of protein used for evaluation with xanthan gum. Preliminary research indicated that when either soy sauce or tamari sauce were mixed with xanthan gum, stable solutions with notable viscosity synergisms resulted. The soy protein and whey protein systems were subsequently prepared with a range of 0 to 5% added sodium chloride. Results indicated that an equilibrium existed between proteins and xanthan gum such that increased sodium chloride initially increased solution stability; but when in excess, the sodium chloride led to a loss of protein - xanthan gum solution solubility and in some cases to precipitation. Precipitation was also noted at the pH extremes of 2,3, and 9 and when xanthan gum was present in excess, or at 0.25%. The effects of sodium chloride, protein type, and pH on the rheological parameters of model solutions were also examined. Higher sodium chloride levels yielded greater viscosity synergisms. Those solutions made.with intact protein were generally higher in apparent viscosity than similar solutions made with hydrolyzed protein. Solutions at pH 5 were generally higher in viscosity than were similar solutions at pH 7. Several factors that appeared to affect the stability, solubility, and the rheological parameters of protein - xanthan gum solutions were sodium chloride concentration, gum concentration, pH, and protein type. / M.S.

Clusters in protein interaction networks can potentially help identify functional relationships among proteins. We study the clustering problem by modeling it as graph cut problems. Given an edge weighted graph, the goal is to partition the graph into a prescribed number of subsets obeying some capacity constraints, so as to maximize the total weight of the edges that are within a subset. Identification of a dense subset might shed some light on the biological function of all the proteins in the subset. We study integer programming formulations and exhibit large integrality gaps for various formulations. This is indicative of the difficulty in obtaining constant factor approximation algorithms using the primal-dual schema. We propose three approximation algorithms for the problem. We evaluate the algorithms on the database of interacting proteins and on randomly generated graphs. Our experiments show that the algorithms are fast and have good performance ratio in practice. / xiii, 71 leaves : ill. ; 29 cm.

Activity-induced immediate-early gene (IEG) transcription foci can be labelled with fluorescent probes, permitting high temporal and spatial resolution in mapping neuronal circuits. Previous quantification approaches have assumed a Boolean function of transcription foci, assuming that cells are either active or inactive. Due to multiple amplification steps in the in situ hybridization process, it was thought that information relating to magnitudes of firing rates was lost. However, the current data suggest that transcription foci actually exhibit non-Boolean intensity and size values which vary according to behavioural condition. Systematic characterization of transcription foci intensity and size revealed incremental variations such that: home-cage < one-environment exposure < five-environment exposure < maximal electroconvulsive shock. Visual differences in transcription foci may result from a quantifiable relationship between spiking patterns and transcription rates. The exact stoichiometry between neuronal spiking and transcription is not yet clear, but these results suggest that Boolean applications of IEG imaging may neglect accurate neuronal activation properties. / xvi, 125 leaves : ill. ; 29 cm

Spider attachrnentdisc silk fibers are spun into a viscous liquid that rapidly solidifies, gluing dragline silk fibers to substrates for locomotion or web construction. Here we report the identification and artificial spinning of a novel attachment disc glue silk fibroin, Pyriform Spidroin 2 (PySp2), from the golden orb weaver Nephila c/avipes. MS studies support PySp2 is a constituent of the pyriform gland that is spun into attachment discs. Analysis of the PySp2 protein architecture reveals sequence divergence relative to the other silk family members, including the cob weaver glue silk fibroin PySpl. PySp2 contains internal block repeats that consist of two sub-repeat units: one dominated by Ser, Gin and Ala, the other Pro-rich. Artificial spinning of recombinant PySp2 truncations shows that the Ser-Gln-Ala-rich sub-repeat is sufficient for the assembly of polymeric subunits and subsequent fiber formation. These studies support that both orb- and cob-weaving spiders have evolved highly polar block-repeat sequence with the ability to self-assemble into fibers, suggesting a strategy to allow fiber fabrication in the liquid environment of the attachment discs.

Spider silk is renowned for its extraordinary mechanical properties, having a balance of high tensile strength and extensibility. To date, the majority of studies have focused on the production of dragline silks from synthetic spider silk gene products. Here we report the first mechanical analysis of synthetic egg case silk fibers spun from the Latrodectus hesperus tubuliform silk proteins, TuSp1 and ECP-2. We provide evidence that recombinant ECP-2 proteins can be spun into fibers that display mechanical properties similar to other synthetic spider silks. We also demonstrate that silks spun from recombinant thioredoxin-TuSp 1 fusion proteins that contain the conserved C-terminal domain exhibit increased extensibility and toughness when compared to the identical fibers spun from fusion proteins lacking the C-terminus. Mechanical analyses reveal that the properties of synthetic tubuliform silks can be modulated by altering the post-spin draw ratios of the fibers . Fibers subject to increased draw ratios showed elevated tensile strength and decreased extensibility, but maintained constant toughness. Wide-angle X-ray diffraction studies indicate that post-drawn fibers containing the Cterminal domain of TuSp 1 have more amorphous content when compared to fibers lacking the C-terminus. Taken together, these studies demonstrate that recombinant tubuliform spidroins that contain the conserved C-terminal domain with embedded protein tags can be effectively spun into fibers, resulting in similar tensile strength but increased extensibility relative to non-tagged recombinant dragline silk proteins spun from equivalently sized proteins.

One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c.

Indiana University-Purdue University Indianapolis (IUPUI) / The addition and removal of phosphate groups is a key regulatory mechanism for many cellular processes. The balance between phosphorylation and dephosphorylation is delicate and must be maintained in order for proper cell functions to be carried out. Protein kinases and phosphatases are the keepers of this balance with kinases adding phosphate groups and phosphatases removing them. As such, mutation and/or altered regulation of these proteins can be the driving factor in disease. Phosphatase of Regenerating Liver (PRL) is a family novel of three dual specificity phosphatases (DSPs) first discovered in the regenerating liver tissue of rats. PRLs have also been shown to act as oncogenes in cell culture and in animal models. However, the physiological substrate and mechanisms of the PRLs are not yet known. Recently, our lab has developed a PRL 2 knockout mouse and found several striking phenotypes all of which correspond to a significant increase in PTEN. We also found that PRL 2 is targetable by small molecular inhibitors that can potentially be used to disrupt tumor growth and spermatogenesis. Furthermore, a PTEN heterozygous mouse model crossed into our PRL 2 knockout line was generated to investigate the relevance of PRL interaction with PTEN in cancer.

Basic helix-loop-helix (bHLH) proteins are involved in the regulation of a multitude of developmental processes including cellular differentiation, cellular proliferation and xenobiotic metabolism. Among the members of the bHLH protein family are the products of the Pan gene Pan-1, Pan-2 and ITF -1. Pan proteins have been demonstrated to be required for proper B cell development, suggesting a unique role for Pan proteins during B cell formation. In our study we tested the function of ARNT (Ah receptor nuclear translocator) at the IgH (immunoglobulin heavy chain) enhancer. We were able to determine that ARNT appears to partially down-regulate activation at the IgH enhancer by Pan-1 in transient transfection assays by cotransfection of the multimerized murine form of the IgH enhancer elements 1-1E2, !-LE3 , and 1-1ES upstream of a luciferase reporter gene, a rodent Pan-1 (human homolog E47) expression vector, and an ARNT expression vector. Furthermore, during our investigation we discovered a putative ARNT -binding ligand that increases DNA-binding activity of the ARNT homodimer. This ligand was partially characterized by UV crosslinking studies and a variety of biochemical studies using electrophoretic mobility-shift assays. Preliminary data suggests that it is hydrophilic, heat-stable, small, and non-protein.