Proteases are important enzymes in the biotechnology due to their specific cleavage of substrates. HRV 3C, sortase A and TEV are some examples of cysteine proteases which become more of use lately in applications as removal of affinity tags (3C/TEV) and labelling of proteins (sortase). Here an investigation was made on the proteases by displaying them on two different prokaryotic hosts; E. coli and S. carnosus and to use these to cleave away affinity proteins (Affibody molecule) from other cells with an incorporated cleavage site. Constructs were cloned and incorporated into expressing strains which were then cultivated and induced. Analysis of surface expression was done by flow cytometer. Cleavage was made by cultivating combinations with cleavable bacteria and bacteria displaying proteases. A functional protease would lead to the presence of Affibody molecules in the supernatant. Flow cytomtery analysis was first made to inevstigate signal difference in Affibody binding by the addition of flurophores. Secondly SDS-PAGE was made on the centrifuged supernatant to investigate the presence of a product. Finally analysis of the bacteria was made by examining the reaction with soluble substrate and comparing activity with soluble enzyme. All of the enzymes were able to be displayed on the surface of bacteria with a clear separation from control. The cleavage analysis showed however varying results yet no clear evidence of product. Best flow cytometer results were seen for 3C but SDS-PAGE/MS did not show any cleaved product. For Sortase SDS-PAGE showed positive result but analysis with MS showed no product. TEV was concluded not to be funcional at all hence the failing to cleave soluble substrate  when condition seemed near optimal and faulty flow cytometer data. Even though the lack of success there is still many further studies that can be done on the proteases in order to prove its absence/presence  of activity.

Le projet de thèse à pour but de produire et caractériser un nouvel allergène de pollen d'ambroisie. Le projet est décliné en cinq axes: production d'une forme recombinante mature et en conformation native, caractérisation structurale, étude de la fonction enzymatique, étude de l'allergenicité et l'immunogénicité et évaluation du potentiel thérapeutique. / The goal of the thesis project is to product and caracterize a novel ragweed pollen allergen.The project have five axes: production of recombinant mature and native form,structurale caracterization, study of enzymatique function, study of allergenicity and immunogenicity and evaluation of therapeutic potential.

Homocysteine is a natural occurring aminothiol. It is an intermediate product in the metabolism of methionine. Methionine is an essential amino acid required for protein synthesis. Metabolic irregularities disrupt homocysteine levels in plasma. Elevated homocysteine levels are directly linked to folate and cobalamin (vitamin B12) deficiencies, and are an independent risk factor for cardiovascular diseases. High homocysteine levels have also been associated with Alzheimer's, osteoporosis, renal failure, cancer, birth defects and pregnancy complications. The association of elevated homocysteine levels with cardiovascular disease and other diseases has generated great interest in the detection of homocysteine. An optical method for the detection of homocysteine has been developed using fluorescein mono- and dialdehydes. Selectivity for homocysteine was achieved based on the characteristic differences between 5- and 6-membered ring heterocyclic amines formed upon the reaction with fluorescein mono- and dialdehydes. 6-membered ring homocysteine-derived thiazinane-4-carboxylic acids were found to be more basic than 5-membered cysteine-derived thiazolidine-4-carboxylic acids. Fluorescence enhancement in response to homocysteine was thus attained by tuning pH and excitation wavelengths. Furthermore, the design and synthesis of a more sensitive fluorophore, fluorescein tri aldehyde has been accomplished based on the aforementioned findings to enable the detection of homocysteine at physiological levels. Calculations of Mulliken charges revealed that the formation of thiazinanes results in modulation of the electron density on the fluorophore leading to higher fluorescence.

Glutathione (GSH) is an important biological thiol, it performs significant biological functions such as serving an antioxidant which protect cells from oxidative stress by trapping free radicals which damage DNA and RNA. It is known that abnormal plasma levels of GSH have been linked to various human diseases. Therefore, the rapid, sensitive and highly selective detection of GSH is of great importance for investigating its functions in diseases diagnosis. Interestingly, we found in cetyl trimethylammonium bromide (CTAB) medium, the resorufin-based probe shows an extremely fast, highly selective response to GSH. The result indicates that this dye can be employed to detect GSH in biological samples such as human plasma. Cysteine (Cys) is another important biological thiol which is involved in a variety of significant cellur functions, including protein synthesis, detoxication, and metabolic process, etc. Abnormal levels of Cys are related to many diseases, such as slowed growth, Alzheimer's disease and cardiovascular disease. Thus, the detection and quantification of Cys in physiological media is of great importance. In this thesis, I am going to present two organic fluorescent probes (Resorufin-based probe and SNF probe) for the detection and quantification of Cys. In addition, we prove that they can directly quantify Cys in human plasma. The chemical mechanisms involved in the detection of Cys are discussed.

Spiders are capable of producing a variety of silk types, each with their own unique protein composition and function. Dragline silk in particular, has been of great interest due to its high tensile strength and extensibility. In the past, synthetically produced dragline fibers have not been able to match the superior properties of natural silk. A recent discovery in the western black widow spider, Latrodectus hesperus, might be the missing link between the current state of synthetic silks and naturally produced fibers. Our research is centered around the discovery a new family of five low-molecular-weight cysteine-rich proteins (CRPs) and their potential function within dragline silk. This study focuses on the characterization of recombinantly expressed CRP1, CRP2, and CRP4. Through structural analysis using circular dichroism, it has been determined that the CRP family members have mostly alpha-helical secondary structure and exhibit small differences in their ability to maintain their structure in the presence of changing environmental conditions. The study also covers the effects of temperature and pH on the folding and unfolding of the CRPs. It appears that pH is the dominant influence on protein unfolding within the major ampullate gland.

Trichomonas vagina/is, one of the most common sexually transmitted diseases, has been shown to increase patients' susceptibility to HIV infection and cervical cancer; moreover, resistance to metronidazole is increasing, and new drug targets must be identified in order to combat resistant strains. T vagina/is expresses cysteine proteases that have been implicated in vaginal epithelial apoptosis as well as immune system evasion. In the past the various cysteine proteases have been studied as a group, and the following work examines, one specific protease, TvCP2, in detail through Western blot analysis, immunofluorescent staining, and recombinant expression. The experiments 5 presented here suggest that aT l-CP2 over-expressing transfectant line processes CP2 and sequesters it in cellular compartments. Previous data gives strong evidence of the secretion of cysteine protease CP4 and hints at the possibility of CP2 secretion as well; however, our results show no co-localization between CP2 and CP4 in T l-CP2 over expressing transfectants, suggesting separate trafficking and different roles. To better characterize CP2 function, we attempted to express active, recombinant protein. Although Pichia pastoris serves as a reliable expression vehicle, a processing event following translation ofTvCP2 appears to have cleaved the pro-domain and, along with it, the a-secretion signal, trapping active TvCP2 within the cellular pellet. A thioreoxintagged version ofTvCP2 has been expressed in E. coli, and preliminary experiments show it may auto-activate under certain conditions, but further experimentation is required to confirm the presence of active CP2 within the fraction purified from these cells.

Spiders spin a wide variety of different silk types with different biological functions that are known for their extraordinary mechanical properties. Dragline silk has predominantly captured the interest of researchers because it exhibits high tensile strength and toughness while maintaining its elasticity. This thesis has focused on the characterization of a family of small molecular weight proteins recently discovered in dragline silk. These proteins were discovered in the western black widow spider, Latrodectus hesperus, and have been termed Cysteine-Rich Proteins (CRPs) due to their high conserved cysteine content. CRP family members were used in protein-protein interaction studies to determine if there is any interaction with the major ampullate spidroins (MaSps). After affinity chromatography and co-expression studies in bacteria, there were no detectable interactions between the CRPs and MaSp1. Further studies which could be an important role in the natural silk assembly process. Further protein interaction studies in different salt and pH conditions can further determine the function of the CRPs in dragline silk formation.

Trichomoniasis affects 170 million people worldwide, and 7.4 million in the USA. There is increasing focus on the role of cysteine proteases in Trichomonas vaginalis because of their role in virulence of other parasitic protozoa. Determining their location and function will provide insight about their role in the pathogenicity of T. vaginalis and their feasibility as a drug target. This study begins to characterize the first sequenced cysteine protease (CP1). E. coli and P. pastoris expression systems were developed to produce CP1 to generate antiserum, and to have enough active protein for biochemical characterization. Secondly, endogenous and epitope tagged CP1 were localized in T. vaginalis vesicles. These vesicles were confirmed to have alkaline phosphatase activity which is a characteristic of lysosomes. Lastly, deletion mutants of CP1 were created to determine the role of the prodomain in targeting CP1 to vesicles.

Indiana University-Purdue University Indianapolis (IUPUI) / The anti-apoptotic protein Survivin and the cyclin-dependent kinase p34Cdc2 are involved in cell cycle progression and apoptosis. Activation of Cdc2 is required for its pro-apoptotic activity, which can be inhibited by phosphorylation at Tyrosine-15 (Tyr15). In transduced IL-3-dependent murine BaF3 hematopoietic cells, over-expression of wild-type-(wt)-Survivin increased Cdc2-Tyr15 phosphorylation, while over-expression of a dominant-negative-(dn)-T34A-Survivin construct decreased its phosphorylation. The increased phospho-Tyr15 levels associated with ectopic Survivin directly correlated with enhanced BaF3 cell survival in the absence of growth factors, and low phospho-Tyr15 levels observed in cells expressing ectopic dn-Survivin correlated with decreased survival. BaF3 cells transduced with Internal Tandem Duplication (ITD) mutations of the Flt3 receptor that results in increased Survivin levels, also contained increased levels of Tyr15 phosphorylated Cdc2. In BaF3 cells over-expressing wt-Survivin, 2-fold higher levels of Wee1 protein were observed compared to cells expressing control vector alone. Treatment of control BaF3 cells with the caspase-3 inhibitor Ac-DEVD-CHO increased both Cdc2-Tyr15 phosphorylation and Wee1 protein levels. In a similar fashion over-expression of wt-Survivin in these cells maintained high levels of Tyr15 phosphorylated Cdc2 and Wee1 protein. In MCF7 human breast cancer cells that lack caspase-3, increase of Tyr15 phosphorylated Cdc2 and Wee1 kinase protein by caspase-3, -7 or a pan-caspase inhibitor was absent, linking Survivin and caspase-3 to the increase of Wee1 and Tyr15 phosphorylation of Cdc2. To further link Survivin and Cdc2, we treated cells with AICAR and 17-AAG that inhibit Hsp90, which is known to be required for Survivin stability. Treatment of BaF3 cells expressing wt-Survivin with AICAR and 17-AAG decreased Cdc2-Tyr15 phosphorylation compared to vehicle-treated control cells. Taken together, these results indicate that Survivin protects the Cdc2-Tyr15-targeting kinase Wee1 from degradation by caspase-3 which leads to increased inhibitory Cdc2-Tyr15 phosphorylation resulting in reduced apoptosis and enhanced survival.

A significant number of known human genetic diseases is associated with nonsense mutations leading to the introduction of a premature termination codon into the coding sequence. A termination codon can be read through by its near-cognate tRNA (tRNA with two anticodon nucleotides base-pairing with a stop codon); potentially generating C-terminally extended protein variants. In yeast, UGA stop codon was described to be read through by tRNA-Trp and tRNA-Cys. Similar was observed for tRNA-Trp in human HEK293T cell line. The aim of this thesis was to investigate if human tRNA-Cys can act as a near-cognate tRNA in human HEK293T cell line. There are two isoacceptors which constitute the tRNA-Cys family, with ACA and GCA anticodon. There are 1 and 23 isodecoders to the ACA and GCA anticodons, respectively. Here, altogether as many as nine tRNA-Cys isodecoders (distinct in their sequence and with varying levels of expression) were tested for their ability to increase UGA readthrough in HEK293T using p2luci and pSGDluc dual-luciferase reporter vectors. In both p2luci and pSGDluc, we observed that at least one tRNA-Cys isodecoder, tRNA-Cys-GCA-4-1, is capable of significantly elevating the UGA readthrough levels when overexpressed in HEK293T. This indicates that similarly to yeast, tRNA-Cys is capable of...