A novel calmodulin (CaM)-binding cysteine-rich protein from Dictyostelium, cyrA, with epidermal growth factor-like (EGFL) repeats was discovered and characterized. Calcium-dependent and –independent CaM-binding was verified. Western blots show that full length cyrA is detected constitutively throughout development. Analyses of the extracellular medium reveal that cyrA is cleaved and that the fragments containing the N-terminus are secreted early in development, while those containing the C-terminus are secreted later. In support of this, GFP and immunohistochemistry studies reveal that cyrA localizes to the endoplasmic reticulum and secretory vesicles of vegetative cells, and to the extracellular matrix (slime sheath) of migrating slugs. The addition of EGFL1 peptides enhanced cell motility and cAMP-mediated chemotaxis. Finally, cyrA cleavage is regulated by extracellular Dictyostelium CaM and by the extracellular EGFL repeats. In total the data suggest that cyrA is a true matricellular protein that mediates cell motility during multicellular development.

A novel calmodulin (CaM)-binding cysteine-rich protein from Dictyostelium, cyrA, with epidermal growth factor-like (EGFL) repeats was discovered and characterized. Calcium-dependent and –independent CaM-binding was verified. Western blots show that full length cyrA is detected constitutively throughout development. Analyses of the extracellular medium reveal that cyrA is cleaved and that the fragments containing the N-terminus are secreted early in development, while those containing the C-terminus are secreted later. In support of this, GFP and immunohistochemistry studies reveal that cyrA localizes to the endoplasmic reticulum and secretory vesicles of vegetative cells, and to the extracellular matrix (slime sheath) of migrating slugs. The addition of EGFL1 peptides enhanced cell motility and cAMP-mediated chemotaxis. Finally, cyrA cleavage is regulated by extracellular Dictyostelium CaM and by the extracellular EGFL repeats. In total the data suggest that cyrA is a true matricellular protein that mediates cell motility during multicellular development.

The flavivirus West Nile virus (WNV) encodes a single polyprotein that is processed into three structural and seven nonstructural proteins. Various WNV bicistronic replicons that direct cap-dependent translation of an N-terminal viral capsid or capsid/Renilla luciferase fusion protein as well as IRES-dependent translation of the nonstructural proteins were constructed. An original replicon consisting of the WNV 5' NCR, the 5' 198 nts of the capsid coding sequence, which included the 5' cyclization sequence (Cyc), and an EMCV IRES followed by the WNV nonstructural genes and 3' NCR was generated. Real time qRT-PCR analysis of intracellular levels of this replicon RNA showed a 4 fold increase by 96 hr after transfection of BHK cells. Increasing the distance between the 5' Cyc and IRES by insertion of a 5' IRES flanking sequence alone or together with a Renilla luciferase reporter did not increase RNA replication. Addition of only a reporter decreased RNA replication. The insertion of an extended capsid coding sequence also did not enhance RNA replication, but did enhance both cap- and IRES-dependent translation of replicon RNA, as indicated by immunofluorescence and Western blot analysis. These results suggest the presence of a translation enhancer in the 3' portion of the capsid coding region. Simian hemorrhagic fever virus (SHFV) is a member of the family Arteriviridae, order Nidovirales. SHFV is unique among Nidoviruses in having three instead of two papain-like cysteine protease (PCP) motifs designated alpha, beta, and gamma, within the N-terminal region of its ORF1a. Mutations of putative PCP cleavage sites showed that the most efficient cleavage was by PCP beta at its downstream cleavage site. A large deletion located between the two catalytic residues of PCP alpha was hypothesized to render this protease inactive. However, processing was observed at the cleavage site following PCP alpha. Mutational analyses confirmed that PCP alpha is an inactive protease, and that the cleavage sites downstream of PCP alpha are cleaved by PCP gamma. When the catalytic residues of PCP gamma were mutated, PCP beta was also able to back cleave at these sites. This "back" cleavage is a previously unreported activity for an arterivirus PCP.

<p>Driven by the development of new technologies and an ever expanding knowledge base of molecular and cellular function, Biology is rapidly gaining the potential to develop into a veritable engineering discipline - the so-called `era of synthetic biology' is upon us. Designing biological systems is advantageous because the engineer can leverage existing capacity for self-replication, elaborate chemistry, and dynamic information processing. On the other hand these functions are complex, highly intertwined, and in most cases, remain incompletely understood. Brazenly designing within these systems, despite large gaps in understanding, engenders understanding because the design process itself highlights gaps and discredits false assumptions. </p><p>Here we cover results from design projects that span several scales of complexity. First we describe the adaptation and experimental validation of protein functional assays on minute amounts of material. This work enables the application of cell-free protein expression tools in a high-throughput protein engineering pipeline, dramatically increasing turnaround time and reducing costs. The parts production pipeline can provide new building blocks for synthetic biology efforts with unprecedented speed. Tools to streamline the transition from the in vitro pipeline to conventional cloning were also developed. Next we detail an effort to expand the scope of a cysteine reactivity assay for generating information-rich datasets on protein stability and unfolding kinetics. We go on to demonstrate how the degree of site-specific local unfolding can also be determined by this method. This knowledge will be critical to understanding how proteins behave in the cellular context, particularly with regards to covalent modification reactions. Finally, we present results from an effort to engineer bacterial cell suicide in a population-dependent manner, and show how an underappreciated facet of plasmid physiology can produce complex oscillatory dynamics. This work is a prime example of engineering towards understanding.</p> / Dissertation

The mammalian embryo is encased in a glycoproteinaceous covering, the zona pellucida (ZP/zona) during preimplantation development. Prior to implantation into the recipient maternal endometrium, the blastocyst has to hatch out of this zona. This is a critical and an important event for the successful establishment of pregnancy. Hatching in mammals is characterised by the expansion of the blastocyst, followed by the nicking of the zona and extrusion of the blastocyst by repeated contraction-expansion cycles, thereby leaving the empty zona behind. In species such as the mouse, cow and primates, the empty zona is left behind in the uterine lumen. However, in the hamsters, the features associated with hatching are characteristic for this species. Firstly, the blastocyst remains predominantly in a deflated state. Secondly, the zona undergoes focal rupture which is followed by the complete dissolution of the zona. Third, trophectodermal projections (TEPs) present in the blastocysts, aid the hatching of the blastocyst. Hence, this study was aimed to identify the molecular players involved in hamster blastocyst hatching and to study their embryo-endometrial expression. Earlier work in the laboratory has demonstrated the involvement of cysteine protease-like factors in hamster blastocyst hatching (Mishra and Seshagiri, 2000a). Broad spectrum cysteine protease inhibitors, E-64 and PHMB, completely blocked the hatching of blastocysts. To identify the class of cysteine proteases involved in this phenomenon, class-specific inhibitors were used in this study. Calpain and caspase inhibitors, calpastatin and Z-VAD- FMK, respectively, did not block hamster blastocyst hatching (Fig 2.2). Cathepsin (cts)-specific inhibitors, cystatin-C and peptidyl diazomethane (PPDM) blocked hatching of embryos in a dose-, time- and embryo-stage dependant manner (Figs 2.5, 2.6, 2.10 and 2.11). Continuous exposure of 1.0 µM cystatin to expanded or deflated blastocysts completely blocked hatching (Fig 2.3Aii, iii), without effecting their viability (Fig 2.3Bii and iii). Deflated blastocysts exposed transiently to 1.0 µM cystatin, for 12 or 6 h failed to hatch, but could overcome the inhibition and exhibited hatching, when transferred to fresh, inhibitor-free medium (Fig 2.6). Effect of the inhibitor was less pronounced in the deflated blastocysts when compared to expanded blastocysts (Figs 2.5 and 2.6). The viability of the cystatin-treated embryos was not affected as assessed by vital-dye staining and also their ability to attach and exhibit trophoblast (TB) proliferation on serum-coated dishes was not compromised. The area of the TB outgrowth of cystatin-treated embryos was similar to that of the untreated embryos (Fig 2.9). The inhibitory effect of PPDM, an irreversible inhibitor of cts, on blastocyst hatching was demonstrated. Expanded and deflated blastocysts exhibited a dose-dependent inhibition in hatching, following the exposure to 0.5 and 1.0 µM of PPDM (Figs 2.10A and 2.11A). When treated with the inhibitor for 6 or 12 h, there was a transient inhibition in hatching, as blastocysts could overcome the inhibition and exhibit hatching following transfer to inhibitor-free, fresh medium. Inhibitor-treated hatched blastocysts, when transferred to serum-coated dishes, attached and exhibited TB outgrowth, similar to untreated embryos (Figs 2.13 and 2.14). A PPDM-interacting protease was localised to the cytoplasm of the embryonic cells in the hamster blastocyst, suggesting that the embryo is the source of the zona lysin. Two forms of the enzyme, a probable variant zymogen of molecular mass 65 k and an active form of molecular mass 32 k were detected in the blastocysts (Fig 2.15). In vitro susceptibility of hamster zona to cathepsins is significantly different from that of other species zonae such as the mouse, rat, monkey and human zonae (Table 2.2). All these lines of evidence unequivocally demonstrate the involvement of cathepsins in hamster blastocyst hatching, which is in sharp contrast to what is observed in the mouse, where serine proteases such as strypsin/hepsin, ISP-1 and -2 are reported to play an important role in blastocyst hatching. However, since extensive inhibitor studies were not performed using embryos from other species, it is possible that cysteine proteases maybe involved in the hatching of blastocysts from other species. Having shown the role of cathepsins in hamster zona dissolution, expression of the cathepsins in preimplantation embryos was investigated. Hamster specific cts–L, -B and –P were amplified from day 14 placenta using mouse primers and the amplicons were found to be highly homologous to the cts of other species (Fig 3.2). Hamster and mouse preimplantation embryos i.e., 8-cell, morula and blastocyst were found to express cts–L, -B and –P transcripts (Figs 3.6 and 3.10). Cts-P, present only in the TBs of the placenta (Fig 3.4), for the first time, was also shown to be present in the preimplantation embryos. The immunoreactive cts-L and -P proteins were detected in blastomeres of 8-cell embryo, in the inner cell mass (ICM) and trophectoderm (TE) of the blastocyst (Figs 3.7 and 3.8). These cathepsins could probably correspond to the PPDM- interacting enzymes of molecular mass 32 and 65 kDa, described above. (fig) Fig 5.1. Overview of the expression and the role of embryo-endometrial cathepsins in blastocyst hatching in the golden hamster. Cathepsins ( ) produced by the inner cell mass ( ) or the trophectoderm ( ) of the blastocyst or the endometrial cells ( ) act on the zona matrix ( ), bringing about its lysis. The cathepsins are secreted into the peri-vitelline space or are carried by trophectodermal projections (TEPs, yellow projections) to the zona. Also shown are endogenous inhibitors and growth factors that can regulate these cathepsins. A striking observation made in this study was the detection of the immunoreactive signals for cathepsins in the zona matrix of blastocysts. Since hamster blastocysts possess extracellular projections (TEPs), it is possible for these projections to participate in the transport of cathepsins from TE cells to the zona; as the localisation of the proteases to these projections was demonstrated (Fig 3.9). Also, since the actin-based projections are highly undulating structures, they might potentiate the mechanical rupture of the zona during hatching, apart from acting as carriers for the proteases. Hence, during hatching of the hamster blastocyst, cathepsins, expressed in the ICM and the TE, might be secreted transiently into the peri-vitelline space, whereby they can act on the ZP. Alternatively, in the absence of any apoptotic cells in the embryo that can release the cell contents (Fig 3.13), the cathepsins may be deposited by TEPs in specific pockets of the zona matrix, thereby causing focal zona lysis. In vivo, the hatching of the blastocysts is brought about by both embryonic and maternal proteases. Cts–L and -B transcripts were detected in the maternal endometrium during different stages of the reproductive cycle and early pregnancy (Fig 4.1 and 4.3). Immunoreactive cts-L protein was detected in the uterine luminal epithelium and the stromal cells (Fig 4.5). In the uterus, the PPDM-interacting 32 kDa form was in abundance compared to the 65 kDa form (Fig 4.6). Hence, uterine cathepsins might play a major role in the remodelling of the extracellular matrix during estrous cycle and pregnancy. However, the role of these cathepsins in causing zona dissolution during blastocyst hatching, along with embryonic proteases cannot be ruled out. Reports of recurrent miscarriages in women with low serum cystatin levels imply a role for cysteine proteases in early pregnancy events like blastocyst development, hatching and implantation. Hence, these studies, described in the thesis, could form a basis to investigate the role of cathepsins in early human development. Taken together, the results demonstrate the involvement of embryo-derived cathepsins in hamster blastocyst hatching. These cathepsins may be secreted into the peri-vitelline space or transported to the zona matrix by TEPs (Fig 5.1). Additionally, in vivo, endometrial cathepsins might aid the embryonic zona lysins in the complete zona dissolution. The regulation of these proteases by growth factors, cytokines and their specific inhibitors needs to be explored. (For figure pl see the original document)

Protein phosphorylation is a post translational modification of proteins in which a serine, a threonine or a tyrosine residue is phosphorylated by an enzyme, kinase. Phosphorylation of proteins is a reversible and very important regulatory mechanism that occurs in both prokaryotes and eukaryotes. Phosphorylation turns many protein enzymes on and off, preventing or causing many diseases such as diabetes, cancer and rheumatoid arthritis. The phosphorylation on tyrosine residues of proteins is essential for transmission of signals for cell growth, proliferation and differentiation. Protein tyrosine phosphatases (PTPs) in concert with protein tyrosine kinases (PTKs) regulate many signal transduction pathways by controlling the degree of phosphorylation of tyrosine residues within the protein. While the roles and mechanisms of protein tyrosine kinases are well documented, our present understanding of protein tyrosine phosphatases is very limited. In this regard we still have much more to learn about PTPs. Here we propose the design and synthesis of novel protein tyrosine phosphatase mimetics and their activity against tyrosine phosphatases. Chapter two describes the synthesis of 2-aminopyrimidine chlorides, sulfonamides and the sequence of reactions to make its amino acid analog. Chapter three describes the synthesis of α-aryl, α,β-epoxy carboxylates, phosphonates and their biological activity against tyrosine phosphatases. These compounds could be very helpful in significantly improving the current understandings about the roles and mechanisms of the PTPs. These proposed tyrosine phosphatase inhibitors are believed to work effectively in treating the diseases by modulating the phosphorylation in signal transductions pathways. Chapter four describes the design and the synthesis of Peptide Nucleic Acids (PNAs) both standard as well as hybrid PNAs with novel cysteine based monomers that are aimed to increase the cellular uptake by introducing positively charged or amphipathic species attached to cysteine thiol functional group.

Gingipaine sind Cysteinproteinasen des wohl in Ätiologie und Pathogenese der chronischen Erwachsenenparodontitis bedeutsamsten bakteriellen Erregers und zugleich die wichtigsten Virulenzfaktoren der Spezies Porphyromonas gingivalis. Die für diese extrazellulären Produkte kodierenden Gene sind rgpA, rgpB und kgp. Deren Produkte sind entsprechend HRgpA, RgpB und Kgp. HRgpA und RgpB verursachen eine Steigerung der Gefäßpermeabilität durch die Aktivierung des Kallikrein/Kinin-Systems und aktivieren die Blutgerinnung, welche potentiell mit der Synthese der Sulkusflüssigkeit und dem Fortschreiten der Entzündung bis hin zum Verlust des alveolären Knochens assoziiert sind. Offenbar wird dieses durch die Aktivierung von Matrixmetalloproteinasen begünstigt. Kgp ist von den dreien die potenteste fibrinogen-und fibrinabbauende Proteinase und bei der Blutungsneigung der erkrankten Stellen involviert. HRgpA aktiviert besonders Blutgerinnungsfaktoren. Gingipaine stören das Komplementsystem und manipulieren den Zytokinhaushalt der Entzündungskaskaden. Die Gingipaine unterstützen die Kolonisierung von P. gingivalis durch die Bindung zu anderen Bakterien des subgingivalen Biofilms und der Bindung zu epithelialen Zellen. Sie vermögen an Laminin, Fibrinogen, Fibronektin, Hämoglobin und an manchen Typen von Kollagen zu binden. Alle können den Rezeptor CD14 auf Makrophagen abbauen und so die Leukozytenaktivierung hemmen. Sie regulieren die Infektionsintensität, den Bakterienhaushalt, die Aminosäurenaufnahme aus Wirtsproteinen und die Fimbrienreifung. Die Genetik, die Chemie und die virulenzverursachenden Eigenschaften der Gingipaine stehen seit Mitte der 90iger Jahre des letzten Jahrhunderts im Blickpunkt des wissenschaftlichen Interesses. Aufgrund ihrer Schlüsselrolle bei der Pathogenese der Parodontitis und der mikrobiellen Infektion sind die Gingipaine Ziele für die mögliche Entwicklung von Hemmstoffen respektive für Immunisierungsstrategien gegen die chronische Parodontitis. / Gingipains are cysteine proteases of the probably most important bacterial pathogen of the adult periodontitis in the field of etiology as well as pathology. At the time, they are the most important virulence factors of the species Porphyromonas gingivalis. The encoding genes of this extracellular products are rgpA, rgpB and kgp. Their products are corresponding HRgpA, RgpB and Kgp. HRgpA and RgpB induce vascular permeability enhancement through activation of the kallikrein/kinin system and activate the blood coagulation, processes potentially associated with gingival crevicular fluid synthesis and progression of inflammation which ultimately can lead to alveolar bone loss. Obviously, this will be favoured by matrix metalloproteinases. Kgp is the most potent fibrinogen/fibrin degrading enzyme of the three gingipains involved in the bleeding tendency at the diseased sites. HRgpA especially activates coagulation factors. Gingipains disturb the complement system and manipulate the cytokine network of the inflammation cascades. Gingipains support colonizing of P. gingivalis due to their connection to other bacteria of the subgingival biofilm and to epithelial cells. They are able to bind to laminin, fibrinogen, fibronectin, hemoglobin and some types of collagen. All of them are able to degrade macrophage CD14 receptor, thus preventing activation of the leukocytes. They regulate the intensity of infection and the bacterial housekeeping, including amino acid uptake from host proteins and fimbriae maturation. Genetics, chemistry and the virulence inducing properties of gingipains are the focus of scientific attention since the middle of the nineties of the last century. Due to their key role in pathogenesis of periodontitis and microbial infection the gingipains are targets for the possible development of inhibitory substances, respectively for immunization strategies against chronic periodontitis.

Carcinogenesis is a progressive process involving multiple genetic alterations in tumor cells and complex interactions in the tumor-host microenvironment. To better understand the contribution of molecular alterations in tumor cells and stromal variables to the development of colorectal cancer (CRC) and identify prognostic factors, in this study we examined the clinicopathological and biological significance of stromal variables, including particularly interesting new cysteine-histidine rich protein (PINCH), inflammatory infiltration, angiogenesis and lymphangiogenesis, as well as hRAD50/hMRE11/hNBS1 proteins and hRAD50 mutation in tumor cell in CRC. PINCH protein expression in the stroma was increased from normal mucosa to primary tumors and further to lymph node metastases. In particular, PINCH expression was most intense at the tumor invasive margin, which was related to low inflammatory infiltration and independently related to an unfavorable prognosis. Low inflammatory infiltration at the tumor invasive margin was related to advanced tumor stage, worse differentiation and microsatellite instability (MSI). Further, it was independently related to an unfavorable prognosis. Increased blood and lymphatic vessel density was observed in the primary tumors compared with the corresponding normal mucosa. However, neither angiogenesis nor lymphangiogenesis was associated with tumor stage and patients’ survival. Moreover, PINCH was present in a proportion of endothelial cells of the tumor vasculature, and PINCH expression in tumor-associated stroma was positively related to blood vessel density. In primary tumor cells of CRC, strong expression of hRAD50, hMRE11 or hNBS1 was related to microsatellite stability (MSS). A high percentage of hMRE11 expression was associated with less local recurrence and high apoptotic activity. Further, we observed that the expression of hRAD50, hMRE11 or hNBS1 among normal mucosa, primary tumors and metastases in MSS CRC differed from that in MSI CRC. In MSS CRC, the expression intensity of hRAD50, hMRE11 and hNBS1 was consistently increased with respect to normal mucosa, but there was no difference between the primary tumors and metastases. In the primary MSS tumors, the expression of individual or combination of hRAD50/hMRE11/hNBS1 was associated with a favorable prognosis in the same series of the CRCs. Moreover, strong/high hRAD50 in MSS primary tumors was related to earlier tumor stage, better differentiation and high inflammatory infiltration, whereas strong hNBS1 expression tended to be independently related to a favorable prognosis in MSS CRC with earlier tumor stage. However, in MSI CRC, there were neither differences in the expression of hRAD50/hMRE11/hNBS1 among normal mucosa, primary tumors and metastases, nor any association of the protein expressions with clinicopathological variables. On the other hand, frameshift mutations of (A)9 at coding region of hRAD50 were only found in MSI CRC. Our study indicates that 1) PINCH is likely a regulator of angiogenesis, and PINCH expression at the tumor invasive margin is an independent prognostic indicator in CRC. 2) Inflammatory infiltration at the tumor invasive margin is also an independent prognostic indicator in CRC. The lack of association between high inflammatory infiltration and MSI may help to explain the non-association of MSI with survival in CRC patients. 3) Angiogenesis and lymphangiogenesis occur in the early stage of CRC development, but do not associate with CRC progression and patients’ prognosis. 4) hRAD50/hMRE11/hNBS1 may act dependently and independently, playing different roles in MSS and MSI CRC development. In MSS CRC, the strong expression of the three proteins, associated with a favorable prognosis, may present the cellular response against tumor progression. Expression of hNBS1 may be a prognostic indicator for MSS CRC patients in the earlier tumor stage. In MSI CRC, the frameshift mutations at the coding region of hRAD50 may contribute to tumor development.

Trypanosoma brucei and T. congolense are protozoan parasites that infect humans, domestic livestock and wildlife in Africa. These parasites undergo complex morphological and biochemical changes, during the various stages of their life cycle. These changes correlate with alterations in the levels of trypanosomal lysosomal cysteine proteases, suggesting a role for transcriptional regulation of the cysteine protease in these parasites. The mechanism of this regulation is not yet understood nor have the promoter regions of the cloned trypanosome cysteine protease genes been investigated. This study involved an attempt to clone the T. brucei and T. congolense DNA fragments containing the promoter regions as the initial step in the investigation of the control elements of the cysteine protease gene. Trypanosomes were isolated from infected rat blood employing a combination of the methods of isopicnic isolation on Percoll gradients and DEAE-cellulose anion exchange resin chromatography. Approximately 5 x 10⁹ viable trypanosome cells were isolated from the infected rat blood and chromosomal DNA (approximately 500 μg) was extracted by alkaline-lysis method. Trypanosome genomic libraries were initially constructed in Eschericia coli HB101 employing the positive selection vector pEcoR251. The Trypanosoma brucei pEcoR251 library contained 6 000 recombinants and the Trypanosoma congolense library contained 15 000 recombinants. Plasmid DNA was then extracted from pools of recombinants, employing the alkaline-lysis method, digested with EcoRl restriction endonuclease and resolved by agarose gel electrophoresis. After Southern hybridisation, the pEcoR251 libraries did not reveal any putative clones containing the fragment of interest when probed with both an oligonucleotide probe and the PCR generated dsDNA probe. Genomic libraries were then constructed in the phagemid pUC119. The T. brucei and T. congolense genomic libraries contained 33 000 and 27 000 recombinants respectively. Recombinants from the T. brucei and T. congolense libraries were pooled in lots of 400 and 300 respectively. Of the 80 T. brucei plasmid pools that were screened 30 pools contained fragments that hybridised with the probe whilst 12 pools from the 90 T. congolense library pools that were screened contained fragments that hybridised with the probe. Putative clones identified appeared to contain inserts, ranging between two and seven kb in size. A partial T. congolense library consisting of approximately 12 pools was screened by colony hybridisation for identification of individual clones and 76 putative clones were identified. After confirmation of these putative clones on a dot blot using a DIG-labelled dsDNA probe, a selection of 30 putative clones were subjected to Southern hybridisation using a DIG-labelled DNA probe. Following Southern hybridisation 23 putative clones were identified to contain DNA inserts of interest in the range of two to seven kb. Five clones, designated pCPC1, pCPC2, pCPC3, pCPC4 and pCPC5 were then selected for further restriction mapping. Clone pCPC4 contains a seven kb fragment of T. congolense genomic DNA. A partial T. brucei library consisting of approximately 30 pools was screened by colony hybridisation for the identification of individual putative clones. Although plasmid pools containing putative clones were identified repeatedly by Southern blotting and DNA/DNA hybridisation, it was not possible to identify individual putative clones following transformation into E. coli MV1184 and colony hybridisation. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1997.

With over a billion people infected world wide, hookworms are considered as important human pathogens, particularly in developing countries which have the highest rates of infections. Hookworms reside in the gastrointestinal tract of the host where they continuously feed on blood, leading to conditions such as chronic irondeficiency anaemia. The majority of blood-feeding parasites rely on proteins found in blood to provide many of their nutritional requirements for growth, reproduction and survival. Of the numerous proteins found in blood, haemoglobin (Hb) is one of the most abundant. In order to acquire amino acids for protein synthesis, it is thought that haematophagous parasites degrade Hb using various classes of endo- and exoproteases, in a manner similar to that which occurs in catabolism of proteins in mammalian cellular lysosomes. This study identified and characterised proteases involved in the Hb degradation process in the human hookworm, Necator americanus, in order to identify potential candidate antigens for a vaccine that interrupts blood-feeding. Red blood cells ingested by hookworms are lysed to release Hb, which is cleaved by various proteases into dipeptides or free amino acids and these are taken up through the gut membrane by amino acid transporters. Proteases expressed in the intestinal tract of hookworms are thought to play a major role in this process and would therefore make good targets for vaccine candidates aimed at interrupting blood-feeding. To identify these proteases, adult hookworms (both N. americanus and Ancylostoma caninum) were sectioned and intestinal tissue was dissected via laser microdissection microscopy. RNA extracted from the dissected tissue was used to generate gut-specific cDNA, which then was used to create plasmid libraries. Each library was subjected to shotgun sequencing, and of the 480 expressed sequence tags (ESTs) sequenced from each species, 268 and 276 contigs were assembled from the N. americanus and A. caninum libraries, respectively. Nine percent of N. americanus and 6.5% of A. caninum contigs were considered novel as no homologues were identified in any published/accessible database. The gene ontology (GO) classification system was used to categorise the contigs to predicted biological functions. Only 17% and 38% of N. americanus and A. caninum contigs, respectively, were assigned GO categories, while the rest were classified as being of unknown function. The most highly represented GO categories were molecular functions such as protein binding and catalytic activity. The most abundant transcripts encoded fatty acid binding proteins, C-type lectins and activation associated secreted proteins, indicative of the diversity of functions that occur in this complex organ. Of particular interest to this study were the contigs that encoded for cysteine and metalloproteases, expanding the list of potential N. americanus haemoglobinases. In the N. americanus cDNA library, four contigs encoding for cathepsin B cysteine proteases were identified. Three contigs from the A. caninum and one contig from the N. americanus cDNA libraries encoded for metalloproteases, including astacin-like and O-sialoglycoprotein endopeptidases, neither of which had previously been reported from adult hookworms. Apart from haemoglobinases, other mRNAs encoding potential vaccine candidate molecules were identified, including anti-clotting factors, defensins and membrane proteins. This study confirmed that the gut of hookworms encodes a diverse range of proteases, some of which are likely to be involved in Hb digestion and have the potential to be hidden (cryptic) vaccine antigens. Four cysteine proteases (Na-CP-2, -3, -4 and -5) were identified from the gut cDNA library of N. americanus. All four proteases belong to the clan CA, family C1, share homology with human cathepsin B and possess a modified occluding loop. Real-time PCR indicated that all transcripts were up-regulated in the adult stage of the hookworm parasite with high levels of mRNA expression detected in gut cDNA. All four proteases were expressed in recombinant form, but only Na-CP-3 was successfully expressed in soluble form in the yeast Pichia pastoris. Proteolytic activity for Na-CP-3 was detected on a gelatin zymogen gel, however no catalytic activity was detected against the class-specific fluorogenic peptides Z-Phe-Arg-AMC and Z-Arg-Arg-AMC. Mass spectrometry analysis of the purified protein suggested that the pro-region had not been processed in trans when the protein was secreted by yeast. Incubation of Na-CP-3 in salt buffers containing dextran sulfate resulted in autoprocessing of the pro-region as detected by Western blot and catalytic activity was detected against Z-Phe-Arg-AMC. Activated Na-CP-3 did not digest intact tetrameric human Hb. The other three cysteine proteases (Na-CP-2, -4, and -5) were expressed in insoluble form in Escherichia coli. Antibodies to all four proteins (Na- CP-2 to 5) immunolocalised to the gut region of the adult worm, supporting mRNA amplification results and strongly indicated that they might play a role in nutrient acquisition. Hb digestion in blood feeding parasites such as schistosomes and Plasmodium spp. occurs via a semi-ordered cascade of proteolysis involving numerous enzymes. In Plasmodium falciparum, at least three distinct mechanistic classes of endopeptidases have been implicated in this process, and at least two classes have been implicated in schistosomes. A similar process is thought to occur in hookworms. An aspartic protease, Na-APR-1, was expressed in P. pastoris and purified protein was shown to cleave the class-specific fluorogenic peptide 7- Methoxycoumarin-4-Acetyl-GKPILFFRLK(DNP)-D-Arg-Amide. Recombinant Na- APR-1 was able to cleave intact human Hb and completely degrade the 16 kDa monomer and 32 kDa dimer within one hour. Recombinant Na-CP-3 was not able to cleave intact Hb, but was able to further digest globin fragments that had previously been digested with Na-APR-1. A clan MA metalloprotease, Na-MEP-1, was identified in gut tissue of N. americanus and was expressed in recombinant form in Hi5 insect cells using the baculovirus expression system. Recombinant Na-MEP-1 displayed proteolytic activity when assessed by gelatin zymography, but was incapable of cleaving intact Hb. However, Na-MEP-1 did cleave globin fragments which had previously been incubated with Na-APR-1 and Na-CP-3. Hb digested with all three proteases was subjected to reverse phase HPLC and peptides were analysed using Liquid Chromatography-Mass Spectrometry (LC-MS). A total of 74 cleavage sites were identified within Hb ƒ¿ and ƒÀ chains. Na-APR-1 was responsible for cleavage of Hb at the hinge region, probably unravelling the molecule so that Na- CP-3 and Na-MEP-1 could gain access to globin peptides. All three proteases were promiscuous in their subsite specificities, but the most common P1-P1Œ residues were hydrophobic and/or bulky in nature, such as Phe, Leu and Ala. Antibodies to all three proteins (Na-APR-1, -CP-3, -MEP-1) immunolocalised to the gut region of the worm, further supporting their roles in Hb degradation. These results suggest that Hb degradation in N. americanus follows a similar pattern to that which has been described in Plasomdium falciparum. Studies conducted in this project have identified a number of potential haemoglobinases and have demonstrated that the gut region of the hookworm contains a multitude of proteases which could be targeted for production of new chemotherapies or as vaccine candidates. Results presented here also suggest that the Hb degradation process occurs in an ordered cascade, similar to those which have been reported in other haematophagous parasites. More importantly, it has been confirmed that Na-APR-1 plays a crucial role in the initiation of the Hb degradation process and therefore targeting this molecule as a vaccine candidate could provide high levels of protection against hookworm infection.