A proteína de matriz do Virus Respiratório Sincicial humano foi o foco deste trabalho. Verificamos que o gene de matriz possui sítios internos de poliadenilação, sinais de instabilidade de RNA, baixo índice de adaptação de codons (CAI) e conteúdo GC, que podem impedir a expressão gênica vitro. Quando clonado sob controle do promotor de CMVie, o gene selvagem não apresenta expressão detectável, enquanto um gene sintético com a sequência do gene de matriz otimizada apresenta altos níveis de expressão em células transfectadas. Esse alto nível de expressão permitiu a confirmação da presença da proteína M no núcleo no início de sua expressão, por análise em microscopio confocal de varredura a laser, além de sua associação a membranas em regiões conhecidas como lipid rafts. Também foi observado que a proteína M é capaz de associar à proteína tropomiosina. Ainda foram analisados os possíveis domínios funcionais através de expressão de variantes da proteína M com deleções de trechos da proteína. Finalmente foi analisada a capacidade de indução de resposta imune. / The Human Respiratory Syncytial Vírus was the focus of this work. We found that matrix gene has internal polyadenilation sites, RNA instability motifs, low codon adaptation index (CAI) and GC content, that may impair its expression in vitro. When cloned under control of the ieCMV promoter, the wild-type M gene expression was not detectable, whereas a synthetic optimized matrix gene was highly expressed in transfected cells. This high level of expression made possible to follow M nuclear localization in the beginning of its expression by confocal laser scanning microscopy, and its association with membranes in regions known as lipid rafts. It has also been found that the matrix protein associates with tropomyosin. It was further analyzed the possible functional through expression of deviations of the M protein that lack portions of the protein. Finally it was analyzed its capacity to induce an immune response.

ABSTRACT The aim of this study is develop and test an inexpensive molecular tool kit to be used for diagnostic PCR for diseases such as Leber hereditary optic neuropathy (LHON) and Cystic fibrosis(CF). By developing and optimizing recombinant Taq polymerase and making a DNA size ladder from plasmids pPSU1 and pPSU2 the financial cost for the tool kit would be reduced significantly compared to the commercial components. With an inhouse method both the recombinant Taq polymerase and the pPSU1 and pPSU2 plasmids were purified from the E.coil strain DH5-α. Thereafter to analyse the components of the tool kit both conventional PCR and Real-time PCR to make sure that the tool kit would work for both types of PCRs.     The homemade Taq polymerase proved to be able to sustain in room temperature for at least 24 h and the polymerase also showed that it works with different primers such as LHON, CF and Beta-globin in both endpoint and probe base real-time PCR. The homemade size marker produced a reliable in agarose gel electrophoresis but requires optimization for continued usage for smaller PCR products.     In conclusion the homemade Taq polymerase will be used in future PCR analysis in the laboratory and the recombinant production process as well. Meanwhile the homemade size marker did not work sufficiency enough to be continuously used with gel electrophoresis in the laboratory without being further modified.

Rabies is a fatal but preventable neurotropic disease of potentially all mammals. The disease is caused by lyssaviruses. Rabies is recognized as the 10th most common lethal infectious disease in the world, rendering it one of the most feared zoonotic diseases known to man. Nevertheless, rabies can be prevented by application of pre- or post exposure treatments. Rabies vaccines have been available since the time of Pasteur, more that one hundred years ago. Since, vaccine research focused on the development of safer and more effective vaccines. Topics of current interest in the field of rabies vaccinology were addressed in this study. A primary concern regarding the disease is human mortalities, in the range of 60 000, reported every year. Most of these are linked to exposure to rabid dogs. In addition, a great number of post exposure treatments are administered each year at great costs. Despite availability of efficacious biologics, several factors influence the optimal use and accessibility of these agents in the countries of interest, with cost and availability being the major contributing factors. A proven approach is mass oral vaccination of target animals, such as dogs, which indirectly infers protection to susceptible hosts, including man. Currently available vaccines present several disadvantages of use though, including issues of safety or doubtful stability. Safer but effective alternative vaccines that could be used in oral baits would be valuable. Here the use of two candidate host restricted poxvirus vaccine vectors were explored, particularly also in regard to oral innocuity. The construction, convenient isolation and use of a recombinant Lumpy skin disease virus (Neethling strain) expressing rabies virus glycoprotein in a mouse model were investigated. In addition, a recombinant Modified Vaccinia virus Ankara expressing rabies virus glycoprotein was prepared and tested as a vaccine in mice, dogs and raccoons. In both cases it was clear that the severe attenuation of these viruses did affect the efficacy of the recombinant vaccines in the non-permissive hosts. With the recombinant MVA a clear dosage effect could be shown, and equivalent humoral responses could only be attained at much higher titers of vaccine virus as with replication competent counterparts. Secondly, the cross-protection of rabies vaccines across the spectrum of lyssaviruses was addressed. Lyssaviruses can be divided into two groups based on sequence analysis and pathogenesis. Viruses belonging to the so-called phylogroup II, are the Mokola, Lagos and West Caucasian Bat viruses. Classic rabies biologics fail to fully protect against the viruses attributed to a lack of cross-neutralization. Here, cross-protection and cross-reactive immune responses induced by recombinant vaccinia viruses expressing rabies, Mokola or West Caucasian Bat virus glycoproteins, in single or dual combinations, were investigated. As expected, there was a lack of cross-protection of rabies and Mokola glycoprotein vaccines. There was also a clear lack of cross-protection of West Caucasian Bat virus glycoprotein vaccine and rabies and Mokola viruses. The dual antigen expressing vaccines did not appear to offer any additional protective effect in the tested model. The Mokola virus glycoprotein vaccines induced neutralizing antibody responses that significantly cross-neutralized Lagos Bat virus. / Thesis (PhD (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

This master's thesis addresses the solubility of recombinant proteins and its prediction. It describes the subject of protein synthesis, as well as the process of recombinant protein creation. Recombinant protein synthesis is of great importance for example to pharmacologic industry. This synthesis is not a simple task and it does not always produce viable proteins. Protein solubility is an important factor, determining the viability of the resulting proteins. It is of course favourable for companies, that take part in recombinant protein synthesis, to focus their effort and their resources on proteins, that will be viable in the end. In this regard, bioinformatics is of great help, as it is capable, with the help of machine learning, of predicting the solubility of proteins, for example based on their sequences. This thesis introduces the reader to the basic principles of machine learning and presents several machine learning methods, used in the field of protein solubility prediction. It deals with the definition of a dataset, which is later used to test selected predictors, as well as to train the ensemble predictor, which is the main focus of this thesis. It also focuses on several specific protein solubility predictors and explains the basic principles upon which they are built, as well as the results of their testing. In the end, it presents the ensemble predictor of protein solubility.

Many diseases display a multitude of relevant factors that contribute to the persistence of the disease and difficulty treating it. The multifactorial characteristics of some diseases lead to the requirement of combination of treatments in order to restore health. The latter may necessitate the mixing of treatments, medications, and therapeutics to first halt the disease, then assist the human body in returning itself to a state of normality. For example, chronic wounds exhibit this multifactor characteristic in which there exist many factors that lead to the body’s inability to properly heal in a timely manner. This presents a further threat to the body, such as exposure to infection and long-term pain. In this example, it is important to look at the ultimate cause of a chronic wound, which may be due to presence of other diseases impairing the body’s ability to properly heal. This may include diabetes, initial antibiotic-resistant infection, autoimmune disorders, and poor vasculature. Furthermore, the mentioned causes for chronic wounds may have associations with one another in a single case of a chronic wound. Treating each interrelated cause with drug combinations may run the risk of adverse side effects or further complications due to mixing drugs in a systemic method. The goal of this study is to develop a point-specific, protein-based therapy that incorporates a single-protein molecule with multifunctional characteristics based on what we know about chronic wounds and infections, as a proof of concept of multifunctional proteins. Multifunctionality of a single therapeutic molecule is desirable because it may eliminate the unknowns of how differing individual chemical or protein therapies may interact when simply mixed. In addition, examples of peptides, such as antimicrobial peptides, are known to have synergy, and creating a single protein platform that consists of two synergistic peptides could be of value in the making of a protein with greater activity by guaranteeing that the synergistic peptides are local to one another. Furthermore, broad spectrum activity can be obtained by combining two differing peptides. This proof of concept was accomplished by targeting two proteinases that are upregulated in chronic wounds: Matrix Metalloproteinase-2 (MMP-2) and Neutrophil Elastase. Recombinant DNA techniques were used to create a fusion protein that incorporates an inhibitor of MMP-2, which is a β-Amyloid Precursor Protein-derived Inhibitory Peptide (APP-IP), and PMP-D2, an inhibitor of Neutrophil Elastase. PMP-D2 was joined to the N-terminus of an Elastin-like peptide, while the APP-IP was joined to the C-terminus of the same Elastin-like peptide. Elastin-like peptides (ELPs) are commonly used as a backbone for recombinant protein production as their distinct thermoresponsive characteristics provide adequate protein purification using an inverse transition cycling [3]. In addition, ELPs can serve as point-specific drug delivery platforms with a transition temperature (Tt) near that of normal body temperature causing low diffusivity [3]. Therefore, when ELPs are applied to a site at their Tt, they will aggregate, which provides diffusional limitations of the protein in the application site, and may decrease the reapplication rate needed for a therapeutic, as well as eliminate adverse side effects by retaining the protein to the specific application site. From this dual fusion, the final resulting protein is PMP-D2٠ELP٠APP-IP. This protein was tested for its inhibitory activity of both MMP-2 and Neutrophil Elastase. It was hypothesized that the fusion protein, PMP-D2٠ELP٠APP-IP, would inhibit MMP-2 just as effectively as APP-IP·ELP unaccompanied by PMP-D2, as well as effectively inhibit Neutrophil Elastase to the same degree as PMP-D2·ELP unaccompanied by APP-IP. Furthermore, an additional dually fused ELP fusion protein was currently made with two synergistic antimicrobial peptides fused to each end of the ELP. The two antimicrobial peptides used were human-derived LL37 and insect-derived Cecropin A. This novel fusion peptide contains synergistic increase in antibacterial activity in which preliminary data suggests.

N-methyl-D-aspartate (NMDA) receptors are glutamatergic ionotropic receptors involved in excitatory synaptic transmission, synaptic plasticity and excitotoxicity. They are heteromeric complexes of GluN1 combined with GluN2A-D and/or GluN3A-B subunits that are activated by glutamate and glycine. Many allosteric modulators can influence the activity of these receptors including neurosteroids. Pregnanolone sulfáte (3α5βS) is an endogenous neurosteroid that inhibits NMDA receptors in a use-dependent manner and has neuroprotective effect. Binding site for 3α5βS on the NMDA receptor molecule is still not indentified. The aim of my work was to contribute to the identification of the biding site by kinetic analysis of rate of response return from 3α5βS inhibition. Using the point mutation we also attempted to identify the amino acids residues that could be involved in the neurosteroid binding. In order to study the effect of 3α5βS on NMDA receptors the electropfysiological recordings on human embryonic kidney 293T cells expressing recombinant GluN1/GluN2B receptors was performed. We confirm that the effect of 3α5βS on GluN1/GluN2B receptors is voltage-independent. The results of my work indicate that steroids can reach the binding site on the NMDA receptors through the membrane rather than directly from the aqueous...

Recombinant preparation of TEAD transcription factor (abstract) The TEAD family transcription factors play an important role during devolopment of organisms, where their main purpose is to regulate organ size by activating expression of proteins involved in cell growth and differentiation and apoptosis inhibition. TEAD proteins activity is regulated by signalling pathways and interactions with coactivators. Disregulation of these mechanisms can lead to development of tumors, which is the reason why TEAD proteins became an interesting target for development of new anticancer drugs based on inhibiting their activity. There are several possibilities how to inhibit activity of a transcription factor including blocking its bond to DNA. To design a new drug that blocks transcription factors binding to DNA the structural basis of interaction of these two molecules has to be known first. In this thesis the DNA binding domain of human protein TEAD1 was prepared using the technique of recombinant expression in bacteria E. coli. Suitable conditions of protein production were found and the DNA binding domain of TEAD1 protein was purified so it will be possible to use it for structural analysis of its intraction with DNA.

Influenza virus A circulates in birds and mammals and causes severe infectious disease that affects from 3 to 5 million people each year. There are two classes of anti-influenza drugs currently available: neuraminidase and M2 channel inhibitors. However, increasing resistance against these two types of inhibitors along with the potential emergence of new viral strains and unpredictability of pandemic outbreaks emphasize an unmet need for new types of inhibitors. RNA-dependent influenza polymerase serves as a novel promising target for the development of anti-influenza medications. The aim of this master thesis is to develop in vitro high-throughput assays for screening of compounds targeting influenza RNA polymerase, particularly, its cap binding and endonuclease domains. For cap-binding domain the screening is based on DIANA (DNA-linked Inhibitor ANtibody Assay) method that was recently developed in our laboratory; for endonuclease domain, the method is based on AlphaScreen technology. For the purposes of the methods development, recombinant cap binding domain of PB2 subunit and N-terminal endonuclease domain of PA subunit of influenza polymerase were expressed with appropriate fusion tags and purified using affinity and gel permeation chromatography. The probes for the screening assays were...

Study of receptor-ligand pair NKR-P1F and Clrg Mouse NKR-P1F:Clr-g receptor:ligand pair is important component of the receptor "zipper" that occurs at the contact between natural killer cell and its target cell, and represents a recently discovered example of lectin-lectin interactions important for recognition among immune cell subsets. In order to study structure of these proteins and interactions between them, we have prepared pET-30a(+) bacterial expression vectors coding parts of extracellular domains of the two receptors. After induction of protein production with IPTG, the proteins precipitated into inclusion bodies, from which they could be refolded in vitro. Refolded proteins were purified using combination of ion exchange and size exclusion chromatography. NKR-P1F construct yielded only small amounts of soluble protein using standard refolding protocols. Furthermore we have experienced difficulties with reproducibility of the refolding results. In the case of Clrg the standard protocols for protein refolding were not sufficient. In order for the Clrg to fold properly, the odd cysteine which does not fit into the pattern usual for this family of receptors was substituted for serine and resulting C148S construct was shown to be more useful. Further, using (benzyldimethylammonio)propanesulfonate in...

The discovery of novel genes involved in hematopoietic development and lymphoid function is necessary for the understanding of these systems. To this end, we utilized transmembrane protein-specific gene trapping in embryonic stem (ES) cells, a method of forward genetics, to identify a novel, complex locus from which several splice variants arise. The trapped locus identified in the KST30 ES cell clone encodes several genes including outer membrane protein 25 (OMP25) and activin receptor interacting protein (ARIP2) and two novel genes, AK74 and AK88. AK74 is highly conserved between human and mouse with 85% identity at the amino acid level. The human homolog was cloned from CD34+ cord blood hematopoietic stem cell progenitors (HSCPs) implying that it may have a role in the hematopoietic system. We generated mice from the gene trapped ES cells, called KST30 mice, to analyze the expression pattern of transcripts from the trapped locus in the hematopoietic system. Utilizing the gene trap LacZ reporter and RT-PCR, we found that AK88 and AK74 are expressed in hematopoietic stem cells and thymocytes and that AK88 and ARIP2 are dramatically up-regulated in activated Band T lymphocytes. In addition, we found restricted expression of the gene trap in most non-lymphoid tissues. Interestingly, the expression pattern of the gene trap coincides with the expression of activin signaling components in many cell types including thymocytes, activated B cells, hematopoietic stem cells and the ductal cells of the pancreas. AK74, AK88 and ARIP2 share two exons that encode a 44 amino acid region. ARIP2 negatively regulates activin signaling through endocytosis of Activin type II receptors. The N-terminal PDZ domain associates with ActRII and mediates endocytosis via association with RalBP1. The region of ARIP2 that associates with RalBP1 encompasses the 44 amino acid region also found in AK74 and AK88, suggesting that these proteins may also associate with RalBP1, perhaps sequestering it from ARIP2. This possibility combined with the similarities between gene trap expression and expression of the components of activin signaling indicates a role of the trapped genes in activin signaling. AK74 and AK88 have a signal sequence and transmembrane domain that are predicted to direct them to mitochondria. To confirm this prediction, we examined the subcellular localization of AK74 and found that it localizes to a punctate, perinuclear structure identified as mitochondria using a mitochondria specific dye. AK74 was not seen in the cytoplasm, nucleus or at the plasma membrane of cells. To determine the function of these novel genes, AK74 was retrovirally over-expressed in a double positive thymoma cell line and examined the global expression profile using Affymetrix gene chip. AK74 changed the expression levels of 36 genes greater than 3-fold compared to vector alone. Of these genes, several are involved in cytoskeletal rearrangement, apoptosis or are regulated by calcium signaling. Using yeast two-hybrid, several candidate binding partners for AK74 were identified, one of which is the receptor for activated protein kinase C (RACK1). RACK1 was also identified as a potential binding partner for AK88. RACK1 is a WD40 domain-containing scaffolding protein that has been implicated in many pathways but most prominently in the protein kinase C signaling pathway. Association with RACK1 by either AK74 or AK88 suggests that they may be involved in RACK1 function. Both RACK1 and PKC are involved with Ca2+ signaling through different mechanisms. This, combined with global gene expression changes in AK74 over-expressing cells suggests a role for AK74, AK88 or ARIP2 in Ca2+ signaling. When we examined the expression of the trapped genes in mice homozygous for the gene-trapped allele (KST30tr/tr) we found that insertion of the gene trap caused a severe decrease in AK88 and ARIP2 but not AK74 transcripts. Analysis of KST30tr/tr mice showed no abnormalities in conventional lymphoid populations and precursors, however, intraepithelial lymphocyte (IEL) populations were altered by the loss of AK88 and/or ARIP2. There was an approximate 2-fold decrease CD8αα+ T cells in the small intestine while CD8αβ+ T cells were largely unaltered. Using gene trap technology, we have identified two novel, mitochondria-localized proteins. The cumulative findings described in this thesis, including the homology between AK74, AK88 and ARIP2, their expression pattern and the phenotype of KST30tr/tr mice, suggest possible roles of AK74 and AK88 in diverse pathways.