El CMC, tamaño y forma de micelas son características importantes en la determinación de sus principales propiedades y campos de aplicación. Esta tesis tiene dos partes, las transiciones de forma de las micelas que se trata con "Single chain Field Theory, /SCMFT)" y simulaciones de Monte Carlo. El SCMFT reveló todas las características esenciales de las transiciones de forma esférica a cilíndrica y esférica a disco de las micelas. MC muestra que las transiciones esfera a cilindro se produce a través de una región en que esferas y cilindros coexisten junto con otras formas intermedias.

Antibodies are proteins capable of specific interactions to a wide range of molecules. These interactions are facilitated by the complementary determining regions (CDR). Carcinomas are the most common of human cancers and they release significant amount of cytokeratins (CK) in the necrotic areas of the tumors. The CKs stay in the tumor, since they have low solubility. The antibody studied in this thesis, the anti-CK 8 antibody TS1, has shown to be effective in tumor targeting and is proposed to be useful in therapy. Single-chain antibodies (scFv) are recombinant antibodies which are much smaller than the intact IgG. This is an advantage when used in tumor therapy, since they can penetrate the tumors more easily than the larger IgG. Moreover, they are expressed by one single gene which make them easy to modify, for example by site-directed mutagenesis. The anti-idiotypic antibody αTS1 can be used to clear the TS1 form the circulation and thereby clear the body from non-tumor bound TS1 in therapy. To be able to modify the binding of an antibody to its antigen and or anti-idiotype, these interactions must be studied. In this study this is accomplished by chemical modifications of the IgGs TS1 and αTS1 and the antigen CK 8. Guided by these results, amino acid residues were mutated by using site-directed mutagenesis in the TS1-218 scFv and the effects were studied. From mutational study results, the functional epitope could be mapped and it was found that there are mainly tyrosines, but also charged residues, serine and a tryptophan that are important for both interactions. The binding of TS1-218 to both αTS1 and CK 8 could be improved by changing the negatively charged side-chains by mutations to their corresponding amide or alanine. Both the IgG and scFv versions of TS1 were administered in vivo. The IgG αTS1 was used to clear the TS1 from the circulation by forming immune complexes. The immune complexes, consisting of four or more antibodies, were mainly metabolized by the liver. The scFv TS1-218 could localize to the tumor in a tumor xenograft mouse model, although a higher uptake would be desired in a therapeutic strategy. The scFv was cleared rapidly by the kidneys, but the clearance could be slowed by pre-formed immune complexes with anti-TS1 scFv in vitro, prior to administration in vivo.

Alzheimer's disease (AD) is the most common cause of dementia. No strong disease-modifying treatments are currently available. Amyloid-beta peptide (Abeta) appears to play a pivotal role in the pathogenesis of AD. We focused our interest on revealing the pathogenesis of the disease and developing novel therapeutic modalities. The thesis consists of three projects: 1. Prevention of AD by intramuscular delivery of an anti-Abeta single chain antibody (scFv) gene: Immunotherapy is effective in removing brain Abetaƒzbut was associated with detrimental effects. In the present study, the gene of an anti-Abeta scFv was delivered in the hind leg muscles of APPSwe/PS1dE9 mice with adeno-associated virus at three months of age. Six months later, we found that brain Abeta accumulation, AD-type pathologies and cognitive impairment were significantly attenuated in scFv-treated mice relative to enhanced green fluorescence protein (EGFP)-treated mice. Intramuscular delivery of scFv gene was well tolerated by the animals. These findings suggest that peripheral application of scFv is effective and safe in preventing the development of AD, and would be a promising non-inflammatory immunological modality for prevention and treatment of AD. 2. Prevention of AD with grape seed derived polyphenols: Polyphenols extracted from grape seeds are able to inhibit Abetaƒnaggregation, reduce Abeta production and protect against Abeta neurotoxicity in vitro. We investigated the therapeutic effects of a polyphenol-rich grape seed extract (GSE) in vivo. APPSwe/PS1dE9 transgenic mice were fed with normal AIN-93G diet (control diet), AIN-93G diet with 0.07% curcumin, or diet with 2% GSE beginning at 3 months of age for 9 months. Total phenolic content of GSE was 592.5 mg/g dry weight, including gallic acid, catechin, epicatechin and proanthocyanidins. Long-term feeding of GSE diet was well tolerated. The Abetaƒnlevels in the brain and serum of the mice fed with GSE were reduced by 33% and 44% respectively compared with the mice fed with the control diet. Amyloid plaques and microgliosis in the brain of mice fed with GSE were also reduced by 49% and 70% respectively. In conclusion, polyphenol-rich GSE is promising to be a safe and effective drug to prevent the development of AD. 3. Roles of p75NTR in the development of AD: P75NTR has been suggested to mediate Abeta induced neurotoxicity. However, its role in the development of AD is undetermined. APPSwe/PS1dE9 transgenic mice were crossed with p75NTR knockout mice to generate APPSwe/PS1dE9 mice with p75NTR gene deleted. P75NTR mainly expressed in the basal forebrain neurons and degenerative neurites in neocortex and hippocampus. Genetic deletion of p75NTR gene in APPSwe/PS1dE9 mice reduced soluble Abeta levels, but increased the insoluble Abeta accumulation and Abeta plaque formation in the brain. P75NTR deletion decreased Abeta production of cortical neurons in vitro. Recombinant extracellular domain of p75NTR attenuated the oligomerization and fibrillation of synthetic Abeta42 peptide in vitro, and reduced local Abeta plaques after hippocampus injection in vivo. Our data suggest that p75NTR plays an important role in AD development and may be a valid therapeutic target for the treatment of AD.

Les cyanobactéries sont des micro-organismes qui préoccupent les autorités de santé publique dans le monde entier, en raison de la toxicité des cyanotoxines qu'elles produisent. Certaines cyanotoxines dont les microcystines (MC) sont des hépatotoxines inhibitrices de protéines phosphatases à sérine/thréonine. Aujourd'hui, plus de 200 variants de MCs ont été identifiés. Il s'agit d'heptapeptides monocycliques synthétisés par voie non-ribosomale dont la MC-LR (cyclo- (D-Ala-L-Leu-D-érythro-β-méthylAsp-L-Arg-ADDA-D-Glu-N-méthyl-hydro-Ala) est le variant le plus étudié en raison de sa fréquence et de sa forte toxicité. L’objectif de cette étude est le développement d'une méthode d'immunoanalyse rapide, sensible et fiable pour détecter les MCs. Le projet vise donc à développer un outil alternatif de détection de la MC-LR, qui serait mieux adapté aux analyses sur le terrain que les méthodes analytiques, biologiques ou les méthodes d'inhibition d'activité enzymatique actuellement disponibles. L'originalité de ce projet réside dans l'utilisation de deux approches différentes pour sélectionner de nouveaux anticorps spécifiques de la MC-LR. La première repose sur l'immunisation d'animaux de laboratoire, la technologie d'hybridation cellulaire et la sélection d'hybridomes sécréteurs d'anticorps monoclonaux. Si la méthodologie mise en œuvre a effectivement permis d'obtenir des immun-sérums spécifiques, la sélection des hybridomes d'intérêt reste à optimiser. La seconde stratégie mise en œuvre est basée sur la technologie du phage display pour sélectionner des fragments d'anticorps spécifiques de MC-LR à partir d'une banque de taille d’environ 109 phages, exprimant en surface des anticorps sous un format scFv (Shahsavarian et al., 2014). Plusieurs méthodes de criblage ont été développées et trois scFv ont été sélectionnés et étudiés, parallèlement à un quatrième scFv identifié dans une étude précédente (McElhiney et al., 2002), tous spécifiques à la MC-LR. Ces scFv ont été produits sous forme libre, soluble et leur spécificité à la MC-LR a été évaluée par ELISA et résonance plasmonique de surface. Les résultats obtenus montrent que les scFv sélectionnés sont tous capables de reconnaître la MC-LR. Néanmoins, ces résultats sont peu reproductibles et remettent en question le protocole de renaturation utilisé. Un travail de fond sur l’optimisation du protocole de renaturation s’avèrerait nécessaire pour les scFv ici sélectionnés, afin d’identifier les paramètres précis aboutissant à la perte ou au gain de leur fonctionnalité. / Cyanobacteria are ubiquitous microorganisms that present a worldwide concern to public health authorities because of the toxicity of the cyanotoxins they produce. Some cyanotoxins are hepatotoxins such as microcystins (MCs). At least 200 variants of MCs have been identified till today. In our study, we focus on MC-LR, a monocyclic heptapeptide (cyclo-(D-Ala-L-Leu-D-erythro-β-methylAsp-L-Arg-ADDA-D-Glu-N-methyldehydro-Ala), since it is the most frequently detected and one of the most toxic. In our study, we are interested in developing a fast, sensitive and reliable method to detect MCs. The project aims to develop an alternative pollution detection method that would be better suited to field measurements than the physicochemical methods currently available. The originality of this project lies in the use of two different approaches to select a panel of antibodies suitable for the development of immunodetection tests. The first one is based on the hybridoma technology for the production of monoclonal antibodies. The second one is based on phage display technique to select antibody fragments that are specific to MC-LR from a library of approximately 109 phages, expressing on the surface scFv fragments (Shahsavarian et al., 2014). Two monoclonal antibodies were selected using the first approach, and their specificity was evaluated using ELISA technique. Along with three scFvs selected from phage display approach. An additional scFv was added to this list: 3A8, selected from a previous study (McElhiney et al., 2002) and also specific to MC-LR. The scFvs were cloned into an expression vector in order to get each clone in its scFv soluble form. Then, their specificity to MC-LR was evaluated using ELISA technique and Surface plasmon resonance. The results show a potential specificity to MC-LR. Nevertheless, these results are not very reproducible and call into question the refolding protocol used. A thorough work on this protocol optimization would be necessary, in order to find the key parameters that control the loss or gain of their functionality

In the medical and veterinary diagnostic fields there is an ongoing need for stable and specific antibodies. There is also a requirement for simple, robust and cost-effective diagnostic assays to be used in the developing world. Recombinant antibodies from phage displayed libraries are economical to produce and can often be engineered to improve affinity, avidity and stability. While recombinant antibody fragments are useful in immunoassays, they are not strictly comparable to normal immunoglobulins and may under-perform in certain assays. Converting monovalent single-chain antibody fragments (scFvs) to bivalent immunoglobulin-like formats could conceivably provide a more suitable molecular scaffold for use in immunoassays. Two scFvs that recognised the 65 kDa heat-shock protein (HSP65) of Mycobacterium bovis were used in this study. They were originally derived from the Nkuku® repertoire, a phage displayed antibody library based on the immune repertoire of the chicken, Gallus gallus. The genes coding for these scFvs were subcloned in expression vectors containing chicken IgY constant-heavy domains, to create bivalent constructs which were designated ‘gallibodies’. Expression of these constructs was attempted in three heterologous systems. While they were successfully produced in adherent mammalian cell cultures, the growth requirements of these cultures complicated subsequent purification. Bacteria and yeasts were investigated as alternative expression systems, but antibodies were not produced in either system. The gallibodies were compared to their monovalent scFv counterparts for stability as well as their applicability in ELISAs and gold-conjugated immunochromatographic lateral-flow assays. As gallibodies, both retained their functionality after exposure to different conditions and they were capable of immunocapture in ELISA. This was in contrast to their performance as scFvs. Furthermore, these antibody-like molecules could be stably conjugated to colloidal gold and used in lateral flow tests where positive and specific signals were obtained. This confirmed that recombinant single-chain monomeric antibody fragments could be reconstituted as bivalent immunoglobulin-like molecules and that they are a potentially useful platform for developing practical, robust immunodiagnostic reagents. It appeared from these experiments that the antibodies could act as a pair in which one captures, and the other detects HSP65. To find out whether they recognised discrete regions on the protein, their epitopes were mapped using a phage displayed peptide library in combination with computer-based algorithms. The presumptive epitope of one was mapped to residues 350 to 370 on HSP65 of M. bovis. The sequences selected from the peptide library by the other corresponded to three separate regions on the target protein. These recombinant antibody recognition sites are analogous to some of those that have been mapped by others using traditional monoclonal antibodies. / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted

Molecular magnets with different dimensionality, whether they are zero-dimensional singlemolecule magnets (SMM) or one-dimensional single-chain magnets (SCM) are very interesting, since they allow probing the fundamental aspects bordering quantum and classical physics at the nanoscale level. This dissertation covers experimental studies of two Mn-based exchangecoupled molecule-based magnets and two Co-based single-chain magnets, using both dc Halleffect magnetometry and electron paramagnet resonance (EPR) techniques. In these multidimensional systems, the spin of the molecule exhibits quantum mechanical behavior at low temperature. It is quite interesting to observe the effect of magnetic exchange interactions on the magnetic properties of various complexes; hence they strongly affect the magnetic behavior. In this dissertation, the research is initiated with the study of low-magnetic-nuclearity molecules, starting with a spectroscopic study of a significantly anisotropic Mn(IV) monomer. At low temperature the molecule possesses easy-plane type anisotropy of a remarkable magnitude. Although the molecule is not a single-molecule magnet, the remarkable anisotropy can initiate synthesis of newer and better molecular magnets with Mn(IV) as the main building block. Furthermore, the interplay between the magnetic anisotropy and the inter-ion exchange interactions (J) within the molecule are probed for a dimer and a trimer where the magnetic core is comprised of two and three ions respectively. In the Mn-based case of the dimer, the low coupling between the atoms leads to significant state mixing, thus making it impossible to assign the individual spin states to the dimer or to the respective individual Mn(II) ions. In the case of iv the trimer, lowering of the symmetry achieved by fine tuning of the inter-ion exchange interactions leads to relieving of frustration in the antiferromagnetic (AF) triangular Mn(III) system, resulting in a well defined ground state and significant zero field splitting. Also a clear hysteretic behavior observed in this system demonstrates its SMM nature at low temperature. Finally, high-field high-frequency magnetic and spectroscopic studies performed on two cobalt-based SCMs reveal that formation of magnetic domains by exchange interactions within the chain are strongly influenced by thermal fluctuations. The chain possesses a uniaxial anisotropy with the quantization axis lying along the length of the chain. Moreover it is shown that modulation of the magnitude of inter- and intra-chain interactions results in a threedimensional dynamics in one of the samples. Interestingly, detailed dc magnetic studies show a tunable crossover between one- and three-dimensional magnetic dynamics as a function of temperature and/or magnetic field sweep rate. Our voyage through several molecular systems of different dimensionality have allowed us to expand our understanding of the role of exchange interactions on the magnetic behavior in molecular magnetism

Interleukin-12 (IL-12), an important immuno-modulator for cell-mediated immunity, shows significant potential as a vaccine adjuvant and anti-cancer therapeutic. However, its clinical application is limited by lack of an effective bioproduction system and by toxicity associated with systemic administration of IL-12. The goals of this research were to determine whether plants can serve as an effective production system for bioactive IL-12, a complex 70kDa glycoprotein cytokine, and whether the plant lectin RTB can facilitate mucosal delivery of IL-12 to immune responsive sites. Transgenic tobacco plants expressing murine IL-12 were generated and characterized. To ensure stochiometric expression of the two separately encoded, disulfide-linked subunits of IL-12 (p35 and p40), a single-chain form of mouse IL-12 (mIL-12) was utilized. Hairy root cultures, as a fast-growing bioproduction system were developed from high expressers of mIL-12. A purification scheme was developed to purify plant-derived mIL-12 from hairy roots and purified mIL-12 was used to assess IL-12 bioactivity in vitro in mouse splenocytes and in vivo in mouse intranasal vaccination trials. Plant-derived mIL-12 triggered induction of interferon-gamma secretion from mouse splenocytes as well as stimulation of cell proliferation with comparable activities to those observed for the animal-cell-derived mIL-12. Mouse vaccination trials using GFP as the antigen and CT as the adjuvant suggested that plant-derived mIL-12 enhanced Th1 immunity and exhibited similar activity to animal-cell-derived mIL-12 in vivo. Plant-derived IL-12 itself was non-immunogenic suggesting conformational equivalency to endogenous mouse IL-12. Ricin B (RTB), the non-toxic carbohydrate-binding subunit of ricin, directs uptake of ricin into mammalian cells and the intracellular trafficking of ricin A, the catalytic subunit of ricin. RTB's function suggests that it may work as a molecular carrier for effective mucosal delivery of IL-12. To prove this hypothesis, transgenic plants producing RTB:IL-12 fusions were generated and characterized. Our results demonstrated that RTB fused to the carboxyl-terminus of IL-12 maintained full lectin activity and IL-12 bioactivity. RTB fused to the amino-terminus of IL-12 did not show lectin activity due to steric hinderance. Purified IL-12:RTB from transgenic plant tissue was tested in an in vitro mucosal-associated lymphoid tissue (MALT) assay. The results indicate that RTB facilitates the binding of IL-12 to the epithelial cells and presentation of IL-12 to immune responsive cells. In conclusion, my research has shown that transgenic plants are capable of producing valuable bioactive proteins, such as IL-12. Plant-derived mIL-12 exhibited similar activity to animal-cell-derived mIL-12 both in vitro and in vivo. Fusion of IL-12 with the RTB lectin facilitates the delivery of IL-12 to mucosal immune responsive cells and thus may serve as a molecular carrier to enhance IL-12 efficacy and reduce the side-effects associated with systemic administration of IL-12. / Ph. D.

Dental plaque forms sequentially, with Fusobacterium nucleatum facilitating the adhesion of pathogenic late colonizers. We hypothesize that a single-chain variable fragment (scFv) antibody library will enable the identification of F. nucleatum adhesins and help elucidate the molecular mechanisms of coaggregation between F. nucleatum and other bacteria. A 4X10^8 clones scFv phage display library was created using spleen RNA from a mouse immunized with F. nucleatum. The library was enriched by biopanning against F. nucleatum 6 times and 292 individual clones tested by ELISA reacted strongly to F. nucleatum. Sixty-two of those clones inhibited F. nucleatum coaggregation with Streptococcus sanguinus. Analysis of select clones revealed differences in coaggregation inhibition, recognition of outer membrane proteins, and BstOI restriction pattern. DNA sequencing showed 6 unique scFvs and of them 3 strongly inhibited interaction with 5 Streptococcus species. These scFvs recognize the outer membrane autotransporter protein RadD (Fn1526), as determined by mass spectrometry. / Farhan Khan placed second in the International Association for Dental Research/Unilever Hatton Competition in the Senior Basic Science Research Category representing Canada, while presenting the research contained in this dissertation. This international competition took place during the 90th General Session & Exhibition of the International Association for Dental Research in Iguaçu Falls, Brazil in June 2012.

Mycolic acids are long chain lipids from the cell walls of Mycobacterium tuberculosis. The Nkuku phage display library was previously used to obtain monoclonal antibody binders to mycolic acids. In total 11 binders were obtained of which one was selected (MAC10) for further investigation by genetic engineering as presented in this dissertation. The antibodies of the Nkuku phage display library are in the format of single chain variable fragments (scFv). ScFv’s constitute only the epitope binding domains of an antibody consisting of the VH and VL domains fused into a single chain by a flexible linker protein. The selected anti-mycolic acid scFv is referred to as mycolic acid clone 10 (MAC10). Genes encoding the scFv’s of the Nkuku phage display library were cloned into the plasmid pHEN-1, a phage display vector. This vector is not commercially available or ideally suited for expression of scFv proteins. Therefore two vectors were investigated as possible targets for subcloning. The plasmids pGE20 and pAK400 were previously used for the expression of scFv antibody proteins. Subcloning into plasmid pAK400 proved to be the more efficient of the two investigated for subcloning. This subcloning yielded the recombinant plasmid pAKJS. Following the subcloning scFv protein expression was attempted using the plasmids pMAC10 (derived from pHEN-1) and pAKJS (derived from pAK400). Expression of MAC10 using plasmid pMAC10 in both Escherichia coli TG-1 and HB2151 was constitutive. This demonstrates that plasmid pHEN-1 is a non ideal vector as expression should not occur unless induced. Expression of MAC10 did not occur when pAKJS and Escherichia coli HB2151 were used. This was due to both the vector and expression host producing inhibitor protein for the Lac Z promoter controlling expression of the scFv. The MAC10 gene was subsequently randomized using the directed evolution method, error prone PCR. Sequence analysis of the five selected mutants indicated an average mutation rate of 8.6 mutations per 1000 base pairs. From the combined total of all five mutants, transversions made up the majority of substitutions. The majority of transversion mutations occurred at A-T base pairs. Transition substation mutations that made up the minority of total mutations occurred mostly at G-C base pairs. / Dissertation (MSc)--University of Pretoria, 2012. / Biochemistry / unrestricted

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