Designing a Luminescence Assay to Measure In Vitro Binding of the Fibronectin Attachment Protein (FAP) of Mycobacterium avium subspecies paratuberculosis to Fibronectin

The attachment and penetration of epithelial cells by Mycobacterium avium subsp. paratuberculosis (MAP) requires a bacterial fibronectin (FN) attachment protein FAP-P, which facilitates binding of FN by MAP. The region of FAP-P that interacts with FN is known, however, the region of FN that is bound by FAP-P is to be determined. Identification of the region of FN to which FAP-P binds would enable the synthesis of a competing peptide that could prevent FN binding by MAP. The first step toward locating this binding site is the design of a reliable FN binding assay. The purpose of this investigation was to determine the optimal conditions (time, temperature, probe concentration, specificity, and color of assay plates) necessary for sensitive, specific binding of FN by FAP-P. Biotinylated peptides representing the FN binding domain of FAP-P and a control peptide were used as probes in a chemiluminescence assay to evaluate the binding of FAP-P to FN. The FAP-P probe bound specifically to FN when probes were incubated for 30 minutes at 37oC in black 96-well microtiter plates. A 10-fold increase in specific FN binding by the FAP-P probe resulted when incubation was increased from 30 minutes to 60 minutes in combination with the use of white microtiter plates over black microtiter plates. A dose dependent binding of FN by the FAP-P probe was observed with increasing probe concentrations (1.0 µg FAP-P, 2.0 µg FAP-P, and 4.0 µg FAP-P) using optimized conditions. The assay thus described can be used to locate the FAP-P binding site on FN in protease-generated FN fragments. This, in turn will enable the design of peptides that could be incorporated into calf management programs that could block FN binding by MAP and reduce the reduce the incidence of Johne’s disease

The Concept of ‘States within a State’ Admist Conflict and Peace Building Ventures in Bafut, Cameroon

This study looks at the perception and manifestation of the concept of states in African communities A state in African context is an organisation of human beings connected by a system of relations Within the states different groups of people exist and different individuals have different roles to play Some exercise special powers or authority capable of giving command which is obeyed by the people they rule In the Bamenda Grassfields of Cameroon present-day North West Region these individuals are called fons and chiefs and they rule fondoms In Westernised societies they would be called kings Since colonial period government administrators refer to them as traditional rulers or natural rulers Amongst these rulers are some who rule over what is commonly referred to as semi-autonomous polities within the fondom

Gender Identity as a Marker of Cultural Crisis in Marriage. A Study of Two West African Novels

A majority of West African women, like most other African women, are victims of society regulated by cultural norms and traditional values, especially in the marital institution. The present comparative study of Mariama Ba’s So long a Letter and Buchi Emechita’s The Joys of Motherhood seeks to explore Gender Identity as a Marker of Cultural Crisis in marriage. This comparative study reveals the increasing attention being accorded to the mediation of gender relations. Most African women are powerless and voiceless victims of ever deepening oppression rooted in layers of male-supremacist tradition. During and after colonization most West African women declined from a position of power and self-sovereignty to becoming a man’s helper. Patriarchy was established firmly in a macho conviviality and a one-dimensional and minimalised presentation of women who assumed peripheral roles. Most male writers in the early phase of African literature encouraged the marginalization of women: the female characters created by male writers were made marginal to the plot of the fiction while a few emerge as credible protagonist. The female gender was trivialized through practices like patriarchy, tradition, gender socialization, polygamy, class religion and domestic enslavement. Using comparative methodology, this thesis shows how African female writers like Mariama Bâ and Buchi Emecheta make the culture of African art relevant to the understanding of gender relations, suggesting how women can be redeemed in contemporary writings, and also how these writers react to sexist depictions of women and gender power or identity. A countervailing set of images change the notion of African women as victims, casting them as assertive and self-reliant heroines. African female writers’ attempt to bridge the gender rift through their writing gives a voice to the silenced, oppressed women.fi=Opinnäytetyö kokotekstinä PDF-muodossa.|en=Thesis fulltext in PDF format.|sv=Lärdomsprov tillgängligt som fulltext i PDF-format

A Novel Mode of Action of C-reactive Protein in Protecting Against Streptococcus pneumoniae Infection and Synergy with Antibiotics

C-reactive protein (CRP) is a part of the innate immune system, is synthesized in the liver, its blood level increases in inflammatory states, and it binds to Streptococcus pneumoniae. The conformation of CRP is altered under conditions mimicking an inflammatory milieu and this non-native CRP also binds to immobilized/aggregated/pathogenic proteins. Experiments in mice have revealed that one of the functions of CRP is to protect against pneumococcal infection. For protection, CRP must be injected into mice within two hours of administering pneumococci, thus, CRP is protective against early-stage infection but not against late-stage infection. It is unknown how CRP protects or why CRP does not protect against late-stage infection. The hypotheses are that the protection requires complement activation by CRP-pneumococci complexes and that CRP cannot protect if pneumococci have time to recruit complement inhibitor factor H on their surface to become complement attack-resistant. To test these hypotheses, we generated CRP mutants by site-directed mutagenesis: a mutant that binds to pneumococci but does not activate complement and a mutant that binds to immobilized factor H. We found that mutant CRP incapable of activating complement was not protective against infection and that mutant CRP capable of binding to factor H was protective against both early and late stage infections. Additional experiments showed that CRP enhances the effects of the antibiotic clarithromycin in reducing bacteremia in infected mice. Moreover, we observed that mutant CRP capable of binding to factor H bound to several proteins immobilized on plastic, suggesting that CRP recognizes a pattern, probably an amyloid-like structure, on immobilized proteins. Indeed, mutant CRP, after binding to amyloid b peptides, prevented the formation of pathogenic amyloid fibrils. Lastly, employing a hepatic cell line, we investigated the mechanism of CRP expression in response to pro-inflammatory cytokines. We found that the transcription factor C/EBPb and two C/EBP-binding sites on the CRP promoter were critical for inducing CRP expression. We conclude that complement activation is necessary for CRP-mediated protection against infection, that CRP functions in two structural conformations, that CRP and clarithromycin act synergistically, that CRP has anti-amyloidogenic properties, and the increased CRP expression requires C/EBPb

Novel Cell Penetrating Peptides Effect Endosomal Escape and Deliver Protein Cargos into Living Cells

Over the last decade a number of peptides that are rapidly internalized by mammalian cells have been discovered or designed. Cell-penetrating peptides (CPPs) are capable of mediating penetration of the plasma membrane, allowing delivery of macromolecular cargoes to the cell interior. We have developed a novel CPP-adaptor protein technology that allows any user-defined cargo delivery and release into the cytoplasm. Our hypothesis is that a CPP-adaptor with a moiety allowing high-affinity but reversible non-covalent cargo binding would lead to more efficient penetration and release than current CPP delivery strategies. Delivery of proteins to the interiors of cells has many applications. In addition to detecting and mapping the location of the components of living cells with fluorescent tags in real time, the availability of our system will likely enable the manipulation of signaling pathways and gene expression by allowing the introduction of components, e.g. constitutively active kinases, repressors or enhancers. CPP-adaptor, TaT-Calmodulin, and cargo proteins (horse radish peroxidase, myoglobin and beta-galactosidase) were expressed and purified from E. coli BL21 (DE3)pLysS. Optical biosensing experiments demonstrated that affinity and kinetics between the novel CPP and cargo proteins did not significantly differ from wild-type interactions; all had subnanomolar affinities. Cargo proteins were labelled with DyLight 550. CPP-cargo complexes or cargo alone were incubated with subconfluent baby hamster kidney, HEK 293T and HT-3 cells. After washing, cells were imaged by fluorescence confocal microscopy. All users define cargos exhibited penetration and release to the cytoplasm whereas cargo-only controls exhibited no measurable penetration (though some adherence to the outside of the cells was observed). Time courses and dose-dependency studies characterizing penetration and release kinetics will be presented as will initial efforts to deliver cargo that may alter cell-signaling pathways. The results presented herein demonstrate the feasibility of delivering a wide variety of cargo proteins to the intracellular environment; creating an array of potential research, diagnostic and therapeutic applications

Comparing methods for modeling longitudinal and survival data, with consideration of mediation analysis

Joint modeling of longitudinal and survival data has received much attention and is becoming increasingly useful. In clinical studies, longitudinal biomarkers are used to monitor disease progression and to predict survival. These longitudinal measures are often missing at failure times and may be prone to measurement errors. In previous studies these two types of data are frequently analyzed separately where a mixed effects model is used for longitudinal data and a survival model is applied to event outcomes. The argument in favor of a joint model has been the efficient use of the data as the survival information goes into modeling the longitudinal process and vice versa. In this thesis, we present joint maximum likelihood methods, a two stage approach and time dependent covariate methods that link longitudinal data to survival data. First, we use simulation studies to explore and assess the performance of these methods with bias, accuracy and coverage probabilities. Then, we focus on four time dependent methods considering models that are unadjusted and adjusted for time. Finally, we consider mediation analysis for longitudinal and survival data. Mediation analysis is introduced and applied in a research framework based on genetic variants, longitudinal measures and disease risk. We implement accelerated failure time regression using the joint maximum likelihood approach (AFT-joint) and an accelerated failure time regression model using the observed longitudinal measures as time dependent covariates (AFT-observed) to assess the mediated effect. We found that the two stage approach (TSA) performed best at estimating the link parameter. The joint maximum likelihood methods that used the predicted values of the longitudinal measures, similar to the TSA, provided larger estimates. The time dependent covariate methods that used the observed longitudinal measures in the survival analysis underestimated the true estimates. The mediation results showed that the AFT-joint and the AFT-observed underestimated the mediated effect. Comparison of the methods in Framingham Heart Study data revealed similar patterns. We recommend adjusting for time when estimating the association parameter in time dependent Cox and logistic models. Additional work is needed for estimating the mediated effect with longitudinal and survival data

The Concept of ‘States within a State’ Amidst Conflict and Peace Building Ventures In Bafut

This study looks at the perception and manifestation of the concept of states in African communities A state in African context is an organisation of human beings connected by a system of relations Within the states different groups of people exist and different individuals have different roles to play Some exercise special powers or authority capable of giving command which is obeyed by the people they rule In the Bamenda Grassfields of Cameroon present-day North West Region these individuals are called fons and chiefs and they rule fondoms In Westernised societies they would be called kings Since colonial period government administrators refer to them as traditional rulers or natural rulers Amongst these rulers are some who rule over what is commonly referred to as semi-autonomous polities within the fondom

Identification of motoneurons innervating individual extraocular muscles within the oculomotor nucleus in human

The oculomotor nucleus (nIII) and trochlear nucleus (nIV) of the midbrain contain motoneurons that innervate extraocular eye muscles. The aim of this study is to identify the motoneuronal subgroups of the human nIII and nIV, which innervate individual extraocular muscles using several histochemical stains, and comparing the results with those obtained in monkey. The nIV innervates only the superior oblique (SO) muscle, whereas nIII contains extraocular motoneurons, which innervate the medial rectus (MR), inferior rectus (IR), superior rectus (SR) and inferior oblique (IO) muscles. The organization of the motoneuron subgroups in human is not clear, and is the main subject of this investigation. In monkey the localization of the motoneuron subgroups for individual muscles is well known as a result of tract tracing studies, and analyses of their differential transmitter inputs. The results in monkey provided a useful basis for this study on human nIII. Human brainstems were fixed in 4% paraformaldehyde, series of frozen and paraffin sections of 40μm, 10μm and 5μm respectively were stained with choline acetyltransferase (ChAT) antibody to identify cholinergic motoneurons of extraocular muscles. The ChAT sections were then immunostained for the inhibitory transmitter GABA with antibodies against glutamate decarboxylase (GAD) or for the calcium-binding protein calretinin (CR). The cytoarchitecture of nIII was visualized with cresyl violet, gallyas stain or antibodies against non-phosphorylated neurofilaments (NP-NF). Six subgroups were delineated in nIII: a dorsolateral (DL), dorsomedial (DM), central (CEN), ventral (VEN), lateral group (LAT) and the nucleus of Perlia (NP), in addition to the central caudal nucleus (CCN) and the urocortin-positive centrally projecting neurons of the Edinger- Westphal nucleus (EWcp). The DL, VEN, LAT, EWcp and NP groups receive a strong supply of GAD-positive terminals as the SO motoneurons in nIV. Strong CR- immunoreactivity was found in the CEN group, NP and CCN, but not in nIV. Based on the staining properties of the subgroups and a comparison to the existing studies on monkey nIII, the CCN was considered to innervate the levator palpebrae; the CEN group was identified as the upgaze motoneuron subgroup for SR and IO, and DL, VEN and LAT showed characteristics of MR motoneurons. For the first time two separate subgroups of motoneurons (DL and VEN) subserving motor pathways for MR were identified in the human nIII, whereby the DL subgroup corresponds to the B-group and the VEN subgroup to the A-group in the monkey nIII. The DM group was considered to innervate IR muscle. The strong CR input to NP revealed characteristics of upgaze motoneurons in nIII. A good correlation was found between monkey and human in CR stains. But surprisingly, there were striking differences between monkey and human nIII with GAD stains. The results indicate that human MR motoneurons may contribute to a specialized function, e.g. during vergence, by its strong GABAergic input.Der Nucleus oculomotorius nIII und Nucleus trochlearis (nIV) im Mittelhirn enthalten die Motoneurone der extraoculären Augenmuskeln. Ziel der vorliegenden Arbeit war die Identifizierung der verschiedenen Motoneuronengruppen im humanen nIII und nIV, welche individuelle Augenmuskeln innervieren. Dies erfolgte anhand verschiedener histochemischer Färbungen, die im Vergleich zu Daten an Affen erhoben wurden. Der nIV innerviert nur den Musculus obliquus superior (SO), während nIII die Motoneurone von Musculus rectus medialis (MR), inferior (IR), superior (SR) und Musculus obliquus inferior (IO) enthält. Die Organisation der Motoneuronengruppen im Menschen ist noch nicht eindeutig geklärt und ist Hauptthema der vorliegenden Studie. Im Affen ist die Lokalisation der einzelnen Motoneuronengruppen individueller Augenmuskeln aufgrund von Trakt-Tracer-Studien etabliert, ebenso wie die Transmittereingänge zu den einzelnen Gruppen, die als Basis für die vorliegende Arbeit am menschlichen nIII dienten. Humane Hirnstämme wurden in 4% Paraformaldehyd fixiert, und Serien von Gefrier- und Paraffinschnitten (40μm, 10μm und 5μm) wurden mit Antikörpern gegen Cholinacetyltransferase (ChAT) gefärbt, um die cholinergen Motoneurone der äußeren Augenmuskeln zu identifizieren. Die auf ChAT angefärbten Schnitte wurden dann mit einer weiteren Immun-Peroxidase-Färbung auf den inhibitorischen Transmitter GABA mit Antikörpern gegen Glutatmatdecarboxylase (GAD) oder das Calcium-bindende Protein Calretinin (CR) angefärbt. Die Zytoarchitektur des nIII wurde mit einer Kresyl-Violett- Färbung, Gallyas-Färbung oder Immunfärbung auf nicht-phosphorylierte Neurofilamente (NF-NP) dargestellt. Sechs Untergruppen konnten im nIII des Menschen voneinander abgegrenzt warden: eine dorsolaterale (DL), dorsomediale (DM), zentrale (CEN), ventrale (VEN), laterale Gruppe (LAT) neben dem Nucleus Perlia (NP) und dem Nucleus centralis caudalis (CCN) und der urocortin-positiven central projizierenden Neurone des Edinger-Westphal Kerns (EWcp). Die Gruppen DL, VEN, LAT, EWcp and NP erhalten einen starken Eingang von GAD-positiven Endigungen, ebenso die Motoneurone des SO im nIV. Eine starke Immunreaktivität für CR wurde innerhalb des nIII nur in der CEN-Gruppe gefunden, sowie dem NP und CCN, nicht aber im nIV. Basierend auf den histochemischen Eigenschaften der einzelnen Motoneuron-Subgruppen im Affen wurde beim Menschen der CCN bestätigt als der Kern, der die Motoneurone des Musculus levator palpebrae enthält; die CEN-Gruppe wurde anhand der selektiven CR- Eingänge als Sitz der Motoneurone von SR und IO identifiziert, die Blickbewegungen nach oben bewirken. Die Gruppen DL, VEN und LAT zeigten die Eigenschaften von MR- Motoneuronen, die DM-Gruppe die von IR-Motoneuronen. Hierbei konnte zum ersten mal gezeigt werden, dass auch beim Menschen der MR in zwei Gruppen innerhalb des nIII repräsentiert ist, wobei DL der B-Gruppe, und VEN der A-Gruppe beim Affen entspricht. Ein weiterer neuer Befund zeigte sich für den NP, der einen starken CR-Eingang erhält und damit eher Eigenschaften von Motoneuronen für Aufwärtsblick (wie SR und IO) aufweist. Insgesamt fand sich eine gute Übereinstimmung in den histochemischen Eigenschaften der Motoneuroneingänge bezüglich CR zwischen Mensch und Affe. Aber überraschenderweise war beim Menschen ein viel stärkerer GAD-Eingang auf die Motoneurone für horizontale Augenbewegungen (MR) als beim Affen präsent. Diese Daten weisen auf eine spezialisierte Rolle der MR-Motoneurone im Menschen, z.B. bei Vergenz, hin

The impact of HIV-1 subtype C Envelope N-glycosylation on DC-SIGN meditated modulation of DC function to facilitate transmission or enhance viral pathogenesis

N-glycosylation plays an important role in Envelope (Env) function and may be involved in the modulation of the immune response to HIV-1 infection. In this study, we hypothesized that Env N-glycosylation may affect viral pathogenesis by influencing Env structure and function. Furthermore, we also postulated that differences in Env glycosylation could affect interactions between Env and DC-SIGN of dendritic cells (DCs), activating alternative signalling pathways which stimulate the release of different immune modulators. We generated pseudovirus of eighteen Env clones (PSVs) with variable number and position of potential N-glycan sites (PNGs) and compared their ability to infect TZM-bl cells, bind to Raji+ DC-SIGN cells, trans-infect TZM-bl cells when captured by either Raji-DC-SIGN cells or monocyte-derived dendritic cells (MDDCs) and modulate MDDC signaling by investigating the release of Interleukin-10 (IL-10) and other immune modulatory cytokines and MAPK activation. Entry efficiency, DC-SIGN binding and trans-infection varied widely across all clones. The level of IL-10 secreted by MDDCs in response to PSV stimulation varied 32-fold. The induction of IL-10 secretion by purified gp140 confirmed that Env was the viral component that stimulated the secretion of IL-10 via interaction with DC-SIGN and potentially other undefined receptors. PSV and purified gp140 stimulated MDDC signaling via ERK and JNK phosphorylation, while p38 was not activated. The addition of recombinant DC-SIGN lowered the levels of secreted IL-10 and ERK /JNK phosphorylation, suggesting that DC-SIGN plays a role in these responses. As Env mannosylation correlated with DC-SIGN binding, five highly conserved Env PNGs (241, 262, 386, 392, and 448) previously identified to carry high mannose type N-glycans and hence thought to be involved in DC-SIGN binding were deleted in two Env clones by site-directed mutagenesis to confirm their importance in Env function. The potential role of these PNGs in Env entry efficiency, DC-SIGN binding, trans-infection, induction of MDDC IL-10 secretion and activation of MAPK phosphorylation was determined. Deletion of these sites significantly affected the entry efficiency, DC-SIGN binding, trans-infection and MDDC IL-10 secretion, with one Env clone proving to be more sensitive to mutation than the other. This suggests that PNGs influence Env function in a clone-specific manner. As deletion of highly conserved PNGs abrogated Env function we used sequence analysis to identify PNGs involved in binding DC-SIGN and inducing MDDC IL-10 secretion. We grouped PSVs based on the presence or absence of specific PNGs in Env sequences and compared entry efficiency, DC-SIGN binding, trans-infection, stimulation of MDDC IL-10 secretion and induction of MAPK phosphorylation. Three Env PNGs were significantly associated with entry efficiency (N356, N392, and N674), and three sites (N289, N356 and 674) were significantly associated with trans-infection while N674 also influenced DCSIGN binding. The majority of MDDC donors secreted higher levels of IL-10 when stimulated with PSVs that carried PNGS at N130 (p = 0.0016) and N332 (p = 0.0039) and lacked N674 (p = 0.033). When Envs were graded on whether they had 0, 1, 2 or 3 of the PNGs (e.g. -130, -332, +674; -130, +332 and +674, etc.) those that carried either one of the PNGs or the entire induction motif (N130+ N332+ N674-) significantly stimulated MDDCs to secrete higher levels of IL-10 than those that completely lacked the motif (p = 0.0335 and p = 0.0304, respectively). As the presence of N674 was linked to reduction in all functions of Env, it is likely that the presence of an N-glycan at this site affected Env structure and could skew the analysis. Excluding N674 indicated that the presence of PNGs at position 130 and 332 was sufficient to induce significantly higher IL-10 release than those that had either none or one of these sites (p = 0.0053). When we determined whether N130 and N332 were enriched in subtype C acute infection Envs, these sequences were not enriched with PNGs at either N130 or N332 compared to chronic infection viruses. However, when IL-10 levels were compared between MDDC donors stimulated with PSV of either acute or chronic infection clones, those from early infection significantly enhanced MDDC secretion of IL-10 (p = 0.0039). This suggests that even though PNGs at 130 and N332 could be involved in inducing MDDC IL-10 secretion, it is not the only requirement for enhanced stimulation. Although Env differentially activated ERK and JNK phosphorylation, ERK phosphorylation did not correlate with IL-10 secretion, suggesting that this MAPK signaling pathway was not solely responsible for triggering the release of MDDC IL-10 and other regulatory cytokines. PSVs also stimulated the release of TNFα, IL-1β, IL-6, IL-8, MIP-1a, and MIP-1b while having no effect on IL-12 levels. This suggests that HIV-1 binding to DCs in the genital tract could change the dynamics of DC immune responses, deregulating their cytokines secretion and destabilising the Th0 cell differentiation to facilitate viral survival and thus productive clinical infection. We therefore conclude that HIV-1 variants differentially stimulate MDDCs to release immunosuppressive IL-10 and that transmitted founders could be better at modulating immune responses in the genital tract compared to chronic infection variants