Dissertations / Theses on the topic 'In vitro antithrombotic properties'

The rising costs of prescription drugs in the maintenance of personal health and wellbeing have increased the interest in medicinal plants. The World Health Organization estimates that 65 percent-80 percent of the world’s population use traditional medicine as their primary form of health care. In this project the focus has been on the use of Leonotis leonurus extracts as a traditional medicine. The major chemical constituent of this plant is marrubiin, which is a diterpenoid labdane lactone formed from a precursor called premarrubiin. Aqueous and acetone extract (AL and OL extract, respectively) of this plant has been found to have an antithrombotic effect, with IC50 values of 3mg/ml and 6mg/ml, respectively. The extracts also have an effect on fibrinolysis, where the lysis time was decreased by more than 50 percent by the organic extract and standard marrubiin. In whole blood ADP-induced platelet aggregation, the organic extract inhibited aggregation by 68 percent at a final concentration of 138μg/ml (equivalent to 7.2μg/ml marrubiin). Marrubiin has also been screened for antithrombotic/anticoagulant activity; no antithrombotic activity has been observed but it increased the rate of fibrinolysis, by decreasing lysis time by 64 percent and also decreasing fibrin formation. From these findings it can be concluded that marrubiin has a fibrinolytic effect and antiplatelet aggregation effect. In the diabetic studies, in hyperglycemic condition, the OL (10μg/ml) extract and standard marrubiin significantly increased insulin secretion by 200 percent (2-fold) and 400 percent (4-fold), respectively, with respect to the control. The OL extract and standard marrubiin stimulated the release of insulin, the stimulatory index was significantly increased by 450 percent (4.5-fold) and 500 percent (5-fold), respectively, with respect to the control. In the apoptotic studies, in the normoglycemic and hyperglycemic conditions, the OL extract decreased the occurrence of apoptosis, in a dose-dependent manner, with the lower concentrations inducing apoptosis significantly higher than the relevant controls. Standard marrubiin did not have an effect on apoptosis in hyperglycemic condition, but it decreased the occurrence of apoptosis by 200 percent (2-fold) under normoglycemic conditions. The OL extract increased proliferation by 148 percent (1.48- fold) and 155 percent (1.55-fold) in normoglycemic and hyperglycemic conditions, respectively. The same effect was observed for standard marrubiin, where, proliferation was increased by 180 percent (1.8-fold) and 200 percent (2.0-fold) in normoglycemic and hyperglycemic conditions, respectively. RT-PCR displayed that standard marrubiin inhibited the expression of insulin by 50 percent under normoglycemic conditions.

The formation of a thrombus stems from the malfunction of a normal physiological function referred to as haemostasis and the activity of blood platelets; such thrombi give rise to debilitating and often fatal strokes. Consequently much effort is associated with the search for pharmacological compounds capable of their prevention or dispersion. · Most of the primary screens associated with such work rely on in-vitro tests and in separating the blood from it's vasculature, the influence and results associated with several naturally occuring moderators may be lost. There therefore exists the incentive to develop more representative in-vivo screening methods. Following an introduction to the underlying physiology and pharmacology and a review of established screening methods, this thesis proceeds to describe the development of a novel technique suitable for such in-vivo studies. It's inception is shown to be a consequence of an amalgamation of ultrasonic methods associated with the clinical detection of occlusions and laser Doppler velocimetry. Both topics are individually surveyed and then brought together through a concept whereby the efficacy of compounds might be evaluated in animal models by measuring the velocity of blood in the fluid jet formed distal to an induced thrombus.The main underlying assumption is that the jet velocity will reflect the degree of encroachment of the thrombus into the vasculature. In accord with the evolved measurement rationale there then follows a description of a specific laser Doppler velocimeter and some associated experiments, designed to qualitatively appraise the validity of the underlying assumptions. The ensuing results in turn give rise to the design of a laser Doppler microscope, an analyser for extracting the required velocity information from the Doppler shift spectrum and an additional series of experiments. Central to this latter stage of validation is the use of a thrombus analogue in a narrow bored glass flow tube. Finally, some preliminary in-vivo experiments and results are presented.

Use of synthetic materials in medical applications is one of the most common practices in modern medicine. Yet occurrence of surface-induced thrombus formation on these materials, especially those associated with cardiovascular applications, generates a need for surface modifications. Limiting thrombus formation on a biomaterial surface represents the ultimate success for blood contacting devices. One interesting approach is to enhance fibrinolysis before the blood clot becomes stabilized. Herein, two synthetic polymers, poly-N- [(2, 2-dimethyl-1, 2-dioxolane) methyl] acrylamide (PDMDOMA) and poly- (N-isopropylacrylamide) (PNIPAm), were tested for this particular antithrombotic property. Surface-grafted PNIPAm samples, brush-PNIPAm and star-PNIPAm, were also tested for the biological activity. We evaluated the influence of these synthetic polymers on blood hemostasis by studying the fibrin polymerization process, the three-dimensional clot structure, and the mechanical properties of blood clot such as its clot strength, clot elasticity and clot fibrinolysis. Both linear PDMDOMA and PNIPAm altered the normal fibrin polymerization by changing the rate of protofibril aggregation and resulting in a 5-fold increase in the overall turbidity. Fibrin clots formed in presence of these synthetic polymers exhibited thinner fibers with less branching and resulted in a more porous and heterogeneous clot structure in scanning electron micrographs. The structural changes in these clots led to significant difference to their mechanical properties. Lower clot strength and clot elasticity were recorded from the thromboelastography study. More interestingly, enhanced clot lysis was measured by thromboelastography when whole blood was clotted in presence of PDMDOMA or PNIPAm. Further evidence of the altered clot structure and clot cross-linking was obtained from the significant decrease in D-dimer levels measured from degraded plasma clot. Similar results were obtained when star-form of PNIPAm was used but not for brush-form PNIPAm. The antithrombotic activity of soluble PDMDOMA and PNIPAm could potentially lead to the development of novel antithrombotic agents that could enhance endogenous fibrinolytic activity by modulating the fibrin clot structure. In the exploratory analysis of surface grafted PNIPAm (brush-PINPAm), brush-PNIPAm showed that the biological activity of attached chains is quite different from soluble polymers and several parameters need to be optimized to generate an antithrombotic coating for biomaterials.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ)
Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA)
Recent researches have emphasized the importance of redox mechanisms for platelet function modulation. The platelet surface contains a large variety of integrin receptors and other molecules presenting functional thiol groups in their structures, which are potential targets for redox regulation. Among these various thiol-containing proteins, integrin αIIbβ3 stands out for being the convergence path of platelet activation induced by various agonists. Activation of αIIbβ3 integrin is catalyzed by protein disulfide isomerase (PDI) through an essential conformational change leading to the exposure of fibrinogen-binding site. Thus, PDI has been shown to be an important target for the development of antiplatelet drugs. In recent years, many studies have described substances from plan (DE A. PAES et al., 2011), as well as synthetics that are capable of inhibiting PDI. In a previous study of our research group has shown that the synthetic peptide CxxC, which contains the redox motif of PDI in its original sequence CGHC, inhibited reductase activity of this enzyme, effect not observed with AxxA peptide, whose cysteines were replaced with alanine and Scr peptide, which contains the same aminoacids from CxxC peptide, but under random sequence. It has been also demonstrated that CxxC peptide was the only to reduce by 30% ADP-induced aggregation (5μM) in platelet rich plasma, an effect apparently mediated by the association of CxxC and PDI at platelet surface. Thus, in this work, we further assessed the effects of CxxC and its control peptides on platelet aggregation. Washed human platelets were incubated with CxxC peptide at concentrations of 3, 6 and 10 μM, resulting in a dose-dependent inhibition of maximum aggregation activated by thrombin (0.02 U/mL) at 25, 60 and 74%, respectively with IC50 of 6.13 ± 1.09 μM. The presence of control peptides did not produce any inhibitory effect. CxxC peptide also reduced the activation of αIIbβ3 integrin at platelet surface, but did not affect the expression of the markers CD 62-P and CD 63. Control peptides did not alter the expression of these markers. Analysis by mass spectrometry of the interaction of recombinant human PDI with the peptide showed that only CxxC peptide associated with the redox Cys400 of a’ motif of PDI, which has been considered essential for platelet aggregation. Together, these results demonstrate that CxxC peptide reduces platelet aggregation by association with PDI and can be further used as a model for the development of new antithrombotic drugs.
Investigações recentes têm enfatizado a importância de mecanismos redox na modulação da função plaquetária. A superfície da plaqueta contém grande variedade de integrinas e outras moléculas receptoras que possuem tióis funcionais em sua estrutura, os quais são alvos potenciais de regulação redox. Dentre estas várias proteínas tiólicas, a integrina αIIbβ3 destaca-se por ser a via de convergência da ativação plaquetária induzida por diversos agonistas. A ativação da integrina αIIbβ3 é catalisada pela proteína dissulfeto isomerase (PDI), essencial à mudança de conformação que leva à exposição do sitio de ligação ao fibrinogênio. Sendo assim, a PDI tem se mostrado como um alvo importante para o desenvolvimento de fármacos reguladores da agregação plaquetária. Nos últimos anos, diversos estudos têm descrito substâncias de origem vegetal, animal e sintéticas que são capazes de inibir a PDI. Em trabalho do nosso grupo de pesquisa (DE A. PAES et al., 2011), demonstrou que o peptídeo sintético CxxC, o qual contém o motivo redox da PDI na sua sequência original CGHC, inibiu a atividade redutase desta enzima; efeito não observado com os peptídeos AxxA, que possui as cisteínas substituídas por alanina e Scr, peptídeo controle contendo os mesmos aminoácidos do peptídeo CxxC, porém com sequência aleatória sem formação de ditiol. Demonstrou-se, também, que apenas o peptídeo CxxC reduziu em 30% a agregação induzida por ADP (5M) em plasma rico em plaquetas, efeito aparentemente mediado pela associação do CxxC com a PDI na superfície plaquetária. Sendo assim, neste trabalho continuamos a avaliação dos efeitos do peptídeo CxxC e seus controles sobre a agregação plaquetária. Para tanto, incubamos lavado de plaquetas humanas com o peptídeo CxxC nas concentrações de 3, 6 e 10 μM, resultando em inibição concentração-dependente da agregação ativada por trombina (0,02 U/mL) em 25, 60 e 74 %, respectivamente, com IC50 de 6,13 ± 1,09 μM. A presença dos peptídeos controle não produziu quaisquer efeitos inibitórios. O peptídeo CxxC reduziu a ativação da integrina αIIbβ3 na superfície da plaqueta, porém não impactou a expressão dos antígenos CD 62-P e CD 63. Os peptídeos controle não alteraram a expressão desses marcadores. A análise por espectrometria de massas da interação da PDI recombinante humana com os peptídeos, mostrou que apenas o peptídeo CxxC associa-se com a Cys400 do motivo redox a’ da hPDI, o qual tem sido considerado fundamental para a agregação plaquetária. Em conjunto, estes resultados demonstram que o peptídeo CxxC reduz a agregação plaquetária via associação com a PDI, podendo ser empregado como modelo para o desenvolvimento de fármacos novos antitrombogênicos.

The three dimensional obturation of root canals is vital and cannot be overstated. The attainment of a fluid-tight seal in a root canal system is very important for the longterm success of endodontic treatment. A number of different materials have been used for obturation. Gutta percha is the most common root filling material in use today. It is used as the core material in combination with sealers of different compositions. Despite gutta percha has enjoyed a long history of use and his success rate; it does not offer all of the properties of an ideal root canal filling materials. New root canal filling materials have been developed. These include resin-based (EndoRez and RealSeal systems) and silicone-based (Guttaflow') filling materials. This study aimed to investigate properties of these new root canal filling materials and compare it to gutta percha and a zinc oxide based sealer (TubliSeal).

Polymers with mucoadhesive properties are universally used in the development of mucoadhesive drug delivery system. Their physicochemical properties as well as the mechanisms related to their adhesive actions draw great attention for the modification of mucoadhesive properties. In this study, relationships between physicochemical properties of hydroxypropyl methylcellulose (HPMC) compacts and mucoadhesive performance were investigated. Different commercial grades of HPMC (K3, E3, E5, E50, K4M, E4M and K15M) were prepared into compacts, and their surface hydrophilicity and hydration behavior were characterized. The in vitro mucoadhesive performance was determined by the tension strength between the compacts and different regions of mucous membrane (buccal, sublingual, stomach, and intestine). Positive correlations were found between: (1) viscosity of HPMC compacts and contact angle values measured by different simulated body fluids; (2) viscosity of HPMC compacts and in vitro mucoadhesive force; (3) contact angle values and in vitro mucoadhesive force. The hydration behavior exhibited improvement with the increasing viscosity of HPMC compacts. Moreover, the polar lipid content of each mucosa was likely an important factor affecting the mucoadhesion phenomenon. Different ratios of ethyl cellulose (EC) was mixed with HPMC grade K15M to form combination compacts for the purpose of modifying the surface property. The mucoadhesive mechanism of both different grades of HPMC compacts and combination compacts were studied via the thermodynamic analysis of Lifshiz-van der Waals interaction and Lewis acid-base interaction. The total free energy of adhesion (〖∆G〗^TOT) provided a prediction of an overall tendency of mucoadhesion, however, the results were showing disagreement with the measured mucoadhesive force. In general, the involving of EC in the combination compacts did not give a boost to the whole mucoadhesive performance.

The purpose of this study was to develop a deeper understanding of the nature of carotenoid metabolism in the human and primate retina. We have sought to do this from two perspectives (1) through preparation and study of a carotenoid diketone that is a candidate metabolic product and (2) through measurement of the carotenoid distribution in the retinas of neonatal macaques. In this thesis we report the synthesis, purification, and characterization of the product using HPLC, UV/Vis, MS, 1H-NMR. The data obtained are all consistent with the proposed β,β-carotene-3,3'-dione. There has been no thorough study of the development of the macular pigment during the earliest stages of life immediately following birth. In this study 30 macaque retinas ranging from 148 days of gestation to 15 years in age were analyzed. The amounts of the carotenoids, lutein, R,R-zeaxanthin, and meso-zeaxanthin were determined using C 18 reversed-phase column and chiral, normal-phase column HPLC.

A HEp-2 cell culture model was used to investigate the invasive properties of Campylobacter species. Two of twenty-five Campylobacter isolates did not invade HEp-2 cells, and one of these isolates did not adhere to the epithelial cells. Penetration of HEp-2 epithelial cells by C. jejuni was significantly (P < 0.05) inhibited with C. jejuni lysates and a MAb (1B4) in competitive inhibition studies. Immunogold electron microscopic studies revealed that the 1B4 MAb bound to the flagella and cell surface of low passage (invasive) C. jejuni M 96, whereas only the flagella of high passage (non-invasive) C. jejuni were labelled. Western blot analysis revealed that the 1B4 MAb identified an epitope on antigens ranging in size from 66 to 44 kDa in invasive and non-invasive organisms. Antigens were also recognized in lysates prepared only from invasive strains from 42 to 38 kDa. Sodium meta-periodate chemical treatment of C. jejuni lysates significantly (P < 0.05) affected its inhibitory capacity. Additionally, proteinase K and sodium meta-periodate treatment of lysates changed the mobility of antigens recognized by the 1B4 MAb. This suggests that the antigens required for epithelial cell penetration by C. jejuni may be glycoprotein in nature and that the functional binding site is dependent upon an intact carbohydrate moiety. Co-infection of HEp-2 epithelial cells with coxsackievirus B3, echovirus 7, polio virus (LSc type 1), porcine enterovirus and Campylobacter isolates was performed to determine if a synergistic effect could be obtained. The invasiveness of C. jejuni was significantly increased for HEp-2 cells pre-infected with echovirus 7, coxsackievirus B3, and UV-inactivated (non-infectious) coxsackievirus B3 particles. Polio and porcine enterovirus had no effect on C. jejuni adherence and invasiveness. C. hyointestinalis and C. mucosalis, two non-invasive isolates, did not invade virus-infected HEp-2 cells. The increase of invasiveness of C. jejuni appears to be the result of specific interactions between the virus and the HEp-2 cell membrane. The data suggest that the invasiveness of Campylobacter is dependent upon the inherent properties of the organism. Virus-induced cell alterations can potentiate the invasiveness of virulent Campylobacter but are not sufficient to allow internalization by non-invasive bacteria.

Orthodontic brackets undergo resistance during sliding that includes classical friction, binding, and notching. Current bracket systems are hampered by these challenging forces. As a result, the clinician usually needs to apply additional forces to overcome the resistance which increases the risk of root resorption and discomfort for the patient. This study evaluated frictional properties of a novel bracket that had polytetrafluoroethylene (Teflon™) coated rollers in its design. Five types of brackets (n = 10, each), including a passive self-ligating bracket, a traditional ligated bracket, a three-dimensionally printed direct metal laser sintering (DMLS) bracket with and without Teflon™ rollers, and computer numeric controlled (CNC) machine milled bracket with Teflon™ rollers were tested. The peak resistance values were assessed at 0°, 4°, and 8° of tip on a 0.019 x 0.025” arch wire. At 8° of tip, the DMLS and the CNC milled bracket systems, both with Teflon™ rollers, exhibited less friction as compared to the other brackets tested (p

Asthma is a chronic inflammatory disease of the airways. The current cornerstone for asthma treatment is glucocorticoids (steroids), and the majority of patients respond to steroids with an improvement in lung function (Steroid Sensitive; SS). However a proportion of asthmatics do not respond to steroids with improvement in lung function (Steroid Resistant; SR), who represent those patients most at risk. Epidemiological studies demonstrate that asthma severity and poor responsiveness to treatment, is associated with vitamin D insufficiency and deficiency. Vitamin D is an immunomodulatory molecule which induces the synthesis of antimicrobial peptides and anti-inflammatory molecules, and inhibits pro-inflammatory cytokine synthesis. The overall goal of this work was to investigate immunological differences between SS and SR asthma patients, and effects of vitamin D that may explain the epidemiological observations. Peripheral blood from a clinically well-characterized cohort of SS (n=14; mean improvement in lung function or FEV1 post 2-weeks oral prednisolone 16.1%) and SR (n=23; no improvement in FEV1) asthmatics was studied by ex vivo flow cytometry. Major findings included evidence for significantly higher frequency of myeloid dendritic cells, and lower frequency of Foxp3+ CD4+ T cells in the peripheral blood of SR as compared to SS asthmatics. The accepted measure of vitamin D status is serum 25-hydroxyvitamin D3, 25(OH)D, and the severe asthmatics had significantly lower levels of serum 25(OH)D as compared to healthy controls. A significant positive correlation between the number of Foxp3+ T cells in the peripheral blood and serum 25(OH)D was observed. Following culture of PBMCs with anti-CD3 activation it was found that severe asthmatics synthesised significantly higher IL-17A levels than healthy controls, which was most striking in the SR patients. The steroid Dexamethasone (Dex) enhanced IL-3 17A production in culture, whereas the active form of vitamin D 1,25-hydroxyvitamin D3 (1,25(OH)2D3) significantly reduced IL-17A production in a Dex-independent manner. This work proposed that IL-17A inhibition by 1,25(OH)2D3 may be partially due to a CD39-dependent mechanism. In vivo evidence for a positive association between serum 25(OH)D and Foxp3+Treg numbers was further investigated in vitro. High concentrations of 1,25(OH)2D3 (10-6M) increased the frequency of Foxp3+ T cells in culture through two main mechanisms: the induction of IL-2, on which Foxp3+Treg are dependent, as well as less inhibition of Foxp3+ over Foxp3- T cell proliferation. Lower, and likely more physiological concentrations of 1,25(OH)2D3 (10-7M), which are known to enhance IL-10 synthesis, failed to significantly increase the frequency of Foxp3+ CD4+ T cells. However upon modulation of the cytokine environment to one of low IL-10 and high TGFβ, this concentration of 1,25(OH)2D3 significantly increased Foxp3+ Treg frequency. Collectively this data highlights additional immunoregulatory properties of 1,25(OH)2D3 including induction of anti-inflammatory cytokines such as IL-10 and TGFβ, inhibition of pro-inflammatory cytokines such as IL-17A and IL-22 as well as the direct induction of Foxp3+ Tregs. In conclusion evidence for a number of immunomodulatory effects of 1,25(OH)2D3 exists that may help to reduce uncontrolled inflammatory responses associated with severe asthma.

This dissertation mainly deals with some biochemical aspects regarding the efficacy of novel platinum anticancer compounds, as part of a broader study in which both chemistry and biochemistry are involved. Various novel diamine and N-S donor chelate compounds of platinum II and IV have been developed in which factors such as stereochemistry, ligand exchange rate and biocompatibility were considered as additional parameters. In the first order testing, each of these compounds was tested with reference to their “killing” potential by comparing their rate of killing, over a period of 48 hours with those of cisplatin and oxaliplatin. Some 80 compounds were tested in this way. Although only a few could be regarded as equal to or even better than cisplatin and oxaliplatin, the testing of these compounds on cancer cells provided useful knowledge for the further development of novel compounds. Four of the better compounds, namely Y9, Y14, Y16 and Lt16.2 were selected for further studies to obtain more detailed knowledge of their anticancer action, including some flow cytometric studies. In addition to the above, cisplatin resistant cells were produced for each of the three different cell lines tested, namely, HeLa, HT29 and MCF7 cancer cell lines, by intermittent and incremental exposure to cisplatin (all the cell lines tested became resistant to cisplatin). Each of the selected compounds were exposed to the cells in the same manner, in order to attempt the induction of resistance against these compounds in the three cell lines tested (i.e. whether these cells will become resistant to the various compounds). Each of these selected platinum containing compounds were subsequently tested against the “cisplatin resistant” cell lines in order to determine their efficacy against such cells. One such compound could be singled out, since cervical cancer cells (HeLa cells) do not become resistant to it. This behaviour is similar to that of oxaliplatin against cervical cancer and colon cancer (HT29) cells (oxaliplatin is the number one treatment for colon cancer at present). This compound also proved to be more active against cisplatin resistant cell lines. It was found that all the compounds induced apoptosis in the cell lines tested as well as inhibit the DNA cycle at one or more phase. Finally, an effort was made to evaluate the different compounds by comparing them with respect to their properties relating to anticancer action.

The electrophysiological properties of dentate granule cells and hippocampal pyramidal neurons were examined with extracellular and intracellular recording techniques in the hippocampal slice. Intracellular analysis revealed that there may exist two populations of granule cells distinguishable by the presence or absence of non-linear current-voltage (I-V) membrane properties (anomalous rectification, AR). The granule cells exhibiting AR also maintained greater resting membrane potentials and action potential (AP) amplitude values. The membrane input resistance (Rn) and time constant (Tc) measurements were similar between the populations in response to hyperpolarizing current injection, but granule cells displaying AR had significantly higher Rn and Tc values in response to depolarizing pulses. Both groups also responded to maintained depolarizing current injection with repetitive AP discharges; however, this response accommodated. Upon termination of the depolarizing current injection, an afterhyperpolarization (AHP) resulted, the amplitude of which appeared to depend on the duration of the depolarizing pulse and not on the number of APs generated during the pulse. Stimulation of either the lateral (LPP) or medial (MPP) perforant paths evoked a monosynaptic EPSP followed by a depolarizing afterpotential (DAP) and a long afterhyperpolarization (LHP). In contrast, antidromic stimulation elicited a depolarizing-IPSP (D-IPSP) and a LHP. Both the DAP and D-IPSP were reversed by membrane depolarization, whereas, the LHP was inverted by membrane hyperpolarization. In all cases, however, the EPSP could not be inverted. Afterpotentials were associated with an increase in conductance, but the change accompanying the LHP was less than the DAP and D-IPSP. In addition, by reducing the [Ca]₀ and increasing the [Mg]₀, the DAP was attenuated and the LHP eliminated. Similar results were also obtained with the GABAB agonist, baclofen. Paired pulse stimulation of either the LPP or MPP resulted in the potentiation of the intracellular EPSP at condition-test (C-T) intervals less than 100 ms; however, simultaneous extracellular records from the granule cell layer (GCL) illustrated depression of the EPSP. The discrepancy between the extra- and intracellular recordings was shown to be related to the presence of the DAP. In addition, the MPP evoked test EPSP at C-T intervals greater than 150 ms exhibited inhibition regardless of whether it was recorded inside or outside the granule cell and this EPSP depression was partially due to the granule cell LHP. The LPP evoked test EPSP potentiated at all C-T intervals less than 1s when recorded from the outer molecular layer (OML) but was inhibited at both the GCL and intracellular recording sites. These data confirmed that postsynaptic processes contribute to the short-term alterations observed with paired pulse stimulation. The typical inhibition-potentiation-inhibition sequence of the perforant path (PP) evoked population spike (PS) was noted at C-T intervals of 20, 80 and 400 ms, respectively. The inhibition of the PS at 20 ms was abolished with perfusion of the GABA antagonist, bicuculline. In contrast, the PS inhibition at 400ms was unaffected by this treatment but was slightly attenuated by the gKca antagonist TEA. A number of factors appeared to contribute to the potentiation of the PS: 1) reduction in AP threshold; 2) the presence of the DAP; and 3) extrasynaptic events. In addition to the PS data from normal tissue, hippocampal slices from chronically kindled rats exhibited depression of the PS at all C-T intervals tested. This augmentation of inhibition was dependent on the presence of hippocampal afterdischarges but not on motor seizures. Perfusing the kindled slices with either bicuculline or lowered [Cl]₀ did not markedly reverse the enhanced inhibition at C-T intervals which displayed dramatic facilitation in normal slices. Intracellular recordings of granule cells obtained from kindled slices also exhibited an increase in the Rn and Tc. Both the alterations in inhibition and membrane characteristics appear to be localized to.the granule cells, since these changes were not observed in CA1 pyramidal neurons. These data indicate that short-term and long-term alterations in granule cell neuronal excitability are partially due to changes in the postsynaptic membrane.
Medicine, Faculty of
Cellular and Physiological Sciences, Department of
Graduate

Thesis (M.S.)--East Tennessee State University, 2003.
Title from electronic t.p. ETSU ETD database URN: etd-0618103-114702. Includes bibliographical references. Also available via Internet at the UMI web site.

Les interactions entre nutriments dans les produits alimentaires peuvent affecter leur bioaccessibilité. La β-lactoglobuline (βlg), protéine majeure du lactosérum, est connue pour lier des ligands hydrophobes tels que des acides gras (AG). Très sensible aux conditions industrielles, la βlg est souvent sous formes non natives dans les aliments transformés. Notre hypothèse est que les changements structuraux au niveau de la protéine modifient l'affinité pour les AG et par conséquent les propriétés biologiques des complexes AG/protéine. L'objectif était d'étudier l'interaction de la βlg bovine sous différentes structures (native, dimère covalent et nanoparticules) avec l’acide linoléique (LA) et l’acide linoléique conjugué (CLA), et l'impact de ces complexes sur leurs activités biologiques in vitro. Par fluorescence intrinsèque et calorimétrie de titration isotherme, nous avons montré que quel que soit l’état d'agrégation de la protéine, LA interagit avec la βlg au niveau de deux classes de sites. En augmentant le niveau d’agrégation de la βlg, la stœchiométrie des complexes LA/βlg augmente mais sans changement des constantes d'association. En présence de LA, la βlg native est plus sensible à la digestion gastrique in vitro en raison de l'augmentation du niveau de dénaturation/agrégation de la βlg. La quantification de l’absorption du LA dans une monocouche cellulaire imitant la barrière intestinale indique que son transport est ralenti en présence de βlg native ce que confirme la microscopie confocale. La cytotoxicité du LA pour les cellules Caco-2 a été réduite lorsque l’AG était lié à la βlg. Le CLA, qui est moins soluble dans l'eau que le LA, est plus cytotoxique lorsqu'il est complexé à la βlg que sous sa forme libre. Par conséquent, la βlg peut moduler le transport et la bioaccessibilité de l’AG en fonction de la solubilité de ce dernier
Food structure can have a profound influence on delivering health benefits. Bioaccessibility of nutrients can be affected by their interaction with food components. The dairy protein β-lactoglobulin (βlg) is known to bind hydrophobic ligands such as fatty acids (FA). However, this protein is highly sensitive to the process conditions used in the dairy industry. Therefore βlg is often present in non-native or aggregated form in processed food. This structural change may modify the protein affinity for FA and the biological properties of the FA/protein complexes. The aim of this thesis was to investigate the interaction of bovine βlg in different structural forms (native, covalent dimer and nanoparticles) with linoleate (C18:2, cis,cis-9,12-octadecadienoic acid) and conjugated linoleic acids (CLA, C18:2), and the impact of those complexes on their biological activity in vitro. Two different sets of binding sites were determined for the interaction between linoleate and βlg, regardless of its state of aggregation, using intrinsic fluorescence spectroscopy and isothermal titration calorimetry. By increasing the level of βlg aggregation, the linoleate/βlg stoichiometry increased but the association constants remained similar for both sets of binding sites. In the presence of linoleate, the native protein was more sensitive to gastric in vitro digestion, due to the increased level of denaturation/aggregation of βlg. Transport of linoleate in Caco-2 cells was decreased in presence of the native βlg as observed by confocal microscopy and a monolayer that mimics the intestinal barrier. Cytotoxicity of linoleate on Caco-2 cells was reduced when the FA was bound to βlg compared to free FA. CLA, which is less water soluble than linoleate, is more cytotoxic when complexed by βlg than in its free form. Therefore, it is proposed that βlg can act as a molecular carrier and alter the bioaccessibility of FA depending on their solubility

Les RAB GTPases sont des régulateurs majeurs du trafic vésiculaire et sont localisées sur des compartiments spécifiques. L'identification des processus moléculaires régulant la localisation des RAB est donc cruciale afin de comprendre les mécanismes de transport intracellulaire. Nous sommes parvenus, pour la première fois, à incorporer des protéines RAB purifiées et prénylées dans des membranes artificielles. Nous avons tout d'abord observé que RAB6 est capable de promouvoir une agrégation de vésicules, phénomène qui n'est pas observé avec RAB1 et RAB5. Nous suggérons un modèle dans lequel RAB6 interagit en trans avec lui-même et par conséquent induit un accolement de membranes. La partie principale de cette étude consistait à identifier les propriétés physicochimiques des membranes requises pour le recrutement des protéines RAB. Nous avons observé que RAB1, RAB5 et RAB6 se lient préférentiellement à des membranes désordonnées et courbées, phénomène qui s'explique par l'insertion du groupement prenyl hydrophobe au niveau de défauts d'agencement de lipides. En revanche, le recrutement de RAB35 requiert la présence de lipides chargés négativement et peut être modulé, dans une moindre mesure, par les défauts d'agencement lipidique. Bien que RAB4 et RAB11 soient recrutées sur des fractions de Golgi purifiées, les charges membranaires et les défauts d'agencement lipidique ne sont pas suffisants pour permettre leur recrutement sur des vésicules synthétiques. Cela suggère que le recrutement de RAB4 et RAB11 nécessite des propriétés membranaires plus complexes. Nos travaux démontrent que les propriétés membranaires sont cruciales pour la localisation spécifique des protéines RAB
RAB GTPases are major regulators of vesicular trafficking and localize to specific compartments. Deciphering the molecular mechanisms governing RAB localization is thus critical to understand intracellular transport processes. We have managed, for the first time, to incorporate purified and prenylated RABs into artificial membranes. By doing so, we observed that RAB6, but not RAB1 or RAB5, is able to promote by itself vesicle tethering. We believe that RAB6 is able to interact in trans with itself and to consequently drive homotypic membrane tethering. In the main part of this study, we investigated the physicochemical membrane requirements necessary for RAB recruitment. RAB1, RAB5 and RAB6 were all found to only localize to disordered membrane domains and to preferentially bind to curved membranes. We demonstrated that this specific recruitment of RAB1, RAB5 and RAB6 is primarily dependent on the hydrophobic insertion of their prenyl group into lipid packing defects. In contrast, RAB35 recruitment was primarily dependent on the presence of negatively charged lipids and was found to be modulated, to a lesser extent, by lipid packing defects. Although RAB4 and RAB11 were effectively recruited to purified Golgi fractions, in an effector-independent manner, membrane charges and lipid packing defects were not sufficient to promote their recruitment to synthetic vesicles; suggesting that RAB4 and RAB11 require more demanding membrane physicochemical properties. Our work demonstrates that the properties of membranes are critical for the regulation of RAB specific membrane targeting

Neural stem cell (NSC) transplantation strategy offers great potential to treat spinal cord injury (SCI). NSCs may replace lost neurons or oligodendrocytes, and act as a source of neurotrophic factors to support the survival of remaining cells. Their efficiency was limited by poor survival after transplantation, and they had more tendencies to differentiate into astrocytes, but not neurons and oligodendrocytes. This project investigated whether activated microglia is a factor that contributes to this phenomenon, and studied the potential role of minocycline, a widely used antibiotic drug, to modify the negative effects of microglia on NSCs. In the first part of this study, we used organotypic spinal cord slice (SCS) culture to mimic in vivo local environment after SCI, and NSCs were grafted on their surface or shared culture medium with them. After specific depletion of microglia with clodronate loaded liposome, more grafted NSCs survived, and in the co-culture system, the NSC neuronal differentiation rate increased while glial differentiation rate decreased, the apoptosis rate also decreased. This suggested that activated microglia may impair NSC survival, and neuronal differentiation, but improve glial differentiation. In the second part of this study, we first tested the direct effects of minocycline on NSC apoptosis, proliferation and differentiation in vitro, to test whether minocycline has any side effect on NSCs. The results showed that at the concentration 10μg/ml or lower, minocycline did not affect NSC survival and proliferation, but impaired neuronal differentiation. Then we treated primary microglia culture with LPS or LPS plus minocycline, and collected the conditioned mediums (CM-LPS and CM-LPSMC) to test their effects on NSC　apoptosis and differentiation. The results showed that compared with CM-LPS, CM-LPSMC resulted in a significantly lower apoptotic rate of NSCs, also allowed NSC neuronal differentiation. This suggested that minocycline may impair the pro-apoptotic effect of activated microglia on NSCs. In conclusion, our study showed that activated microglia may impair NSC survival and neuronal differentiation. This indicated that in NSC transplantation strategy for SCI, microglia would be a target to be manipulated to improve graft survival and neuronal differentiation. Although minocycline may suppress NSC differentiation towards neurons, it has the potential to protect NSCs from the toxic effects of activated microglia. This showed the therapeutic potential of minocycline in NSC transplantation strategies for SCI.
published_or_final_version
Anatomy
Doctoral
Doctor of Philosophy

The endothelial glycocalyx is a thin layer of macromolecular matrix on the luminal surface of vascular endothelial cells. It determines fluid and solute transport across the vessel wall; affects the mechanotransduction of endothelial cells and contributes to vascular patho-physiology. This thesis investigates the spatiotemporal development of the endothelial glycocalyx in vitro using fluorescence confocal microscopy. The Young’s modulus of the glycocalyx is evaluated using AFM indentation. The glycocalyx on cultured HUVECs shows temporal development: up to day 5 after cell seeding, it covers predominantly the edge of cells and appears on the apical membrane of cells as time progresses. After day 14, the entire cell membrane is covered by the glycocalyx. The thickness of this layer is estimated to be between 300nm and 1μm. AFM indentation result reveals the Young’s modulus of the cell membrane decreases with time. The Young’s modulus of the glycocalyx is deduced from Young’s moduli of cell membranes with and without the glycocalyx layer. The results show the glycocalyx on cultured HUVECs has a Young’s modulus of ~0.39kPa. The thesis further investigates the distribution of the glycocalyx on the endothelial cell membrane following shear flow stimulation. Both the percentage area of the cell membrane that is covered with the glycocalyx and the fluorescence intensity ratio between the apical and edge areas on endothelial cells are used to measure the glycocalyx distribution. It is observed that the glycocalyx appears near the edge of the endothelial cell following shear stimulation and develops towards the apical area with time. The speed of the recovery of the glycocalyx layer is faster in the earlier period, i.e. 0hr - 4hrs after shear stimulation, than that in the later period, i.e. 8hrs - 24hrs. Additionally, the recovery of the glycocalyx following neuraminidase degradation.

Magister Chirurgiae Dentium - MChD (Prosthodontics)
The aim of this study was to evaluate and compare the physical properties of two core build-up materials (ParaCore and CoreXflow) and compare this to conventional composite material (Filtek Supreme Plus and SDR Flow) used as core build-up material.

Sublingual administration of drugs offers advantages including avoidance of first pass metabolism and quick absorption into the systemic circulation. In spite of being one of the oldest routes of drug delivery, there is dearth of literature on characterization of the barrier properties of the sublingual mucosa. Therefore, the aim of this research was to gain an insight into the barrier properties of the porcine sublingual mucosa. The studies conducted in this dissertation research focused on an important aspect of sublingual permeation, the dependence of permeability on different physicochemical properties of the permeant such as the degree of ionization, distribution coefficient and molecular weight/size on drug transport across sublingual mucosa. Further the data from the sublingual permeation of model compounds was used in development of a predictive model which provided us with some understanding regarding the important descriptors required for sublingual drug delivery. A series of β-blockers were employed as the model drugs to study the dependence of permeability on lipophilicity across the sublingual mucosa. Eighth different β-blockers with log D (distribution coefficient) values ranging from -1.30 to 1.37 were used in this study. The most hydrophilic drug atenolol showed the lowest permeability (0.19 ± 0.04 x 10 -6 ) cm/sec and the most lipophilic drug propranolol showed the highest permeability (38.25 ± 4.30 x 10 -6 ) cm/sec. The log-log plot of permeability coefficient and the distribution coefficient showed a linear relationship. It was concluded that the increase in lipophilicity results in improved partitioning across the lipid bilayers of sublingual mucosa which results in increased permeation for the drugs. As the sublingual mucosa contains a significant amount of the polar lipids bonded with water molecules, therefore, it was hypothesized that the hydrophilic or ionized permeants will have significant permeation across the sublingual mucosa. The objective of this research was to study the effect of ionization on permeation across sublingual mucosa using a model drug nimesulide. Based on the relationship between the permeability coefficient and distribution coefficient of nimesulide at different pH, the lipoidal route was suggested as the dominant transport route for nimesulide across the sublingual mucosa. The contribution of individual ionic species of nimesulide to the total drug flux was quantitatively delineated. It was observed that the ionized species of nimesulide contributes significantly to the total flux across the sublingual mucosa. The contribution of the ionized species to total flux was almost (90%) at a pH where the drug was almost completely ionized. Polyethylene glycols (PEGs) were used as the model permeants to study the dependence of permeability on molecular weight. An inverse relationship between molecular weight and permeability coefficients was observed. This relationship was used to estimate the molecular weight cut off for the sublingual mucosa. The molecular weight cut off was estimated to be around 1675 daltons. Further, the Renkin function was used to estimate the theoretical pore size of the sublingual mucosa and the pore size of the sublingual mucosa was estimated to be around 30–53 Å based on two separate calculations using the radius of gyration and Stokes-Einstein radius for PEG molecules, respectively. No specific model is present in literature to predict the in vitro sublingual drug permeability. In this dissertation a specific model was developed and validated by performing permeation studies of 14 small molecules across the porcine sublingual mucosa. It was shown that the lipophilicity (logD 6.8 ) and the number of hydrogen bond donors (HBD) were the most significant descriptors affecting sublingual permeability. Research conducted in this dissertation provided an in-depth understanding about the barrier properties of the porcine sublingual mucosa and role of different physicochemical properties on sublingual transport. Such an understanding will hopefully expand the suitable lead candidates for sublingual delivery.

Philosophiae Doctor - PhD
The fluoride release and chemical adherence to tooth structure remain the most desirable features of glass ionomer restorative cements (GICs). Although the physical properties for multi-surface restorations are well-defined, even with the introduction of newer GICs not all demands have been met. Yet, increased use of GICs will only be possible if clinicians change their perceptions of the low survival rate of GICs. The lower clinical success rate of GICs is partly due to the marginal integrity and wear over time, which has often been recorded in the literature as restoration failure. The current, well-established restorative options for the primary dentition are Resin Modified Glass Ionomers (RMGICs) and Compomer resins. There is a paradigm shift towards materials that are more biologically favourable. Areas of research for dental materials include antibacterial properties in conjunction with ion release to maintain healthy restored teeth. If a GIC can provide adequate physical properties with the inclusion of the aforementioned features, GICs might become a more viable permanent restorative solution.

Endogenous opiates, the enkephalins, have been identified in the brain and intestine. Their physiological role in the gut has yet to be determined, but since opiates have anti-diarrhoeal actions it was thought possible that they might be involved in the control of mucosal ion transport in addition to their known effects on motility. This possibility was studied using the lq'in vitro' technique of Ussing and Zerahn. Morphine (10^-6 to 10^-4M) induced a significant fall in potential difference (PD) and short-circuit current (I_sc) across stripped rabbit ileal mucosa, with no change in tissue resistance. A maximal electrical response was dependent on the presence of Na, Cl and HCO₃ in the bathing medium. A significant increase in Cl absorption due to a decrease in serosa to mucosa flux was provoked in response to morphine (2 x 10^-5M), accompanied by an increase in residual ion flux (J^R/_net), possibly due to HCO₃ secretion. Na transport was unaffected. Dextromoramide (10^-5M) mimicked the response to morphine, but the inactive isomer laevomoramide (10^-5M) had no effect. Naloxone (10^-6M) inhibited the response to morphine (2 x 10^-5M) and this inhibition was competitive in nature supporting the existence of mucosal opiate receptors. The enkephalin analogue Me-Tyr-D-Met-Gly-Phe-Pro-NH₂ also decreased the PD and I_sc and produced a similar increase in Cl absorption and J^R/_net. This analogue had a more rapid action and provoked a response at a lower dose (10^-8M) than morphine. Tetrodotoxin (10^-7M) inhibited the response to morphine (10^-4M) but blockade of cholinergic, α and β adrenergic and dopaminergic mechanisms had no effect. Morphine (10^-4M) inhibited the secretion produced by three secretagogues, prostaglandin E₂ (10^-5M), acetylcholine (10^-4M) and cholera toxin (1 μg/ml). Adenyl cyclase and cyclic AMP levels were unaffected by morphine, but the electrical response to morphine was increased by omitting Ca^+ + from the medium. Thus these studies provide evidence for the presence of mucosal opiate receptors which may have a physiological role, and demonstrate that opiates enhance Cl absorption and inhibit secretion provoked by three secretagogues. The mechanism of action may be related to an antagonism to intracellular calcium.

Magister Scientiae Dentium - MSc(Dent) (Restorative Dentistry)
Dental composite is a synthetic resin that is used as a tooth coloured restorative material in dentistry. It became the material of choice in the dental field due to its superiority in strength and aesthetics (Garcia et al., 2006). Composite resin has favourable physical properties such as high wear resistance and shade stability. The main disadvantages are as follows: polymerization shrinkage which leads to marginal leakage; discoloration of the filling; postoperative sensitivity; and recurrent caries (Garcia et al., 2006). In order to decrease the application time of incremental layering techniques in conventional resin composite restorations, bulk-fill composites were introduced to the dental market with modifications in physical and mechanical properties. BulkFill composite can be applied in a single one-step increment layer of 4 - 5mm, saving considerable time during the clinical procedure when compared to the conventional composite layering technique of 2 mm (Leprince et al., 2014).

Magister Chirurgiae Dentium (MChD)
Background: This study aimed to determine the association between dimensional change and surface roughness (Ra) of Vitremer, Activa and Cention N after immersing them into two different media: acidic and artificial saliva media for the period of a year. Measurements were made at 10 time intervals during the observation period. Methodology: This was a quantitative and qualitative study. For the quantitative part, a total of 60 specimens were tested, 20 specimens for each material. The 20 specimens were further divided into 10 specimens. Ten were immersed in acidic media and the rest in saliva media. A measurement of the weight, height, and Ra was carried out as follows: day 0, day 1, day 2, day 7, day 21, day 28, day 60, day 90, day 180 and day 365. Scanning electron microscopy (SEM) was used to examine the surface of each material qualitatively pre and post immersion in the two media. For fluoride measurements, an additional five samples from each material were left suspended in the de-ionized water by the use of dental floss. The materials were moved to new specimen jars after the completion of day 1, 2, 3, 4, 5, 6, 7, 14, 21 and 28. All the specimen jars had been kept for the fluoride measurements. Results: Non-parametric tests were used to analyze the data. Linear regression analysis was used to measure the association between weight, height or surface roughness (Ra) and immersion time for a year. The result of this test showed that Vitremer had a significant association between the weight (p = 0.000), height (p = 0.007) and Ra (p = 0.001) when it was immersed in acidic media. On the other hand, when Vitremer was immersed in saliva media, only the weight variable showed a significant association (p = 0.002). For Cention N, significant association was found for only Ra when immersed in acidic media (p = 0.000). Finally, for Activa, all the studied associations; the weight, height and Ra in both media were found to be insignificant. For saliva media, there was a significant weight change between the three materials during all 10 periods of time (p = 0.000). In the first six months, Cention N demonstrated a significant increase in weight changes followed by Vitremer, then Activa. Yet, after a year, the difference between Cention N and Vitremer became insignificant and Activa showed the least weight changes. There was not a significant difference between the materials in terms of height and Ra measurements. The fluoride experiment was not successful due to technical issues during pH measurements of the collected solutions. For comparison of the studied parameters between the three materials, the Kruskal-Wallis test was used. In acidic media, there was a significant difference between the materials in term of weight change in 10 periods of time (p = 0.000). In particular, after a two month period, Cention N had the highest weight, followed by Vitremer and then by Activa. The difference between Vitremer and Activa became insignificant throughout the rest of the experimental time frame. All the height measurements between the three materials were found to be insignificant except for day 365 (p = 0.048), where both Activa and Cention N were found to be significantly higher than Vitremer. For the Ra comparison, in the first two weeks, particularly day 1, 7 and 14, Cention N had significantly the lowest Ra among the other materials. As the three materials aged in the acidic media (day 180), Vitremer had significantly the highest Ra values. Cention N showed higher Ra values than Activa; nonetheless this difference was not significant. The SEM images showed loss of some particles in all post-experimental images of the materials in acidic media. Vitremer showed the widest cracks with the loss of fillers. In saliva media, there was also loss of particles but to a lesser extent than in acidic media. Yet, the post-experimental image of Activa in saliva resembled the pre-experimental one. Conclusion: Within the limitations of the study, the best material to resist Ra from prolonged acidic attack was Activa followed by Cention N and then Vitremer. Except for Vitremer, no significant changes in the Ra of the other materials were detected when the three materials were immersed in saliva media in the long term. In acidic media Vitremer tended to lose weight and height faster than Cention N and Activa over a year. Cention N is the best material to resist dimensional change. However, in artificial saliva Vitremer gained water rapidly. Activa did not absorb a lot of water and did not reject a lot of water; Activa demonstrated good dimensional stability and this property may be beneficial when compared to the other two materials tested. The clinical significance of the study: All the materials studied were subjected to dimensional and Ra changes following long-term exposure to acidic substances, but the newer materials (Cention N and Activa) seemed to be more dimensionally stable and resistant to Ra changes than the older, well-known material (Vitremer). This may influence a clinician’s choice of restorative material for use in pediatric dentistry.

Magister Theologiae - MTh
This study aimed to determine the association between dimensional change and surface roughness (Ra) of Vitremer, Activa and Cention N after immersing them into two different media: acidic and artificial saliva media for the period of a year. Measurements were made at 10 time intervals during the observation period.

Polyelectrolyte complexes (PECs) between chitosan, a mucoadhesive, intestinal mucosal permeability-enhancing polysaccharide, and cellulose nanocrystals, rod-like cellulose nanoparticles with sulfate groups on their surface, have potential applications in oral drug delivery. The purpose of this research was to develop an understanding of the formation and properties of chitosan–cellulose nanocrystal PECs and determine their in vitro drug release properties, using caffeine and ibuprofen as model drugs. Cellulose nanocrystals were prepared by sulfuric acid hydrolysis of bleached wood pulp. Chitosans with three different molecular weights (81, 3â ¢103, 6â ¢103 kDa) and four different degrees of deacetylation (77, 80, 85, 89%) were used. PEC formation was studied by turbidimetric titration. PEC particles were characterized by FT-IR spectroscopy, scanning electron microscopy, dynamic light scattering, and laser Doppler electrophoresis. The formation and properties of chitosan–cellulose nanocrystal PEC particles were governed by the strong mismatch in the densities of the ionizable groups. The particles were composed primarily of cellulose nanocrystals. Particle shape and size strongly depended on the mixing ratio and pH of the surrounding medium. The ionic strength of the surrounding medium, and the molecular weight and degree of deacetylation of chitosan had a minor effect. Release of caffeine from the chitosan–cellulose nanocrystal PEC particles was rapid and uncontrolled. Ibuprofen-loaded PEC particles showed no release in simulated gastric fluid and rapid release in simulated intestinal fluid. Further evaluation studies should focus on the expected mucoadhesive and permeability-enhancing properties of chitosan–cellulose nanocrystal PEC particles.
Ph. D.

El virus de la Pesta Porcina Africana (VPPA) és l’amenaça principal per a la indústria porcina. L’absència de vacunes enfront del virus complica el control i erradicació de la malaltia que provoca: la Pesta Porcina Africana (PPA). El VPPA infecta tant a porcs domèstics com a senglars euroasiàtics (ambdós Sus Scrofa), cursant amb diferents quadres clínics, que varien des de la PPA aguda o hiperaguda, amb taxes de mortalitat de fins al 100%, fins a la infecció crònica. Malgrat el VPPA és capaç d’infectar també als porcs salvatges africans, incloent al facoquer, el virus no provoca signes clínics aparents, convertint-se de fet en reservoris naturals del VPPA. Aquesta Tesi s’ha sustentat en dues observacions realitzades al nostre laboratori. D’una banda, la infecció de porcs lliures de patògens específics (SPF) amb una soca atenuada de VPPA, provoca un quadre agut de PPA, mentre que aquesta mateixa infecció resulta innòcua per als porcs domèstics convencionals. D’altra banda, estudis comparatius d’espècies de porcs amb molt diferent susceptibilitat a la VPPA van permetre demostrar que la composició de la microbiota intestinal, és influenciada tant per factors genètics com ambientals. Aquests resultats, juntament amb el coneixement sobre el paper clau de la microbiota intestinal en el manteniment de l’equilibri homeostàtic de l’organisme, la maduració del sistema immunològic i la resistència als patògens, ens va conduir a plantejar com a objectiu general d’aquesta tesi, investigar el paper potencial que podria exercir la microbiota fecal del facoquer en la seva resistència a la PPA. Els objectius específics van ser: 1) Establir un model de trasplant de microbiota fecal (TMF) de facoquers en porcs domèstics; 2) utilitzar aquest model per comparar la seva susceptibilitat a la PPA, després de la infecció experimental amb soques virulentes o atenuades de VPPA; 3) aïllar bacteris individuals de la microbiota fecal del facoquero per caracteritzar la capacitat immunoestimulatòria i microbicida in vitro, i 4) inocular porcs domèstics in vivo amb components seleccionats amb l’objectiu de mimetitzar els efectes del TMF. Els principals resultats van ser que el TMF de facoquers: (1) no és perjudicial per als garrins domèstics deslletats, (2) modifica la composició de la microbiota del receptor, (3) millora la immunitat de mucoses del porc domèstic trasplantat i (4) confereix protecció parcial contra una soca atenuada de VPPA, mostrant una reducció molt significativa de virus en sèrum, excreció nasal i signes clínics. No obstant això, el TFM de facoquer no va resultar eficient enfront la infecció intramuscular amb una soca altament virulenta de VPPA. (5) L’aïllament de bacteris a partir de femtes de facoquer va permetre la caracterització de components individuals de la seva microbiota in vitro. Es van caracteritzar bacteris de facoquer amb propietats beneficioses sobre el creixement in vitro d’organoides d’ili i colon del porc. Es va caracteritzar un grup de bacteris capaços d’inhibir el creixement in vitro de bacteris patògens del porc: Clostridium perfringens, Salmonella enterica, Salmonella entérica Typhimurium, Escherichia coli K88 i Streptococcus suis (soques virulentes i apatogèniques). Finalment, es va caracteritzar un aïllat capaç d’estimular la secreció in vitro d’IFNγ de cèl·lules del teixit limfoides associats a l’intestí. (6) La inoculació intragástrica de 15 soques seleccionades, va millorar la immunitat de mucoses en els animals receptors confirmat per un augment en la producció total d’IgA en el sèrum i d’IgA específica enfront del VPPA. Aquests resultats han obert una nova via d’investigació en la recerca d’estratègies que permetin lluitar no només contra el VPPA, sinó també contra altres patògens del porcí. El desemmascarament del paper biològic de la microbiota intestinal dels facoquers podria suposar un gran benefici pel futur.
El Virus de la Peste Porcina Africana (VPPA) es la amenaza principal para la industria porcina. La ausencia de vacunas frente al virus complica el control y erradicación de la enfermedad que este virus provoca: la Peste Porcina Africana (PPA). El VPPA infecta tanto a cerdos domésticos como a jabalíes euroasiáticos (ambos Sus Scrofa), cursando con distintos cuadros clínicos, que varían desde la PPA aguda o hiperaguda, con tasas de mortalidad de hasta el 100%, hasta la infección crónica. A pesar el VPPA es capaz de infectar también a los cerdos salvajes africanos, incluyendo al facóquero, el virus no provoca signos clínicos aparentes, convirtiéndose de hecho en reservorios naturales del VPPA. Esta Tesis se ha sustentado en dos observaciones realizadas en nuestro laboratorio. Por un lado, la infección de cerdos libres de patógenos específicos (SPF) con una cepa atenuada de VPPA, provoca un cuadro agudo de PPA, mientras que esa misma infección resultaba inocua para los cerdos domésticos convencionales. Por otro lado, estudios comparativos de especies de cerdos con muy distinta susceptibilidad a la VPPA permitieron demostrar que la composición de su microbiota intestinal, venía marcada por factores genéticos como ambientales. Estos resultados, junto con el conocimiento sobre el papel clave de la microbiota intestinal en el mantenimiento del equilibrio homeostático del organismo, la maduración del sistema inmunológico y la resistencia a los patógenos, nos condujo a plantear como objetivo general de esta tesis, investigar el papel potencial que podría desempeñar la microbiota fecal de facóquero en su resistencia a la PPA. Los objetivos específicos fueron: 1) Establecer un modelo de trasplante de microbiota fecal (TMF) de facóqueros en cerdos domésticos; 2) utilizar este modelo para comparar su susceptibilidad a la PPA, tras la infección experimental con cepas virulentas o atenuadas del VPPA; 3) aislar bacterias individuales de la microbiota fecal del facóquero para caracterizar la capacidad inmunoestimulatoria y microbicida in vitro, y 4) inocular cerdos domésticos in vivo con componentes seleccionados con el objetivo de mimetizar los efectos del TMF. Los principales hallazgos fueron el TMF de facóqueros: (1) no es perjudicial para los lechones domésticos recién destetados, (2) modifica la composición de la microbiota del receptor, (3) mejora la inmunidad de mucosas del cerdo doméstico trasplantado. (4) y confiere protección parcial contra una cepa atenuada del VPPA, mostrando una reducción muy significativa del virus en suero, excreción nasal y signos clínicos. Sin embargo, el TFM de facóquero no resultó eficiente frente la infección intramuscular con una cepa altamente virulenta del VPPA. (5) El aislamiento de bacterias a partir de heces de facóquero permitió la caracterización de componentes individuales de su microbiota in vitro. Se caracterizaron bacterias de facóquero con propiedades beneficiosas sobre el crecimiento in vitro de organoides del íleon y colon del cerdo. Se caracterizó un grupo de bacterias capaces de inhibir el crecimiento in vitro de bacterias patógenas del cerdo: Clostridium perfringens, Salmonella enterica, Salmonella entericaTyphimurium, Escherichia coli K88 y Streptococcus suis (cepas virulentas y apatogénicas). Finalmente, se caracterizó un aislado capaz de estimular la secreción in vitro de IFNγ de células del tejido linfoides asociados al intestino. (6) La inoculación intragástrica de 15 cepas seleccionadas, mejoró la inmunidad de mucosas en los animales receptores indicada por un aumento en la producción total de IgA en el suero y de la IgA específica frente al VPPA. Estos resultados han abierto una nueva vía de investigación en la búsqueda de estrategias que permitan luchar no sólo contra el VPPA, sino también contra otros patógenos del porcino. El desenmascaramiento del papel biológico de la microbiota intestinal de los facóqueros podría suponer un gran beneficio para el futuro.
African swine fever virus (ASFV) is the number one threat for the pig industry. Until today, there is no commercial vaccine or treatment available, thus complicating the control and eradication of African swine fever (ASF). ASFV can infect domestic pigs (DPs) and Eurasian wild boars (both being Sus Scrofa), resulting in different clinical disease courses, varying from acute ASF with 100 % mortality rate to chronic infection. Conversely ASFV can infect African wild pigs, including warthog, bushpig and giant forest hog, without causing apparent disease. The work here presented is based on two observations preliminary obtained in our laboratory. Firstly, specific-pathogen-free (SPF) pigs infected with attenuated ASFV strains, developed acute ASF dying in a matter of two weeks, while with same ASFV strains conventional DPs perfectly overcame the infection, despite sharing identical genetic background than the SPF pigs. This definitively demonstrated that together with genetic differences, environmental factors could also play roles in ASF susceptibility. Furthermore, fecal microbiota comparisons between two swine species (pigs and warthogs), grown in diverse environmental conditions, confirmed that microbiota composition varies depending on genetic and environmental factors. With this data at hand and taking into account that gut microbiota is one of the key players driving body homeostasis equilibrium, immune system maturation and pathogen resistance, the main objective here was to investigate the potential role of warthog fecal microbiota in ASF resistance. We proposed four specific objectives: 1) To establish a fecal microbiota transplantation (FMT) model in DPs using microbiota from DPs or warthogs. 2) To use this animal model to compare the ASF susceptibility after experimental challenge with virulent or attenuated ASFV strains. 3) To isolate individual bacteria from warthog fecal microbiota for further characterization of their in vitro microbicidal or immunostimulatory capabilities, and 4) to inoculate in vivo DPs with selected components of the in vitro characterized microbiota, aiming to mimic the effects observed after FMT. The main findings obtained can be summarized as follows: (1) Transplantation of fecal microbiota from warthog is not harmful to domestic weaned piglets. (2) FMT from warthog modifies the microbiota composition of transplanted DPs. (3) FMT from warthog improves the mucosal immunity of transplanted DPs, with higher levels of total IgA in sera. (4) FMT from warthog to DPs confers partial protection against intramuscular infection with E75CV1, an attenuated strain. Thus, pigs transplanted with fecal microbiota from warthog, showed a very significant reduction of virus in serum, nasal shedding and clinical signs, while FMT from pigs to pigs did not. No effect was observed against intramuscular challenge with E75, a highly virulent strain. (5) Isolation of individual bacteria from warthog feces allowed the characterization of individual microbiota components in vitro. Therefore, some bacteria showed beneficial properties on pig ileum and colon organoids, others showed microbicidal properties against different pig pathogenic bacteria, including: Clostridium perfringens (type B), Salmonella enterica, Salmonella enterica serovar Typhimurium monophasic variant, Escherichia coli K88 and Streptococcus suis (virulent and apathogenic strains). Several bacteria were able to stimulate in vitro IFNγ secretion by gut associated lymphoid tissues, a key cytokine involved in ASFV protection. (6) The intragastric inoculation of 15 selected isolates strains, improved the mucosal immunity in the recipient animals denoted by the increase in total IgA production in sera and the ASFV-specific IgA in both serum and nasal swabs upon E75CV1 challenge. The results obtained during the present doctoral thesis will open new avenues for the future fighting not only against ASFV, but also against other pathogens.”

Hypothalamic neurotransmitter systems are likely to play an important role in bringing about the changes in neuroendocrine function which occur throughout development. In particular, the dopaminergic neurones intrinsic to the hypothalamus are widely implicated in the regulation of the reproductive and growth axes which exhibit clear ontogenetic changes in their endocrine profiles. Therefore, using foetal rat hypothalamic cells in primary culture, maintained in a serum-free defined medium, we have investigated the morphological and functional development of the intrahypothalamic dopaminergic systems. Immunocytochemical studies demonstrated the presence of three morphologically distinct sub-types of tyrosine hydroxylase-immunopositive (THIP) neurones. On day three in vitro unipolar, bipolar and multipolar cell types were apparent. The latter two sub-types persisted to later stages in culture and increased both in perikarya size and neurite length. All subtypes have been shown to have correlates in vivo. Biochemical studies employing tritiated dopamine ([3H]DA) demonstrated a time- and temperature- dependent uptake mechanism within the cultures which was significantly attenuated by uptake inhibitors such as benztropine and nomifensine in a dose - dependent manner. The degree of uptake into the hypothalamic cells in culture increased as a function with time reflecting a maturation of the uptake mechanism in play. Certain experiments suggest that the uptake may also be influenced by dopamine receptor modulation. [3H]DA was released under both basal and potassium (56mM) - stimulated conditions and the magnitude of the response was reduced by exclusion of calcium from the release medium. The ability of the cells to release pH]DA also increased with the age of the culture again indicating a functional maturation of the DA containing neurones within this preparation. Functional maturation of these systems was also reflected in studies involving the measurement of endogenous DA using high performance liquid chromatography coupled with electrochemical detection (HPLC - EC). The neurones displayed an inherent maturation as shown by an increase in the magnitude of release (basal and potassium - stimulated) of endogenous catecholamine with advancing cultivation time. Potassium - depolarization was seen to enhance the rate of development/improve the efficiency of the release mechanism. In addition, we found that cultures initiated from tissue collected at different gestational ages showed differential behaviour in terms of DA output. The role of 17 6 - oestradiol (E2) in regulating hypothalamic dopaminergic function was also investigated both indirectly with the use of pH]DA and by direct measurement of endogenous DA. Both uptake and release of [^HJDA and release of endogenous DA were significantly modulated by the concentration of the steroid in the defined medium. The optimal concentration for uptake ([^HJDA) and release (pH]DA and endogenous DA) were 10 and 10 respectively. These levels of oestrogen are likely to be within the physiological range. The parameters under investigation were also influenced by progesterone, but in a manner which was distinctly different from that of oestrogen. The effect of prolactin and gonadotrophin - hormone - releasing hormone on the release of endogenous DA was also studied to investigate whether intrahypothalamic dopaminergic systems might be involved in negative feedback regulation of hormone release. While prolactin was ineffective in modulating DA output GnRH was a potent secretagogue for the dopaminergic systems within the culture model. GABA-ergic neurones within the cultures were also studied in order to test a) the specificity of the results with the DA neurones and b) a functional interaction between hypothalamic GABA-ergic and dopaminergic neurones. Studies on the uptake and release of [^HJGABA in hypothalamic cells in culture highlighted marked differences in behaviour of GABA-ergic and dopaminergic systems both in terms of their pattern of maturation and their responsiveness to gonadal steroids. Bicuculline (GABAA receptor antagonist) significantly (p < 0.005) enhanced the release of endogenous DA at concentrations ranging from to 10"^M and both GABAA and GABAB agonists (muscimol and baclofen respectively) were also found to modulate the release of endogenous DA. Marked differences in the behaviour of [^H]DA release compared with that of the endogenous amine were noted and suggest caution when interpreting pharmacological studies using cells pre-loaded with [^H] - labelled neurotransmitters. In conclusion, these results demonstrate that hypothalamic dopaminergic neurones in primary culture undergo a pre-programmed morphological and functional maturation which have several correlates in vivo. At least one sub-population of these neurones is responsive to gonadal steroids and influenced by hypothalamic GABAergic systems. As hypothalamic dopaminergic pathways are essential components in the regulation of neuroendocrine activity we propose that this model may serve as a valuable tool for the investigation of the ontogeny of DA systems intrinsic to the hypothalamus and also their interaction with other hypothalamic neurotransmitters and neuropeptides.

15N-based nuclear magnetic resonance techniques are considered very powerful to study the molecular properties of platinum-containing anticancer agents, these properties being responsible for the efficacy of the compounds, but also for the understanding of resistance mechanisms and toxicity. Therefore, the first part of the present work aimed to develop a new method for synthesizing 15N-labeled, chiral platinum compounds. A theoretical discussion on the nucleophilic ring-opening of aziridines has also been envisaged, rationalizing an interesting regiochemistry question. Indeed, a surprising inversion of regiochemistry arose during the development of the above-mentioned synthetic pathway, and density functional theory calculation brought a rational framework to the experimental findings.

Infrared spectroscopy probes the global chemical composition of a sample and has been used to produce a snapshot of cancer cells contents after treatment with platinum coordinates. Indeed, in vitro studies focused here on the use of modern spectroscopic methods to fingerprint the cellular impact of platinum complexes. These drug signatures help to classify and select promising compounds. It makes no doubt that such systemic approaches for compound discovery are helpful technologies. Also, we made the use of the COMPARE algorithm from the NCI, which analyzes similarity between any active compounds previously tested by the NCI large scale in vitro screening program of anticancer agents.

The last chapter aimed to study the interactions between a series of platinum coordinates and DNA. Binding mode to telomeric-like sequences and binding kinetics to genomic-like sequences were assessed to investigate any differences between the compounds and to gain insight into structure-activity relationships.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Invariant natural killer T (iNKT) cells are endowed with features of both innate and adaptive immunity. They are activated by the recognition of glycolipid agonists presented by CD1d which makes them excellent candidates for cellular therapies. In order to investigate the ability of iNKT cells to suppress experimental acute Graft versus host disease (GVHD), iNKT cells were first expanded in vitro, which is likely to be required prior to their use as a cellular therapeutic. Interestingly, the expanded cells showed increased frequencies of IL-10, IL-13 and IL-17 producing cells and were found to robustly suppress alloreactive T cell proliferation in vitro compared to freshly isolated cells. However, in a model of acute GVHD induced by alloantigen-reactive TCR-transgenic T cells neither freshly isolated nor expanded iNKT cells suppressed GVHD, although some survival benefit was seen following the activation of host iNKT cells. These data indicate that iNKT cells can be expanded ex vivo, that they can acquire different functional properties and that such cells robustly suppress alloreactive T cell proliferation in vitro. Therefore, further investigation into the suppressive behaviour of these cells is warranted despite a failure to suppress acute GVHD in the current study.

Thesis (M.S.)--Biomedical Engineering, Georgia Institute of Technology, 2005.
Sacks, Michael, Committee Member ; Vito, Raymond, Committee Member ; Guldberg, Robert, Committee Member ; Yoganathan, Ajit, Committee Chair. Includes bibliographical references.