Antagonism of tetherin restriction of HIV-1 release by Vpu involves binding and sequestration of the restriction factor in a perinuclear compartment

The Vpu accessory protein promotes HIV-1 release by counteracting Tetherin/BST-2, an interferon-regulated restriction factor, which retains virions at the cell-surface. Recent reports proposed beta-TrCP-dependent proteasomal and/or endo-lysosomal degradation of Tetherin as potential mechanisms by which Vpu could down-regulate Tetherin cell-surface expression and antagonize this restriction. In all of these studies, Tetherin degradation did not, however, entirely account for Vpu anti-Tetherin activity. Here, we show that Vpu can promote HIV-1 release without detectably affecting Tetherin steady-state levels or turnover, suggesting that Tetherin degradation may not be necessary and/or sufficient for Vpu anti-Tetherin activity. Even though Vpu did not enhance Tetherin internalization from the plasma membrane (PM), it did significantly slow-down the overall transport of the protein towards the cell-surface. Accordingly, Vpu expression caused a specific removal of cell-surface Tetherin and a re-localization of the residual pool of Tetherin in a perinuclear compartment that co-stained with the TGN marker TGN46 and Vpu itself. This re-localization of Tetherin was also observed with a Vpu mutant unable to recruit beta-TrCP, suggesting that this activity is taking place independently from beta-TrCP-mediated trafficking and/or degradation processes. We also show that Vpu co-immunoprecipitates with Tetherin and that this interaction involves the transmembrane domains of the two proteins. Importantly, this association was found to be critical for reducing cell-surface Tetherin expression, re-localizing the restriction factor in the TGN and promoting HIV-1 release. Overall, our results suggest that association of Vpu to Tetherin affects the outward trafficking and/or recycling of the restriction factor from the TGN and as a result promotes its sequestration away from the PM where productive HIV-1 assembly takes place. This mechanism of antagonism that results in TGN trapping is likely to be augmented by beta-TrCP-dependent degradation, underlining the need for complementary and perhaps synergistic strategies to effectively counteract the powerful restrictive effects of human Tetherin.

Polymeric nanoparticles : from microporosity and drug release kinetics to cellular interactions

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Role of nucleotides on lung epithelial cells : mechanism of release and development of a side-view microscopic chamber to study nucleotide-dependent airway surface liquid height

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Increased extracellular brain glutamate in acute liver failure: decreased uptake or increased release?

Glutamatergic dysfunction has been suggested to play an important role in the pathogenesis of hepatic encephalopathy (HE) in acute liver failure (ALF). Increased extracellular brain glutamate concentrations have consistently been described in different experimental animal models of ALF and in patients with increased intracranial pressure due to ALF. High brain ammonia levels remain the leading candidate in the pathogenesis of HE in ALF and studies have demonstrated a correlation between ammonia and increased concentrations of extracellular brain glutamate both clinically and in experimental animal models of ALE Inhibition of glutamate uptake or increased glutamate release from neurons and/or astrocytes could cause an increase in extracellular glutamate. This review analyses the effect of ammonia on glutamate release from (and uptake into) both neurons and astrocytes and how these pathophysiological mechanisms may be involved in the pathogenesis of HE in ALF.

Acute insult of ammonia leads to calcium-dependent glutamate release from cultured astrocytes, an effect of pH.

Hyperammonemia is a key factor in the pathogenesis of hepatic encephalopathy (HE) as well as other metabolic encephalopathies, such as those associated with inherited disorders of urea cycle enzymes and in Reye's syndrome. Acute HE results in increased brain ammonia (up to 5 mM), astrocytic swelling, and altered glutamatergic function. In the present study, using fluorescence imaging techniques, acute exposure (10 min) of ammonia (NH4+/NH3) to cultured astrocytes resulted in a concentration-dependent, transient increase in [Ca2+]i. This calcium transient was due to release from intracellular calcium stores, since the response was thapsigargin-sensitive and was still observed in calcium-free buffer. Using an enzyme-linked fluorescence assay, glutamate release was measured indirectly via the production of NADH (a naturally fluorescent product when excited with UV light). NH4+/NH3 (5 mM) stimulated a calcium-dependent glutamate release from cultured astrocytes, which was inhibited after preincubation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester but unaffected after preincubation with glutamate transport inhibitors dihydrokainate and DL-threo-beta-benzyloxyaspartate. NH4+/NH3 (5 mM) also induced a transient intracellular alkaline shift. To investigate whether the effects of NH4+/NH3 were mediated by an increase in pH(i), we applied trimethylamine (TMA+/TMA) as another weak base. TMA+/TMA (5 mM) induced a similar transient increase in both pH(i) and [Ca2+]i (mobilization from intracellular calcium stores) and resulted in calcium-dependent release of glutamate. These results indicate that an acute exposure to ammonia, resulting in cytosolic alkalinization, leads to calcium-dependent glutamate release from astrocytes. A deregulation of glutamate release from astrocytes by ammonia could contribute to glutamate dysfunction consistently observed in acute HE.

High-amylose carboxymethyl starch matrices for oral sustained drug-release : in vitro and in vivo evaluation

Les amidons non modifiées et modifiés représentent un groupe d’excipients biodégradables et abondants particulièrement intéressant. Ils ont été largement utilisés en tant qu’excipients à des fins diverses dans des formulations de comprimés, tels que liants et/ou agents de délitement. Le carboxyméthylamidon sodique à haute teneur en amylose atomisé (SD HASCA) a été récemment proposé comme un excipient hydrophile à libération prolongée innovant dans les formes posologiques orales solides. Le carboxyméthylamidon sodique à haute teneur en amylose amorphe (HASCA) a d'abord été produit par l'éthérification de l'amidon de maïs à haute teneur en amylose avec le chloroacétate. HASCA a été par la suite séché par atomisation pour obtenir le SD HASCA. Ce nouvel excipient a montré des propriétés présentant certains avantages dans la production de formes galéniques à libération prolongée. Les comprimés matriciels produits à partir de SD HASCA sont peu coûteux, simples à formuler et faciles à produire par compression directe. Le principal objectif de cette recherche était de poursuivre le développement et l'optimisation des comprimés matriciels utilisant SD HASCA comme excipient pour des formulations orales à libération prolongée. A cet effet, des tests de dissolution simulant les conditions physiologiques du tractus gastro-intestinal les plus pertinentes, en tenant compte de la nature du polymère à l’étude, ont été utilisés pour évaluer les caractéristiques à libération prolongée et démontrer la performance des formulations SD HASCA. Une étude clinique exploratoire a également été réalisée pour évaluer les propriétés de libération prolongée de cette nouvelle forme galénique dans le tractus gastro-intestinal. Le premier article présenté dans cette thèse a évalué les propriétés de libération prolongée et l'intégrité physique de formulations contenant un mélange comprimé de principe actif, de chlorure de sodium et de SD HASCA, dans des milieux de dissolution biologiquement pertinentes. L'influence de différentes valeurs de pH acide et de temps de séjour dans le milieu acide a été étudiée. Le profil de libération prolongée du principe actif à partir d'une formulation de SD HASCA optimisée n'a pas été significativement affecté ni par la valeur de pH acide ni par le temps de séjour dans le milieu acide. Ces résultats suggèrent une influence limitée de la variabilité intra et interindividuelle du pH gastrique sur la cinétique de libération à partir de matrices de SD HASCA. De plus, la formulation optimisée a gardé son intégrité pendant toute la durée des tests de dissolution. L’étude in vivo exploratoire a démontré une absorption prolongée du principe actif après administration orale des comprimés matriciels de SD HASCA et a montré que les comprimés ne se sont pas désintégrés en passant par l'estomac et qu’ils ont résisté à l’hydrolyse par les α-amylases dans l'intestin. Le deuxième article présente le développement de comprimés SD HASCA pour une administration orale une fois par jour et deux fois par jour contenant du chlorhydrate de tramadol (100 mg et 200 mg). Ces formulations à libération prolongée ont présenté des valeurs de dureté élevées sans nécessiter l'ajout de liants, ce qui facilite la production et la manipulation des comprimés au niveau industriel. La force de compression appliquée pour produire les comprimés n'a pas d'incidence significative sur les profils de libération du principe actif. Le temps de libération totale à partir de comprimés SD HASCA a augmenté de manière significative avec le poids du comprimé et peut, de ce fait, être utilisé pour moduler le temps de libération à partir de ces formulations. Lorsque les comprimés ont été exposés à un gradient de pH et à un milieu à 40% d'éthanol, un gel très rigide s’est formé progressivement sur leur surface amenant à la libération prolongée du principe actif. Ces propriétés ont indiqué que SD HASCA est un excipient robuste pour la production de formes galéniques orales à libération prolongée, pouvant réduire la probabilité d’une libération massive de principe actif et, en conséquence, des effets secondaires, même dans le cas de co-administration avec une forte dose d'alcool. Le troisième article a étudié l'effet de α-amylase sur la libération de principe actif à partir de comprimés SD HASCA contenant de l’acétaminophène et du chlorhydrate de tramadol qui ont été développés dans les premières étapes de cette recherche (Acetaminophen SR et Tramadol SR). La modélisation mathématique a montré qu'une augmentation de la concentration d’α-amylase a entraîné une augmentation de l'érosion de polymère par rapport à la diffusion de principe actif comme étant le principal mécanisme contrôlant la libération de principe actif, pour les deux formulations et les deux temps de résidence en milieu acide. Cependant, même si le mécanisme de libération peut être affecté, des concentrations d’α-amylase allant de 0 UI/L à 20000 UI/L n'ont pas eu d'incidence significative sur les profils de libération prolongée à partir de comprimés SD HASCA, indépendamment de la durée de séjour en milieu acide, le principe actif utilisé, la teneur en polymère et la différente composition de chaque formulation. Le travail présenté dans cette thèse démontre clairement l'utilité de SD HASCA en tant qu'un excipient à libération prolongée efficace.

Effect of polymer architecture on Curcumin 1 encapsulation and release from PEGylated polymer nanoparticles: toward a drug delivery nano-platform to the CNS

We developed a nanoparticles (NPs) library from poly(ethylene glycol)–poly lactic acid comb-like polymers with variable amount of PEG. Curcumin was encapsulated in the NPs with a view to develop a delivery platform to treat diseases involving oxidative stress affecting the CNS. We observed a sharp decrease in size between 15 and 20% w/w of PEG which corresponds to a transition from a large solid particle structure to a “micelle-like” or “polymer nano-aggregate” structure. Drug loading, loading efficacy and release kinetics were determined. The diffusion coefficients of curcumin in NPs were determined using a mathematical modeling. The higher diffusion was observed for solid particles compared to “polymer nano-aggregate” particles. NPs did not present any significant toxicity when tested in vitro on a neuronal cell line. Moreover, the ability of NPs carrying curcumin to prevent oxidative stress was evidenced and linked to polymer architecture and NPs organization. Our study showed the intimate relationship between the polymer architecture and the biophysical properties of the resulting NPs and sheds light on new approaches to design efficient NP-based drug carriers.

Decomposition of and nutrient release from ruminant manure on acid sandy soils in the Sahelian zone of Niger, West Africa

In ago-pastoral systems of the semi-arid West African Sahel, targeted applications of ruminant manure to the cropland is a widespread practice to maintain soil productivity. However, studies exploring the decomposition and mineralisation processes of manure under farmers' conditions are scarce. The present research in south-west Niger was undertaken to examine the role of micro-organisms and meso-fauna on in situ release rates of nitrogen (N), phosphorus (P) and potassium (K) from cattle and sheep-goat manure collected from village corrals during the rainy season. The results show tha (1) macro-organisms played a dominant role in the initial phase of manure decomposition; (2) manure decomposition was faster on crusted than on sandy soils; (3) throughout the study N and P release rates closely followed the dry matter decomposition; (4) during the first 6 weeks after application the K concentration in the manure declined much faster than N or P. At the applied dry matter rate of 18.8 Mg ha^-1, the quantities of N, P and K released from the manure during the rainy season were up to 10-fold larger than the annual nutrient uptake of pearl millet (Pennisetum glaucum L.), the dominant crop in the traditional agro-pastoral systems. The results indicate considerable nutrient losses with the scarce but heavy rainfalls which could be alleviated by smaller rates of manure application. Those, however, would require a more labour intensive system of corralling or manure distribution.

Effects of Different Feeding Regimes on the Digestibility and Faecal Excretion of Nitrogen, Soluble Carbohydrates and Fibre Fractions in Water Buffaloes kept under Subtropical Conditions

In the course of the ‘Livestock Revolution’, extension and intensification of, among others, ruminant livestock production systems are current phenomena, with all their positive and negative side effects. Manure, one of the inevitable secondary products of livestock rearing, is a valuable source of plant nutrients and its skillful recycling to the soil-plant interface is essential for soil fertility, nutrient - and especially phosphorus - uses efficiency and the preservation or re-establishment of environmentally sustainable farming systems, for which organic farming systems are exemplarily. Against this background, the PhD research project presented here, which was embedded in the DFG-funded Research Training Group 1397 ‘Regulation of soil organic matter and nutrient turnover in organic agriculture ’ investigated possibilities to manipulate the diets of water buffalo (Bubalus bubalis L.) so as to produce manure of desired quality for organic vegetable production, without affecting the productivity of the animals used. Consisting of two major parts, the first study (chapter 2) tested the effects of diets differing in their ratios of carbon (C) to nitrogen (N) and of structural to non-structural carbohydrates on the quality of buffalo manure under subtropical conditions in Sohar, Sultanate of Oman. To this end, two trials were conducted with twelve water buffalo heifers each, using a full Latin Square design. One control and four tests diets were examined during three subsequent 7 day experimental periods preceded each by 21 days adaptation. Diets consisted of varying proportions of Rhodes grass hay, soybean meal, wheat bran, maize, dates, and a commercial concentrate to achieve a (1) high C/N and high NDF (neutral detergent fibre)/SC (soluble carbohydrate) ratio (HH), (2) low C/N and low NDF/SC ratio (LL); (3) high C/N and low NDF/SC ratio (HL) and (4) low C/N and high NDF/SC (LH) ratio. Effects of these diets, which were offered at 1.45 times maintenance requirements of metabolizable energy, and of individual diet characteristics, respectively, on the amount and quality of faeces excreted were determined and statistically analysed. The faeces produced from diets HH and LL were further tested in a companion PhD study (Mr. K. Siegfried) concerning their nutrient release in field experiments with radish and cabbage. The second study (chapter 3) focused on the effects of the above-described experimental diets on the rate of passage of feed particles through the gastrointestinal tract of four randomly chosen animals per treatment. To this end, an oral pulse dose of 683 mg fibre particles per kg live weight marked with Ytterbium (Yb; 14.5 mg Yb g-1 organic matter) was dosed at the start of the 7 day experimental period which followed 21 days of adaptation. During the first two days a sample for Yb determination was kept from each faecal excretion, during days 3 – 7 faecal samples were kept from the first morning and the first evening defecation only. Particle passage was modelled using a one-compartment age-dependent Gamma-2 model. In both studies individual feed intake and faecal excretion were quantified throughout the experimental periods and representative samples of feeds and faeces were subjected to proximate analysis following standard protocols. In the first study the organic matter (OM) intake and excretion of LL and LH buffaloes were significantly lower than of HH and HL animals, respectively. Digestibility of N was highest in LH (88%) and lowest in HH (74%). While NDF digestibility was also highest in LH (85%) it was lowest in LL (78%). Faecal N concentration was positively correlated (P≤0.001) with N intake, and was significantly higher in faeces excreted by LL than by HH animals. Concentrations of fibre and starch in faecal OM were positively affected by the respective dietary concentrations, with NDF being highest in HH (77%) and lowest in LL (63%). The faecal C/N ratio was positively related (P≤0.001) to NDF intake; C/N ratios were 12 and 7 for HH and LL (P≤0.001), while values for HL and LH were 11.5 and 10.6 (P>0.05). The results from the second study showed that dietary N concentration was positively affecting faecal N concentration (P≤0.001), while there was a negative correlation with the faecal concentration of NDF (P≤0.05) and the faecal ratios of NDF/N and C/N (P≤0.001). Particle passage through the mixing compartment was lower (P≤0.05) for HL (0.033 h-1) than for LL (0.043 h-1) animals, while values of 0.034 h-1 and 0.038 h-1 were obtained for groups LH and HH. At 55.4 h, total tract mean retention time was significantly (P≤0.05) lower in group LL that in all other groups where these values varied between 71 h (HH) and 79 h (HL); this was probably due to the high dietary N concentration of diet LL which was negatively correlated with time of first marker appearance in faeces (r= 0.84, P≤0.001), while the dietary C concentration was negatively correlated with particle passage through the mixing compartment (r= 0.57, P≤0.05). The results suggest that manure quality of river buffalo heifers can be considerably influenced by diet composition. Despite the reportedly high fibre digestion capacity of buffalo, digestive processes did not suppress the expression of diet characteristics in the faeces. This is important when aiming at producing a specific manure quality for fertilization purposes in (organic) crop cultivation. Although there was a strong correlation between the ingestion and the faecal excretion of nitrogen, the correlation between diet and faecal C/N ratio was weak. To impact on manure mineralization, the dietary NDF and N concentrations seem to be the key control points, but modulating effects are achieved by the inclusion of starch into the diet. Within the boundaries defined by the animals’ metabolic and (re)productive requirements for energy and nutrients, diet formulation may thus take into account the abiotically and biotically determined manure turnover processes in the soil and the nutrient requirements of the crops to which the manure is applied, so as to increase nutrient use efficiency along the continuum of the feed, the animal, the soil and the crop in (organic) farming systems.

Diversity and nutritional properties of Pakistani dates: implications for sustainable value chain and decent living perspectives of rural households

Date palm (Phoenix dactylifera L.) occupies almost three percent of the total worldwide cultivated area, with an annual production of seven million tonnes (t). Pakistan is an ideal place for the cultivation of date palm due to its sandy loam soil and semi-arid climate. In 2012, Pakistan produced 600,000 t of dates, on an area of 95,000 ha. Baluchistan province is the country’s top date producer, followed by Sindh, Punjab and Khyber Pakhtunkhwa (KPK) provinces. More than 300 date varieties are known to exist in Pakistan and some commercially important cultivars are: Karbalaen, Aseel, Muzawati, Fasli, Begum Jhangi, Hillawi, Dashtiari, Sabzo, Koharaba, Jaan Swore, Rabai and Dhakki. Six districts from the four provinces of Pakistan (Jhang, Muzaffargarh and Bahawalpur (Punjab), Dera Ismail Khan (KPK), Khairpur (Sindh) and Panjgur (Baluchistan)) with largest area under date palm cultivation were selected to conduct socio-economic surveys including the income sources of date palm growers. A structured questionnaire with open-ended and closed questions was used for face-to-face interviews of 170 date palm growers. At each location after selection of a first farmer through a local guide, the former was requested to provide names and addresses of three other date growers in his area. From these three names, one was randomly selected for the next sampling. Additionally, date palm fronds and fruits of all available cultivars were collected for morphological and nutritional analyses. Soil samples were collected from the groves for subsequent chemical and physical analyses. Almost all farmers used dates as a food item for their families and some were using low quality dates as a feed for their livestock. Apart from dates, other date palm components (trunk, spadix, frond, inflorescence and seed) were used by date palm growers as a raw material for making many by-products for their families. Date palm had a major contribution in the income of households, 24% received 91-100% of their income from date palms. More than half of the surveyed farmers had date palm groves, but scattered plantations, home gardens and intercropping systems with cereal and other fruits were also present. Dhakki, Muzawati, Aseel, and Karbalaen were the most important commercial cultivars grown in the provinces of KPK, Baluchistan, and Sindh. Aseel, Karoch, Haleni, Karbalaen, and Muzawati cultivars had the most firm fruit and good total soluble sugar, calcium and magnesium contents. The amount of magnesium found in dates of studied cultivars ranged from 0.143 to 0.876 mg g-1. A great variation in frond morphology was recorded among the cultivars. Fruit length and fruit weight was highest in Dhakki date, making it visually more attractive for customers in addition to its good nutritional properties. The seed weight of the studied cultivars ranged from 0.7-2.0 g, while Desi dates had largest seed, making them less attractive for marketing. However, in terms of nutritional value and fruit size, most of the investigated varieties can compete with globally important commercial dates.

Apatite-Polymer Composite Particles for Controlled Delivery of BMP-2: In Vitro Release and Cellular Response

Bone morphogenetic protein-2 (BMP-2) has the ability to induce osteoblast differentiation of undifferentiated cells, resulting in the healing of skeletal defects when delivered with a suitable carrier. We have applied a versatile delivery platform comprising a novel composite of two biomaterials with proven track records – apatite and poly(lactic-co-glycolic acid) (PLGA) – to the delivery of BMP-2. Sustained release of this growth factor was tuned with variables that affect polymer degradation and/or apatite dissolution, such as polymer molecular weight, polymer composition, apatite loading, and apatite particle size. The effect of released BMP-2 on C3H10T1/2 murine pluripotent mesenchymal cells was assessed by tracking the expression of osteoblastic makers, alkaline phosphatase (ALP) and osteocalcin. Release media collected over 100 days induced elevated ALP activity in C3H10T1/2 cells. The expression of osteocalcin was also upregulated significantly. These results demonstrated the potential of apatite-PLGA composite particles for releasing protein in bioactive form over extended periods of time.

Subcutaneous study on the controlled release of Etanidazole and Taxol for the treatment of Glioma

BALB/c nude mice 6 weeks old were inoculated with glioma C6 cell-line and the efficacy of the different amount of Etanidazole-discs and Taxol-microspheres was investigated. Poly (D,L-lactic-co-glycolic acid) (PLGA) was used as the main encapsulating polymer and polyethylene glycol was added to increase the porosity. The 1% drug loading microspheres of each drug were produced by spray drying and the discs were obtained by compressing the Etanidazole-microspheres. Intra-tumoral injection followed by irradiation resulted in high systemic dosage and thus systemic toxicity. Tumors grown for 6 days, 9 days and 16 days were implanted with 0.5 mg or 1.0 mg or 1.5 mg of the drug. A radiation dosage of 2 Gy each time for a number of times was given for animals implanted with Etanidazole and no irradiation was given for animals implanted with Taxol. Increasing the number of doses clearly decreased the rate of tumor growth. The increase in the amount of drug on smaller sized tumors controlled the tumor better and there was agglomeration of the microspheres resulting in deviation of release profile of the drug as compared to the in vitro studies. It was observed that 1.0 mg of Taxol given to a tumor grown for 6 days was able to suppress the tumor for a total period of approximately two months and no tumor resurrection was observed during the second month.

Fabrication of Controlled Release Devices Using Supercritical Antisolvent Method

In this study, the supercritical antisolvent with enhanced mass transfer method (SASEM) is used to fabricate micro and nanoparticles of biocompatible and biodegradable polymer PLGA (poly DL lactide co glycolic acid). This process may be extended to the encapsulation of drugs in these micro and nanoparticles for controlled release purposes. Conventional supercritical antisolvent (SAS) process involves spraying a solution (organic solvent + dissolved polymer) into supercritical fluid (CO[subscript 2]), which acts as an antisolvent. The high rate of mass transfer between organic solvent and supercritical CO[subscript 2] results in supersaturation of the polymer in the spray droplet and precipitation of the polymer as micro or nanoparticles occurs. In the SASEM method, ultrasonic vibration is used to atomize the solution entering the high pressure with supercritical CO[subscript 2]. At the same time, the ultrasonic vibration generated turbulence in the high pressure vessel, leading to better mass transfer between the organic solvent and the supercritical CO₂. In this study, two organic solvents, acetone and dichloromethane (DCM) were used in the SASEM process. Phase Doppler Particle Analyzer (PDPA) was used to study the ultrasonic atomization of liquid using the ultrasonic probe for the SASEM process. Scanning Electron Microscopy (SEM) was used to study the size and morphology of the polymer particles collected at the end of the process.