Myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) play an essential role in the immunosuppressive networks that contribute to tumor immune evasion. The mechanisms by which tumors promote the expansion and/or function of these suppressive cells and the cross-regulation between MDSC and Treg remain incompletely defined. The current work evaluates the influence of MDSC, expanded in two mouse cancer models, on immunosuppressive Treg. We demonstrate that tumor-induced MDSC endowed with the potential of suppressing conventional T lymphocytes surprisingly impair TGF-β1-mediated generation of induced Treg (iTreg) from naïve CD4⁺ T lymphocytes. Suppression of iTreg generation by MDSC occurs early in the differentiation process, and is cell contact dependent. This inhibition of FoxP3-expressing T lymphocyte differentiation by MDSC does not depend on arginase 1, cystine/cysteine depletion, iNOS/NO, or PD-1/PD-L1 signaling. These findings therefore indicate that MDSC from tumor-bearing hosts have the heretofore unreported ability to restrict some immunosuppressive Treg subpopulations.

Cell-based therapies involving adipose-derived stem cells (ASCs) have shown promise in stimulating cardiovascular regeneration, including in the treatment of myocardial infarction (MI) and ischemic heart disease. However, previous studies involving the delivery of ASCs following MI have indicated that therapeutic efficacy has been limited by low survival and/or poor retention of the transplanted cells at the site of injury. To address these limitations, the goal of this thesis was to develop a more effective delivery strategy incorporating an injectable biomaterial combined with chemotactic growth factor delivery to enhance ASC retention within the gel. Working towards future in vivo analysis in a rat model, multilineage characterization studies confirmed that ASCs isolated from the epididymal fat pad of male Wistar rats could differentiate in vitro along the adipogenic, osteogenic, and chondrogenic lineages. Subsequently, the chemotactic response of the rat ASCs (rASCs) to varying concentrations of stromal derived factor-1 α (SDF-1α) and hepatocyte growth factor (HGF) was analyzed using a modified Boyden chamber assay. The results demonstrated that SDF-1α and HGF, at 20, 50, and 100 ng/mL elicited significant migratory responses under normoxic (21%) and hypoxic (5%) culture conditions. RT-PCR analysis was conducted to assess the expression of the two chemotactic growth factors and their associated receptors in the rASCs, and secreted SDF-1α protein expression was quantified by ELISA. Moving towards the development of the biomaterials-based delivery approach, the viability of rASCs encapsulated by photopolymerization in methacrylated glycol chitosan (MGC) hydrogels modified with various degrees of arginine-glycine-aspartic acid (RGD)-peptide modification was examined. More specifically, rASCs were encapsulated in MGC hydrogels with 0%, 4%, and 7% RGD modification and cultured for up to 14 days. Viability staining results indicated that rASC viability was enhanced in the 4% and 7% RGD-modified MGC hydrogels in comparison to the MGC hydrogels with no peptide modification. Pre-loading the gels with 50 ng/mL of SDF-1α had no significant effects on cell viability over 14 days. Overall, the results demonstrate that peptide modification to promote cell adhesion within the MGC hydrogels is key to improving cell viability and thereby improving the therapeutic potential of ASCs. / Thesis (Master, Chemical Engineering) -- Queen's University, 2012-10-24 23:54:37.126

The common view of brand choice in consumer marketing is that brands compete against each other within a specified product category. For example, different coffee brands are compared and evaluated by consumers and the most preferred brand is selected. Is this the only adequate way of demonstrating how consumers make brand choices? This thesis challenges the common view on brand choice and brand choice determinants in consumer markets on several accounts. First, brand choice is made in goal-derived categories (e.g., "foods to eat while on a diet"), and research on goal-derived categories and consumption goals suggests that consumers often choose between brands from different product categories. For example, a consumer may choose between brands of coffee, tea, and soft drinks to fulfill a consumption goal. Second, there is the question of complementarity. Are brands always chosen one by one? We argue in this thesis that consumers often choose brand constellations from complementary product categories in goal-derived categories (e.g., hamburgers and soft drinks when on a short lunch break). The thesis consists of four articles based on empirical studies. The articles cover single-brand choice and brand constellation choice in goal-derived categories, and the use of goal-derived categories by marketing practitioners. The general conclusion is that consumers evaluate more aspects than just brand-related ones when they choose brands in goal-derived categories. Product category associations (i.e., how typical a product category is perceived in a goal-derived category) are a more important determinant of brand (constellation) choice than brand associations. Also, in brand constellation choice, complementarity (i.e., perceived fit between brands) is more strongly related to brand choice than attitude towards individual brands. / <p>Diss. Stockholm : Handelshögsk., 2003</p>

Major depressive disorder (MDD) and bipolar disorder (BD) are psychiatric diseases that affect behavior and impair cognition. A gene important to these disorders is the brain derived neurotrophic factor (BDNF) which is involved in processes controlling neuroplasticity. Previous studies have suggested that BDNF expression levels have to be finely regulated for normal mental health and cognition. This study therefore aimed to identify cis-regulatory elements that regulate BDNF promoter IV (BP4), which plays a role in mood and cognition, and investigated how polymorphisms in these cis-regulatory elements might alter BP4 activity contributing to MDD and BD. BP4-LacZ transgenic mice and primary neuron cultures were used to show that BP4 was active in the hippocampus, cortex and amygdala and responded to PKC, KCl and Wnt signaling activation. Using comparative genomics, two highly conserved regions were identified, BE5.1 and BE5.2, which contain the rs10767664 and rs12273363 polymorphisms respectively. Reporter gene assays in primary cultures derived from these brain structures showed that BE5.1 and BE5.2 were responsible for “filtering” or “gating” the effects of different combination of activated signal transduction pathways on BP4. Thus, BE5.1 increased BP4 response to forskolin in cortical cultures while abolishing BP4 response to PMA in hippocampal cultures. Similarly, BE5.2 permitted BP4 response to KCl and combined forskolin and PMA treatment, but not individual forskolin and PMA treatment nor LiCl in cortical cultures. Significantly, the minor allele of rs12273363, which has been associated with MDD and BD susceptibility, acted as a more potent repressor of BP4 response to neuron depolarization by KCl and PKA/PKC activation in different primary cultures. The possible relevance of these findings to the role of altered BDNF expression in MDD and BD are discussed.

Estrogen is the natural agonist of the estrogen receptor (ER). However, certain plant-derived compounds or phytoestrogens have been identified that mimic estrogens and act as agonists and/or antagonists of ERs, depending on subtype and target tissue. Understanding how phytoestrogens interact with ERs, and therefore effect the estrogenic response, may prove beneficial in hormone replacement therapy and in the prevention and treatment of hormone-related diseases. Using Thin Layer Chromatography, gas chromatography/mass spectrometry (GC/MS), and proton nuclear nagnetic resonance (HNMR), I identified 4-ethoxymethylphenol (4EM) found in Maclura pomifera. While most phytoestrogens are heterocyclic compounds, 4EM is a simple phenol that acts as an agonist of ER-alpha and -beta in HeLa and MCF-7 cells. To study the effect of 4EM on ER-alpha and -beta activity, I performed transient transfection assays and showed that 4EM activates ER dependent gene transcription in a dose dependent manner in both ER subtypes. Further, 4EM- mediated transcription in ER-alpha, like estrogen, was enhance in the presense of co-activators, SRC-1 (steroid receptor coactivator-1), CBP (CREB binding proteins), and E6-AP (E6-associated protein) and inhibited by trans-4- hydroxytamoxifen (4HT). I found that 4EM was specific for ER and did not activate transcription of the progesterone receptor in HeLa cells.

<p>The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (hMSC) chondrogenesis. We combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in hMSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce hMSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.</p><p> Following this, we modified this anti-inflammatory engineered cartilage to incorporate rabbit MSCs and evaluated this therapeutic strategy in a pilot study in vivo in rabbit osteochondral defects. Rabbits were fed a custom doxycycline diet to induce gene expression in engineered cartilage implanted in the joint. Serum and synovial fluid were collected and the levels of doxycycline and inflammatory mediators were measured. Rabbits were euthanized 3 weeks following surgery and tissues were harvested for analysis. We found that doxycycline levels in serum and synovial fluid were too low to induce strong overexpression of hIL-1Ra in the joint and hIL-1Ra was undetectable in synovial fluid via ELISA. Although hIL-1Ra expression in the first few days local to the site of injury may have had a beneficial effect, overall a higher doxycycline dose and more readily transduced cell population would improve application of this therapy. </p><p> In addition to the 3D woven PCL scaffold, cartilage-derived matrix scaffolds have recently emerged as a promising option for cartilage tissue engineering. Spatially-defined, biomaterial-mediated lentiviral gene delivery of tunable and inducible morphogenetic transgenes may enable guided differentiation of hMSCs into both cartilage and bone within CDM scaffolds, enhancing the ability of the CDM scaffold to provide chondrogenic cues to hMSCs. In addition to controlled production of anti-inflammatory proteins within the joint, in situ production of chondro- and osteo-inductive factors within tissue-engineered cartilage, bone, or osteochondral tissue may be highly advantageous as it could eliminate the need for extensive in vitro differentiation involving supplementation of culture media with exogenous growth factors. To this end, we have utilized controlled overexpression of transforming growth factor-beta 3 (TGF-β3), bone morphogenetic protein-2 (BMP-2) or a combination of both factors, to induce chondrogenesis, osteogenesis, or both, within CDM hemispheres. We found that TGF-β3 overexpression led to robust chondrogenesis in vitro and BMP-2 overexpression led to mineralization but not accumulation of type I collagen. We also showed the development of a single osteochondral construct by combining tissues overexpressing BMP-2 (hemisphere insert) and TGF-β3 (hollow hemisphere shell) and culturing them together in the same media. Chondrogenic ECM was localized in the TGF-β3-expressing portion and osteogenic ECM was localized in the BMP-2-expressing region. Tissue also formed in the interface between the two pieces, integrating them into a single construct. </p><p> Since CDM scaffolds can be enzymatically degraded just like native cartilage, we hypothesized that IL-1 may have an even larger influence on CDM than PCL tissue-engineered constructs. Additionally, anti-inflammatory engineered cartilage implanted in vivo will likely affect cartilage and the underlying bone. There is some evidence that osteogenesis may be enhanced by IL-1 treatment rather than inhibited. To investigate the effects of an inflammatory environment on osteogenesis and chondrogenesis within CDM hemispheres, we evaluated the ability of IL-1Ra-expressing or control constructs to undergo chondrogenesis and osteogenesis in the prescence of IL-1. We found that IL-1 prevented chondrogenesis in CDM hemispheres but did not did not produce discernable effects on osteogenesis in CDM hemispheres. IL-1Ra-expressing CDM hemispheres produced robust cartilage-like ECM and did not upregulate inflammatory mediators during chondrogenic culture in the presence of IL-1.</p> / Dissertation

<p>Articular cartilage is a connective tissue that lines the surfaces of diarthrodial joints; and functions to support and distribute loads as wells as facilitate smooth joint articulation. Unfortunately, cartilage possesses a limited capacity to self-repair. Once damaged, cartilage continues to degenerate until widespread cartilage loss results in the debilitating and painful disease of osteoarthritis. Current treatment options are limited to palliative interventions that seek to mitigate pain, and fail to recapitulate the native function. Cartilage tissue engineering offers a novel treatment option for the repair of focal defects as well as the complete resurfacing of osteoarthritic joints. Tissue engineering combines cells, growth factors, and biomaterials in order to synthesize new cartilage tissue that recapitulates the native structure, mechanical properties, and function of the native tissue. In this endeavor, there has been a growing interest in the use of scaffolds derived from the native extracellular matrix of cartilage. These cartilage-derived matrix (CDM) scaffolds have been show to recapitulate the native epitopes for cell-matrix interactions as well as provide entrapped growth factors; and have been shown to stimulate chondrogenic differentiation of a variety of cell types. Despite the potent chondroinductive properties of CDM scaffolds, they possess very weak mechanical properties that are several orders of magnitude lower than the native tissue. These poor mechanical properties lead to CDM scaffolds succumbing to cell-mediated contraction, which dramatically and unpredictably alters the size and shape of CDM constructs. Cell-mediated contraction not only prevents the fabrication of CDM constructs with specific, pre-determined dimensions, but also limits cellular proliferation and metabolic synthesis of cartilage proteins. This dissertation utilized collagen crosslinking techniques as well as ice-templating in order to enhance the mechanical properties of CDM scaffolds and prevent cell-mediated contraction. Furthermore, the decellularization of CDM was investigated in order to remove possible sources of immunogenicity. This work found that both physical and chemical crosslinking techniques were capable of preventing cell-mediated contraction in CDM scaffolds; however, the crosslinking techniques produced distinct effects on the chondroinductive capacity of CDM. Furthermore, the mechanical properties of CDM scaffolds were able to be enhanced by increasing the CDM concentration; however, this led to a concomitant decrease in pore size, which limited cellular infiltration. The pore size was able to be rescued through the use of an ice-templating technique that led to the formation of large aligned grooves, which enabled cellular infiltration. Additionally, a decellularization protocol was developed that successfully removed foreign DNA to the same order of magnitude as clinically approved materials, while preserving the native GAG content of the CDM, which has been shown to be critical in preserving the mechanical properties of the CDM. Altogether, this body of work demonstrated that dehydrothermal crosslinking was best suited for maintaining the chondroinductive capacity of the CDM, and given the appropriate scaffold fabrication parameters, such as CDM concentration and ice-templating technique, dehydrothermal treatment was able to confer mechanical properties that prevented cell-mediated contraction. To emphasize this finding, this work culminated in the fabrication of an anatomically-relevant hemispherical scaffold entirely from CDM alone. The CDM hemispheres not only supported chondrogenic differentiation, but also retained the original scaffold dimensions and shape throughout chondrogenic culture. These findings illustrate that CDM is a promising material for the fabrication of tailor-made scaffolds for cartilage tissue engineering.</p> / Dissertation

In the efforts to create sustainable production of heat and power and to reduce the net CO2 emissions to the atmosphere, alternative fuels are today being utilised. These fuels are, for example, biofuels and waste derived fuels such as different residues from the agricultural sector and the pulp and paper industry, municipal sewage sludge and municipal sorted solid waste. These fuels put new demands on the combustion facilities due to their chemical composition and this in turn calls for methods of prediction for the evaluation of their combustion behaviour. Most significant for the majority of these fuels are the high alkali and chlorine concentrations which cause bed agglomeration, deposit formation and corrosion on heat transfer surfaces. These problems can be solved if sufficient knowledge is obtained of the specific fuel or fuel mix. In this work, chemical fractionation, a step by step leaching method, was used on fuels, fuel mixes and fly ashes from co-combustion in a fluidised bed combustor. In addition, XRD and SEM-EDX were used for the fuel and fly ash characterisation. Different alkali chloride reducing additives i.e. kaolin, zeolites and sulphur were investigated as was the influence of various bed materials: silica sand, olivine sand and blast furnace slag (BFS). Some of the new, alternative fuels, such as municipal sewage sludge and meat and bone meal (MBM) contain high concentrations of phosphorus which is a very important nutrient essential in many biological processes. Phosphorus rock used as raw material in the phosphate industry is a depleting natural resource estimated to last for only 30-200 years according to different sources. The combustion of municipal sewage sludge enriches the phosphorus in the ashes while hazardous components such as pathogens and organic pollutants are rendered harmless after combustion. However, toxic heavy metals are also enriched in the ashes. One aim of the work was to find a sufficiently effective and low cost method for phosphorus extraction from fly ashes derived from municipal sewage sludge combustion. Two types of municipal sewage sludges were investigated using different chemicals for the phosphorus cleaning step in the waste water treatment plants. The first sewage sludge derived from a plant using iron sulphate as flocculant to precipitate phosphorus as iron phosphate. The second sludge meanwhile came from a plant using aluminium sulphate as flocculant to precipitate phosphorus as aluminium phosphate. Both sewage sludges were dewatered prior to combustion and co-combusted with wood pellets. At pH 1 nearly all the phosphorus was released from the fly ash derived from the sewage sludge where aluminium sulphate was used as a phosphorus precipitation agent. Iron sulphate as precipitant inhibited the phosphorus extraction from the ashes, resulting in only 50-80% of the phosphorus being released. Furthermore, the mobility of heavy metals to the leachates was investigated to establish whether the leachates were suitable as fertilisers. Only minor fractions of Pd, Hg, Cr, Cu, Mn, Co, Ni, As, Sb, V and Zn were found in the leachates, all well within the legislated limitations for fertilisers. However, one exception was Cd that was nearly totally dissolved in the leachate. Thus a decadmiation of the leachate is necessary prior to any utilisation of the ashes and reuse of phosphorus as fertiliser. / <p>Akademisk avhandling för avläggande av teknologie doktorsexamen vid Chalmers tekniska högskola försvaras vid offentlig disputation den 15 oktober 2008</p>

Les cellules T invariantes associées aux muqueuse (MAIT) sont des lymphocytes innés caractérisés par l'expression d'un récepteur des cellules T semi-invariant (iTCR) et restreints par la molécule du complexe majeur d'histocompatibilité de classe Ib, MR1. Chez l'homme, les cellules MAIT sont abondantes dans le sang (1 à 10%), l'intestin (3 à 5%) et le foie (20 à 40%) et réagissent contre des métabolites microbiens. En raison de leur rareté dans les souris de laboratoire classiques, les études sur les cellules MAIT murines ont été principalement effectuées sur des souris transgéniques (Tg) pour des TCR MAIT. Cependant, ces cellules MAIT Tg ne récapitulent pas de manière adéquate le phénotype des cellules MAIT humaines. Ici, nous décrivons une souche de souris congénique que nous avons générée qui possède des cellules MAIT qui ressemblent aux cellules MAIT humaines. Nous utilisons cet outil pour étudier les caractéristiques des cellules MAIT murines. L'étude de souches de souris consanguines d'origine sauvage montre que la souche CAST/Ei présente une fréquence des cellules MAIT nettement supérieur à celle retrouvée dans la souche C57BL/6. Un seul locus est impliqué et a été localisé dans la région TCRα. Ceci a permis la génération d'une souche "MAIT" congénique, qui ont été en outre croisé à une souris Tg pour un rapporteur GFP du facteur transcriptionnel RORγt sur la base de données antérieurs montrant que les MAITs humaines expriment ce facteur. Grâce à cet outil, nous montrons que les MAITs murines sont CD4−CD8−/lo, ont un phénotype mémoire effecteurs (CD44+) et coexpriment PLZF et RORγt. Ces MAITs murines sont orientées vers une localisation tissulaire (CCR6+CCR7−) et résident préférentiellement dans les tissus non lymphoïdes périphériques, y compris les poumons, le foie et la peau. Après stimulation du TCR, les MAITs produisent des cytokines TH1/2/17 et sont aussi activées par de antigènes bactériens (par exemple semi-purifié fraction bactérienne ou 5-OP-RU) d'une manière dépendant de MR1. Les MAITs ont une forte expression de récepteurs de cytokines (IL-7R, IL-18Rα, IL-12Rβ) et peuvent ainsi répondre à des cytokines innées. Lors d'une infection expérimentale des voies urinaires, les MAITs migrent vers la vessie et ont une activité protectrice anti-bactérienne. Au total, nos résultats démontrent que les cellules MAIT murines ressemblent étroitement à leurs homologues humains. Ce nouveau modèle murin sera un outil puissant pour faire avancer notre compréhension de la biologie des cellules MAIT en situation normale et pathologique. / Mucosal-associated invariant T cells (MAIT) are innate lymphocytes that express a semi-invariant T cell receptor (iTCR) and are restricted by the major histocompatibility complex (MHC) related molecule, MR1. In human, MAIT cells are abundant in the blood (1-10%), gut (3-5%), and liver (20-40%). They react against microbial-derived riboflavin metabolites that are common in bacteria and yeast. Due to the paucity of MAIT cells in classical inbred laboratory mice, studies on mouse MAIT cells were mostly performed in TCR-transgenic (Tg) mice. However, these Tg MAIT cells do not adequately recapitulate the phenotype of human MAIT cells. Herein, we present a recently generated congenic mouse strain harboring MAIT cells that closely resemble human MAIT cells and use this tool to study the characteristics of natural mouse MAIT cell. An analysis of wild-derived inbred mouse strains revealed that CAST/Ei strain has increased frequency of MAIT cells than C57BL/6 mice. This was linked to a locus on the TCRα region. Introduction of such locus into C57BL/6 mice generated a “MAIT” congenic strain, which were further crossed to Rorc(γt)-GfpTG reporter strain based on previous findings of RORγt expression on human MAIT cells. Using this tool, we show that natural mouse MAIT cells are CD4−CD8−/lo, display an effector memory phenotype (CD44+), and coexpress the transcription factors PLZF and RORγt. They exhibit tissue-homing properties (CCR6+CCR7−) and preferentially reside in peripheral non-lymphoid tissues, including lung, liver, and skin. Upon TCR ligation, MAIT cells produce TH1/2/17 type cytokines and react to bacterial-derived antigens (i.e. semi-purified bacterial fraction or 5-OP-RU) in an MR1-dependent manner. They have high expression of cytokine receptors (IL-7R, IL-18Rα, IL-12Rβ) and may respond to the corresponding innate cytokines. During experimental urinary tract infection, MAIT cells migrate to the bladder and display a protective anti-bacterial activity. Altogether, our results demonstrate that mouse MAIT cells resemble their human counterparts more closely than previously recognized and therefore this new mouse model will be a powerful tool for advancing our understanding of MAIT cell biology in health and disease.

Stress is a major risk factor for substance abuse. Intermittent social defeat stress increases drug self-administration (SA) and elevates brain-derived neurotrophic factor (BDNF) expression in the ventral tegmental area (VTA) in rats. Intra-VTA BDNF overexpression enhances social defeat stress-induced cross-sensitization to psychostimulants and induces nucleus accumbens (NAc) ΔFosB expression. Therefore, increased VTA BDNF may mimic or augment the development of drug abuse-related behavior following social stress. To test this hypothesis, adeno-associated virus (AAV) was infused into the VTA to overexpress either GFP alone (control) or GFP + BDNF. Rats were then either handled or exposed to intermittent social defeat stress before beginning cocaine SA training. The SA acquisition and maintenance phases were followed by testing on a progressive ratio (PR) schedule of cocaine reinforcement, and then during a 12-h access "binge" cocaine SA session. BDNF and ΔFosB were quantified postmortem in regions of the mesocorticolimbic circuitry using immunohistochemistry. Social defeat stress increased cocaine intake on a PR schedule, regardless of virus treatment. While stress alone increased intake during the 12-h binge session, socially-defeated rats that received VTA BDNF overexpression exhibited even greater cocaine intake compared to the GFP-stressed group. However, VTA BDNF overexpression alone did not alter binge intake. BDNF expression in the VTA was also positively correlated with total cocaine intake during binge session. VTA BDNF overexpression increased ΔFosB expression in the NAc, but not in the dorsal striatum. Here we demonstrate that VTA BDNF overexpression increases long-access cocaine intake, but only under stressful conditions. Therefore, enhanced VTA-BDNF expression may be a facilitator for stress-induced increases in drug abuse-related behavior specifically under conditions that capture compulsive-like drug intake.