Parasites have a major impact on host condition and fitness and thereby represent a strong selective force for individuals in wild populations. The main defence against parasite infection and associated morbidity is the host immune response, and consequently it is expected for there to be strong selection eroding genetic variation underlying immune responses in natural populations. However, studies in the wild have found considerable heritable variation underlying immune responses. Few studies have investigated the genetic variants underlying immunity in wild populations and are able to examine how genetic variation is maintained in the face of natural selection. The aim of this thesis is to investigate the selection on, and genetic variation underlying, immunity in a wild Soay sheep population by looking at antibody responses to the prevalent parasite Teladorsagia circumcincta. Anti- T. circumcincta antibody levels (IgA, IgE, IgG) were measured in neonatal plasma samples taken soon after birth, representing maternally-derived antibodies, and in samples from August yearly from four month old lambs and adults, representing endogenous antibodies. All three endogenously produced antibody measures in lambs and adults were repeatable and heritable. In addition, a genome wide association study run on the three antibody traits on August lamb and adult measures found associations between anti-T. circumcincta IgA levels and single nucleotide polymorphisms in a region on chromosome 24. There was evidence for age- and isotype- dependent negative associations between antibody isotypes and strongyle faecal egg counts (FEC). Further, there was evidence for age-dependent selection via positive associations between anti-T. circumcincta IgG and survival in females and annual fecundity in males. In comparison, there was no additive genetic variance underlying maternally-derived (neonatal) anti-T. circumcincta antibody levels in neonates, but maternal and maternal genetic effects explained a considerable proportion of the variance in these traits. There was evidence for associations between neonatal anti-T. circumcincta IgG and later offspring phenotype and fitness, independent of total antibody (IgG) transferred. We found that neonatal anti-T. circumcincta IgG levels positively predicted survival to four months old, as well as weight in August. In addition, neonatal anti-T. circumcincta IgG levels were associated with reduced strongyle FEC in August, and were associated with improved survival over the first winter. In early life, maternally-derived anti-helminth antibodies are important for early growth, survival, and parasite resistance, as well as first winter survival, while fitness benefits in adulthood were associated with higher endogenous anti-helminth antibody levels. This thesis illustrates that maternal effects and genetic variation can have strong effects on variation in immunity in the wild, and this variation in turn can have health and fitness consequences for individuals.

In the United Kingdom 1 in 6 - 8000 children are born missing one or both of their ears. The surgical technique most commonly used to reconstruct ears requires surgeons to remove ribs from the patient, and the cartilage from the ribs is used to carve a new ear. This procedure involves many risks including significant pain, punctured lung and chest deformity. Therefore the ability to ‘grow’ an ear would be a major advancement. Stem cells show huge promise in tissue engineering and regenerative medicine. Approved stem cell technology must be evaluated with regards to safety, purity, identity, potency and efficacy prior to biologic licensing and clinical use. Therefore, access to ethically sourced tissue for research is fundamental to the successful delivery of novel therapies. Adipose tissue provides an abundant and accessible source of stem cells for clinical translation. Within the first section of this thesis, the perceptions and attitudes of patients towards the donation and use of adipose tissue for research are sought. Based on this information, a tissue bank with all appropriate ethical approval to collect, process, store and distribute adipose tissue and adipose derived stem cells is established. The second part of this thesis demonstrates the specific identity, location and frequency of stem cells within adipose tissue; revealing them to reside in a perivascular niche. Using this data, protocols to rapidly purify stem cells from adipose tissue using Fluorescence Activated Cell Sorting are developed. The frequency of cells, and both the patient and procedure based variables that can affect this yield are also examined. The final section of this thesis uses a high-throughput microarray platform to screen thousands of polymers to identify potential substrates that can support the attachment, stable proliferation and subsequent differentiation of stem cells purified from adipose tissue. From the initial screen, 5 distinct polymers have been identified, characterised and their effects on the stem cells examined and quantified. Combined together, these elements provide significant advances in our understanding, and the basis for on going research to deliver a tissue engineered ear for use in human ear reconstruction.

No presente estudo, desenvolveu-se um método de análise de especiação de As inorgânico em alimentos derivados de arroz, mediante a técnica de geração de hidretos (HG) associada à espectrometria de absorção atômica (AAS). As condições para a geração seletiva dos hidretos de As(III) e As inorgânico, e As total foram HCl 10 mol L-1 e NaBH4 0,1% (m/v), HCl 6 mol L-1 e NaBH4 0,5% (m/v), respectivamente. Vinte amostras de alimentos à base de arroz comercializadas no Rio Grande do Sul foram analisadas, incluindo bolacha, massa, leite em pó, farinha, mistura para bolo e arroz para sushi; também foram analisadas amostras líquidas tais como vinagre, cerveja, vinho e bebidas lácteas. Nessas amostras, o As total foi quantificado após extração alcalina com K2S2O8 2% (m/v) e HNO3 10% (v/v) sob aquecimento em banho-maria a 90 °C, seguido da determinação do As por HG-AAS. O limite de detecção do As total foi 6,8 ng g-1. As espécies de As nas amostras sólidas foram extraídas utilizando-se HNO3 0,28 mol L-1 e aquecimento em banho-maria a 95 °C, sendo que, nessas condições, não ocorre a interconversão das espécies. Para a quantificação do As inorgânico, foi necessária a pré redução do As, com o emprego de solução de KI/ácido ascórbico, em meio ácido. Os limites de detecção do As(III) e As inorgânico foram 5 ng g-1 e 11 ng g-1, respectivamente. As concentrações de As(III) e As inorgânico nas amostras analisadas variaram, respectivamente, de 9,5 ± 0,6 a 71,8 ± 6,1 ng g-1 e 23,7 ± 6,9 a 82,9 ± 4,7 ng g-1. A concentração de As(V) foi obtida por diferença. A concentração de As total variou de 20,3 a 124 ng g-1. As amostras líquidas, para determinação do As total e do As (III) presente, foram simplesmente diluídas com HNO3 0,28 mol L-1, uma vez que testes preliminares mostraram melhor exatidão mediante diluição das amostras, ao invés da extração com K2S2O8 2% (m/v) e HNO3 10% (v/v). Os limites de detecção do As total variaram de 0,34 a 1,70 μg L-1, enquanto que os do As(III) variaram de 0,50 a 2,50 μg L-1. Com o intuito de se avaliar a exatidão do método foram realizados testes de recuperação do analito. Foi também analisada farinha de arroz certificada, sendo as concentrações de As total e As inorgânico concordantes com os valores de referência. / The present study is about chemical speciation analysis of inorganic As (iAs) in rice products using hydride generation-atomic absorption spectrometry (HG-AAS). Selective generation of arsine of As(III) is achieved with 0.1% (m/v) NaBH4 and 10 mol L-1 HCl. Inorganic arsenic (iAs) is pre reduced and determined in the same conditions used for As(III). Samples of rice products such as flour, cookies, cracker, pasta, milk powder, cakes, rice for sushi, dairy beverage, vinegar, wine and beer were analyzed. Total arsenic (tAs) in solid samples was extracted with 2% (m/v) K2S2O8 and 10% (m/v) HNO3 and heating in water bath at 90 °C for 3 h, while arsenic species were extracted with 0.28 mol L-1 HNO3 and heating in water bath at 95 °C for 1.5 h. The limits of detection (LODs) of the method for As(III), iAs and tAs were 5 ng g-1, 11 ng g-1, and 6.8 ng g-1 respectively. Concentrations of As(III), iAs, and tAs in the solid samples ranged from 16.5 ± 7.2 to 71.8 ± 6.1 ng g-1, 26.7 ± 0.3 to 82.9 ± 4.7 ng g-1, and 35.8 ± 0.4 to 136 ± 2 ng g-1, respectively. Liquid samples were simply diluted in 0.28 mol L-1 HNO3. Arsenic (III) and tAs were determined in these samples. The LODs of As(III) and t(As) were in the range of 0.28 to 0.85 g L-1 and 0.5 to 2.5 g L-1, depending on the sample dilution. The concentration of As(III) in liquid samples was up to 11.4 ± 0.4 5 g L-1 while that of tAs ranged from 1.35 ± 0.12 to 24.6 ± 0.5 g L-1.

Tang Yin Nei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves xv-xxv). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.i / ABBREVIATIONS --- p.ii-iii / INDEX FOR FIGURES --- p.iv-v / INDEX FOR TABLES --- p.vi / ABSTRACT (Chinese and English) --- p.vii-xi / TABLE OF CONTENTS --- p.xii-xiv / Chapter CHAPTER I 226}0ؤ --- INTRODUCTION --- p.1 / Chapter 1.1 --- Background --- p.2 / Chapter 1.2 --- Tendon / Chapter 1.2.1 --- Structure and function --- p.3 / Chapter 1.2.2 --- Tendon fibroblast --- p.6 / Chapter 1.2.3 --- Components of the extracellular matrix --- p.7 / Chapter 1.2.3.1 --- Collagen --- p.8 / Chapter 1.2.3.2 --- Proteoglycan --- p.9 / Chapter 1.2.3.3 --- Non-collagenous structural glycoprotein --- p.10 / Chapter 1.3 --- Inflammation disorders of tendon / Chapter 1.3.1 --- Inflammation --- p.11 / Chapter 1.3.2 --- Treatment --- p.12 / Chapter 1.3.2.1 --- Glucocorticoid as an anti-inflammatory agent --- p.12 / Chapter 1.3.2.2 --- Dexamethasone --- p.14 / Chapter 1.3.3 --- Clinical occurrence of tendon rupture --- p.15 / Chapter 1.3.4 --- Animal research related to glucocorticoids and tendon rupture --- p.18 / Chapter 1.4 --- Platelet-derived growth factor isoform B (PDGFBB) / Chapter 1.4.1 --- Structure and function --- p.21 / Chapter 1.4.2 --- PDGFbb effects on connective tissue --- p.22 / Chapter CHAPTER II 226}0ؤ --- AIM OF THE STUDY --- p.23 / Chapter 2.1 --- Limitations of the past researches --- p.24 / Chapter 2.2 --- Hypothesis of this study --- p.25 / Chapter 2.3 --- Objectives --- p.26 / Chapter 2.4 --- Long term significance --- p.26 / Chapter CHAPTER III 226}0ؤ --- METHODOLOGY --- p.27 / Chapter 3.1 --- Chemicals and materials used / Chapter 3.1.1 --- Chemicals --- p.28 / Chapter 3.1.2 --- Materials --- p.28 / Chapter 3.2 --- Specimen collection and preparation / Chapter 3.2.1 --- Collection --- p.29 / Chapter 3.2.2 --- Preparation and isolation --- p.30 / Chapter 3.2.3 --- Cell culture --- p.31 / Chapter 3.3 --- Reagent preparation / Chapter 3.3.1 --- Charcoal-stripped serum --- p.32 / Chapter 3.3.2 --- Phenol-red free DMEM --- p.33 / Chapter 3.3.3 --- MTT --- p.33 / Chapter 3.3.4 --- Dexamethasone --- p.34 / Chapter 3.3.5 --- PDGFbb --- p.34 / Chapter 3.3.6 --- Trypan blue --- p.35 / Chapter 3.3.7 --- TCA/Tannic acid --- p.35 / Chapter 3.3.8 --- Collagenase buffer --- p.35 / Chapter 3.4 --- Morphology / Chapter 3.4.1 --- Inverted phase contrast light microscopy --- p.36 / Chapter 3.4.2 --- Scanning electron microscopy --- p.36 / Chapter 3.5 --- Biological assays / Chapter 3.5.1 --- "MTT (3-[4,5-Dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) assay" --- p.38 / Chapter 3.5.1.1 --- Correlation between MTT assay and trypan blue dye method --- p.38 / Chapter 3.5.1.2 --- Growth kinetics for tendon fibroblasts --- p.41 / Chapter 3.5.1.3 --- Cell viability --- p.43 / Chapter 3.5.2 --- Brdu (5-bromo-2'-deoxyuridine) assay --- p.44 / Chapter 3.5.3 --- Flow cytometry --- p.45 / Chapter 3.5.4 --- Apoptosis --- p.47 / Chapter 3.5.5 --- 3H-Proline incorporation assay --- p.48 / Chapter 3.5.6 --- 35Sulfate incorporation assay --- p.51 / Chapter 3.5.7 --- Immunocytochemistry (PDGF-β receptor) --- p.54 / Chapter 3.6 --- Statistical analysis / Chapter 3.6.1 --- Dose-response curve of dexamethasone on cell viability and proliferation --- p.55 / Chapter 3.6.2 --- Comparison among various treatments of fibroblasts --- p.55 / Chapter CHAPTER I´Vؤ --- RESULTS --- p.56 / Chapter 4.1 --- In vitro effect of dexamethasone on rat tendon fibroblasts / Chapter 4.1.1 --- Viable cell number between two sexes --- p.57 / Chapter 4.2 --- In vitro effect of dexamethasone and PDGFBB on human tendon fibroblasts / Chapter 4.2.1 --- Gross morphology --- p.58 / Chapter 4.2.2 --- Cell cycle --- p.60 / Chapter 4.2.3 --- Apoptosis --- p.61 / Chapter 4.2.4 --- Viable cell number / Chapter 4.2.4.1 --- Effect of dexamethasone --- p.62 / Chapter 4.2.4.2 --- Effect of PDGFBB --- p.63 / Chapter 4.2.5 --- Cell proliferation / Chapter 4.2.5.1 --- Effect of dexamethasone --- p.65 / Chapter 4.2.5.2 --- Effect of PDGFbb --- p.67 / Chapter 4.2.6 --- Collagen synthesis --- p.68 / Chapter 4.2.7 --- Proteoglycan synthesis --- p.72 / Chapter 4.2.8 --- PDGF-rβ expression --- p.74 / Chapter CHAPTER V 226}0ؤ --- DISCUSSION --- p.75 / Chapter 5.1 --- Dexamethasone and PDGFBB induced change of cell morphology --- p.77 / Chapter 5.2 --- Dexamethasone retarded cell growth of human tendon fibroblast --- p.80 / Chapter 5.3 --- Dexamethasone inhibited collagen synthesis --- p.82 / Chapter 5.4 --- Dexamethasone inhibited proteoglycan synthesis --- p.86 / Chapter 5.5 --- PDGFbb could counteract the inhibitory effects of dexamethasone --- p.88 / Chapter 5.6 --- Expression of PDGF-(3 receptor is regulated by dexamethasone and PDGFBB --- p.90 / Chapter 5.7 --- Limitations of this study / Chapter 5.7.1 --- Not enough sample to differentiate different between two sexes --- p.92 / Chapter 5.7.2 --- Small sample size and few assays --- p.92 / Chapter 5.7.3 --- Limitations of the cell culture model --- p.93 / Chapter 5.7.4 --- Difficult to further in vivo study on human --- p.93 / Chapter 5.8 --- Contributions of this study / Chapter 5.8.1 --- Improve the limitation of the past research --- p.94 / Chapter 5.8.1.1 --- Human tendon specimen --- p.94 / Chapter 5.8.1.2 --- In vitro system --- p.94 / Chapter 5.8.2 --- Understand the effect of dexmaethasone on human tendon fibroblasts --- p.95 / Chapter 5.8.3 --- Counteract the deleterious effects of dexamethasone by PDGFBB --- p.95 / Chapter CHAPTER VÍؤ --- CONCLUSION & FUTURE STUDY --- p.96 / Chapter 6.1 --- Conclusion --- p.97 / Chapter 6.2 --- Future study --- p.98 / Chapter 6.2.1 --- Study the balance between matrix synthesis and degradation --- p.98 / Chapter 6.2.2 --- Determine collagen typing --- p.99 / Chapter 6.2.3 --- Further explore the effect of glucocorticoid in organ culture model --- p.100 / Chapter 6.2.4 --- Investigate molecular mechanism of dexamethasone and PDGFBB --- p.100 / REFERENCES --- p.xv-xxv / APPENDIX --- p.xxvi

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / O pi à um nÃmero de natureza singular, pois muitos homens em diversos momentos histÃricos se detiveram a calculÃ-lo e estudÃ-lo. CÃrculos podem ser vistos em quase todos os lugares, e como consequÃncia, o pi tambÃm. Por estar tÃo presente na realidade, muitos foram os matematicos que se dedicaram ao estudo desse nÃmero e de seu valor numÃrico. Este trabalho, fruto de muita pesquisa, mostrarà muitos dos diversos caminhos que os matemÃticos fizeram para encontrarem uma aproximaÃÃo para pi . Trataremos tambÃm neste trabalho as curiosas descobertas envolvendo este nÃmero, os famosos problemas em torno dele, assim como tambÃm os diversos mÃtodos que foram usados para calculÃ-lo. A busca pelo valor numÃrico de pi levou os matemÃticos a suporem sua irracionalidade, que posteriormente fora provada e tambÃm serà feita aqui. Finalizaremos tratando de como podemos calcular de uma maneira diferenciadana sala de aula. / pi is a number of singular nature because several men in different historical moments lingered themselves to calculate and study it. Circles can be seen in almost all places, and as a consequence, so can pi. Due to being so present in the reality, a huge number of mathematicians devoted themselves to the study of this number and its numerical value. This work, result of much research, will show many of the different ways that the mathematicians took to find an approximation for pi. We will also approach in this work the curious founds involving this number, the famous problems around it as well as the diverse methods which were used to calculate it. The search for the numerical value took the mathematicians to assume its irrationality which was proved afterwards and will be done here. We will finish approaching how we can calculate pi in the classroom in a different way.

Nas redes de distribuição de energia, as cruzetas sofrem uma exposição direta a intempéries, particularmente à ação da umidade, o que acelera seu processo de degradação. A higroscopicidade da madeira facilita a absorção de água, com significativa perda de suas características mecânicas e elétricas. Para minorar os problemas relacionados a absorção de umidade foi desenvolvido um novo processo de revestimento e impregnação da madeira e, neste trabalho, é apresentada uma investigação das características mecânicas e elétricas desse novo compósito. Para tanto, ensaios mecânicos de flexão estática foram executados em três materiais: na madeiras em estado natural (sem tratamento), na madeira resinada e no novo compósito; e, com intuito de estudar as características elétricas, ensaios de resistividade superficial e volumétrica foram realizados em amostras secas e molhadas. Também fez parte deste estudo o desenvolvimento de uma prensa mecânica para duas toneladas, cujo programa computacional pode ser facilmente adaptado para outras cargas. / In the electrical distribution system, the crossarms suffer direct exposure to weather conditions especially humidity, which can accelerates the degradation process. The hygroscopicity of wood facilitates the absorption of water, with significant loss of its mechanical and electrical characteristics. To reduce the problems listed above; we develop a new process of coating and impregnation of wood. Thus, we present in this work several studies on mechanical and electrical properties of these new composite. With this in mind, bending mechanical tests were performed in three materials: wood in natural state (no treatment), resin-impregnated wood also on the new composite. In addition, to study the electrical characteristics, tests of surface and volume resistivity were carried out in dry and wet samples. Furthermore, a two-ton mechanical press (whose computer program can be easily adapted for other tonnages) development was part of this work.

Apresentamos neste trabalho uma definição de complexidade na categoria derivada de complexos (limitados superiormente) de módulos sobre uma k-álgebra de dimensão finita. Um dos resultados que conseguimos foi uma relação entre a complexidade de objetos indecomponíveis e a noção de dimensão global forte. Mais especificamente, mostramos que a existência de um objeto indecomponível na categoria derivada limitada superiormente com complexidade não nula é condição suficiente para que a respectiva álgebra tenha dimensão global forte infinita. Também investigamos se existe uma relação entre as dimensões global e global forte da classe das álgebras shod (Coelho e Lanzilotta, 2009). Fomos motivados pela caracterização da classe das álgebras quase inclinadas (Happel, Reiten e Smalo, 1996) em termos da sua dimensão global forte, dada por D. Happel e D. Zacharia (2008), e pelo fato das álgebras shod serem uma generalização das álgebras quase inclinadas. Nossa conclusão foi que não existe, em geral, uma caracterização das álgebras shod em termos de sua dimensão global forte. Isto é, mostramos que para cada inteiro d > 2 existe uma álgebra shod estrita cuja dimensão global forte é igual a d. / We introduce in this thesis a definition of complexity in the derived category of bounded above complexes of modules over a finite dimensional k-algebra. One of our result shows a relationship between the complexity of indecomposable objects and the notion of strong global dimension. More specifically, we prove that the existence of an indecomposable object in the category derived bounded above whose complexity is not zero is a sufficient condition for corresponding algebra being of infinite strong global dimension. We also investigate the existence of a relationship between the global dimension and the strong global dimension of shod algebras (Coelho and Lanzilotta, 1999). Our motivation came from characterization of quasitilted algebras (Happel, Reiten and Smalo, 1996) by its strong global dimension, given by D. Happel and D. Zacharia (2008), and from the fact that shod algebras are a generalization of quasitilted algebras. Our conclusion was that there is not in general a characterization of shod algebras in terms of its strong global dimension. This conclusion comes from the fact that we showed that for each integer d > 2 there exists a strictly shod algebra whose strong global dimension is d.

In [Bri07], Bridgeland introduced the notion of stability conditions on the bounded derived category D(X) of coherent sheaves on an algebraic variety X. This topic is originally inspired by concepts in string theory and mathematical physics and has many interesting applications in algebraic geometry. In the first part of the thesis, we provide a direct proof of an important result in [Bri08, BMS16] which states there is a two dimensional family of weak Bridgeland stability conditions on the bounded derived category D(X) of coherent sheaves on a variety X. As a first application of this result, we prove an effective restriction theorem which provides sufficient conditions on a stable locally free sheaf on a projective variety such that its restriction to a hypersurface remains stable. Secondly, we extend and complete Mukai's program to reconstruct a K3 surface from a curve on that surface. We show that the K3 surface containing the curve can be obtained uniquely as a Fourier-Mukai partner of a suitable Brill-Noether locus of vector bundles on the curve.

The difference between the number of patients needing transplantation for chronic liver disease and the number of organ donors is growing, drawing attention to the urgent requirement for novel therapies. Chronic liver injury is commonly caused by viral hepatitis, alcohol consumption, obesity and metabolic disorders. Prolonged liver injury leads to fibrosis, hepatic scarring and eventually cirrhosis. This project is based on previous studies demonstrating the therapeutic effects of bone marrow-derived macrophages (BMDM) in a murine model of liver fibrosis. BMDM facilitated fibrosis regression and improved liver regeneration. Pro-resolution macrophages exhibited increased expression of MMPs, growth factors and phagocytosis-related genes. However, macrophages derived from bone marrow are inherently heterogeneous and difficult to genetically manipulate. To overcome this limitation, our laboratory has established a protocol whereby pure populations of macrophages can be produced in significant numbers from murine embryonic stem cells (ESC) in vitro, providing an essentially limitless source of macrophages. The first goal of this project was to compare macrophages derived from ESCs (ESDM) with classical BMDM. ESDM displayed characteristic macrophage morphology, could be activated and responded to different cytokines in vitro, and were functionally phagocytic. However, they displayed some differences in their gene expression profile, and were found to be less phagocytic than BMDM. We then assessed whether ESDM could be used in the treatment of a murine model of hepatic injury induced by carbon tetrachloride administration. ESDM therapy helped in the regression of liver fibrosis, down-regulated the number of fibrogenic myofibroblasts, and activated liver progenitor cells. However, a higher number of ESDM compared to BMDMs were required to exert that effect. To assess whether ESDM may be similar to yolk sac derived tissue-resident macrophages, rather than monocyte-derived, we compared their behaviour in a Kupffer cell repopulation assay. Macrophages were depleted using liposomal clodronate treatment then animals were transplanted with either ESDM or BMDM. We demonstrated that ESDM were more efficient than BMDM at repopulating the Kupffer cell compartment and reversing the effects of liposomal clodronate treatment in mice. It is well known that macrophages are very difficult to genetically modify. So our strategy was to genetically modify ESC and then differentiate them to macrophages that carry the modification. By genetically modifying ESCs, we attempted to produce pro-fibrolytic ESDM that over-express MMP12 which is a member of the matrix metalloproteinase family of genes that mainly degrades elastin, an extracellular matrix component. We initially employed a Tet-On 3G expression system to create an ESC line where MMP12 could be expressed in an inducible manner in differentiated macrophages. However, although this inducible strategy functioned in undifferentiated ESCs we could not induce the expression of MMP12 in differentiated macrophages. In an attempt to overcome possible gene-silencing issues, we designed and constructed an expression strategy such that Mmp12 was expressed specifically in macrophages. The ESC line was built such that Mmp12 expression would be driven by the promoter of macrophage colony stimulating factor-1 receptor gene (Csf-1r or c-fms). Using the CRISPR/Cas9 strategy, we successfully targeted the Mmp12 cDNA to the Csf-1r locus but ESDM that were differentiated from targeted ESC lines did not express Mmp12. Thus, despite having adopted two independent strategies, we have failed to generate genetically modified macrophages. As a first step to translate the therapeutic effects of macrophages into the clinical setting, we optimized a feeder- and serum-free protocol to efficiently generate macrophages from human induced pluripotent stem cells.

The purpose was to analyze the role of dopamine D1 and D2 receptors in conditioned place preference (CPP) of a relatively high dose (5 mg/kg) of methylphenidate (MPH) in adolescent male and female rats, as well as the role of these receptors in the effects of MPH on brain-derived neurotrophic factor (BDNF). The primary mechanism of MPH in the brain is the blockade of the dopamine transporter, yielding an increase of dopamine in the synaptic cleft and is the basis for the rewarding properties of MPH. An initial preference given on postnatal day (P)32 yielded no preference for any context in a three-chambered shuttle box with removable dividers, thus, a biased procedure was used. Conditioning began the day after the initial preference test on P33. On conditioning trials, animals were first administered saline or their respective antagonist (D1 antagonist: 0.1 or 0.2 mg/kg SCH-23390; D2 antagonist: 0.01 or 0.03 mg/kg Eticlopride HCl), followed by methylphenidate (MPH; 5mg/kg). Approximately 10 min after MPH administration, rats were placed into the paired context for a 10 min trial. The choice of the paired context was balanced across animals. In a separate session, all animals received saline in the opposing context. One day post-conditioning on P38, a preference test was administered with dividers removed. Preference was determined through the amount of time spent in the paired context as compared to time spent in the unpaired context on the post-conditioning preference test. One day following the preference test on P39, brain tissue was removed, and nucleus accumbens and striatum analyzed for BDNF. Results showed that MPH produced an increased preference on the post-conditioning preference test that was blocked by either dose of SCH-23390, but was not affected by either dose of eticlopride. Additionally, the higher dose of SCH-23390 resulted in a conditioned place aversion in males, which may be due to the increased presence of dopamine D1 receptors in adolescent males. MPH produced a robust significant increase in BDNF in both nucleus accumbens and striatum, and this increase was alleviated by SCH-23390, but the effect on BDNF is still to be analyzed relative to D2 antagonism. These results show that MPH results in a conditioned place preference in adolescent male and female rats, and these effects appear to be mediated by the dopamine D1 receptor. Further, MPH results in a significant increase of BDNF in drug reward areas of the brain, which has implications towards synaptic plasticity in these regions in response to MPH.