Esta tese é estruturada em dois capítulos. O primeiro capítulo explora os retrotransposons com LTR (LTR-RT) em cana-de-açúcar e grande parte de seus resultados foram publicados no artigo \'\'Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns\'\'. Nossos resultados mostraram que as diferentes famílias de LTR-RT em cana-de-açúcar possuem estruturas e regulação distintas. O segundo capítulo desta tese visa responder a perguntas que surgiram durante a primeira metade deste trabalho, mas ao invés de focar no genoma de uma planta optamos por trabalhar com linhagem Del de LTR-RT em dez genomas de angiospermas sequenciados. Os resultados desta parte do trabalho foram submetidos para publicação no artigo intitulado \'\'Virus-like attachment sites and plastic CpG islands: landmarks of diversity in plant Del retrotransposons\'\'. Os resultados mostraram que a LTR é uma região dinâmica e importante para a evolução dos LTR-RTs. Nós especulamos que mudanças nas LTR atuem como gatilhos para a diversificação dos LTR-RTs. / This doctoral thesis is structured in two chapters. In the first chapter we explore the LTRretrotransposons (LTR-RT) in sugarcane, these results were published in an article entitled \'\'Analysis of plant LTR-etrotransposons at the fine-scale family level reveals individual molecular patterns\'\'. In this paper we show that different sugarcane LTR-RT families have distinct structure and are differentially regulated. In the second chapter we try to find answers to questions that came up in the first half of this work, but instead of focusing in one plant genome we chose to work with the Del lineage of LTR-RT in tem angiosperm sequenced genomes. These results are submitted to publication as an article entitled \'\'Virus-like attachment sites and plastic CpG islands: landmarks of diversity in plant Del retrotransposons\'\'. Our results indicate that the LTR region is dynamic and important in the evolution of LTR-retrotransposons, we speculate that it is a trigger for retrotransposon diversification.

The association between extent of chronic cannabis use (CCU-extent) and cognitive impairment among adolescents has been the subject of controversial debate. Linking DNA methylation to CCU-extent could help to understand cannabis associated changes in cognitive performance. We analyzed cognitive task performances, CpG methylation in peripheral whole-blood samples and self-reported past-year CCU-extent of n = 18 adolescents (n = 9 psychiatric outpatients with chronic cannabis use (CCU), n = 9 without) who were matched for age, gender and psychiatric disorders. Patients with CCU were at least 24 h abstinent when cognitive tasks were performed. A Principal Component Analysis (PCA) was carried out to identify group differences in whole genome DNA methylation. Mediation analyses were performed between CCU-extent associated CpG sites and CCU-extent associated variables of cognitive tasks. PCA results indicated large differences in whole genome DNA methylation levels between the groups that did not reach statistical significance. Six CpG sites revealed reduced methylation associated with CCU-extent. Furthermore, CCU-extent was associated with lower scores in verbal learning. All six CpG sites mediated the effects between CCU-extent and verbal learning free recall. Our results indicate that CCU is associated with certain patterns in the methylome. Furthermore, CCU-extent associated impairments in memory function are mediated via differential methylation of the six CCU-associated CpG sits. Six identified CpG are located in genes previously described in the context of neurodegeneration, hippocampus-dependent learning and neurogenesis. However, these results have to be carefully interpreted due to a small sample size. Replication studies are warranted.

Doctor of Philosophy / Department of Anatomy and Physiology / Elizabeth G. Davis / Activation of innate immunity through Toll-like receptor (TLR) signaling can also enhance antigen-specific adaptive immunity. TLR9 is an endosomal receptor for unmethylated bacterial and viral cytosine-phosphate-guanine DNA (CpG-DNA). West Nile virus (WNV) infection may result in meningitis and encephalitis in humans and horses, especially aged and immunocompromised individuals. Using flow cytometric analyses and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we investigated equine cell-mediated immunity (CMI) to an inactivated West Nile virus vaccine in healthy yearling and adult horses. We also studied the potential of enhancing equine adaptive immunity to viruses and other pathogens by activation of innate immunity though TLR9 signaling pathway. We found vaccination with inactivated WNV vaccine induced strong WNV-specific T helper type 1 (Th1) and Th2 CMI with a Th1 bias, also effectively induced WNV-specific CTLs in yearling horses. In adult horses, the pre-existing Th1 CMI bias against WNV was enhanced following booster vaccination with inactivated WNV vaccine. Molecular characterization and flow cytometric analysis of TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in neutrophils (PMNs), CD4[superscript]+ and CD8[superscript]+ T cells and other leukocytes. Conservation of equine TLR9 and a high expression profile among leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity. Unmethylated CpG-DNA can significantly activate equine PMNs. It also induces expression of interferon (IFN)-[Alpha], IFN-[Beta], IFN-[Gamma], and interleukin (IL)-12p35 in PBMCs, as well as IFN-[alpha] and IFN-[gamma] in monocyte-derived DCs. Enhanced expression of IFNs in immune cells by CpG-DNA is not only crucial for host viral clearance, but also important in mediating host immune responses due to IFNs' anti-inflammatory effects. Compared to the relatively weaker activation of equine innate immunity by inactivated WNV, the tested CpG-DNA species showed potential as vaccine adjuvants for enhancement of CTLs and Th1 CMI against intracellular pathogens, characterized by significant induction of type I IFNs and Th1-specific cytokines such as IL-12p35 and IFN-γ. These data provide a basis for further investigation of these CpG-DNA species as potentially effective vaccine adjuvants in horses.

A principle challenge of modern biology is to understand how the human genome is organised and regulated within a nucleus. The field of chromatin biology has made significant progress in characterising how protein and DNA modifications reflect transcription and replication state. Recently our lab has shown that the human genome is organised into large domains of altered DNA helical twist, called DNA supercoiling domains, similar to the regulatory domains observed in prokaryotes. In my PhD I have analysed how the maintenance and distribution of DNA supercoiling relates to biological function in human cells. DNA supercoiling domains are set up and maintained by the balanced activity of RNA transcription and topoisomerase enzymes. RNA polymerase twists the DNA, over-winding in front of the polymerase and under-winding behind. In contrast topoisomerases relieve supercoiling from the genome by introducing transient nicks (topoisomerase I) or double strand breaks (topoisomerase II) into the double helix. Topoisomerase activity is critical for cell viability, but the distribution of topoisomerase I, IIα and IIβ in the human genome is not known. Using a chromatin immunoprecipitation (ChIP) approach I have shown that topoisomerases are enriched in large chromosomal domains, with distinct topoisomerase I and topoisomerase II domains. Topoisomerase I is correlated with RNA polymerase II, genes and underwound DNA, whereas topoisomerase IIα and IIβ are associated with each other and over-wound DNA. This indicates that different topoisomerase proteins operate in distinct regions of the genome and can be independently regulated depending on the genomic environment. Transcriptional regulation by DNA supercoiling is believed to occur through changes in gene promoter structure. To investigate DNA supercoiling my lab has developed biotinylated trimethylpsoralen (bTMP) as a DNA structure probe, which preferentially intercalates into under-wound DNA. Using bTMP in conjunction with microarrays my lab identified a transcription and topoisomerase dependent peak of under-wound DNA in a meta-analysis of several hundred genes (Naughton et al. (2013)). In a similar analysis, Kouzine et al. (2013) identified an under-wound promoter structure and proposed a model of topoisomerase distribution for the regulation of promoter DNA supercoiling. To better understand the role of supercoiling and topoisomerases at gene promoters, a much larger-scale analysis of these factors was required. I have analysed the distribution of bTMP at promoters genome wide, confirming a transcription and expression dependent distribution of DNA supercoils. DNA supercoiling is distinct at CpG island and non-CpG island promoters, and I present a model in which over-wound DNA limits transcription from both CpG island promoters and repressed genes. In addition, I have mapped by ChIP topoisomerase I and IIβ at gene promoters on chromosome 11 and identified a different distribution to that proposed by Kouzine et al. (2013), with topoisomerase I maintaining DNA supercoiling at highly expressed genes. This study provides the first comprehensive analysis of DNA supercoiling at promoters and identifies the relationship between supercoiling, topoisomerase distribution and gene expression. In addition to regulating transcription, DNA supercoiling and topoisomerases are important for genome stability. Several studies have suggested a link between DNA supercoiling and instability at common fragile sites (CFSs), which are normal structures in the genome that frequently break under replication stress and cancer. bTMP was used to measure DNA supercoiling across FRA3B and FRA16D CFSs, identifying a transition to a more over-wound DNA structure under conditions that induce chromosome fragility at these regions. Furthermore, topoisomerase I, IIα and IIβ showed a pronounced depletion in the vicinity of the FRA3B and FRA16D CFSs. This provides the first experimental evidence of a role for DNA supercoiling in fragile site formation.

Mammalian genomes are characterised by unique regions of non-methylated DNA known as CpG islands (CGIs). These genomic elements are characterised by a high density of CpGs and an elevated GC content compared to the surrounding, bulk of the genome. CGIs are prevalently associated with the 5’ end of genes and represent key nucleation sites where specific transcription factors and chromatin modifiers are recruited to impact on gene function. This thesis is focused at understanding the biochemical properties of the recently discovered H3K36-specific histone demethylase, KDM2A. This enzyme is specifically recruited to CGIs but how it interfaces with local chromatin in vivo remains unknown. Using defined chromatin templates in vitro, this study demonstrates that KDM2A binding to DNA relies on a zinc finger CXXC domain that preferentially recognizes non-methylated CpGs. In particular, nucleosomes represent a major barrier to KDM2A binding and chromatin substrates are interpreted by the CXXC domain through specific interaction with CpGs within linker DNAs. Moreover, the adjacent PHD domain does not contribute to KDM2A binding to chromatin. Together these observations suggest that sequence, methylation status and accessibility of DNA define how CGI chromatin is interpreted by CXXC domain proteins. In particular, the precise targeting of KDM2A to CGIs contributes to the creation of a unique chromatin architecture that highlights gene regulatory regions within large and complex mammalian genomes.

Cheung Kin Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 87-105). / Abstracts in English and Chinese. / Abstract (English version) --- p.i / Abstract (Chinese version) --- p.iii / Acknowledgments --- p.vi / Table of Contents --- p.v / List of Tables --- p.viii / List of Figures --- p.iv / List of Abbreviations --- p.xi / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.3 / Chapter 2.1 --- Multiple myeloma --- p.3 / Chapter 2.2 --- Epidemiology of MM --- p.3 / Chapter 2.3 --- Risk factors --- p.4 / Chapter 2.4 --- Pathophysiology of MM --- p.5 / Chapter 2.5 --- Clinical presentations and diagnosis --- p.6 / Chapter 2.5.1 --- Diagnosis --- p.6 / Chapter 2.5.1.1 --- Laboratory testing of blood and urine --- p.6 / Chapter 2.5.1.2 --- Radiographic evaluations --- p.1 / Chapter 2.5.1.3 --- Bone marrow biopsy --- p.7 / Chapter 2.6 --- Staging and classification --- p.9 / Chapter 2.6.1 --- Staging --- p.9 / Chapter 2.6.2 --- Classification --- p.11 / Chapter 2.6.2.1 --- Monoclonal gammopathy of undetermined significance --- p.11 / Chapter 2.6.2.2 --- Asymptomatic MM --- p.12 / Chapter 2.6.2.3 --- Smouldering MM --- p.12 / Chapter 2.6.2.4 --- Indolent MM --- p.12 / Chapter 2.6.2.5 --- Symptomatic MM --- p.12 / Chapter 2.7 --- Treatment --- p.14 / Chapter 2.8 --- Epigenetics: DNA methylation --- p.15 / Chapter 2.9 --- Fundamental aspects of DNA methylation --- p.16 / Chapter 2.9.1 --- CpG islands --- p.16 / Chapter 2.9.2 --- Roles of DNA methylation --- p.16 / Chapter 2.9.3 --- Proposed mechanisms of transcriptional repression mediated by methylation --- p.18 / Chapter 2.10 --- Possible mechanisms to initiate aberrant DNA methylation --- p.21 / Chapter 2.11 --- DNA methylation in tumorigenesis --- p.22 / Chapter 2.11.1 --- Oncogenic point C → T mutation --- p.22 / Chapter 2.11.2 --- Global DNA hypomethylation --- p.23 / Chapter 2.11.3 --- Regional DNA hypermethylation --- p.23 / Chapter 2.12 --- Aberrant DNA methylation in MM --- p.25 / Chapter 2.12.1 --- Self-sufficiency in growth signals --- p.25 / Chapter 2.12.2 --- Evading apoptosis --- p.26 / Chapter 2.12.3 --- Insensitivity to antigrowth signals --- p.26 / Chapter 2.12.4 --- Tissue invasion and metastasis --- p.27 / Chapter 2.12.5 --- Infinite replicative potential --- p.28 / Chapter 2.12.6 --- Genome instability --- p.30 / Chapter 2.13 --- Methodologies of DNA methylation analysis --- p.32 / Chapter 2.13.1 --- Genome wide screening method: MS.AP-PCR --- p.32 / Chapter 2.13.2 --- Combined bisulfite restriction analysis --- p.34 / Chapter 2.13.3 --- Cloned bisulfite genomic sequencing --- p.36 / Chapter 2.13.4 --- Treatment with demethylating agent --- p.36 / Chapter CHAPTER 3 --- MATERIALS AND METHODS --- p.38 / Chapter 3.1 --- MM specimens --- p.38 / Chapter 3.1.1 --- MM samples --- p.38 / Chapter 3.1.2 --- MM cell lines --- p.38 / Chapter 3.2 --- Magnetic cell sorting of CD138-positive plasma cells --- p.39 / Chapter 3.3 --- Isolation of nuclear pellet from PB --- p.41 / Chapter 3.4 --- "DNA extraction from MM cell lines, MM plasma cells and PB" --- p.41 / Chapter 3.5 --- MS.AP-PCR --- p.42 / Chapter 3.5.1 --- Restriction enzyme digestion of genomic DNA --- p.42 / Chapter 3.5.2 --- Arbitrarily primed polymerase chain reaction --- p.42 / Chapter 3.5.3 --- Isolation of differentially methylated DNA fragments --- p.43 / Chapter 3.6 --- Cloning of differentially methylated DNA fragments --- p.46 / Chapter 3.6.1 --- TA cloning --- p.46 / Chapter 3.6.2 --- Heat shock transformation --- p.46 / Chapter 3.6.3 --- Screening of positive clones by PCR --- p.46 / Chapter 3.6.4 --- Alkaline lysis for plasmid DNA preparation --- p.47 / Chapter 3.7 --- MS.AP-PCR sequence analysis --- p.47 / Chapter 3.7.1 --- Nucleotide sequencing --- p.47 / Chapter 3.7.2 --- CpG islands analysis of differentially methylated sequences --- p.48 / Chapter 3.8 --- DNA methylation analysis --- p.48 / Chapter 3.8.1 --- Sodium bisulfite modification --- p.48 / Chapter 3.8.2 --- Combined bisulfite restriction analysis --- p.49 / Chapter 3.8.3 --- Cloned bisulfite genomic sequencing --- p.49 / Chapter 3.9 --- Gene expression analysis --- p.50 / Chapter 3.9.1 --- RNA extraction --- p.50 / Chapter 3.9.2 --- Reverse transcription PCR --- p.50 / Chapter 3.9.3 --- 5'-aza-2'-deoxycytidine treatment --- p.51 / Chapter CHAPTER 4 --- RESULTS --- p.53 / Chapter 4.1 --- Generation of DNA methylation patterns by MS.AP-PCR --- p.53 / Chapter 4.1.1. --- Global methylation content in MM samples and normal PB lymphocytes --- p.56 / Chapter 4.1.2. --- Differential methylation in MM --- p.56 / Chapter 4.2 --- UCSC BLAT analysis of differentially methylated DNA fragments --- p.60 / Chapter 4.3 --- Identification of two candidate genes with downregulated expression --- p.60 / Chapter 4.4 --- Zinc fingers and homeoboxes 2 (ZHX2) --- p.62 / Chapter 4.4.1 --- ZHX2 CpG islands BLAT search analysis --- p.62 / Chapter 4.4.2 --- Hypermethylation of ZHX2 in MM cell lines --- p.63 / Chapter 4.4.3 --- Downregulated expression of ZHX2 in methylated MM cell lines --- p.66 / Chapter 4.4.4 --- Restoration of ZHX2 expression by 5-Aza-dC treatment --- p.67 / Chapter 4.4.5 --- Unmethylation of ZHX2 in primary MM tumors --- p.68 / Chapter 4.5 --- Ring finger protein 180 (RNF180) --- p.69 / Chapter 4.5.1 --- RNF180 CpG islands BLAT search analysis --- p.69 / Chapter 4.5.2 --- Hypermethylation of RNF180 in MM cell lines --- p.70 / Chapter 4.5.3 --- Downregulated expression of RNF180 in methylated MM cell lines --- p.73 / Chapter 4.5.4 --- Restoration of RNF180 expression by 5-Aza-dC treatment --- p.74 / Chapter 4.5.5 --- Methylation of RNF180 in primary MM tumors --- p.75 / Chapter CHAPTER 5 --- DISCUSSION --- p.76 / Chapter 5.1 --- Importance of methylation in MM --- p.76 / Chapter 5.2 --- Genome-wide screening approach by MS.AP-PCR --- p.76 / Chapter 5.3 --- Sample selection in MS.AP-PCR --- p.78 / Chapter 5.4 --- Methylation patterns in MM --- p.79 / Chapter 5.5 --- Candidate genes selection strategies --- p.81 / Chapter 5.6 --- Zinc fingers and homeoboxes 2 --- p.81 / Chapter 5.7 --- Ring finger protein 180 --- p.83 / Chapter 5.8 --- Limitations --- p.84 / Chapter CHAPTER 6 --- CONCLUSION --- p.86 / REFERENCES --- p.87

La compréhension de la chimie prébiotique et la recherche de matière organique d’origine extraterrestre qui lui est associée sont parmi les thématiques fortes de la branche de l’astrobiologie qui concerne notamment la recherche de traces de vie dans notre Système Solaire. C’est avec cet objectif que nous nous sommes intéressés à deux objets du Système Solaire : le satellite saturnien Titan, pour ses aérosols organiques, et Mars, pour son habitabilité avérée et la recherche de matière organique in situ. A ces fins, nous avons mis en œuvre deux techniques permettant d’étudier la matière organique de ces objets : la simulation expérimentale qui permet de reproduire en laboratoire des conditions environnementale extraterrestres et la préparation d’analyse in situ grâce à des laboratoires entièrement automatisés implémentés sur des véhicules mobiles, à savoir les rovers Curiosity/MSL actuellement en activité à la surface de Mars et le futur rover Pasteur de la mission ExoMars. La première partie de cette étude est ainsi consacrée à la simulation expérimentale appliquée à l’étude des aérosols organiques de Titan. Nous avons synthétisé en laboratoire des analogues (tholins) de ces aérosols, puis nous avons étudié leur composition moléculaire par pyrolyse et chromatographie en phase gazeuse couplée à la spectrométrie de masse (Pyr-GC-MS). Nous avons tout particulièrement investigué l’influence de la composition du mélange gazeux permettant la synthèse de nos tholins (taux de méthane dans du diazote) sur leur composition moléculaire. Une étude systématique par Pyr-GC-MS nous a permis d’estimer les conditions optimales d’analyse dont la température de pyrolyse. Dans un second temps, nous nous sommes intéressés à l’analyse in situ du sol martien par les instruments SAM-GC-MS et MOMA-GC-MS à bord du rover Curiosity et du futur rover Pasteur respectivement. Ces deux instruments ont la possibilité de mettre en œuvre des techniques de prétraitements des échantillons par chimie humide (dérivatisation) pour faciliter l’extraction, la volatilisation, la préservation et l’identification de la matière organique présente dans le sol martien. Parmi ces techniques, nous avons optimisé les conditions analytiques de la thermochimiolyse en présence de TMAH sur un sol analogue du sol martien pour assurer le succès des futures analyses in situ du sol de Mars par les instruments SAM et MOMA. / The understanding of prebiotic chemistry, and the search for organic matter of extraterrestrial origin associated with it, are among the strong themes of the astrobiology branch, which concerns in particular the search for traces of life in our Solar System. It is with this objective in mind that we have taken an interest in two objects of the Solar System: the Saturn satellite Titan, for its organic aerosols, and Mars, for its proven habitability and research of organic matter in situ. To this end, we have implemented two techniques to study the organic matter of these objects: experimental simulation, which allows the reproduction of extraterrestrial environmental conditions in the laboratory, and preparation of in situ analysis thanks to fully automated laboratories implemented on the Curiosity/MSL rover currently in operation on the surface of Mars, and the future Pasteur rover of the ExoMars mission. The first part of this study is thus devoted to the experimental simulation applied to the study of organic aerosols from Titan. We synthesized analogs (tholins) of these aerosols in the laboratory, then studied their molecular composition by pyrolysis and gas chromatography coupled to mass spectrometry (Pyr-GC-MS). We investigated in particular the influence of the composition of the gas mixture allowing the synthesis of our tholins (methane content in nitrogen) on their molecular composition. A systematic study by Pyr-GC-MS allowed us to estimate the optimal conditions of analysis including pyrolysis temperature. In a second phase, we were interested in the in situ analysis of the Martian soil by the SAM-GC-MS and MOMA-GC-MS instruments aboard the Curiosity rover and the future Pasteur rover respectively. These two instruments have the possibility of using wet chemistry (derivatization) techniques to pre-treat samples to facilitate extraction, volatilization, preservation and identification of the organic matter present in the Martian soil. Among these techniques, we have optimized the analytical conditions of thermochemolysis in the presence of TMAH on a terrestrial analog of the Martian soil to ensure the success of future in situ analyses of the Mars soil by the SAM and MOMA instruments.

Dong Shumin. / "February 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 187-220). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Cancer is a group of diseases caused by aberrant continuously proliferating cells capable of metastasis. Despite significant advances in preventive, diagnostic and treatment measures, cancer is one of the major causes of death in the United States, second only to heart diseases. Main treatment approaches are surgery, radiotherapy, chemotherapy, and the recently expanding immunotherapeutic approaches. The main challenge in treating cancer is the ability of cancer cells to mutate and develop resistance to drug treatments therefore lowering the efficacy of chemotherapy in preventing metastatic tumors. Cancer vaccines are a treatment modality that employs the potential of the immune system to recognize and eliminate tumor cells by unmasking tumor cell antigens and generating an effective anti-tumor immune response with an immune memory capable of preventing metastases formation. This dissertation describes and evaluates an innovative cell-particle hybrid cancer vaccine construct involving irradiated tumor cell surface-engineered with polymeric particles using streptavidin-biotin cross-linking. The tumor cells were biotinylated indirectly using biotin-linked antibodies targeting a surface integrin and the particles were loaded with an immune adjuvant and coated with streptavidin. The tumor cells served as the source of tumor antigens and the anchored particles served to confine loaded immune adjuvant to the tumor cells. The vaccine construct was designed to co-deliver tumor antigens and the immune adjuvant to the same antigen presenting cell, a criteria that has been suggested recently to be important for optimal cancer vaccine potency. The first report on this cell-particle construct was published in my master’s thesis defended in May 2013. In that report, the feasibility of assembling the cell-particle hybrid was demonstrated. However, loading of the immune adjuvant, CpG ODN (cytosine phosphate guanine oligonucleotide), into streptavidin-coated particles was not optimal. In the current studies, this problem was addressed and the cancer vaccine potential of the cell-particle construct was assessed. We first evaluated a new TLR4 (toll like receptor 4) agonist, PET lipid A (pentaeryhtritol lipid A), for its potential use in cancer vaccines with the intention to incorporate it in the cell-particle hybrid. PET lipid A is a fully synthetic lipid A analog that has been demonstrated to have immunostimulatory properties. We evaluated the potential use of PET lipid A in cancer vaccine applications and the effect of particulate formulations on its adjuvant properties. Results showed improved in vitro immunostimulatory properties for particle based formulations. Upon testing the immunostimulatory properties of PET lipid A in vivo, moderate enhancement in antigen specific cytotoxic T cells stimulation was observed when PET lipid A was delivered in particles, which then translated into a corresponding trend toward increased survival in a prophylactic tumor study. PET lipid A was concluded to be a weak potential cancer vaccine adjuvant and was not chosen as the immune adjuvant to use in the cell-particle hybrid assembly. Instead, CpG ODN (TLR9 agonist) was chosen due to its strong record of efficacy as a cancer vaccine adjuvant. The second part of this research project aimed at addressing the challenges we encountered previously in achieving acceptable CpG ODN loading of the final streptavidin-coated PLGA (Polylactic-co-glycolic acid) particles. The approach taken was to modify the method used earlier to make the particles in order to circumvent CpG ODN loss. In the modified method the number of steps required to make streptavidin-coated CpG ODN-loaded PLGA particles was reduced and the fabrication media was altered to allow simultaneous particle fabrication and activation of surface carboxyl groups. The modified method resulted in 5-fold higher loading in the final streptavidin-coated particles compared to the original method. Subsequent to establishing the feasibility of constructing the cell-particle hybrid and characterizing the assembled hybrid in vitro, the in vivo cancer vaccine potential of the designed construct was examined. Two independent murine tumor models were chosen for this purpose, namely prostate cancer and melanoma. The proposed cell-particle hybrid vaccine construct had significant therapeutic outcomes in the prostate cancer tumor model where mice vaccinated with cell-particle hybrids were the only group to show significant improvement in survival compared to untreated controls whereas no other vaccine formulation had such an effect. Unfortunately, no prophylactic benefit was observed from any of the vaccine formulations used in the melanoma tumor model involving irradiated GM-CSF (granulocyte macrophage colony stimulating factor)-secreting B16.F10 cells. In vitro examination of the immunostimulatory properties of all cell lines used in these studies revealed that transfected and parent B16.F10 cells (representing murine melanoma) were possibly immunoinhibitory whereas RM11 (representing murine prostate cancer) cells lacked such immunosuppressive effect in vitro. Our objective was to design and evaluate a new cancer vaccine construct that improved the immunostimulatory properties of irradiated tumor cell based vaccines. The approach taken was to surface engineer tumor cells with immune adjuvant loaded polymeric particles. We reported a simple method for fabricating streptavidin-coated PLGA particles and a versatile method of tumor cell surface engineering. We found that the efficacy of tumor cell-based vaccines can be inconsistent across tumor models and the in vitro immunosuppressive effect of tumor cells might be a contributing factor.

Vaccines have become an important approach for the treatment of cancer. Cancer vaccines help the immune system to detect and eradicate tumor cells. Also, cancer vaccines are designed to stimulate an effective immune response that can create long-term immune memory to prevent tumor recurrence. This treatment approach involves the administration of a vaccine comprising or encoding an antigen and can often be combined with an adjuvant to further promote the immune response. The goal of this research was to study the effect of the polyanhydride composition of prophylactic cancer vaccine formulations on the tumor-specific immune response. To achieve this goal, three different amphiphilic polyanhydride copolymers were generated comprising different ratios of 1,6-bis-(p-carboxyphenoxy)-hexane (CPH) and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) or sebacic anhydride (SA) monomers. These copolymers were used to fabricate particles encapsulating a model antigen, ovalbumin (OVA), using a double emulsion solvent evaporation technique. The ability of the three different compositions of amphiphilic polyanhydride copolymers (50:50 CPTEG:CPH, 20:80 CPTEG:CPH, and 20:80 CPH:SA) encapsulating OVA to elicit immune responses was investigated. Further, the impact of soluble unmethylated oligodeoxynucleotides containing deoxycytidyl-deoxyguanosine dinucleotides (CpG ODN), an immunologic adjuvant, on the immune response to the three formulations was also studied. The immune response to cancer vaccines was measured after treatment of C57BL/6J mice with two subcutaneous injections, seven days apart, of 50 μg OVA encapsulated in particles composed of different polyanhydride copolymers with or without 25 μg CpG ODN. In vivo studies showed that 20:80 CPTEG:CPH particles encapsulating OVA significantly stimulated the highest level of CD8+ T lymphocytes, generated the highest serum titers of OVA-specific IgG antibodies, and produced longer survival in comparison to formulations involving the other polyanhydride copolymers. The results also revealed that supplementing the vaccine formulations with CpG ODN did not enhance the immunogenicity of OVA. These results accentuate the crucial role of the copolymer composition of polyanhydrides in stimulating the immune response and improving cancer vaccine efficacy.