A infecção por HPV de alto risco é considerada um dos principais fatores de risco para o desenvolvimento do carcinoma do colo uterino, um das neoplasias mais freqüentes em mulheres de todo o mundo. As oncoproteínas E6 e E7 de HPV-16 são capazes de induzir a degradação dos genes supressores de tumor p53 e pRb, respectivamente. Mais do que isso, a expressão dessas oncoproteínas está relacionada a alterações na via de PI3K/AKT/mTOR. A proteína quinase mTOR apresenta importante papel no controle da tradução de proteínas e é considerada o principal mediador entre crescimento celular e proliferação. A ativação de mTOR é correlacionada à fosforilação das proteínas eIF4G1 e 4EBP1, aumentando assim a taxa de síntese de proteínas. A Rapamicina é um inibidor específico de mTOR e seus análogos apresentam potente atividade antiproliferativa em um grande número de células tumorais e tumores gerados em animais. Uma vez que as proteínas E6 e E7 são capazes de interagir com diversas proteínas da via que controla a atividade de mTOR optamos por investigar o efeito da rapamicina na proliferação de culturas organotípicas de queratinócitos expressando esses genes. Também avaliamos o efeito dos genes E6 e E7 na atividade de mTOR após o tratamento com essa droga. Para geração de culturas organotípicas de queratinócitos infectamos essas células com vetores retrovirais recombinantes contendo os genes E6 e E7 de HPV-16 em conjunto ou separadamente. Nós também avaliamos o papel da degradação de p53 e pRb na resposta à rapamicina através da utilização de mutantes de E6 e E7 incapazes de induzir a degradação dessas proteínas celulares. Após a infecção dos queratinócitos, os mesmos foram semeados em uma matriz de colágeno. Após 6 dias as culturas foram tratadas com 100ng/ml de rapamicina e permaneceram 60h em contato com a droga. Para análise por imunohistoquímica os tecidos foram fixados em formalina tamponada e emblocados em parafina. A reação de imunohistoquímica foi realizada utilizando os anticorpos contra BrdU, p-4EBP1 (ser 65), p-eIF4G1 (ser 1188) e pAKT (ser 473). Os resultados obtidos ilustram que a rapamicina apresenta efeito antiproliferativo em culturas de queratinócitos contendo o vetor vazio. Por outro lado, culturas contendo o gene E7 são resistentes ao efeito antiproliferativo dessa droga. Essa resistência parece estar relacionada à capacidade de E7 induzir a degradação da proteína pRb, uma vez que em queratinócitos expressando o mutante de E7, incapaz de induzir a degradação dessa proteína, não foi observada resistência. Além disso, a fosforilação de eIF4G e 4EBP1 indica que a expressão de E7 impede que a rapamicina seja capaz de inibir a atividade de mTOR. Esses resultados mostram, pela primeira vez, que o efeito antiproliferativo da rapamicina pode ser superado pela expressão de uma proteína viral, no caso a proteína E7 de HPV-16. / High-risk HPV infection has a major etiologic role in development and progression of cervical cancer, one of the most frequent forms of cancer among women worldwide. HPV-16 E6 and E7 oncoproteins are able to induce degradation of p53 and pRb tumor suppressor proteins respectively. Moreover, the expression of these oncoproteins is related to alterations in the PI3K/AKT/mTOR pathway. The cellular kinase mammalian target of Rapamycin (mTOR) is an important regulator of the cellular protein synthesis machinery and has emerged as a principal mediator of cell growth and proliferation. mTOR activation has been shown to stimulates eIF4G1 and 4EBP1 phosphorylation, thus increasing the rate of protein synthesis. Rapamycin is a specific inhibitor of mTOR signaling pathway and its analogues have demonstrated impressive activity against a broad range of human cancer derived cell lines in culture and in human tumor xenograft models. Since E6 and E7 target several proteins controlling the mTOR pathway we aimed to investigate the effect of Rapamycin in the proliferation of organotypic raft cultures expressing these genes. We also evaluated the effect of E6 and E7 genes in mTOR activity after rapamycin treatment. To generate organotypic culture of keratinocytes we infect these cells with recombinant retroviruses containing HPV-16 E6 and E7 together or separately. We also analyzed the role of p53 and pRb degradation in rapamycin responsiveness by using E6 and E7 mutants lacking the hability to inactivate these cellular proteins. After infection, keratinocytes were seeded on to a collagen matrix. After 6 days, these cultures were treated with 100ng/ml of Rapamycin for 60 hours. BrdU was added in the last 12 hours to evaluate proliferation. For immunohistochemistry analysis tissues were fixed in buffered formalin and embedded in paraffin. Immunohistochemistry reactions against BrdU, p-4EBP1 (ser 65), p-eIF4G1 (ser 1188) and p-AKT (ser 473) were performed The results show that proliferation of organotypic cultures of keratinocytes transduced with empty vector is inhibited by Rapamycin. On the other hand, cultures generated with keratinocytes transduced with E7 gene were completely resistance to the antiproliferative effect of Rapamycin. Moreover, we found that this antiproliferative effect was dependent of Rb degradation since the cells transduced with E7 mutant unable do induce Rb degradation were sensitive. In addition, eIF4G and 4EBP1 phosphorylation indicates that E7 expression impairs mTOR inhibition by rapamycin. AKT phosphorilation indicates that rapamycin resistance could be dependent of Rb inactivation induced by E7 expression. These results show for the first time that the Rapamycin antiproliferative effect is bypassed by the expression of a viral oncogene, in this case the HPV-16 E7. Moreover, E7 expression impairs rapamycin to inactivate mTOR.

Vital autonomic and cardiovascular functions are susceptible to dysfunction after spinal cord injury (SCI), with cardiovascular dysregulation contributing to morbidity and mortality in the SCI population. Autonomic dysreflexia (AD) is a condition that develops after injury to the sixth thoracic spinal segment or higher and is characterized by potentially dangerous and volatile surges in arterial pressure often accompanied with irregular heart rate, headache, sweating, flushing of the skin, and nasal congestion. These symptoms occur in response to abnormal outflow of sympathetic activity from the decentralized spinal cord typically triggered by noxious, yet unperceived nociceptive stimulation beneath the level of lesion. Maladaptive plasticity of primary afferents and spinal interneurons influencing sympathetic preganglionic neurons is known to contribute to the development of AD. However, there are currently no treatments capable of targeting this underlying pathophysiology. The goal of this work was to test pharmacological agents for their potential to modify intraspinal plasticity associated with AD in order to prophylactically prevent the development of this condition altogether. We first tested whether the drug rapamycin (RAP), a well-studied inhibitor of the growth promoting kinase “mammalian target of rapamycin” (mTOR), could prevent aberrant sprouting of primary c-fiber afferents in association with reduced indices of AD severity. Naïve and T4-transected rats undergoing 24/7 cardiovascular monitoring were treated with rapamycin (i.p.) for 4 weeks before tissue collection. RAP attenuated intraspinal mTOR activity after injury, however it also caused toxic weight loss. RAP treated SCI rats developed abnormally high blood pressure both at rest and during colorectal distension (CRD) induced AD, as well as more frequent bouts of spontaneous AD (sAD). These cardiovascular alterations occurred without altered intraspinal c-fiber sprouting. Our finding that rapamycin exacerbates cardiovascular dysfunction after SCI underscores the importance of screening potential pharmacological agents for cardiovascular side effects and suggests that the mTOR pathway plays a limited or dispensable role in c-fiber sprouting after SCI. We next examined the effects of the antinociceptive drug gabapentin (GBP) on AD development. Our previous work demonstrated that a single acute administration of GBP can reduce the severity of AD. The mechanism of action, however, remains unclear. Emerging evidence suggests that GBP may act by blocking de novo synaptogenesis. We investigated whether continuous GBP treatment could attenuate the development of AD by modifying synaptic connectivity between primary afferents and ascending propriospinal neurons. SCI rats were treated with GBP every six hours for four weeks. We found that GBP reduced blood pressure during CRD stimulation and prevented bradycardia typically observed during AD. However, GBP treated rats also had a higher sAD frequency and failed to return to pre-injury body weight. Moreover, SCI reduced the density of putative excitatory (VGLUT2+) and inhibitory (VGAT+) synaptic puncta in the lumbosacral cord, although GBP did not alter these parameters. Our results suggest that continuous GBP treatment alters hemodynamic control after SCI and that decreased synaptic connectivity may contribute to the development of AD. These studies demonstrate the need for further research to better understand the cellular signaling driving maladaptive plasticity after SCI as well as the complex and dynamic changes in intraspinal synaptic connectivity contributing to the development of AD. Moreover, GBP treatment may offer clinical benefit by reducing blood pressure during AD, however the optimal dosage must be identified to avoid undesired side-effects.

Apomixis, asexual or clonal seed production in plants, can decrease the cost of producing hybrid seed and enable currently open pollinated crops to be converted to more vigorous and higher yielding hybrids that can reproduce themselves through their own seed. Sexual reproduction may be triggered by a programmed stress signaling event that occurs in both the meiocyte, just prior to meiosis, and later in the egg just prior to embryo sac maturation. The prevention of stress signaling and the activation of a pro-growth signal prior to meiosis triggered apomeiosis, the first half of apomixis. The same approach was used prior to embryo sac maturation to trigger parthenogenesis, the second half of apomixis. This discovery suggests that apomixis exists as a program that can be activated by the appropriate metabolic signal at the appropriate developmental stages. Therefore, apomixis may be alternative mode of reproduction rather a ‘broken’ form of sexual reproduction.

Cell biomass and chemicals (e.g. bioactive compounds) can be produced by fermentation. Optimising a fermentation system involves optimizing many variables such as determining the effect of inoculum quality and media components, and selecting the most appropriate fermenter design and operating conditions (such as agitation aeration and fermentation mode). Identifying the optimal media is very important because it can significantly affect product concentration, yield and productivity. However, the media contains many components so many trials need to be done, which makes the process laborious, expensive, open-ended, and often time-consuming. The data generated from the many trials can be difficult to analyse. This study developed a rapid, inexpensive small-scale technique to identify how media components affected the growth of Streptomyces hygroscopicus and its production of a secondary metabolite, the anti-tumour agent rapamycin. A method was developed using microtitre plates to screen the effect of three concentrations of nine media components on cell growth and rapamycin production using the Box-Behnken experimental design. Firstly, the methodology for microtitre plates was developed, which involved characterizing the physical parameters of a fermentation system, identifying the incubation time to minimize evaporation, modifying the assay method to deal with the small sample volumes, and developing an alternative method to determinate the rapamycin concentration that was cheaper than the HPLC method. Data from shake flasks trials (the normal screening method) were used to validate the microtitre method and to assess the latter's usefulness in predicting scale-up effects. Six media components - sodium chloride (NaCl), di-potassium orthophosphate (K2HPO4), l-aspartic acid, l-arginine, l-histidine and salt (formula 1) solution - significantly affected culture growth and/or rapamycin concentration. The regression tree method was used to indicate the importance and critical concentration range of each factor. The Pearson's product-moment value indicated a good correlation between data from microtitre plates and shake flasks (cell growth: r=0.75 p=0.016 n=8; rapamycin concentration r=0.92 p=0.08 n=6). The speed of the microtitre plate and shake methods were compared by assessing the total cycle time and the time required for various stages in the method. Performance of each method was assessed as cost of media and equipment. Using microtitre plates to screen and optimise media in terms of biomass and secondary metabolite production is faster and cheaper than using shake flasks. Labour efficiency for the numerous, repetitive, small-scale experiments was substantially increased. Trials could be run without well-to-well cross contamination. The regression tree statistics methodology successfully showed the effect of input variables on target variables and identified effective medium component concentrations and any interactions. It is recommended that the microtitre plate procedure developed in this research may be applied to any study investigating the optimum media composition for growing other Streptomyces spp. strains, in screening studies when searching for new bioactive molecules, or for characterizing natural or recombinant/mutated micro-organisms.

<p>Multiple myeloma (MM) is a hematological tumor of plasma blast/plasma cell origin heterogeneous with respect to the morphological differentiation stage of the tumor cells, genetic alterations and course of disease. A challenge in MM research is to overcome resistance to therapy, which inevitably arises. In this thesis, we have used different strategies to sensitize MM cells to apoptosis and explored possible mechanisms of apoptotic control by the insulin-like growth factor-1 receptor (IGF-1R) survival pathway.</p><p>mTOR is a key molecule in the regulation of translation activated by survival signaling pathways in MM. We demonstrate that the mTOR-inhibitor rapamycin alone induced apoptosis in primary MM cells. In addition, rapamycin sensitized MM cells to apoptosis induced by dexamethasone, a glucocorticoid frequently used in MM therapy. MM survival factors IGF-1 and IL-6 could neither restore phosphorylation of the mTOR target p70S6K, nor cell growth inhibited by rapamycin and dexamethasone.</p><p>To study the regulation of inhibitors of apoptosis (IAP), we induced apoptosis and cell cycle arrest with dexamethasone and simultaneously abrogated IGF-1R signaling using the antagonistic antibody αIR3 or the selective IGF-1R inhibitor picropodophyllin (PPP). Dexamethasone transiently up-regulated c-IAP2. The subsequent down-regulation of c-IAP2 and XIAP was associated with the onset of apoptosis. c-IAP2 and XIAP levels further decreased when enhancing dexamethasone-induced apoptosis using αIR3 or PPP indicating a role for IAPs in regulating resistance to apoptosis in MM.</p><p>Finally, we explored glycogen synthase kinase (GSK)3 as a possible pro-apoptotic molecule and its role in regulating sensitization to apoptosis. We show that inhibition of GSK3 counteracts growth inhibition induced by dexamethasone alone and in combinatorial treatments with inhibitors against PI 3-kinase, mitogen-activated protein kinase (MEK), mTOR and IGF-1R. CT99021 also reversed cell cycle arrest induced by LY294002 or rapamycin. Importantly, the GSK3 inhibitor CT99021 sustained viability in untreated and dexamethasone-treated primary MM cells.</p>

Multiple myeloma (MM) is a hematological tumor of plasma blast/plasma cell origin heterogeneous with respect to the morphological differentiation stage of the tumor cells, genetic alterations and course of disease. A challenge in MM research is to overcome resistance to therapy, which inevitably arises. In this thesis, we have used different strategies to sensitize MM cells to apoptosis and explored possible mechanisms of apoptotic control by the insulin-like growth factor-1 receptor (IGF-1R) survival pathway. mTOR is a key molecule in the regulation of translation activated by survival signaling pathways in MM. We demonstrate that the mTOR-inhibitor rapamycin alone induced apoptosis in primary MM cells. In addition, rapamycin sensitized MM cells to apoptosis induced by dexamethasone, a glucocorticoid frequently used in MM therapy. MM survival factors IGF-1 and IL-6 could neither restore phosphorylation of the mTOR target p70S6K, nor cell growth inhibited by rapamycin and dexamethasone. To study the regulation of inhibitors of apoptosis (IAP), we induced apoptosis and cell cycle arrest with dexamethasone and simultaneously abrogated IGF-1R signaling using the antagonistic antibody αIR3 or the selective IGF-1R inhibitor picropodophyllin (PPP). Dexamethasone transiently up-regulated c-IAP2. The subsequent down-regulation of c-IAP2 and XIAP was associated with the onset of apoptosis. c-IAP2 and XIAP levels further decreased when enhancing dexamethasone-induced apoptosis using αIR3 or PPP indicating a role for IAPs in regulating resistance to apoptosis in MM. Finally, we explored glycogen synthase kinase (GSK)3 as a possible pro-apoptotic molecule and its role in regulating sensitization to apoptosis. We show that inhibition of GSK3 counteracts growth inhibition induced by dexamethasone alone and in combinatorial treatments with inhibitors against PI 3-kinase, mitogen-activated protein kinase (MEK), mTOR and IGF-1R. CT99021 also reversed cell cycle arrest induced by LY294002 or rapamycin. Importantly, the GSK3 inhibitor CT99021 sustained viability in untreated and dexamethasone-treated primary MM cells.

<p>Fungal cells sense the amount and quality of external nutrients through multiple interconnected signaling networks, which allow them to adjust their metabolism, transcriptional profiles and developmental programs to adapt readily and appropriately to changing nutritional states. In organisms ranging from yeasts to humans, the Tor signaling pathway responds to nutrient-derived signals and orchestrates cell growth. While in the baker's yeast <italic>Saccharomyces cerevisiae</italic> Tor responds to nutrient-derived signals and orchestrates cell growth and proliferation, in <italic>Schizosaccharomyces pombe</italic> Tor signaling modulates sexual differentiation in response to nutritional cues. Thus, these differences provide a framework to consider the roles of Tor in other fungal organisms, in particular those that are pathogens of humans. </p><p>In this dissertation, I demonstrate that in the human fungal pathogen <italic>Candida albicans</italic>, Tor signaling also functions to promote growth. This study also uncovered a novel role for the Tor molecular pathway in promoting hyphal growth of <italic>C. albicans</italic> on semi-solid surfaces and in controlling cell-cell adherence. Gene expression analysis and genetic manipulations identified several transcriptional regulators (Bcr1, Efg1, Nrg1, and Tup1) that together with Tor compose a regulatory network governing adhesin gene expression and cellular adhesion. While the Tor kinases are broadly conserved, these studies further demonstrate the contrasting strategies employed by fungal organism in utilizing the Tor signaling cascade.</p><p>While extensive studies have focused on elucidating functions for the Tor signaling cascades among ascomycetes, little is known about the pathway in basal fungal lineages, in particular among zygomycetes and chytrids. Moreover, given that the Tor pathway is the target of several small molecule inhibitors including rapamycin, a versatile pharmacological drug used in medicine, there is considerable interest in Tor signaling pathways and their function. Capitalizing on emerging genome sequences now available for several basal fungal species, we show a remarkable pattern of conservation, duplication, and loss of the Tor signaling cascade among basal fungal lineages. Targeting the pathway with rapamycin results in growth arrest of several zygomycete species, indicating a conserved role for this pathway in regulating fungal growth. In addition, we show a potential therapeutic advantage of using rapamycin in a heterologous model of zygomycosis. Taken together, the Tor signaling cascade and its inhibitors provide robust platforms from which to develop novel antimicrobial therapies, which may include less immunosuppressive rapamycin analogs.</p> / Dissertation

This research was conducted to test the hypothesis that select nutrients including glucose, leucine, arginine and glutamine stimulate conceptus development by activating mTOR (mammalian target of rapamycin; HGNC approved gene name: FRAP1, FK506 binding protein 12-rapamycin associated protein 1) signaling pathway. First, temporal changes in quantities of select nutrients (glucose, amino acids, glutathione, calcium, sodium and potassium) in uterine lumenal fluid from cyclic (Days 3 to 16) and pregnant (Days 10 to 16) ewes were determined. Total recoverable glucose, Arg, Gln, Leu, Asp, Glu, Asn, His, beta-Ala, Tyr, Trp, Met, Val, Phe, Ile, Lys, Cys, Pro, glutathione, calcium and sodium was greater in uterine fluid of pregnant compared to cyclic ewes between Days 10 and 16 after onset of estrus. Of note were remarkable increases in glucose, Arg, Leu and Gln in uterine flushings of pregnant ewes between Days 10 and 16 of pregnancy. Second, effects of the estrous cycle, pregnancy, progesterone (P4) and interferon tau (IFNT) on expression of both facilitative (SLC2A1, SLC2A3 and SLC2A4) and sodium-dependent (SLC5A1 and SLC5A11) glucose transporters, cationic amino acid transporters (SLC7A1, SLC7A2 and SLC7A3), neutral amino acid transporters (SLC1A4, SLC1A5, SLC3A1, SLC6A14, SLC6A19, SLC7A5, SLC7A6, SLC7A8, SLC38A3, SLC38A6 and SLC43A2) and acidic amino acid transporters (SLC1A1, SLC1A2 and SLC1A3) in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy as well as in conceptuses from Days 13 to 18 of pregnancy were determined. Among these genes, SLC2A3 and SLC7A6 were detectable only in trophectoderm and endoderm of conceptuses. The abundance of mRNAs for SLC2A1, SLC2A4, SLC5A1, SLC5A11, SLC7A1, SLC7A2, SLC1A4, SLC1A5, SLC43A2 and SLC1A3 changed dynamically in ovine uterine endometria according to day of the estrous cycle and early pregnancy. Expression of mRNAs for SLC2A1, SLC5A11 and SLC7A1 in endometria was induced by P4 and further stimulated by IFNT with shortterm treatment (12 days), while expression of SLC7A1 and SLC1A5 in endometria required long-term treatment (20 days) with P4 and IFNT. Third, effects of the estrous cycle, pregnancy, P4 and IFNT on expression of nitric oxide synthase (NOS1, NOS2 and NOS3), GTP cyclohydrolase (GCH1), ornithine decarboxylase 1(ODC1), insulin-like growth factor II (IGF2), FRAP1 complexes (FRAP1, LST8, MAPKAP1, RAPTOR, RICTOR), regulators (TSC1, TSC2, RHEB) and an effector (EIF4EBP1) of FRAP1 signaling in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy as well as in conceptuses from Days 13 to 18 of pregnancy were determined. All of these genes were expressed in ovine uterine endometrium and conceptuses. Among these genes, expression of NOS1, IGF2, RHEB and EIF4EBP1 changed dynamically due to day of the estrous cycle and early pregnancy. Progesterone stimulated NOS1 and GCH1 expression while IFNT inhibited NOS1 expression in uterine endometria, and P4 and IFNT stimulated expression of RHEB and EIF4EBP1 in uterine endometria. Collectively, these results indicate that: 1) the availability of select nutrients in the ovine uterine lumen increases to support the rapid growth and elongation of the conceptus during the peri-implantation stage of pregnancy; 2) P4 and/or IFNT stimulate(s) glucose and amino acid transporters to facilitate their transport from maternal tissues and/or blood into the uterine lumen during early pregnancy; 3) the FRAP1 cell signaling pathway mediates interactions between the maternal uterus and peri-implantation conceptus and both P4 and IFNT affect this pathway by regulating expression of RHEB and EIF4EBP1. Expression of NOS, ODC1 and IGF2 appear to be linked to FRAP1 signaling in both uteri and peri-implantation conceptuses.

Dendritic cells (DC) are potent antigen presenting cells involved in the initiation of the alloimmune response and organ transplant rejection. This thesis, has investigated pharmacological and genetic approaches to manipulate DC in order to generate tolerogenic DC which elicit poor allostimulatory activity as potential cell therapy agents to treat allograft rejection. In the first aspect of this study, human monocyte-derived DC were used to study the influence of Rapamycin (RAPA) on DC phenotype and function. This study showed that RAPA when added to monocytes prior to DC differentiation or after DC maturation generated tolerogenic DC as evidenced by the ability of these cells to induce T cell hyporesponsiveness. However, T cell hyporesponsiveness was associated with downregulation of costimulatory molecules only when added prior to differentiation and surprisingly was not influenced by the induction of CD4 ⁺FoxP3 ⁺ T cells. To assess the effects of RAPA on DC function in the transplant setting an in vivo chimeric model of ovine vascularised skin allograft transplantation was established in immunocompromised NOD/SCID mice as a host. This model was established as a preliminary model to acquire in vivo data prior to testing the effect of pharmacologically modified DC in the preclinical ovine model of renal allograft transplantation, also established in the host laboratory. Firstly, comparison of ovine DC obtained from cannulation of the prefemoral lymphatic vessels in sheep demonstrated that RAPA-modified ovine DC acted as poor stimulators of allogeneic ovine T cells similar to human DC treated with RAPA. Secondly, in NOD/SCID mice engrafted with ovine skin, the infusion of allogeneic ovine T cells together with RAPA-modified ovine DC reduced histological rejection in comparison to control DC. In the second aspect of this study, the effects of genetic manipulation of DC were investigated. In order to investigate the effects of genetic modification of DC, two isoforms of the human HLA-G molecule, HLA-G1 (membrane bound) and HLA-G5 (soluble isoform) were used to generate adenoviral vectors. Unexpectedly, both HLA-G isoforms expressed by human DC transfectants were unable to induce allogeneic T cell hyporesponsiveness in the mixed lymphocyte reaction (MLR). Surprisingly, in the MLR the allogeneic T cells acquired HLA-G1, but not HLA-G5, indicating that direct cell contact and membrane transfer from DC to T cells occurred (Trogocytosis). In addition to HLA-G1, costimulatory molecules (CD40, CD80, CD86 and MHC Class II) were also cotransferred from DC to allogeneic T cells. Accordingly, in secondary proliferation assays T cells immunoselected after co-culture with allogeneic untransfected DC (TUT) demonstrated potent antigen presenting activity when used as stimulators of autologous T cells (analogous to the indirect pathway of antigen presentation). In contrast to TUT, immunoselected T cells that acquired HLA-G1 (THLA-G1) upon co-culture with DCtransfectants showed poor stimulatory capacity. Thus the data reported in this thesis supports the proposed novel concept that HLA-G acquired by T cells through genetically modified DC, functions to autoregulate T cells via T-T cell interaction through the HLA-G receptor ILT2 (negative signalling receptor) expressed on T cells. In conclusion, this thesis has firstly provided supportive evidence that the pharmacological modification of human and ovine DC with RAPA has potential therapeutic effects on allograft rejection. Secondly, the genetic modification of DC to induce expression of HLA-G has specifically allowed the transfer of this molecule to T cells by trogocytosis and the inhibition of alloreactive T cell expansion. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2009

Thesis (Ph.D.)--Indiana University, 2010. / Title from screen (viewed on July 21, 2010). Department of Biochemistry and Molecular Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Ronald C. Wek, Howard J. Edenberg, Peter J. Roach, Martin Bard. Includes vitae. Includes bibliographical references (leaves 125-132).