Introduction Yearly, 90 million people are infected with C. trachomatis. Even though it is easily treated with antibiotics the often-asymptomatic infection often spreads prior to detection. A vaccine is therefore of great interest. A chimeric major outer membrane protein (MOMP) of C. trachomatis has in earlier studies proved to contain the epitopes necessary for immunization. In this thesis the chimeric MOMP gene was cloned and expressed in E. coli. Furthermore, the expression of the protein was analyzed in previously transformed A. thaliana. Materials and Methods The chimeric MOMP gene was cloned into E.coli. Following vector amplification, the gene was expressed and the protein purified by affinity chromatography.  Seeds from different lines of previously transformed A. thaliana were screened by PCR. Hits were then analyzed by western blot.  Results The results show successful cloning and expression of the chimeric MOMP gene in E. coli. The following protein purification did result in purified protein, however in low concentration. For the A.thaliana lines, the presence and correct orientation of the gene was verified in some of the lines screened. The B7 line was verified to express the protein. Discussion The low concentration of purified protein in E.coli was probably due to un-optimized imunnoprecipitation conditions. In expression analysis of A. thaliana, purification of plant samples by immunoprecipitation prior to running western blot gave results, whereas running un-purified samples in urea buffer did not, probably due to interfering proteins in wild type plants.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Aeglidae is the only freshwater anomuran family. Fossil evidences relate a marine origin for aeglids. However, genus Aegla invaded the freshwater environment of southern South America, becoming endemic and widespread in that Neotropical region. Besides, the group is widely diversified, consisting of approximately 70 recognized species. Despite the species richness, aeglids have experienced progressive habitat loss and reduction in their populations due to the steam environments alterations. In order to enable future works on Aegla diversity, this study proposed to develop microsatellite markers for Aegla longirostri. Because of their high polymorphism, such molecular markers have been widely used for establishing genetic structure, dynamic and relationship within and among populations. A total of 42 true microsatellite loci was isolated from the library enriched for (CA)n repeats, besides 17 chimeric sequences attained from technical artifacts. The repeats ranged from 3 to more than forty units. However, primer pairs could be developed for thirteen sequences. Successful PCR amplifications were obtained for loci AlCA135 and AlGA138 up to the moment. This microsatellite loci have been screening in 15 individuals of a population from the central region of Rio Grande do Sul State, Brazil, where that species is endemic, in order to evaluate polymorphism. The other developed primer pairs are still undergoing optimization. These markers are promising for investigating population genetic structure for this species. Besides, they revealed cross-species amplification when tested with Aegla uruguayana. Chimeric PCR products attained in this study, which correspond to 29% of the captured sequences, were object of a deep study about artifacts in microsatellite isolation. Moreover, it was proposed a mechanism to explain their formation and implications. / Aeglidae é a única família de crustáceos anomuros cujos representantes vivos são habitantes exclusivos de água doce. A origem marinha do grupo é evidenciada pelos registros fósseis. Entretanto, o gênero Aegla conquistou o ambiente dulcícola na região temperada do sul da América do Sul onde é endêmico, amplamente distribuído e diversificado, constituindo-se de aproximadamente 70 espécies descritas. Apesar da riqueza de espécies, os eglídeos têm sido vítimas de progressiva perda de habitat e declínio populacional devido à degradação dos ecossistemas límnicos sul-americanos. Visando possibilitar trabalhos futuros voltados à avaliação da diversidade do gênero, este estudo propôs o desenvolvimento de marcadores microssatélites para Aegla longirostri. Devido ao seu alto grau de polimorfismo, estes marcadores têm sido amplamente utilizados para avaliação de estrutura populacional, dinâmica e relações entre e dentro de populações dos mais variados organismos. Foram obtidos 42 locos verdadeiros de microssatélites com a construção de uma biblioteca genômica enriquecida com microssatélites (CA)n para A. longirostri, além de 17 seqüências quiméricas oriundas de artefatos da técnica. As repetições encontradas variaram de três a mais de 40 unidades. Desse total, 13 permitiram a projeção de primers. Condições ideais de amplificação foram estabelecidas para os locos AlCA135 e AlGA138 até o momento, os quais estão sendo testados em 15 indivíduos de uma população da região central do estado do Rio Grande do Sul, onde esta espécie é endêmica, para avaliação do polimorfismo. Testes de amplificação seguem sendo feitos para os demais locos desenvolvidos. Estes marcadores são promissores para investigação de estruturação genética em populações desta espécie. Além disso, estes locos apresentaram amplificação cruzada positiva quando testados para a espécie Aegla uruguayana. Para as seqüências quiméricas obtidas, as quais correspondem a 29% do total capturado, sugeriu-se um mecanismo para explicar sua causa e avaliaramse as implicações deste evento em construções de biblioteca genômica enriquecida com microssatélites.

No description available.

Glioblastoma multiforme (GBM) is the most common malignant brain cancer that invades normal brain tissue and impedes surgical eradication, resulting in early local recurrence and high mortality. In addition, most therapeutic agents lack permeability across the blood brain barrier (BBB), further reducing the efficacy of chemotherapy. Thus, effective treatment against GBM requires tumor specific targets and efficient intracranial drug delivery. With the most recent advances in immunotherapy, genetically engineered T cells with chimeric antigen receptors (CARs) are becoming a promising approach for treating cancer. By transducing T lymphocytes with CAR constructs containing a tumor-associated antigen (TAA) recognition domain linked to the constant regions of a signaling T cell receptor, CAR T cells may recognize a predefined TAA with high specificity in a non-MHC restricted manner, and is independent of antigen processing. Active T cells can travel across the BBB, providing additional advantage for drug delivery and tumor targeting. Here we review the CAR design and technical innovations, the major targets that are in pre-clinical and clinical development with a focus on GBM, and multiple strategies developed to improve CAR T cell efficacy.

In the nervous system galanin primarily displays a modulatory role. The galaninergic system consists of a number of bioactive peptides with a highly plastic expression pattern and three different receptors. The lack of receptor subtype selective ligands and antibodies have severely hampered the charac-terization of this system. Therefore, most of the knowledge has been drawn from experiments with transgenic animals, which has given some major conclusions, despite the compensatory effects seen in several animal studies. Therefore, the production of subtype selective ligands is of great importance to delineate the galanin system and slowly experimental data from receptor subtype selective ligand trials is emerging. This thesis aims at studying galanin receptor-ligand interactions and to increase and improve the utilized tools in the galanin research field, espe-cially the development of novel galanin receptor subtype selective ligands. Paper I demonstrates the potential to N-terminally extend galanin ana-logues and the successful development of a galanin receptor 2 (GalR2) selec-tive ligand. In addition, a cell line stably expressing galanin receptor 3 (GalR3) was developed, to improve and simplify future evaluations of sub-type selective galanin ligands. Paper II measures the affinities of M617 and M871 to GalR3 and demon-strates that M871 preferentially binds GalR2. Furthermore, the relatively high affinity of M617 was evaluated by assessing the contribution in recep-tor interaction of individual amino acid residues in the C-terminal part of the M617. In conclusion, this thesis has provided a novel design strategy for galanin receptor ligands and increased the understanding of ligand interactions with the GalR3. Furthermore, M1145 has together with new analogues proven to be highly GalR2 specific, holding promises to future delineation of the galaninergic system as a therapeutic target.

Through the years, cancer therapies have progressed rapidly, pouring out novel treatments such as gene therapy, small molecule therapies and immunotherapy. One such immunotherapy, adoptive cell transfer (ACT), augmented through the addition of a chimeric antigen receptor (CAR), has proven success in treatment of hematological malignancies. Additionally, oncolytic viruses (OV) and OV-based (OVV) therapies, have shown promising results in both clinical and pre-clinical studies. In most instances, when applied as a monotherapy, the aforementioned treatment methods are incapable of inducing complete tumour remission. The Wan lab has developed an approach combining ACT with OVV therapies that dramatically increase therapeutic benefit resulting in complete regression of well-established solid tumours. Despite promising results, certain tumours can still escape this combination therapy through antigen loss resulting in antigen negative relapse (ANR). To further augment the therapy, the addition of a secondary receptor (CAR) provides the ACT multiple avenues of attack to prevent ANR. In this dissertation, we define culture conditions that promote strong expression of the CAR alongside confirmation of function in an in vitro setting. Following, it is demonstrated that OVV boosted dual-targeting T cells carry strong T cell activity by measure of cytokine release in vivo. Despite promising T cell activity data, dual-specific T cells are unable to improve tumour control and survival once relapse occurs. The failure to control relapse remains unclear however evidence points towards lack of T cell persistence, poor CAR function in vivo and a lack of endogenous T cell response leading to compounding effects that prevent dual-targeting T cells from preventing ANR. Although dual specific therapies have shown poor efficacy in preventing ANR, further study must be completed to identify areas of improvement – such as persistence, as the potential for success in using dual-targeting T cells coupled with OVVs still lies untapped. / Thesis / Master of Science (MSc)

Cancer immunotherapy is emerging as a powerful tool in the treatment of cancer. Multiple clinical trials have established that infusion of tumour-specific T cells can cause regression of advanced tumours and prevent tumour relapse. While tumour-specific T cells are typically rare, engineering methods have been developed to introduce tumour-specific receptors into T cells and engender peripheral blood T cells with the ability to kill tumour cells. These engineering successes notwithstanding, tumour cells demonstrate variable sensitivity to T cell attack. Therefore, to maximize the impact of the engineered T cells, it is necessary to develop therapeutic strategies that render tumour cells sensitive to immune attack. For my thesis research, I sought to develop a high throughput screening assay that would allow me to screen chemical libraries for agents that sensitize tumour cells to T cell attack. My ultimate goal is to define chemical agents that effectively sensitize tumour cells to T cell attack but display a better toxicity profile than existing chemotherapies. To this end, I developed a screen where resistant tumour cells were exposed to T cells engineered with chimeric antigen receptors and positive hits were defined as agents that could enhance killing of the tumour cells. My work explored both murine and human systems and I ultimately decided to use human cells for my screen. Multiple methods for measuring tumour cell killing were evaluated, many tumour lines were screened and I optimized the conditions for generating large numbers of engineered T cells for the screen. The net result of my thesis work is a miniaturized assay that is ready for high throughput screening. / Thesis / Master of Science in Medical Sciences (MSMS)

At least 10% of all cancer deaths are attributable to local tumor disease burden. In select cases, existing treatments such as surgery, chemotherapy, radiation, and immunotherapy demonstrate efficacy in halting or slowing cancer progression to improve survival. However, these strategies remain insufficient for most patients depending on cancer stage and histological characteristics, resulting in locoregional spread, locoregional recurrence, or progression to metastatic disease while also inducing dose-limiting toxicity. Biomaterials enable localized delivery of small molecules and proteins to improve safety and efficacy of cancer therapies. To leverage the safety and efficacy of biomaterials for local cancer treatment, we developed electrospun, hydrophobic nanofiber meshes that deliver small molecules and proteins. The meshes are biodegradable, biocompatible, and mechanically robust, making them ideal delivery vehicles for intraoperative care in the context of local tumor resection. We optimized the meshes to deliver the chemotherapeutic doxorubicin via drug encapsulation within the nanofibers and within hydrophobic coatings made of poly(glycerol mono-stearate-co-caprolactone) (PGC-C18). The meshes, called doxorubicin-eluting meshes (DoMs), prevent local sarcoma recurrence while avoiding cardiotoxicity associated with systemic doxorubicin therapy. We next utilized this mesh formulation to encapsulate grazoprevir (GZV), an FDA approved drug recently repurposed as an inducer molecule for a logic chimeric antigen receptor (CAR) T cell. Using the meshes, we can modulate release over time and control cytokine expression in vitro. Finally, we designed meshes that adsorb proteins and release them in a tunable manner through wetting modulation, named ZipCAR Adaptor Modulation using a BiOdegradable Nanofiber Implant (ZAMBONI). We used the meshes to deliver universal CAR T cell adaptor proteins, which connect CAR T cells to cancer cells in a modular fashion. We demonstrate improved safety and efficacy in models of ovarian carcinoma and mesothelioma without evidence of on-target, off-tumor toxicity, a common mechanism of CAR T cell toxicity. Together, these results demonstrate a novel and robust approach to improve safety and efficacy in local cancer therapy. / 2025-07-19T00:00:00Z

京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第24292号 / 人健博第107号 / 新制||人健||8(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 藤井 康友, 教授 岡 昌吾, 教授 滝田 順子 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

An important obstacle to a functional cure for HIV/AIDS is the persistence of viral reservoirs found throughout the body in various cells and tissues. Reservoirs can be latently infected cells, or in the case of follicular dendritic cells (FDC), non-infected cells that trap infectious virus on their surface through immune complexes (HIV-IC). Although several strategies have been employed to target and eliminate viral reservoirs, they are short-lived and ineffective. In an attempt to provide a long-term approach, chimeric antigen receptor T (CAR-T) cells were designed to eliminate native HIV on FDCs. Although effective at eliminating HIV-infected cells, and halting spreading infection, their ability to eliminate the viral reservoir found on (FDCs) remains unclear. We used a novel second-generation CAR-T cell expressing domains 1 and 2 of CD4 followed by the mannose binding lectin (MBL) to allow recognition of native HIV envelope (Env) to determine the capacity to respond to the viral reservoir found on FDCs. We employed a novel fluorescent lysis assay, the Carboxyfluorescein succinimidyl ester (CFSE) release assay, as well as flow cytometric based assays to detect functional CAR-T activation through IFN-γ production and CD107a (i.e., LAMP1) membrane accumulation to test cytolytic capacity and functional activation of CD4-MBL CAR-T cells, respectively. We demonstrated their efficacy at eliminating HIV-infected cells or cells expressing gp160. However, these CAR-T cells were unable to lyse cells bearing surface bound HIV-IC. We found that failed lysis was not a unique feature of a resistant target, but a limitation in the CAR-T recognition elements. CAR-T cells were inactive in the presence of free HIV or in the presence of concentrated, immobilized virus. Further experiments determined that in addition to gp120 recognition by the CAR-T, the adhesion molecule ICAM-1 was necessary for efficient CAR-T cell killing of HIV-infected cells. CAR-T cell activity and killing were inhibited in the presence of ICAM-1 blocking antibody. These results suggest that other factors, such as adhesion molecules, play a vital role in CAR-T responses to HIV-infected cells. In addition, our findings highlighted the necessity to consider all models of HIV reservoirs, including FDCs, when evaluating therapeutic efficacy.