Regensburg, Univ., Diss., 2005.

München, Techn. Univ., Diss., 2004.

Techn. Universiẗat, Diss., 2001--Braunschweig.

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Pathology. Includes bibliographical references.

Tierärztl. Hochsch., Diss., 2003--Hannover.

Universiẗat, Diss., 2004--Jena.

Copies of author's previously published articles inserted behind back end-papers. Bibliography: leaves 139-160. Identifies and characterises specific residues of biGFBP-2 that comprises the IGF binding site.

Molecular imaging involves the development of probes which can specifically label a certain object in the body at cellular or subcellular level. This thesis consists of three parts, each involving the development of novel labelling methods for viruses or virus-like particles with specific applications. Virus-like particles (VLP) derived from the E. coli bacteriophage Qβ are widely employed as a nano-carrier for drugs and vaccines, but a powerful method for tracing its circulation without affecting its structure is yet to be developed. In the first part of the thesis, the electrophilic fluorine source ¹⁹F-Selectfluor^TM was employed for introducing single fluorine atoms on Qβ VLPs. For the 'tag-and-modify' approach, site-selective electrophilic C-F bond formation was achieved on the dehydroalanine (Dha) amino acid tag of VLPs under aqueous conditions. Chemoselective electrophilic aromatic fluorination on tyrosine residues were also achieved using the same reagent by manipulating the amino acid sequence. Similar results were observed in conditions required for ¹⁸F-Selectfluor™ reaction, indicating the potential of this technique for positron emission tomography (PET) imaging. In addition, there is a lack of in situ technique for tracking the functional status of Qβ VLPs and hence the release of cargos. In the second part of the thesis, a simple way to monitor the disassembly of ¹⁹F-labelled Qβ VLPs by ¹⁹F NMR spectrosocpy is reported. Analysis of resonances, using experiments under a range of conditions, allowed determination not only of the intact particle but also the disassembled multimeric species and even smaller peptides upon digestion by cells. This in turn allowed mutational redesign of disassembly and testing in both bacterial and mammalian systems as a strategy for the creation of putative, targeted-VLP delivery systems. In the third part of the thesis, a new type of rhodamine B fluorescent dye functionalised with a 2-imino-2-methoxyethyl (IME) group is reported. The amidine linkage formed between the IME group and lysine residue retains the pKaH of the original side chain, which cannot be achieved using commercially available conjugating dyes. This in turn minimises the change in net charge hence virus infectivity following virus labelling. By employing adenovirus (AV) as an example, the IME dye was shown to be a better choice in retaining virus infectivity compared to dyes linked with other coupling groups. In addition, preliminary experiments on dengue virus with the synthesised dyes were also performed.

Toll-like receptors (TLR) are a family of pattern recognition receptors crucial for pathogen pattern recognition. Upon activation, TLRs induce innate immune responses such as cytokine production. However irregular TLR activities can provide fatal, hence fine tuning of the TLR induced responses are necessary. The TLR mediated immune responses are controlled by the positive/negative regulation of TLR signalling pathways, relocation of TLR proteins and modulation of TLR transcription. Systematic analyses of the agonist-induced transcriptional changes of TLRs were shown for the first time in my thesis. In my experiments, I have shown that each agonist induced a unique pattern of TLR transcription. Following PAM stimulation, mRNA levels of the cognate TLR1/2 increased whereas mRNA levels of the cross-regulating TLR4, 7/8/9 reduced in both cell lines and splenic macrophages from different mice strains. Through investigation of the signalling pathways responsible for mediating such TLR transcriptional changes, I then discovered the balancing effect between NFÎoB and MAPK signalling pathways. PAM induced TLR transcriptional changes were controlled by the additive and/or antagonistic interference between MAPK signalling cascades, ERK, JNK, P38 and NFÎoB signalling pathways. This was the first time that signalling synergy between MAPK and NFÎoB pathways were shown. Furthermore, PAM induced transcription of TLR1 and TLR8 may be partially regulated by the indirect feedback mediated by protein production. Importantly, the maintenance of the basal TLR mRNA expression also required activation of both MAPK and NFÎoB signalling pathways. In addition, signalling control for TLR transcription induced by different agonists (PAM vs. LPS) or in different species (chicken vs. mice) was compared. LPS induced transcriptional changes of the cross-regulating TLR1/2 and 3 but not the cognate TLR4 in RAW cells. The LPS induced TLR transcriptional changes required activation of a combination of MAPK and NFÎoB signalling pathways which shared both similarities and differences to the PAM induced signalling activation. In chicken, PAM induced more potent signalling activation, regulating the TLR transcriptional changes at a lower concentration than in mice. Overall, this thesis demonstrates that the transcriptional regulation of TLRs is complex, mediated by the coordination between MAPK and NFÎoB signalling pathways. These studies have significant implications in providing detailed insight of TLR transcriptional regulation which plays an important role in the regulation of TLR mediated innate immune responses. Please watch the following videos that I made for: A short introduction about TLR regulation - https://youtu.be/LTDdEZ3S97o A short explanation about TLR signalling - https://youtu.be/51IY5XhdJR8.

Made available in DSpace on 2015-12-06T23:47:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2007 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A pineal é aceita como reguladora da reprodução em mamíferos, por sua capacidade de interagir com as gonadotrofinas e influenciar as gônadas e promover a ciclicidade estral em algumas espécies. Contudo, sua função em espécies não foto-periódicas (camundongo, rato, macaco e ser humano) ainda não está totalmente esclarecida. Por este motivo, propusemo-nos a avaliar a histomorfologia e a histomorfometria do sistema reprodutor (ovários e útero), bem como os níveis plasmáticos de IGF-I e dos esteróides sexuais em ratas pinealectomizadas ou mantidas sob estímulo luminoso contínuo e após o tratamento com melatonina. Foram utilizadas ratas albinas, adultas, virgens, pesando aproximadamente 250g, da linhagem EPM - 1 Wistar. As ratas foram divididas, aleatoriamente, em seis grupos, a saber: GI – controle, tratado com veículo (n=20); GII – falsamente pinealectomizado, sham, tratado com veículo (n=20); GIII – pinealectomizado e tratado com veículo (n=20); GIV - pinealectomizado e tratado com melatonina (n=20); GV - submetido à luz contínua e tratado com veículo (n= 20); GVI – submetido à luz contínua e tratado com melatonina (n=20). O tratamento com melatonina (GIV e GVI) foi realizado por 90 dias consecutivos. Na noite anterior ao término do estudo, os animais foram colocados em gaiolas metabólicas, coletandose a urina noturna. Posteriormente, o sangue foi retirado pelo plexo ocular nos animais para determinação hormonal. Em seguida, as ratas foram sacrificadas e os ovários e o útero foram removidos para análise histomorfológica e histomorfométrica. Observou-se que a redução dos níveis séricos da melatonina (pela pinealectomia ou luz contínua, grupos III e V, respectivamente) determinou elevação sérica de estrogênio e redução significante de progesterona e IGF-I. Já a reposição com melatonina, reverteu este estado nos animais pinealectomizados, mas não nos animais sob luz contínua (grupo VI). Houve diminuição dos níveis androgênicos nos animais que receberam melatonina (grupos IV e VI) em relação os animais com baixos níveis de melatonina (grupos III e V). A diminuição sérica de melatonina (grupos III e V) determinou a proliferação do endométrio. Este efeito não foi totalmente bloqueado nos animais submetidos à luz contínua ou à pinealectomia e que foram tratados com melatonina. A diminuição dos níveis de melatonina determina alterações histomorfológicas e histomorfométricas nos ovários, sendo que a reposição de melatonina restaura essas modificações nos folículos ovarianos, bem como no epitélio e no estroma (células intersticiais). Contudo, o efeito desse hormônio tem menor impacto no epitélio superficial do ovário dos animais sob luz contínua. Nossos dados sugerem que a melatonina interfere com a produção hormonal e o sistema reprodutor, mas não é efetiva em animais sob luz contínua; possivelmente outros mecanismos que possam estar envolvidos sejam independentes da pineal. / The pineal gland is accepted as the reproduction modulator in mammals but its function in non-photoperiodic species (mouse, rat and primate) is still not thoroughly clear considering the endocrine interactions are fairly complex. For this reason, we have proposed to evaluate the histomorphology of the reproductive system (ovary and uterus) as well as the plasmatic levels of IGF-I and the sexual steroids in pinealectomized female rats or kept under continuous luminous stimulus and after the treatment using melatonin. The albino virgo female rats weighed approximately 250g from the EPM lineage – 1 Wistar were randomly divided into six groups: GI – control treated with vehicle (n=20); GII – falsely pinealectomized, sham, and treated with vehicle (n=20); GIII – pinealectomized and treated with vehicle (n=20); GIV – pinealectomized and treated with melatonin (n=20); GV – submitted to continuous light and treated with vehicle (n=20); GVI – submitted to continuous light and treated with melatonin (n=20). The treatment using melatonin for these groups lasted 90 consecutive days. The night before the end of the study, the animals were put in metabolic cages to collect urine. Later, the blood was taken through the ocular plexus for the hormonal determination. Then, both the ovary and the uterus of the female rats were removed for a histomorphological and histomorphometric analyses. The reduction of the serum levels of melatonin (pinealectomy or continuous light) as followed by an increase in estrogen levels and a significant reduction in progesterone and IGF-I blood levels. Differently, the replacement with melatonin reverted this condition in the pinealectomized animals but this did not happen with the animals exposed to continuous light. There was a reduction in the androgenic levels when compared to animals with low levels of melatonin. The reduction in serum melatonin is related to the endometrial proliferation. This effect is not totally blocked in animals submitted to continuous light or pinealectomy, which were treated with melatonin. Also, the decrase in the melatonin levels determined histomorphologic changes in the ovaries and the melatonin replacement restored these alterations in the ovarian follicle as well as in the epithelium and in the ovarian stroma (interstitial cells). Nevertheless, the effect of this hormone has a smaller impact in the ovarian superficial epithelium in animals under continuous light. Our data suggest that melatonin interferes with the hormone production and the reproductive system, but this is not the case in animals under continuous light; it is possible that other mechanisms, which may be involved are regardless of the pineal. / BV UNIFESP: Teses e dissertações