Parabiosis is the surgical union of two organisms resulting in the development of a single, shared circulatory system. When animals of different ages are conjoined (i.e. heterochronic parabiosis), blood-borne factors from the parabionts can affect the physiology of the other parabiont. This is manifested sometimes by beneficial, rejuvenating impact upon the older animal's tissues and organs (anti-geronic effect), and sometimes by younger animal's tissues regressing and appearing old-like (pro-geronic effect). These effects, and the ability to identify individual factors that could recapitulate pro- and anti-geronic effects, have made heterochronic parabiosis a very attractive approach to studying biology of aging and rejuvenation.cHowever, heterochronic parabiosis has not been widely used to investigate the aged immune system. An important question to be answered is whether the cellular defects involved in the aged immune system are due to intrinsic defects or if they can be rescued by extrinsic factors. Heterochronic parabiosis is ideal to test cellular migration patterns, interrogate the mechanisms driving migration defects that occur with aging, establish if these defects can be rejuvenated and identify molecules that are targets for intervention. Here, we provide evidence of the importance of reducing differences in the background genetics of different C57BL/6 substrains prior to parabiosis. This improvement allowed us to improve survival and confirm robust lymphocyte equilibration across secondary, but not primary, lymphoid tissues. We found no evidence for rejuvenation of the old immune cells, whereas results suggested that adult peripheral lymph nodes (pLN) lost mass and cellularity, potentially indicating the presence of a pro-geronic factor(s) in the old circulation that affects pLN function. Adult and old immune cells were present in equal frequencies in both adult and old secondary lymphoid tissues, indicating that there was no restriction of cellular migration due to the age of the cell or age of the tissue. The propensity of adult immune cells (i.e. large naïve compartment) to occupy lymph nodes and old immune cells (i.e. large memory compartment) to occupy bone marrow was retained following heterochronic parabiosis. Finally, parabiosis separation experiments illuminated the peripheral survival advantage of old T cells over adult T cells. These results highlight the power of heterochronic parabiosis in studying immune aging and provide hypothesis-generating data for future mechanistic studies of peripheral T cell maintenance with aging.

Orientadores: Sérgio Roberto Peres Line, Alexandre Augusto Zaia / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-19T14:41:36Z (GMT). No. of bitstreams: 1 Frozoni_MarcosRobertodosSantos_D.pdf: 22284992 bytes, checksum: f3df32a5434f64642e63697df5e27202 (MD5) Previous issue date: 2011 / Resumo: Na polpa saudável, os odontoblastos pós-mitóticos responsáveis pela secreção de dentina primária e secundária sobrevivem durante a toda a vida do dente e são responsáveis pelas respostas às injúrias externas através da produção de dentina terciária focalmente abaixo do local da agressão. Os odontoblastos sobreviventes à injúria secretam uma matriz de dentina reacional, entretanto, os irreversivelmente danificados, são substituídos por uma segunda geração de células com as mesmas características morfofisiológicas. Estas células são derivadas do recrutamento, proliferação e diferenciação de células pulpares que possuam propriedades de células tronco, para formar uma nova matriz de dentina reparativa como parte do processo de reparo do complexo dentino pulpar. O mecanismo de reparo que segue após uma injúria dental é fundamental para a sobrevivência da polpa e envolve uma série de processos que precisam ser completamente esclarecidos mais precisamente a origem das células precursoras da nova geração de odontoblastos que irão secretar dentina reparativa. Embora evidências sugiram que esta nova geração de odontoblastos tenha origem de células do tecido conjuntivo pulpar, a exata origem destas células ainda não está completamente esclarecida. O objetivo deste estudo foi utilizar camundongos transgênicos que expressam green fluorecent protein (GFP) em todas as células do corpo, com exceção dos eritrócitos, e a técnica cirúrgica de parabiose para unir dois camundongos isogênicos, um GFP e outro não-GFP de maneira a formar um par parabiótico, que possa compartilhar a mesma circulação sanguínea cruzada (células marcadas do camundongo GFP passam para a corrente circulatória do camundongo não-GFP). Após a parabiose foi realizada uma exposição pulpar, devidamente capeada, nos molares dos camundongos não-GFP para estimular a produção de dentina terciária reparativa e conseqüentemente a diferenciação de novos odontoblastos. Os animais foram sacrificados e os molares dos camundongos não-GFP, processados para análise em microscopia de fluorescência. Foi observada a presença de células GFP (células verdes ao microscópio de fluorescência), originárias do sangue periférico (SP) de camundongos GFP, participando do processo de dentinogênese reparativa nos molares de camundongos não-GFP. Este estudo sugere a primeira evidência da participação de células troco mesenquimais do sangue periférico (CTM-SP) no processo de diferenciação de novos odontoblastos durante a dentinogênese reparativa / Abstract: In the healthy pulp, the post-mitotic odontoblasts responsible for secretion of primary and secondary dentin survives during the tooth life and are able to respond to injuries with the production of tertiary dentin focally beneath the site of the injury. If the odontoblast survives the injury it secrets a reactionary dentin matrix but if it is irreversibly damaged, it is replaced by a second generation of odontoblast-like cells, with the same morphophysiological profile. Those cells are derived from recruitment, proliferation, and differentiation of pulp cells, which can have stem cell properties to form a new reparative dentin matrix as part of repair process in the dentin-pulp organ. The repair mechanism that follows a tooth injury is critical to the pulp survival and involves a series of processes that need to be completely understood more precisely the origin of the odontoblast-like cells that will secrete reparative dentin. Although evidences suggesting that odontoblast-like cells originate from cells within dental pulp connective tissue the exact origin of these odontoblast-like cells is not clearly defined. The aim of this study was to use transgenic mice expressing green fluorescent protein (GFP) in all body cells except erythrocytes and parabiosis surgical technique for joining two inbred mice, a GFP and other non-GFP to form one parabiotic pair, who can share the same cross-circulation (labeled cells from GFP mice move into the bloodstream of non-GFP mice After parabiosis a pulp exposure, properly capped, was performed on the molar of the recipient non-GFP mice to stimulate the production of tertiary reparative dentin and hence differentiation of odontoblastlike cells. The animals were sacrificed and the molars of the non-GFP mice processed for fluorescence microscopy. It was observed the presence of GFP cells (green cells to the fluorescence microscope), originating from peripheral blood (PB) from GFP mice, participating in the reparative dentinogenesis process in the molars of non-GFP mice. This study suggests the first evidence of the participation of mesenchymal stem cells from PB (MSC-PB) in the differentiation process of odontoblast-like cells during reparative dentinogenesis / Doutorado / Endodontia / Doutor em Clínica Odontológica

Adipose tissue is a crucial organ that sits at the nexus of organismal metabolism. Evolutionary systems seemingly developed to regulate weight such that the risk of starvation accompanying low weight was balanced against the risk of predation accompanying high weight, but the molecular underpinnings of these systems have not been fully elucidated. The modern obesogenic diet has led these processes to become dysregulated, resulting in increased rates of obesity and associated metabolic disorders, making a full understanding of the mechanisms underlying weight regulation even more important. Parabiosis experiments support the existence of an uncharacterized anorectic factor that opposes weight gain. A previously established system of murine overfeeding recapitulates the defense of body weight against rapid weight gain and uncovers a non-inflammatory adipose tissue environment in the setting of obesity. Building on this past work, this thesis sets out to characterize the protein secretome of adipose tissue from overfed mice in order to provide insight into possible candidate anorectic factors and better understand the physiology of adipose tissue in this experimental form of obesity.In doing so, we uncover a previously unappreciated phenomenon of mitochondrial secretion from adipose tissue depending on metabolic state. We find that mitochondria are secreted in greater number from overfed adipose tissue and that these mitochondria are enriched for enzymes related to de novo lipogenesis. We also demonstrate that mitochondria are released intact. We find that some of these phenotypes are shared in genetically obese db/db mice, pointing toward potential physiological roots. We also characterize the plasma proteomes of overfed mice, finding that in overfed mice, inflammatory pathways are increased in the absence of induction of canonical inflammatory cytokines and in the absence of inflammation in the adipose tissue. Collectively, this work demonstrates the utility and importance of using experimental models in order to better disentangle phenomena of feeding, obesity, and inflammation. It offers direction for future studies that can positively identify an adipocyte-secreted anorectic factor peptide and work to define the manner in which local and systemic inflammation can be uncoupled from adipose tissue hypertrophy.

Besides age, inheritance of the apoE-E4 allele is the main risk factor for late-onset AD. To determine the role of apoE in amyloid deposition, we studied mice expressing both mutant human amyloid [beta]-protein precursor (APP) and presenilin 1 (PS1) that were either normal or knocked-out for apoE. By 7 months, amorphous A[beta] deposition developed equally in both lines, indicating that A[beta] alone is sufficient for deposition to occur. In contrast, filamentous amyloid deposition was catalyzed at least 3000 fold by apoE. Electron micrographs further illustrate the filamentous nature of these plaques. These results and other, behavioral, data indicate that the primary function of apoE in AD is to promote the polymerization of A[beta] into mature, neurotoxic, amyloid. ApoE is also synthesized in the liver and is crucial in cholesterol metabolism, for mice lacking apoE exhibit hypercholesterolemia. We investigated neuropathology in mice using an uncommon technique, parabiosis, to determine whether apoE in the peripheral circulation influences brain amyloid formation. This surgical procedure allows exchange of proteins via peripheral circulation. We show that plasma apoE is found in parabiosed PS/APP/apoE-KO mice, rescuing their hypercholesterolemia. Unexpectedly, amyloid deposition is reduced in parabiosed PS/APP/apoE-KO mice compared to PS/APP controls. ApoE in the periphery seems to slightly reduce amyloid burden, by likely promoting efflux of A[beta];from the brain. These findings reinforce that the mechanisms whereby apoE affects A[beta] metabolism are complex, and the modulation of peripheral apoE metabolism is not likely to impact AD neuropathology. Since cognitive stimulation is associated with lower risk of AD, we sought to investigate the preventative potential of environmental enrichment (EE) using our mouse model.

Thesis (Ph.D.)--University of South Florida, 2005. / Includes vita. Includes bibliographical references (leaves 138-156).