Thesis (MHSc--Health Science) -- Auckland University of Technology, 2004. / Also held in print (218 leaves, col. ill., 30 cm.) in Akoranga Theses Collection (T 616.46200996 SIM)

Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 116-140).

Thesis (Ph. D.)--University of Lund, 1994. / "ISRN LUMEDW / MEMM-1037-SE."

Thèse (M.Sc.)--Université Laval, 2004. / Bibliogr.: f. [102]-111. Publié aussi en version électronique.

Thesis (Ph. D.)--University of Lund, 1994. / "ISRN LUMEDW / MEMM-1037-SE."

Orientador: Iracilda Zeppone Carlos / Banca: Idomeo Bonetti Filho / Banca: Fábio Luiz Camargo Villela Berbert / Banca: Ivaldo Gomes de Moraes / Banca: Abílio Albuquerque Maranhão de Moura / Resumo: Os macrófagos constituem uma população celular do sistema imune. Estas células podem ser ativadas por uma variedade de estímulos e suas principais funções incluem a fagocitose de partículas estranhas, apresentação de antígenos, produção de citocinas e compostos intermediários do nitrogênio (NO) e do oxigênio (H202). Os cimentos endodônticos são capazes de promover uma estimulação do sistema imune. Neste estudo, foram analisados os níveis de quantificação das citocinas, além do mediador óxido nítrico, como uma medida de estimulação de macrófagos peritoneais de camundongos. Através de análise estatística dos dados, foram observados os níveis de citotoxicidade dos macrófagos de camundongos estimulados pelos diferentes cimentos endodônticos, meio RPMI-1640 (grupo controle -) e LPS (grupo controle +). Os diferentes cimentos testados apresentaram concentrações com diferentes citotoxicidades: Sealapex 35ug/ml, Polímero de Mamona 8,75 ug/ml, do Epiphany 17,5 ug/ml, do Epiphany + Primer 17,5 ug/ml, do Primer 35 ug/ml, do EndoRez 17,5 ug/ml e do AH Plus 70 ug/ml. Após a adequação das concentrações viáveis dos cimentos testados conclui-se que o material que mais estimulou a liberação de NO foi Primer, seguido do Endorez, AH Plus, Ephiphany, Sealapex, Epiphany + Primer. O Polímero de Mamona foi o que estimulou a uma menor produção de NO. Em relação à produção de TNF-alfa o material que estimulou maior produção foi o Primer, seguido de Epiphany, AH Plus, Epiphany + Primer, Sealapex e Polímero de Mamona. O EndoRez não foi capaz de estimular a produção de TNF-alfa. Nenhum dos cimentos testados induziu à liberação de IFN-y, sugerindo que outro mediadores tais como IL-1 e IL-12 possam estar envolvidos na liberação de NO observada no presente estudo. / Abstract: It was evaluated the citotoxicity of the sealers, Sealapex, Polímero de Mamona, Epiphany, EndoRez and AH Plus in relation to the release of Nitric Oxide, Tumor Necrotic Factor-Alpha and Interferon Gamma in murine cells culture. After the ideal concentration was found, according to MTT test, it was conduded that the sealers with higher release were Polímero de Mamona, EndoRez, Epiphany + Primer, Epiphany, Primer do Epiphany = Sealapex and AH Plus. All sealers reached lower levels of citotoxicity than control. / Doutor

Une immunité concomitante à long terme est mise en place lors d’infections persistantes avec des parasites protozoaires intracellulaires responsables, par exemple, de la leishmaniose et du paludisme. Dans un modèle murin de leishmaniose, il a ainsi été démontré que les cellules T régulatrices CD4+CD25+ sont impliquées dans la persistance des leishmanies aux sites d’infection cutanés et protègent l’hôte contre une ré-infection.Le protozoaire Toxoplasma gondii est également à l’origine d’une infection chronique liée à l’installation du parasite dans le cerveau et les muscles de l’hôte dans des formes kystiques. Il était donc pertinent de s’intéresser à l’implication des cellules T régulatrices dans l’installation et la persistance du toxoplasme.Pour atteindre cet objectif, nous avons utilisé l’anticorps monoclonal anti-CD25 dans des expériences de déplétion pendant la phase aiguë de la toxoplasmose après infection de souris non consanguines avec une souche de toxoplasmes de type II. Aucune différence significative que ce soit en terme de mortalité ou de charge parasitaire cérébrale n’a été observée entre les souris infectées et déplétées et les souris infectées non déplétées. En complément de ces expériences, nous avons pu montrer que les cellules régulatrices CD4+CD25+Foxp3+ (Treg) sont une cible potentielle de l’anticorps anti-CD25 ainsi que les cellules T effectrices CD4+CD25+Foxp3- (Teff); cellules qui expriment le marqueur CD25 en phase aiguë de l’infection. / Long term concomitant immunity is developed in case of persistant infections with intracellular protozoan parasites like for example in leishmaniosis and malaria. In a murine model of leishmaniosis, it has been demonstrated that CD4+CD25+ regulatory T (Treg) cells are involved in the persistance of leishmania parasites at cutaneous sites of infection and protect the host against re-infection.The protozoan parasite Toxoplasma gondii is also responsible for a chronic infection associated with the settlement of parasite in the brain and the muscles of the host in the form of cysts. It was therefore pertinent to know about the implication of Treg cells in the development and the persistance of toxoplasma. To attain this objective, we have used a monoclonal antibody anti-CD25 in depletion experiments during the acute phase of toxoplasmosis after infection of outbred mice with a type II toxoplasma strain. No significant difference was found in terms of mortality or in brain cyst load between depleted mice and non-depleted mice. In addition to these experiments, we have shown that not only the CD4+CD25+Foxp3+ regulatory T (Treg) cells but also the CD4+CD25+Foxp3- T effector (Teff) cells are a potential target of anti-CD25 antibody-depletion. These cells are induced to express CD25 during acute phase of the infection.

Restriction Modification (R-M) systems prevent the invasion of foreign genetic material into bacterial cells and are therefore important in maintaining the integrity of the host genome. The spread of antibiotic resistance, which is proposed to occur via the transfer of foreign genes to the bacterial genome, makes the subject of R-M systems extremely relevant. R-M systems are currently classified into four types (I to IV) on the basis of differences in composition, target recognition, cofactors and the manner in which they cleave DNA. Kennaway et al (2012) proposed that there is an evolutionary link between Types I and II. Comparing the structures of examples from two of the subfamilies of Type II systems (IIB and IIG) to those of Type I structures, similarities can be observed. Due to the fact that Type II R-M systems cut DNA at fixed positions, they can be used to obtain genetic material selectively. They have therefore proven to be invaluable in molecular biology. One aspect of this project aims to create a novel R-M system, a pseudo-Type II system, by removing the molecular motors from the restriction subunit of a Type I system and fusing the remaining nuclease domain to a known Type I methyltransferase (MTase). This will not only provide evidence to support the theory that evolution has produced a pared down form of the Type I systems in the Type II systems, but it may also become a useful biological tool. This thesis describes the several attempts at doing this and how the subsequent constructs were expressed, purified and assayed to varying degrees of success. An important characteristic of the Type I systems is their ability to methylate DNA, and it is the mechanism via which host DNA is protected from restriction. This is another subject investigated in this project. As with the nuclease activity of the Type I systems, the site at which DNA is methylated is dictated by the HsdS subunit. It is described here how this subunit can be altered to change the sequence of DNA that is recognised by the system. Again, using Type II system subtypes as a reference, various mutations were made to the HsdS subunit of an MTase from Staphylococcus aureus. This is in an effort to bring about a new mode of action, but also to provide further evidence for an evolutionary link between the two system types. The HsdM and HsdS subunits are expressed from two separate genes at the same locus. There is a frameshift between the genes where the start of the hsdS gene occurs a few base pairs upstream from the stop codon of the hsdM gene. This work shows that removing this frameshift creates an MS fusion product, and in vivo studies show that this product has methylase activity and can form an active restriction complex when the HsdR subunit is added. The product can also be over-expressed and purified, and shows in vitro restriction activity on addition of the HsdR subunit protein. The HsdS subunit is composed of two target recognition domains (TRDs), each dictating one part of the bipartite recognition sequence. These TRDs can be altered, bringing about a change in the sequence of DNA recognised by the enzyme. In this thesis, it is shown that the C-terminal TRD can be removed and that the subsequent “Half S” enzyme possesses both methylase and restriction activity in vivo and that its recognition sequence is different from that of the wild-type enzyme. After the successful creation of both “MS fusion” and “Half S” recombinant proteins of the Sau1, Type I system from a CC398 strain of Staphylococcus aureus, a further construct was produced. This possesses both in vivo and in vitro activity. The novel “M Half S Fusion” enzyme not only links the two aspects of this project but also creates a structure similar to some seen in the Type II systems. This shows that the Type I systems can be manipulated to change their mode of action but also supports the idea that Types I and II are evolutionarily linked. By making the alterations in a step-wise fashion identifies that these structural changes can create viable enzymes, and that they could have occurred through the process of evolution.

Base teórica: A Mucopolissacaridose II (MPS II) é uma doença genética recessiva ligada ao X causada por mutações no gene IDS. Como consequência, há acúmulo dos glicosaminoglicanos (GAGs) no lisossomo, fato que é responsável pelo fenótipo de MPS II. Anormalidades articulares e ósseas são conhecidas nos pacientes com MPS II e os tratamentos existentes não são eficientes para sanar tais anormalidades, portanto, realizamos este estudo de caracterização da doença articular e óssea, buscando evidenciar possíveis mecanismos responsáveis pela progressão da doença. Objetivo: Avaliar a progressão das alterações osteoarticulares em animais com MPS II dos dois aos oito meses de idade. Métodos: Foram utilizados camundongos nocaute B6N.Cg-Idstm1Muen/J, adquiridos do Jackson’s Lab. Os machos foram genotipados para compor o grupo controle (normal) ou o grupo de animais com MPS II. Ambos foram avaliados aos 2, 4, 6 e 8 meses de idade. Foi realizada análise histológica da articulação tíbio-femural, avaliando presença de infiltrado inflamatório, reabsorção óssea, reabsorção cartilaginosa e proliferação fibrocartilaginosa. Também foi realizada a mensuração do tamanho total da placa de crescimento e suas zonas e avaliação de anormalidades ósseas mediante exame de imagem por Raio-X dos ossos fêmur e zigomático. Resultados: Nos animais MPS II foi observado que o focinho era menos afilado (mais arredondado) e, em comparação com os animais controle, os animais MPS II apresentaram peso significativamente maior a partir dos 4 meses de idade. O escore histológico teve como principal característica a presença de reabsorção cartilaginosa, presente em 80% (4/5 animais) dos animais aos 8 meses, outras anormalidades encontradas neste tempo foram presença de infiltrado inflamatório (2/5 animais aos 8 meses) e proliferação fibrocartilaginosa (1/5 animais) Não houve diferença significativa entre animais normais e MPS II no tamanho das zonas de cartilagem da placa de crescimento ósseo. As medidas em diâmetro do osso zigomático apresentaram-se significativamente superiores nos animais MPS II aos 4, 6 e 8 meses. Quanto ao comprimento do fêmur não houve diferença significativa entre os grupos. Já, na medida da espessura do fêmur, os animais MPS II do grupo de 6 meses de idade mostraram diferença significativa. Conclusões: Anormalidades na articulação tíbio-femural foram detectadas nos animais aos 8 meses de idade. Não foram encontradas anormalidades óbvias na estrutura da placa de crescimento. Foi observado aumento na espessura do fêmur e do zigomático nos animais MPS II, sem alterações do tamanho do fêmur. / Background: Mucopolysaccharidosis II (MPS II) is a recessive X-linked genetic disease caused by mutations in the IDS gene. Consequently, there is an accumulation of glycosaminoglycans (GAGs) in the lysosome, fact that is responsible by the MPS II phenotype. Joints and bone abnormalities are known in MPS II patients and existing treatments are not efficient in correcting these abnormalities. Therefore, this study was performed to evidence bone and joint disease description in the animal model, searching for mechanisms responsible for disease progression. Objective: To evaluate the progression of osteoarticular changes in animals with MPS II from two to eight months of age. Methods: Male animals from the MPS II colony (B6N.Cg-Idstm1Muen/J) were genotyped to form the control (normal) group or MPS II group. Both groups were evaluated at the 2, 4, 6 or 8 months. Histological analysis of knee joint (presence of inflammatory infiltrate, bone resorption, cartilaginous reabsorption and fibrocartilaginous proliferation), measurement of total growth plate size and its zones, and evaluation of bone abnormalities by X-ray imaging of the femur and zygomatic bones were performed. Results: MPS II mice presented progressive abnormal features, such as a more rounded snout and a significative increased weight from 4 months of age. The main histological alteration was the presence of cartilage reabsorption, present in 80% (4/5 animals) of the eighth-month old animals. Other abnormalities found at this period were the presence of inflammatory infiltrate (2/5 animals at the eighth months old) and fibrocartilaginous proliferation (1/5 animals). There was no significant difference in the growth plate between normal and MPS II animals. The zygomatic bone diameter was increased in MPS II at fourth, sixth and eighth months There were no significant differences in femur length between groups. Thickness of the femur was increased in MPS II at six months. Conclusions: Abnormalities in the joint were detected in the animals at 8 months of age. No obvious abnormalities were found in the growth plate structure. An increase in femur and zygomatic thickness was observed in MPS II animals, with no changes in femur size.

abstract: Sb-based type-II superlattices (T2SLs) are potential alternative to HgCdTe for infrared detection due to their low manufacturing cost, good uniformity, high structural stability, and suppressed Auger recombination. The emerging InAs/InAsSb T2SLs have minority carrier lifetimes 1-2 orders of magnitude longer than those of the well-studied InAs/InGaSb T2SLs, and therefore have the potential to achieve photodetectors with higher performance. This work develops a novel method to measure the minority carrier lifetimes in infrared materials, and reports a comprehensive characterization of minority carrier lifetime and transport in InAs/InAsSb T2SLs at temperatures below 77 K. A real-time baseline correction (RBC) method for minority carrier lifetime measurement is developed by upgrading a conventional boxcar-based time-resolved photoluminescence (TRPL) experimental system that suffers from low signal-to-noise ratio due to strong low frequency noise. The key is to modify the impulse response of the conventional TRPL system, and therefore the system becomes less sensitive to the dominant noise. Using this RBC method, the signal-to-noise ratio is improved by 2 orders of magnitude. A record long minority carrier lifetime of 12.8 μs is observed in a high-quality mid-wavelength infrared InAs/InAsSb T2SLs at 15 K. It is further discovered that this long lifetime is partially due to strong carrier localization, which is revealed by temperature-dependent photoluminescence (PL) and TRPL measurements for InAs/InAsSb T2SLs with different period thicknesses. Moreover, the PL and TRPL results suggest that the atomic layer thickness variation is the main origin of carrier localization, which is further confirmed by a calculation using transfer matrix method. To study the impact of the carrier localization on the device performance of InAs/InAsSb photodetectors, minority hole diffusion lengths are determined by the simulation of external quantum efficiency (EQE). A comparative study shows that carrier localization has negligible effect on the minority hole diffusion length in InAs/InAsSb T2SLs, and the long minority carrier lifetimes enhanced by carrier localization is not beneficial for photodetector operation. / Dissertation/Thesis / Doctoral Dissertation Electrical Engineering 2016