O isolamento do vírus da raiva (genótipo 1) a partir de morcegos não hematófagos está se tornando cada vez mais freqüente nas grandes cidades do Brasil. Este trabalho foi delineado para investigar a patogenicidade de um isolado do vírus da raiva, do morcego insetívoro Lassiurus ega, procedente do município de Presidente Prudente SP, em comparação ao vírus fixo da raiva, amostra CVS/32 (Challenge Vírus Standard). Os vírus foram reativados por meio de inoculação intracerebral em camundongos e os experimentos de patogenicidade foram realizados em camundongos e hamsters, desafiando-os pelas vias intramuscular (IM), intradérmica (ID), intranasal (IN) e abrasão superficial da pele (AP). A presença do vírus nos cérebros de animais que haviam manifestado sinais compatíveis à raiva, foi confirmada mediante a prova de imunofluorescência direta. Foram considerados para a avaliação da patogenicidade o quadro clínico, proporção de mortos, e a duração dos períodos de incubação (PI) e clínicos (PC) em dias. Em hamsters, o isolado de L. ega exibiu um quadro furioso, com proporção total de mortos de 2.60%; assim discriminada: 2.08% IM (PI: 11 dias; PC: 6 dias) e 8.33% IN (PI:10.66 ± 1.15 e PC: 7.33 ± 1.54). A presença do vírus no SNC foi detectada apenas em animais inoculados por essas vias. Por outro lado, o vírus CVS produziu um quadro paralítico com mortalidade total de 39.84%, com a seguinte distribuição: 62.50% IM (PI 7.50 ± 2.33; PC: 5.13 ± 1.89) 78.12% ID (PI 9.13 ± 2.23; PC 3.88 ± 2.23) 18.75% IN (PI 12.00 ± 2.77; PC 7.14 ± 2.54). Em camundongos, o isolado do L. ega manifestou sinais de agressividade e a raiva foi confirmada em animais inoculados IM e IN. A proporção de mortos observados em camundongos foi de 50.00% (PI 16.80 ± 2.20; PC 1.4 ± 0.54) e 30% (PI 14 ± 4.35; PC: 2.66 ± 0.57) respectivamente e o vírus CVS produziu mortalidade de 45.00% (PI: 6.30 ± 0.67; PC: 1.5 ± 0.70), 70% (PI: 7.14 ± 1.34; PC 2.28 ± 1.25) e 30% (PI:10.00; PC:1) pelas vias mencionadas acima, com quadro clínico de paralisia. O isolado de L. ega mostrou diferenças na proporção de mortos e quadro clínico furioso quando comparado com o CVS nos dois modelos animais. Os resultados sugerem que o contato com os morcegos insetívoros infectados pelo vírus da raiva representa um risco de transmissão da doença, por meio de ferimentos superficiais da pele provocadas pelas mordeduras ou ainda pela via respiratória, supostamente por meio de aerossóis. Pelo sequenciamento completo da proteína G viral do isolado do L. ega, foram observadas substituições na seqüência de aminoácidos nos sítios antigênicos AI, AII, assim como no domínio de fusão dependente de baixo pH. Os resultados obtidos, sugerem que as diferenças no comportamento biológico podem estar associadas às substituições encontradas na sequencia de aminoácidos da proteína G. / The isolation of rabies virus (genotype 1) from the non-hematophagous bats is becoming frequent in heavy urbanized areas in Brazil. This work intended to investigate the pathogenicity of a Brazilian rabies virus isolate from the insectivorous bat Lassiurus ega, from PresidentePrudente-SP, comparing with the CVS/32 (Challenge Virus Standard) fixed rabies virus strain. The viruses were reactivated through intracerebral inoculation into mice and the pathogenicity experiments were made in hamsters and mice, challenged by intramuscular (IM), intradermal (ID) and intranasal (IN) routes and by superficial abrasion of skin. The presence of virus in the brain of animals manifesting the signs compatible with rabies was confirmed by the direct immunofluorescence test. For the evaluation of the pathogenicity, the clinical manifestations, incubation (IP) and clinical (CP) periods in days and the mortality were considered. In hamsters, the isolate of L. ega exhibited a furious form of rabies with total mortality rate of 2.60%, with following distribution: 2.08% IM (IP: 11 days; CP: 6 days) and 8.33% IN (IP: 10.66 ± 1.15 and PC: 7.33 ± 1.54). The presence of rabies virus in the CNS was detected only in animals inoculated through IM and IN routes. The CVS strain has provoked paralytic disease with a total mortality rate of 39.84% as the follow: 62.50% IM (IP 7.50 ± 2.33; CP: 5.13 ± 1.89), 78.12% ID (IP 9.13 ± 2.23; CP 3.88 ± 2.23) and 18.75% IN (IP 12.00 ± 2.77; CP 7.14 ± 2.54). In mice, the isolate of L. ega manifested signs of aggressiveness and rabies was confirmed in animals that were inoculated intramuscularly and intranasally. The total mortality rate observed in mice was 20%, by the IM route was 50% (IP 16.80 ± 2.20; CP 1.4 ± 0.54) and 30% by the intranasal route (IP 14.00 ± 4.35; CP: 2.66 ± 0.57) respectively. The CVS strain showed a total mortality rate of 45.00% and by the IM route, 100% (PI: 6.30 ± 0.67; CP: 1.5 ± 0.70), by the ID route, 70% (IP: 7.14 ± 1.34; CP 2.28 ± 1.25) and by IN, 30% (IP: 10, 00; C: 1.00) showing signs of paralysis. Compared to the CVS strain, the isolate of L. ega showed difference in mortality rate and signs of aggressiveness were found both in hamster and mouse model. The results suggest that the contact with the insectivorous bats infected with rabies virus would represent a risk of disease transmission, by means of superficial wounds of the skin inflicted by bites or by inhalation of aerosols. By the complete sequencing of the viral G protein of the isolate of L. ega, sequencings of amino acids substitutions were observed at antigenic sites AI, AII, as well as the in domain of fusion dependent on low pH. According to the results, differences in the biological behavior may be associated to the substitutions found in the amino acids sequence of the G protein.

The zona-pellucida (zona, ZP) is an adhesion-refractory, acellular coat enclosing the rapidly growing, free-living mammalian preimplantation embryo which undergoes successive cleavage divisions to form the blastocyst, composed of ICM-cells surrounded by outer TE-cells. For further development, the blastocyst must ‘hatch’ or ‘escape’ out of the zona before it can implant into the endometrium for further development (Fig. 5.1A). Hence, the event of hatching or ‘zona escape’ assumes critical importance for the establishment of a successful pregnancy. The golden-hamster blastocyst offers a very unique paradigm to understand hatching, whereby upon attainment of a fully-expanded state, the blastocyst undergoes a dramatic (and molecularly unexplained) deflation event, followed by appearance of TE-derived dynamic cellular projections called TE-projections, whose appearance in an embryonic-stage and -time dependant manner suggest an intimate association with the hatching phenomenon (Fig. 5.1B). Thirdly, embryo-derived zonalytic proteases have been shown to bring about a focal-lysis of the ZP followed by global zona dissolution. Earlier work in the laboratory had demonstrated the intimate involvement of signaling molecules like LIF, HB-EGF, TGF-β and (ER)-α with hatching (Seshagiri et al., 2002, 2009). Investigations also revealed the involvement of cysteine-proteases of the cathepsin (cts) family, especially cts-L, -B and-P to be involved in zona lysis (Sireesha et al., 2008). In order to achieve a better understanding of mammalian preimplantation development, especially hatching, it was important to investigate the role and impact of other critical regulators of developmental and reproductive physiology. COX-2 is one such key signaling moiety and it was decided to investigate the role, if any, of COX-2 and its derived PGs in hamster peri-implantation events. COX-2 transcripts and immunoreactive COX-2 protein were detected in the different preimplantation stages, from 8-cell onwards. COX-2 protein was abundant in both the ICM and TE, but was especially enriched in the TE-cells of the late blastocyst. In order to investigate the function of this enzyme in preimplantation development and hatching, two very-specific inhibitors of COX-2 catalytic action, NS-398 and CAY-10404, were tested in identical concentrations of 25, 50 and 75 μM on in vitro cultured hamster blastocysts. In order to assess the impact of COX-2 inhibition on an embryo-stage and time-dependant manner, inhibitors were tested on freshly recovered 8-cell embryos or early blastocysts, continuously for 72 h or 48 h, respectively. COX-2-selective inhibitors inhibited hamster blastocyst hatching in a dose-dependant manner with maximum inhibition observed in the 75 μM dose. Surprisingly, there was a profound dose-dependent failure of deflation of late-blastocysts upon inhibitor treatment and embryos which hatched, did so in an inflated state and retained intact zonae in cultures. Moreover, embryos subjected to NS-398 treatment phenocopied those subjected to CAY-10404 treatment. Results demonstrate that the effect of inhibitors, and hence the need for COX-2 mediated signaling events is more pronounced in 8¬cell embryos than with early-blastocysts, indicating that COX-2 dependant molecular and cellular processes required for blastocyst morphogenesis and ZP-lysis may have been initiated prior to compaction and cavitation. The reversal of effects of COX-2 inhibition on hatching with exogenous addition of PGE2 and Iloprost (a stable PGI2 analogue) to inhibitor cultures, show that COX-2-derived eicosanoids could, in effect bring about hamster hatching, which is in agreement with previous reports (Davis et al., 1999) and augment peri-implantation development including hatching (Huang et al., 2003). Additionally, it has been successfully demonstrated that PGE2 was superior to PGI2 in augmenting blastocyst hatching in inhibitor-cultures. In this study, the modulation of critical cts-L, -B, -P proteases in COX-2 mediated hamster zona hatching has been verified by quantifying cts in transcripts in control and inhibitor-subjected embryonic samples which was further substantiated by the decreased intra-embryonal protein levels of cts-L and -P. These results demonstrate that COX-2 mediated signaling components directly and effectively modulate hamster preimplantation development, especially zona-hatching phenomenon by transcriptional-regulation of the critical zonalytic proteases. Another potential hatching-associated molecule i.e., NF-κB which is known to exert a great deal of influence on overall reproductive and developmental biology, was investigated in this study. Its specific effects on mammalian preimplantation development, especially hatching, remain totally uninvestigated. This formed the rationale to investigate the reach and impact of NF-κB signaling network in the modulation of peri-hatching events. Transcripts and immunoreactive NF-κB protein of crucial pathway-components like IKK, IκB-β and RelA were detected from 8-cell embryo to the zona-free blastocyst. In order to ascertain the impact of NF-κB signaling on peri-hatching events, two very-specific inhibitors of the NF-κB pathway, BAY-11-7082 and JSH-23 were employed which acted at two strategic signaling points. In order to assess the impact of NF-κB inhibition on an embryo-stage and time-dependant manner, inhibitors were tested on freshly recovered 8-cell embryos or early blastocysts, continuously for 72 h or 48 h, respectively. NF-κB-selective inhibitors inhibited blastocyst hatching in a dose-dependent manner. Interestingly, a profound dose-dependent failure of deflation of late-blastocysts upon inhibitor treatment was observed and embryos which hatched did so in an inflated state and also retained intact zonae in cultures. Moreover, embryos subjected to BAY-11-7082 treatment phenocopied those subjected to JSH-23 treatment, indicating specificity of inhibitor action. Time-course experiments demonstrated that the need for efficient NF-κB mediated signaling is distinctively more for 8-cell embryos than early-blastocysts, indicating that NF-κB dependant molecular and cellular processes required for blastocyst morphogenesis and ZP-lysis may have been initiated prior to compaction and cavitation. Moreover, modulation of zonalysins cts-L, -B and -P by NF-κB-signaling, during the event of zona lysis, both by real-time quantitation of its transcripts and intracellular protein levels has been demonstrated. These results demonstrate that NF¬κB mediated signaling components directly and effectively modulate hamster preimplantation development, especially zona-hatching phenomenon by transcriptional-regulation of the critical zonalytic proteases. The profound inhibition of hatching and effects on blastocyst morphogenesis observed by inhibition of COX-2 and NF-κB signaling systems demonstrate a fundamental need of the growing embryo for these critical signaling moieties. Moreover, the underlying similarity of consequences obtained upon inhibition of both signaling networks i.e., NF-κB and COX-2, perhaps indicate a linear mode of signaling between these principles. It remains to be tested, though, if it really is the case. A striking observation made in this study was the detection of immunoreactive signals for critical signaling moieties like ER-α, COX-2 and RelA onto TEPs of the deflated hamster blastocyst, in addition to earlier TEP-localisation of cathepsins. A B C (Figure) ENDOMETRIUM ENDOMETRIUM ENDOMETRIUM Fig 5.1. Schematic representation of the role of molecular and cellular factors in the regulation of concordant phenomena of mammalian blastocyst hatching and endometrial implantation. (A) Depicts a zona-intact well-formed blastocyst. Preimplantation embryo development and blastocyst formation involves close cooperation between several molecular principles (discussed in sections 1.3.1 to 1.3.3), (B) as the embryo prepares to hatch, prior to implantation, it initiates egression from the non-adhesive ZP coat by cathepsin (cts) protease-mediated lysis of zona (pink circles); there is concomitant appearance of cellular principles such as TEPs (undulating projections shown in green). Of interest is the intimate association of hatching-promoting molecules such as COX-2, NF-κB, ER-α, Cts etc. with the TEPs. (C) depicts a zona-free, TEP-rich blastocyst initiating implantation into the maternal endometrium. It is possible, that the embryonic TEPs with the associated hatching-regulatory molecules are also critical for implantation phenomena during the embryo-maternal recognition and implantation during the establishment of early pregnancy. Preliminary results indicate that TEPs could be the site of membrane lipid-rafts, focal points of membrane-based signaling. The definitive role of TEPs in peri-hatching events is yet to be confirmed, but it is presumed that these actin-based undulating structures, harboring several key molecules involved in peri-implantation events in the embryo as well as the maternal uterus could be instrumental in successfully bringing about the concomitant processes of hatching and implantation. Interestingly, during rodent implantation (hamster, guinea-pig, mouse and rat), the blastocyst orients in such a way that the ICM is oriented away from the endometrium and, at least in the hamster, the TEP-carrying abembryonic (mural) pole remains closest to the luminal epithelium (LE) (Gonzales et al., 1996b; Seshagiri et al., 2009; Fig. 5.1C). In contrast, in humans and other primates, the embryonic pole is closest to LE before implantation (Kirby, 1971; Lee and DeMayo, 2004). Although direct evidence is lacking, but these observations gives rise to a possibility that both hatching and implantation could be intimately related to the polar appearance of TEPs in the embryo. Several key signaling molecules like ER-α, LIF, HB-EGF and TGF-β have been already demonstrated to play crucial roles in mammalian hatching. In this thesis, we have exemplified the need for COX-2 mediated prostanoid signaling and the pleotropic NF-κB signaling system in bringing about mammalian blastocyst hatching. How exactly do these molecular entities communicate among themselves and with cellular principles like TEPs thereby effectively enabling peri-implantation development, remain to be understood. Taken together, these results demonstrate, for the first time, the involvement of embryo-derived signaling molecules, like COX-2 and NF-κB in an embryo stage-and time-dependant manner in mammalian peri-implantation events, especially blastocyst hatching. The association of TEPs with key molecules common to embryonic and maternal preparation for hatching and implantation, respectively, indicates towards a molecular and cellular continuity between the concomitant events. These fundamental findings on hamster blastocyst biology have profound clinical implications in the management of human infertility. (For figures pl see the abstract file).

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Quantitative relationships between blood flow velocity, vessel diameter, width of the peripheral plasma layer and induction of thrombus formation were studied in small blood vessels of the retrolingual membrane and intestinal mesentery of the frog, Rana Pipiens, and of the cheek pouch of the golden hamster, Mesocricetus auratus. Blood flow velocity was measured by a modification of the technique described by Hugues (Arch. Int. de Physiol. 61: 565, 1953). Internal vessel diameters and widths of the total peripheral plasma layer were measured with an ocular micrometer. Thrombus thresholds were determined by graded electrical stimulation. Determinations were made on 202 vessels in retrolingual membranes of frogs prepared by single pithing. In 95 arterioles (diameters from 10 to 40 microns), the mean flow velocity was 2.84 mm/sec. and the mean volume flow rate, 1.68xl0-3 cu mm/sec. The mean width of the total peripheral plasma layer was 3.6 microns and its ratio to mean vessel diameter was 1:6. In 107 venules (diameters from 10 to 50 microns), the mean flow rate was 1.20 mm/sec. The mean volume flow rate was 1.02x10-3 cu mm/sec. The mean width of the total peripheral plasma layer was 4.1 microns and its ratio to mean vessel diameter was 1:7.4. The ratios of mean arterial to mean venous velocity and volume flow were 1:2.3 and 1:1.6 respectively. Measurements were made on 100 mesenteric vessels of frogs prepared by single pithing. In 50 arterioles (diameters from 15 to 45 microns), the mean velocity was 3.77 mm/sec. The mean volume flow was 3.05xlo-3 cu mm/sec. The mean width of the total peripheral plasma layer was 2. 7 microns and its ratio to mean vessel diameter was 1:11.5. In 50 venules (diameters from 20 to 50 microns), the mean velocity was 1.55 mm/sec. and the mean volume flow rate, 1.76x10-3 cu mm/sec. The mean width of the total peripheral plasma layer was 6.7 microns and its ratio to mean vessel diameter was 1:5.3. The ratios of mean arterial to mean venous velocity and volume flow were 1:2.3 and 1:1.7 respectively. A total of 109 mesenteric vessels were studied in frogs anesthetized with urethane. In 55 arterioles (diameters from 15 to 45 microns), the mean velocity was 3.28 mm/sec. The mean volume flow was 2.87x10-3 cu mm/sec. The mean width of the total peripheral plasma layer was 3.2 microns and its ratio to mean vessel diameter was 1:9.3. In 54 venules (diameters from 20 to 50 microns), the mean flow rate was 1.61 mm/sec. and the mean volume flow, 1.83x10-3 cu mm/sec. The mean width of the total peripheral plasma layer was 5.6 microns and its ratio to mean vessel diameter was 1:5.9. The ratios of mean arterial to mean venous velocity and volume flow were 1:2.0 and 1:1.5 respectively. In these three series of studies in the frog, no relationship was clearly apparent between velocity and either vessel diameter or width of the peripheral plasma layer in arterioles. Suggestions of a direct relationship between velocity and peripheral plasma layer in veins, however, were evident. In all cases, and in both arterioles and venules, vessel diameter and peripheral plasma layer were clearly and directly related. The effects of flow velocity changes on width of the total peripheral plasma layer in individual vessels were studied in 26 arterioles of the hamster cheek pouch. Blood flow was varied by means of a cuff described by Copley (Biorheology 1: 3, 1962). A direct relationship between velocity and width of the peripheral plasma layer was clearly demonstrated. Thrombus thresholds were determined in mesenteric vessels of the frog. In 103 arterioles (diameters from 20 to 140 microns), the mean strength of stimulus necessary to produce a platelet thrombus was 24.8 volts with an amperage of 0.18 milliamperes. In 100 venules (diameters from 20 to 140 microns), the mean strength of stimulus necessary was 20.7 volts with an amperage of 0.12 milliamperes. Possible relationships of thrombus thresholds to flow velocity in mesenteric arterioles and venules are discussed. / 2031-01-01

by Ng Yuen Keng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 141-148). / Table of contents --- p.1 / List of figures --- p.5 / List of tables --- p.12 / List of abbreviations --- p.13 / Abbreviation table for amino acids --- p.16 / Chapter Chapter 1 --- Literature Review --- p.17 / Chapter 1.1 --- Introduction --- p.17 / Chapter 1.2 --- The Hematopoietin/cytokine receptor superfamily --- p.20 / Chapter 1.3 --- The PRLR protein --- p.22 / Chapter 1.3.1 --- The receptor size --- p.22 / Chapter 1.3.2 --- Primary structure --- p.22 / Chapter 1.3.3 --- Structure of the extracellular domain --- p.26 / Chapter 1.3.4 --- Structure of the cytoplasmic domain --- p.30 / Chapter 1.3.5 --- Characteristics of specific PRL binding to PRLR --- p.32 / Chapter 1.5 --- The PRLR gene --- p.33 / Chapter 1.6 --- Heterogeneity of PRLR --- p.33 / Chapter 1.7 --- Signal transduction of PRLR --- p.35 / Chapter 1.7.1 --- JAK: a novel family of cytoplasmic protein tyrosine kinases --- p.35 / Chapter 1.7.2. --- Interaction between JAK2 and PRLR --- p.37 / Chapter 1.7.3 --- STAT proteins: mediators of PRL-dependent gene transcription --- p.37 / Chapter 1.7.4 --- Other signaling pathways of PRLR --- p.38 / Chapter 1.7.5 --- Future prospects on PRLR signaling --- p.38 / Chapter 1.8 --- Regulation of PRLR gene expression --- p.39 / Chapter 1.9 --- Objective of cloning the PRLR cDNA in male Syrian golden hamster --- p.42 / Chapter Chapter 2 --- PCR cloning of hamster PRLR cDNA fragment from adult male hamster liver --- p.44 / Chapter 2.1. --- Introduction --- p.44 / Chapter 2.2. --- Materials and Methods --- p.45 / Chapter 2.2.1 --- Primer design and PCR strategy --- p.45 / Chapter 2.2.2 --- Collection of liver --- p.46 / Chapter 2.2.3 --- Reverse transcription of polyadenylated RNA --- p.46 / Chapter 2.2.4 --- Nested PCR --- p.47 / Chapter 2.2.5 --- Southern analysis of the PCR products --- p.48 / Chapter 2.2.6 --- Subcloning of PCR product --- p.49 / Chapter 2.2.7 --- Sequence determination of the positive recombinant clone --- p.49 / Chapter 2.2.8 --- Sequence alignment and homology comparison --- p.50 / Chapter 2.3 --- Results --- p.55 / Chapter 2.3.1 --- Nucleotide sequence alignment and primer design --- p.55 / Chapter 2.3.2 --- Nested PCR --- p.55 / Chapter 2.3.3 --- Subcloning of the PCR product --- p.56 / Chapter 2.3.4 --- Analysis of nucleotide and predicted amino acid sequences --- p.56 / Chapter 2.4 --- Discussion --- p.66 / Chapter Chapter 3 --- Nucleotide sequence determination of the 5' and the 3' ends of hamster PRLR cDNA --- p.69 / Chapter 3.1 --- Introduction --- p.69 / Chapter 3.2 --- Materials and Methods --- p.71 / Chapter 3.2.1 --- Collection of liver --- p.71 / Chapter 3.2.2 --- Total RNA preparation and poly (A) + RNA isolation --- p.72 / Chapter 3.2.3 --- Double stranded cDNA synthesis --- p.73 / Chapter 3.2.4 --- Adaptor ligation --- p.74 / Chapter 3.2.5 --- 5´ة and 3' RACE PCR --- p.74 / Chapter 3.2.6 --- Cloning of the RACE PCR products --- p.76 / Chapter 3.2.7. --- Sequence determination of the RA CE PCR products --- p.77 / Chapter 3.2.8. --- Sequence analysis of the RACE PCR products --- p.78 / Chapter 3 .2.9 --- Northern blot analysis of hamster PRLR mRNA in male hamster tissues --- p.79 / Chapter 3.3 --- Results --- p.79 / Chapter 3.1.1 --- RNA preparation and double stranded cDNA synthesis --- p.79 / Chapter 3.3.2 --- RACE PCRfor the 5' and the 3' ends of hamster PRLR cDNA --- p.84 / Chapter 3.3.3 --- Cloning of the 5' and 3'RACE PCR products --- p.92 / Chapter 3.3.4 --- Sequence determination of the RACE PCR products --- p.92 / Chapter 3.3.5 --- Nucleotide sequence analysis of hamster PRLR full length cDNA --- p.101 / Chapter 3.3.6 --- Northern blot analysis of hamster PRLR --- p.101 / Chapter 3.4 --- Discussion --- p.106 / Chapter Chapter 4 --- Attempts to study the PRLR gene expression in male hamster tissues --- p.113 / Chapter 4.1 --- Introduction --- p.113 / Chapter 4.2 --- Materials and Methods --- p.115 / Chapter 4.2.1 --- Collection of tissues --- p.115 / Chapter 4.2.2 --- Total RNA preparation and poly (A)+ RNA isolation --- p.116 / Chapter 4.2.3 --- Reverse Transcription --- p.116 / Chapter 4.2.4 --- Polymerase chain reaction for detecting the presence of hamster PRLR cDNA in various tissues --- p.117 / Chapter 4.2.5 --- Nested PCR for detecting heterogeneity in PRLR cDNA sizes in various tissues --- p.117 / Chapter 4.2.6 --- Analysis and quantitation of PCR products --- p.118 / Chapter 4.3 --- Results --- p.119 / Chapter 4.4 --- Discussion --- p.134 / Chapter Chapter 5 --- General Discussion --- p.137 / References --- p.141 / Appendices --- p.149 / Chapter I. --- "Stock solution preparation (Sambrook et al., 1989)" --- p.149 / Chapter II. --- List of primers --- p.152 / Primers for sequence determination --- p.152 / "Primer for first strand cDNA synthesis and 3' RACE PCR (Frohman et al., 1988 and Loh et al.,1989)" --- p.152 / "Primers for amplifying the actin cDNA fragment (Chan et al.,1995)" --- p.152 / Primers used for PCR-cloning and semi-quantitative analysis of hamster PRLR cDNA --- p.153 / Chapter III. --- "First strand cDNA synthesis primer, cDNA adaptor and adaptor primers used in the 5' and3' end sequence determinations of hamster PRLR cDNA" --- p.154 / Chapter IV. --- "Multiple cloning sites of the pCRII (Invitorgen), pUC 18 (Pharmacia) and pBluescript SK+ vectors (Clontech)" --- p.155 / Chapter VI. --- Nucleic acid molecular weight size markers --- p.158

A Toxoplasmose é uma zoonose cosmopolita que ocorre principalmente em indivíduos imunodeprimidos, gestantes e em pacientes em terapia anti-câncer. Na Medicina Veterinária, animais como ovelhas, cabras e porcos são seriamente afetados podendo ocorrer abortos e com isto perdas significativas em termos econômicos. Atualmente não se dispõem de vacinas contra a toxoplasmose, mas tem-se desenvolvido modelos animais em ratas e camundongos para o ensaio imunológico contra a enfermidade. Os modelos atualmente utilizados, possuem limitações, por exemplo, não é possível ensaiar no modelo rata um imunógeno de Toxoplasma gondii, uma vez que todas as linhagens de ratas disponíveis são resistentes. Portanto não se pode ensaiar desafios vacinais altamente exigentes neste modelo. Por isso é aconselhável contar com experimentos efetuados em diversas espécies animais. Está sendo proposto testar o Hamster (Mesocricetus auratus), com o intuito de estabelecer se é ou não um modelo que se aproxima das respostas esperadas nos humanos.Antes de se comprometer a custos muito elevados com ensaios finais de vacinações, o aconselhável é contar com ensaios em diversas espécies, pois os animais respondem de formas dIferentes aos mesmos imunógenos. Na transmissão congênita crônica dez Hamsters foram inoculados com 40 oocistos por via oral da cepa Me-49 do T.gondii e após sessenta dias foram postas para cruzarem com os machos. Obteve-se resultados promissores uma vez que a transmissão congênita aos seus filhotes nesta fase crônica da infecção foi nula. Na transmissão congênita aguda da infecção, obteve-se 42,9% de taxa de transmissão de T.gondii. Não foi observada a transmissão lactogênica, do toxoplasma, na fase aguda da infecção. / Taxoplasmosis is a cosmopolitan disease that causes a lot of damage to immunocompromised people, pregnant women, patients who have had transplants and people undergoing anticancer therapy. Animals like sheep, goats and pigs are seriously affected causing abortions and consequently significant economic losses. This is why, the importance of studing the transmission, the mechanisms of transmission and the forms of infection. Currently, vaccines against toxoplasmosis are not available, but animals’ models, in rats and mice, have been developed to an immune assay against this disease. These models considered to the study have some limitations, however it is not possible to apply a toxoplasma immune assay on a rat model, because it is a susceptible race and it has already been proven that rats are resistant to toxoplasma. We proposed to test the hamster (Mesocrisetua auratus) with the intention of establishing if it is or not one model that approaches the same results expected on human beings, therefore before compromising high costs with final assays of vaccinations, the advisable thing to do is to turn to assays on diverse species, because animals have different answers to the same immune. We intended to test hamster (Mesocrisetus auratus), with the intention of establishing if it is or no a model that approaches the expected ansewer from diferential ways to the same strains. In the chronic congenital transmit, in Hamsters was obtained promising results because yours creates didn’t present title when their organs (liver and lung), they were bioassayed in mice and twenty five days after they were bled and their serum tested with direct agglutination of Desmonts and Remington (1980), confirming that the passage wasn’t made during the gestation of females infected before the conception. In the sharp congenital transmission of the infection, it was obtained 42,9% that passed to their descendants antibodies of T. gondii. In the lactogenic transmission, wasn’t have title for T.gondii

No description available.

Four in vitro models were used to measure the antioxidant activity of 11 citrus phytochemicals. The citrus limonoids and bergapten showed very weak antioxidant activity. The flavonoids demonstrated mild, to moderate, to strong antioxidant activity. In addition to some other commonly accepted structural features our data indicated that the hydroxyl group in position 6 of ring A could also increase the antioxidant activity of flavonoids. Compared with the active flavonoids, limonoids are highly oxygenated triterpenoids, with fewer hydroxyl groups to stabilize unpaired electrons (or scavenge free radicals). Bergapten lacks a hydroxyl group. This is the first report on the antioxidant activity of limonoids and neoeriocitrin. A feeding study using Syrian hamsters was followed to determine the effect of citrus limonoids and flavonoids on plasma cholesterol. Hamsters fed with limonin, limonin 17-Beta-D-glucopyranoside and grapefruit pulp significantly inhibited the increase of LDL/HDL-cholesterol (36.6%, 52.9% and 57% respectively) compared with the basal control (65.8%) and the pectin control (70%). Furthermore, hamsters fed with limonin had significantly larger LDL particle size (21.21 nm) compared with the control group (19.96 nm). Further studies demonstrated that LDLs from hamsters fed with limonin and limonin 17-Beta-D-glucopyranoside were less susceptible to oxidation. These data suggest that limonin, limonin 17-Beta-D-glucopyranoside and grapefruit pulp have potential inhibitory effects against atherogenesis. Supercritical CO2 (SC-CO2) was attempted to extract limonoids from grapefruit seeds and molasses. Limonin aglycone was successfully extracted with SC-CO2 directly from grapefruit seeds with the yield of 6.3 mg/g seeds at 48.3 MPa, 50˚C and 60 min with CO2 top feeding; and the limonin glucoside was extracted using SC-CO2 and ethanol as co-solvent from the defatted seeds with the yield of 0.73 mg/g seeds at 42 MPa, 52˚C, 45% ethanol (XEth=0.45) and 40 min with CO2 top feeding; and limonin glucoside also was extracted using SC-CO2 and ethanol with the yield of 0.61mg/g grapefruit molasses at 48.3 MPa, 50˚C and 10% ethanol (XEth=0.1), 40 min with CO2 top feeding. CO2 flow rate was around~5 l/min in experiments. The results demonstrated SC-CO2 extraction of limonoids from citrus juice industry byproducts has practical significance for future commercial production.

Whey protein isolates (WPI) have been indicated to have potent cholesterol lowering and antioxidative properties. Such effects, however, are not consistently observed, which could be the result of major differences in the processing, isolation and composition of WPI. Moreover, the mechanisms of action or the bioactive component(s) in WPI are poorly understood although the relatively high cysteine content in WPI has been suggested to play an important role. Although high dietary cysteine has been shown to lower plasma homocysteine concentrations, the impact of WPI in this regard has not been investigated. The overall objective of this thesis was to examine the antioxidative and plasma cholesterol and homocysteine lowering properties of two WPI that were produced via different industrial processing and isolation techniques with the milk protein, casein, used as the control protein. We also examined for the mechanism(s) of action of WPI in terms of possible antioxidative, and plasma cholesterol and homocysteine lowering effects. In this regard, the intake of bovine serum albumin (BSA), a major cysteine-rich whey protein was also studied since this protein has been implicated as a key bioactive component for the antioxidant effects of WPI. Four studies were performed. The first involved the characterization of a variety of commercially prepared WPI by high performance capillary electrophoresis for identification of two WPI products that showed major differences in protein composition for subsequent feeding trials. Most of the WPI had similar characteristic electrophoretic profiles, however, significant differences in protein and macronutrient (Ca, Mg, P) composition were noted in two commercial WPI that were chosen as the test proteins in subsequent feeding trials. In the first two feeding studies, hamsters were fed different commercial WPI or milk protein (BSA or casein) containing diets that were either matched or unmatched in terms of macro

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