Pneumocystis carinii is an opportunistic fungal pathogen that causes lifethreatening pneumonia in immunocompromised individuals. Infants appear to be particularly susceptible to Pneumocystis (PC) pulmonary infections. The higher incidence of PC as well as other pulmonary infections among infants is likely due to an immature immune system. The neonatal lung environment is deficient immunologically in preterm as well as term infants (1, 2). Decreased phagocytic capacity of macrophages in newborns may increase the risk of infection from inhaled pathogens (1, 2). We have previously demonstrated that there is approximately a 3-week delay in the clearance of PC organisms from pup mouse lungs compared to adults. Herein, we demonstrate that there is also a 1-week delay in the infiltration of AMs in pup compared to adult PC-infected mice. We go on to show that there is a delay in pup versus adult lung macrophage phenotypic expression and cytokine production in response to PC organisms. We demonstrated that pup AMs are competent to produce cytokine in response to LPS and that stimulation with zymosan generates cytokine production in pup AMs that is comparable to adult cytokine production. These data indicate that pup lung macrophages are specifically poorly responsive to PC organisms and likely require exogenous stimulation to mount a significant immune response and expedite clearance of the organism. We go on to show that heat-killed Escheriae coli improves cytokine response, cellular infiltration and reduces organism burden in PC-infected pup mice. The clinically relevant cytokine, GM-CSF, has been used to improve the clearance of several pulmonary infections, including PC in adult animal models. We show that monotherapy with GM-CSF is insufficient to improve PC clearance in pup mice; however, when combined with TMP/SMX it improves PC clearance and maintains a reduced PC burden following discontinuation of therapy. Furthermore, we have shown that GM-CSF improves the ability of human infant lung macrophages to phagocytose PC organsms without generating an increased inflammatory response. These data suggest that combination therapy with TMP/SMX and GM-CSF may be a viable treatment option for infants failing or intolerant to standard therapy.

The current edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR; American Psychiatric Association, 2000) defines personality disorders as categorical entities that are distinct from themselves and from normal personality traits. However, many scientists now believe that personality disorders can best be conceptualized using a dimensional model of traits that span normal and abnormal personality, such as the Five-Factor Model (FFM). Many research studies have indicated that the current personality disorder system can be adequately conceptualized using the FFM. However, if the FFM or any dimensional model is to be considered as a credible alternative to the current model, it must first demonstrate an increment in the validity of the assessment offered within a clinical setting. Thus, the current study extended previous research by comparing the convergent and discriminant validity of the current DSM-IV-TR model to the FFM across four assessment methodologies. Eighty-eight individuals that were currently receiving ongoing psychotherapy were assessed for the FFM and the DSM-IV-TR personality disorders using self-report, informant report, structured interview, and therapist ratings. The results indicated that the FFM had an appreciable advantage over the DSM-IV-TR in terms of discriminant validity and, at the domain level, convergent validity. Implications of the findings for future research are discussed.

Replication of the nonsegmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. In my thesis I describe (i) the effect of phosphorylation of p33, (ii) the RNA chaperone-like activity of p33, and (iii) the role of HSP70s a host proteins in the viral replication. To test the effect of phosphorylation on p33 function, I used in vitro phosphorylated p33. I found that phosphorylation inhibited the ability of p33 to bind to the viral RNA. Phosphorylation-mimicking mutations rendered p33 nonfunctional in plant protoplasts and in yeast. Based on these results, I propose that the primary function of phosphorylation of p33 is to regulate its RNA binding capacity, which could affect the assembly of new viral replicase complexes, recruitment of the viral RNA template into replication and/or release of viral RNA from replication. Thus, phosphorylation of p33 might help in switching the role of the viral RNA from replication to other processes, such as viral RNA encapsidation and cell-to-cell movement. Small plus-stranded RNA viruses do not code for RNA helicases that would facilitate the proper folding of viral RNAs during replication. Instead, small RNA viruses might use RNA chaperones for replication as shown here for the p33 replication protein. In vitro experiments demonstrated that the purified recombinant p33 facilitated RNA synthesis on plusstranded and double-stranded (ds)RNA templates up to 5-fold. In addition, p33 rendered dsRNA templates sensitive to single-strand specific S1 nuclease, suggesting that p33 can destabilize highly structured RNA. Altogether, the RNA chaperone activity of p33 might perform similar biological functions to the helicases. SSa a yeast HSP70 found in the viral replication complex and shown to facilitate viral replication (Serva and Nagy, 2006)To dissect the mode of action of SSA in the viral replication I used temperature sensitive and deletion mutants. Both showed miss localization of p33 compared to the wild type. Purified SSA rendered non functional bacterial expressed p92 functional in an in vitro replication assay. SSa might play a role in the transportation and assembly of viral replication proteins.

Psychopathy is associated with several behavioral and psychophysiological deficits. Lynam (2002) has argued that the use of an overarching conceptualization of psychopathy can provide a parsimonious explanation of psychopathic pathology. The current study examined relations between tasks used to explore psychopathic pathology and dimensions from the Five Factor Model of personality. Undergraduate participants completed the NEO PI-R, the BART, a go/no-go task, an emotional morph task, and provided physiological responses to stimuli. While hypothesized relations to FFM psychopathy composites were generally unsupported, other interesting relations to traits were identified. Results indicated that hypoarousal to negative stimuli was negatively related to pan-impulsivity. Maladaptive risk taking was positively related to panimpulsivity and high self-directed negative affect. Response modulation deficits were negatively related to pan-impulsivity, low self-directed negative affect, and facets of openness. Deficits in empathic responding were positively related to other-directed negative affect and self-directed negative affect, and negatively related to pan-impulsivity and interpersonal assertiveness. Although it remains unclear whether the failure to support hypotheses was related to the study variables or population, results indicate that the FFM can provide additional information with regard to what deficit tasks assess.

在這篇論文中，我們將探討整函數與半純函數之分解。並且著重於質函數與擬質函數之研究。同時，我們證明某類多項式其次數為合成數且為質函數。 / In this thesis, we study some factorizations of entire and meromorphic functions, in particular, prime and pseudo-prime functions. Also, we obtain a new class of prime polynomials of composite degree.

La transplantation d’hépatocytes est une nouvelle approche thérapeutique pour le traitement des maladies métaboliques. Elle peut être proposée en alternative à la transplantation de foie entier ou, à tout le moins, en attente de celle-ci chez les patients instables, à risque de décompensation métabolique. Les essais cliniques effectués chez 9 patients aux cliniques St Luc ainsi que ceux publiés dans la littérature démontrent l’intérêt de la transplantation de cellules hépatiques à court et moyen terme. La qualité de la suspension cellulaire transplantée reste le premier facteur limitant pour le développement clinique de la technique. La cryopréservation reste le moyen le plus approprié pour la conservation à long terme des cellules. Elle permet de constituer une banque de cellules pouvant être utilisées à tout moment. Nous avons d’abord analysé les protocoles de cryopréservation décrits dans la litérature, ainsi que leurs limites tant au niveau de la préservation de la qualité cellulaire après décongélation in vitro qu’après transplantation in vivo. Dans ce travail, nous avons démontré l’intérêt d’utiliser des cellules cryopréservées/décongelées, afin de stabiliser des patients atteints de maladies du cycle de l’urée, avant la greffe de foie entier. Les tests de contrôle de qualité effectués sur ces cellules ont cependant montré une altération aux niveaux biochimique et cellulaire, après décongélation. Nous avons ainsi démontré une chute des concentrations intracellulaires d’ATP, signe d’une atteinte mitochondriale. Nos travaux ont également permis de mettre en évidence une diminution de la consommation d’oxygène des hépatocytes en suspension, due plus particulièrement à une atteinte du complexe 1 de la chaîne respiratoire. Cette atteinte mitochondriale peut déjà être observée après l’incubation de la suspension cellulaire à –20°C. Aux alentours de cette température critique se fait le passage de l’état aqueux à l’état cristallin suggérant que les dégâts mitochondriaux observés sont dès lors vraisembablement dus à la formation de glace intracellulaire durant le processus de cryopréservation ou de décongélation. Diverses tentatives visant à améliorer les paramètres mitochondriaux affectés par le processus de congélation/décongélation par l’addition d’agents protecteurs du complexe 1 (Bilobalide), d’ inhibiteurs du pore de transition de perméabilité (Ciclosporine A), d’ anti-oxydants ou encore de solutions hyperosmotiques à la solution de cryopréservation, n’ont pas permis d’améliorer la qualité cellulaire. Le tri de sous-types de populations hépatocytaires ou l’isolement de foies hépatectomisés n’ont pas permis de révéler de différences de capacité de résistance à la cryopréservation. Toujours dans le but d’améliorer le rendement de la transplantation d’hépatocytes et d’augmenter l’efficacité d’implantation dans le parenchyme receveur, nous avons démontré dans la deuxième partie de la thèse la capacité des hépatocytes isolés (fraîchement isolés ou cryopréservés/décongelés) à induire un phénomène de coagulation dépendant du facteur tissulaire. Cette activité pro-coagulante, inhibée in vitro par lea N-acetyl-L-cystéine, pourrait être le point de départ d’une réaction inflammatoire aspécifique influençant ainsi la réussite de la transplantation cellulaire. En conclusion, nous proposons dans ce travail différentes stratégies en vue de l’amélioration du rendement de la thérapie cellulaire. La vitrification, autre technique de cryopréservation, permettrait d’éviter la formation d’eau intracellulaire. Enfin la modulation de l’activité pro-coagulante par la N-acetyl-L-cystéine, due à la transplantation cellulaire, constitue une piste intéressante pour essayer d’améliorer l’implantation des cellules transplantées et ainsi le rendement de la greffe. / Liver cell transplantation provides clinical benefit to patients with congenital metabolic abnormalities and currently represents an alternative to orthotopic liver transplantation or at least an interim measure for unstable patients awaiting transplantation. Our team and others have already demonstrated that transplanted hepatocytes can achieve metabolic control in the short or medium term. The quality of transplanted cells remains the first limiting factor for the success of liver cell transplantation. Because the use of freshly isolated cells is restricted by contemporary organ donation, cryopreservation remains necessary for long-term storage and permanent availability of the cells. In this thesis, we have first reviewed and discussed established hepatocyte cryopreservation protocols, especially the cooling procedure, and have focussed on the in vitro and in vivo assays used for the evaluation of post-thawing hepatocyte quality. Amongst 9 cell transplanted patients in our center, several received exclusively or predominantly cryopreserved/thawed hepatocytes. We demonstrated post-transplantation benefits of using these cells in control patients with congentital abnormalities in the urea cycle, particularly with respect to clear evidence of cell engraftment and de novo appearance of enzyme activity. However, despite these clinical benefits, we found an in vitro relationship between the low post-thawing quality of cryopreserved /thawed hepatocytes and an alteration in their mitochondrial function. This post-thawing mitochondrial damage was already evident after the first −20°C cryopreservation step of our protocol, suggesting it occurrs early in the process, around the nucleation point, by intracellular ice formation. Cellular impairment could therefore be possibly explained by mechanical alteration of mitochondria due to water crystallisation during the cryopreservation process or thawing procedure. We also observed a poor efficacy of cryopreserved/thawed hepatocytes (as compared to freshly isolated cells) when used liver engraftment in two mice transplantation models. The marked reductions in intracellular ATP concentrations and the decreases in oxygen consumption by hepatocytes were therefore used as markers for the evaluation of the effects of several compounds such as bilobalide, hyperosmotic or anti-oxidant molecules, pore transition permeability inhibitors, and for the evaluation of the resistance of selected hepatocyte subtypes to cryopreservation protocols. We also demonstrated that isolated hepatocytes exert tissue factor-dependent pro-coagulant activity, which may contribute to the early loss of infused cells. We observed that the addition of N-acetyl-L-cysteine to hepatocyte suspensions inhibits coagulation activation. In conclusion, this work has identified several ways to improve the clinical benefit of liver cell transplantation, including new cryopreservation strategies, such as vitrification. In addition, modulation of the pro-coagulant activity induced by cell infusion with N-acetyl-L-cysteine might beneficially enhance cell engraftment.

No description available.

NMR studies have long been used as a tool to derive structural and dynamic information. Such information has a wide range of applications, and notably is used in the study of structure-activity relationships. The aims of this work were to use NMR spectroscopy to derive structures of the molecules inhibiting the activation of the alternative pathway of the complement portion of the innate immune system (namely, the N-terminus of factor H (FH) and two small peptides, Compstatin 10 and Compstatin 20) and to consider the interdomain dynamics of proteins consisting of three modules theoretically (in silico) and experimentally (for the three N-terminal domains of FH). We focused on the three N-terminal complement control protein (CCP) domains of the important complement regulator, human factor H (i.e. FH1-3). Its three-dimensional solution structure was derived based on nuclear Overhauser effects and residual dipolar couplings (RDCs). Each of the three CCP modules in this structure was similar to the corresponding CCP in the previously derived C3b-bound structure of FH1-4, but the relative orientations of the domains were different. These orientations were additionally different from the interdomain orientations in other molecules that interact with C3b, such as DAF2-4 and CR1-15-17. The measured RDC datasets, collected under three different conditions in media containing magnetically aligned bicelles (disk-like particles formed from phospholipids), were used to estimate interdomain motions in FH1-3. A method in which the data was fitted to a structural ensemble was used to analyze such interdomain flexibility. More than 80% of the conformers of this predominantly extended three-domain molecule exhibit flexions of < 40°. Such segmental flexibility (together with the local dynamics of the hypervariable loop within domain 3) could facilitate recognition of C3b via initial anchoring, as well as eventual reorganization of modules into the conformation captured in the previously solved crystal structure of a C3b complex with FH1-4. The NMR study of the Compstatin analogues revealed unique structural features that had not before been observed in this group of peptides. These features included two b-turns per peptide, neither of which was located in the ‘canonical’ regions in which b-turns were observed in previous molecular dynamics and NMR studies. The structures of Compstatin 10 and Compstatin 20 derived here were consistent with the isothermal calorimetry (ITC) and surface plasmon resonance (SPR) data recorded previously. In the in silico study of interdomain motion of three-domain proteins carried out here, the domains were represented as vectors attached to one another in a linear fashion. They were allowed to undergo Brownian motion biased by the potentials between the sequential vectors. The resulting trajectories were analyzed using model-free and extended model-free formalism. The degree of coupling of the interdomain motion with overall motion was determined, along with a representation of the overall motion. The similarity between the trajectories of the vectors transformed to this overall motion frame and the results obtained from the model-free analysis was determined.

No description available.

No description available.