Mycobacterium tuberculosis (Mtb) is the causative agent of the disease tuberculosis (TB). While approximately one third of the world’s population is infected with this pathogen, only a small minority of these individuals has active TB infection, where these individuals are able to transmit the pathogen to others. In previous microarrays performed in our lab from lung tissue of non-human primates (NHPs), it was noted that animals undergoing the activation of TB showed greatly increased expression of lymphocyte activation gene 3 (LAG-3). This protein performs immunomodulatory roles, which include: increased function of regulatory T cells, decreased function of Th1 effector T cells, and decreased monocyte differentiation. When studied in rhesus macaques infected with Mtb, RNA expression and protein levels of LAG-3 in lung tissue of active TB animals was found to be greatly increased when compared to lung from animals with latent TB. Interestingly enough, there was a bimodal distribution of LAG-3 expression in animals undergoing reactivation of the disease; the animals with greater levels of LAG-3 were the fast reactivators. LAG-3 expression in the lung tissue of animals with Mtb infections was mainly isolated to the outer periphery of the Mtb induced lung granuloma, where predictably, LAG-3 was expressed by lymphocyte populations of immune cells; mainly NK cells and various populations of T cells. To gain a greater understanding of the function of LAG-3, we created a co-culture system where CD4 T cells derived from blood and lung of Mtb infected NHPs were supplemented to Mtb infected differentiated monocytes. With this co-culture model, we utilized short interfering RNA (siRNA) to silence LAG-3. We observed a decreased bacterial burden, as well as decreased frequencies of IL-10 and IFN-γ producing CD4 T cells. This illustrates that the silencing of LAG-3 in CD4 T cells resulted in increased bacterial clearance, not due to up-regulation of IFN-γ. We believe that the bacterial reduction may be due to increased T cell proliferation, along with production of another proinflammatory cytokines. In the near future, we will utilize cytokine assays and microarrays to better understand the mechanism of action through which increased bacterial killing is occurring. / acase@tulane.edu

Magister Scientiae - MSc / Male infertility in humans has increased in the last few decades and could be as high as 40%, while up to 50% of these men have ''unexplained'' (idiopathic) infertility. Although newly developed molecular techniques have great value in detecting subtle causes of male infertility, more detailed sperm functional tests are required to identify compromised fertility, especially in a clinical set-up. Since ethical constraints often preclude the pursuit of many basic research questions in humans, non-human primates (NHPs) have been identified as key models in human-related studies. NHPs are often used in studies on male fertility/infertility, IVF or assisted reproductive technology (ART) procedures, male contraception and reproductive toxicology. However, comparing results of NHP and human studies require that techniques used for assessment must be objective, standardized and sensitive to recognize compromised sperm function. The aim of this study was to evaluate standard sperm functional tests and develop new functional tests using NHP sperm, specifically from vervet monkeys (Chlorocebus aethiops), chacma baboons (Papio ursinus) and rhesus monkeys (Macaca mulatta), for application in human and NHP studies and to ultimately develop a basic primate model. The sperm functions investigated included sperm motility, longevity, vitality, DNA integrity, acrosome reaction, and hyperactivation. The sperm functional tests evaluated were: CASA motility analysis; Sperm Longevity test; Eosin-Nigrosin and Hoechst and Propidium Iodide staining, as well as the use of WST-1 cytotoxicity assay for vitality; the TUNEL assay for DNA integrity; Acrosome Intactness Test; and induction of hyperactivation via stimulants. The validity of each test was investigated by inhibiting sperm function through the use of copper sulphate and cadmium chloride. All functional tests were successfully performed across all three species, except the TUNEL assay for DNA integrity, and was further used for validation testing. Validation testing proved that all sperm functional parameters were significantly affected by the highest concentrations of the chemicals (250 µg/ml CuSO4 and 500 µg/ml CdCl2) and if not significant, trends of reduction were seen. The tests employed were therefore sensitive to the inhibitory effect of the metals. By evaluating these established sperm functional tests we found that primates would serve as good models for research study. Furthermore, we optimized and modified techniques for sperm and functional analysis in these three primate species and this study will standardize protocols for use in future studies on male infertility. Additionally, comparing human and NHP sperm function can possibly reveal or explain the high infertility rates in humans.

The effects of testosterone (T) extend beyond reproductive behavior to the areas of cognitive and emotional functioning. While T effects on cognition have been extensively investigated, less is known about the role of T in the processing of emotional stimuli. Considering the role that T plays in aggressive behavior and dominance status, it is of particular interest to determine whether T modulates the processing of social threat. Due to their similarities to humans in brain organization, reproductive endocrinology and affective regulation, rhesus monkeys (macaca mulatta) provide an excellent model to investigate this relationship. In a within-subjects design, six male rhesus monkeys underwent treatment to suppress endogenous T and received either T or oil replacement. Tests of anxiety, attention and memory for social and non-social emotional stimuli, and risk-taking were administered to animals during both treatments. Data analyses indicate that T treatment resulted in faster response times, but had no effect on anxiety, attention or memory for emotional stimuli, or on risk-taking behavior. There are several limitations to this study that may account for the lack of effect of T and therefore, further investigation of the relationship between T and emotional processing is warranted.

No description available.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / The incidence of male infertility is increasing, with up to 50% of infertile males having “unexplained” (idiopathic) infertility. Newly developed molecular techniques have great value in detecting subtle causes of male infertility, as compared to idiopathic infertility which may be explained by standardizing and optimizing sperm functional and structural tests in non-human primate (NHP) sperm. The aim of the study was to evaluate sperm functionality utilizing the sperm of two NHP species, i.e.1) the rhesus monkey (Macaca mulatta) and 2) the vervet monkey (Chlorocebus aethiops), and further evaluate the effect of physiological media (including commonly used, and newly formulated sperm wash and sperm capacitating media) on NHP sperm functionality. Sperm functionality was evaluated by investigating the following sperm functions i.e.: sperm motility, vitality, acrosome reaction (AR), hyperactivation, and mitochondrial membrane potential (MMP). Sperm functional tests included computer-aided semen analysis (CASA), motility analysis, BrightVit staining for sperm vitality, flourescenin isothiocyanate (FITC)- conjugated peanut agglutinin (PNA) staining for sperm acrosome integrity, induction of hyperactivation by stimulants (sperm preparation media containing capacitating ingredients), and mitochondrial inhibitor (Oligomycin-A) for testing MMP. All functional and structural tests were investigated in both species, except for acrosome integrity, mitochondrial inhibition and functional tests compared over time that could not be successfully completed and investigated in the rhesus species. Motility analysis tests proved that within the vervet species, the use of different physiological media results in statistically significant differences in motility and kinematic parameters over a 1 hour time period. Hyperactivation tests proved that capacitating physiological media produced significantly higher percentages hyperactivation when compared to sperm wash media within the vervet species over a 1 hour time period. Furthermore, within both NHP species, sperm structural analysis (vitality and acrosome integrity) results showed that no significant differences are present when making use of different physiological media over a period of 1 hour incubation. The incubation of vervet sperm with different concentrations of mitochondrial inhibitor, Oligomycin-A (0 μM, 5 μM, and 25 μM), resulted in motility inhibition over a 1 hour incubation period. By the evaluation of these tests it was found that the use of different sperm wash [Human tubal fluid (HTF), Ham‟s F-10® and HD Sperm Wash Plus (HDSWP)] and sperm capacitation media [Human tubal fluid with added caffeine (HTFC) and HD Sperm Capacitating Plus (HDSCP)] resulted in significantly different results within sperm functional tests as compared to sperm structural tests. The study indicates that the composition of media, varying from simple to more complex, used for semen preparation plays an important role in determining NHP sperm functionality. Based on these findings further investigation in larger NHP sample groups and human sperm are required to evaluate the role of certain ingredients in the development of more cost-effective media producing satisfactory results in terms of sperm functionality for artificial reproductive technologies (ART).

Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (p. 80-97).

Submitted by Wesley José dos Santos null (thedeiwz@hotmail.com) on 2016-07-20T20:08:55Z No. of bitstreams: 1 SANTOS WJ - dissertacao 2016.pdf: 3762980 bytes, checksum: 70891a0132a17b5b79842e9ec8acd7bb (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-07-22T19:05:40Z (GMT) No. of bitstreams: 1 santos_wj_me_bot.pdf: 3762980 bytes, checksum: 70891a0132a17b5b79842e9ec8acd7bb (MD5) / Made available in DSpace on 2016-07-22T19:05:40Z (GMT). No. of bitstreams: 1 santos_wj_me_bot.pdf: 3762980 bytes, checksum: 70891a0132a17b5b79842e9ec8acd7bb (MD5) Previous issue date: 2016-07-15 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os animais silvestres, tanto os de vida livre como os de cativeiro, podem ser reservatórios e portadores de diversos protozoários, como os integrantes do gênero Trypanosoma. Trypanosoma cruzi é um dos mais conhecidos tripanosomas, agente etiológico da doença de Chagas. Manifesta-se clinicamente com um caráter multifacetado, afetando humanos e várias espécies de animais domésticos e silvestres, como os primatas não humanos, que podem constituir-se em reservatórios do parasito. Em relação aos primatas neotropicais, ocorrem diferentes infecções por tripanosomas que também acometem humanos. No presente estudo, foram coletadas amostras de sangue de 39 primatas não humanos procedentes do Parque Zoológico Municipal de Bauru para pesquisa sorológica anti-T. cruzi e molecular de T. cruzi utilizando-se primers da região de kDNA de T. cruzi (TCZ1 e TCZ2) e da região codificadora da proteína de choque térmico (hsp70). Todos os animais foram negativos ao teste sorológico anti-T. cruzi e molecular para T. cruzi com os primers da região de kDNA. Porém, com a utilização de primers para hsp70, 11 dos 39 (28,2%) animais foram positivos. Os resultados do sequenciamento apresentaram similaridade para protozoários do gênero Trypanosoma. Apesar das dificuldades em se chegar à espécie circulante de tripanosoma entre os primatas, conclui-se que vetores triatomíneos e/ou moscas hematófagas podem estar circulando no ambiente dos animais e que ações de vigilância e busca dos mesmos são altamente recomendadas. / Wild animals, both free-living and the captive ones, can be reservoirs and carriers of various protozoa, such as members of the genus Trypanosoma. Trypanosoma cruzi, the etiologic agent of Chagas disease, is one of the most known trypanosomes. Chagas disease is manifested clinically with a multifaceted character, affecting humans and several species of wild and domestic animals, such as non-human primates, which can be reservoirs of this parasite. Regarding the Neo-tropical primates, different infections occur by trypanosomes that also can affect humans. In this study, 39 blood samples were collected from nonhuman primates from Zoo Park Municipal of Bauru, São Paulo, Brazil, by serological and molecular analysis for T. cruzi infection using specific primers for kDNA region (TCZ1 and TCZ2) and the region encoding the heat shock protein (hsp70). All animals were negative to the serological test for anti-T. cruzi and molecular for T. cruzi with primers for kDNA region. However, with the use of primers for hsp70, 11 of 39 (28.2%) animals were positive. The sequencing results showed similarity to the genus Trypanosoma. Despite the difficulties in stablish the species of trypanosome among these primates, it is concluded that triatomine vectors and/or bloodsucking flies may be present in the animal environment; so, the entomologic vigilance for these insects are highly recommended. / FAPESP: 2014/12186-4

Chez les humains et les animaux, l’exposition répétée à une séquence de stimulus conduit à la création de représentations mentales isomorphes aux régularités statistiques de cette séquence. Cette thèse étudie la dynamique avec laquelle les unités et sous-unités sont extraites, s’influencent et s’organisent lors de l’apprentissage séquentiel chez l’humain et le primate non-humain (babouin Papio papio). Nos résultats montrent que lors de l’apprentissage d’une séquence régulière ABC, la sous-unité finale BC est apprise plus rapidement que la sous-unité initiale AB chez les humains comme les primates non- humains. Avec une séquence plus longue ABCD, cet effet ne s’étend pas au-delà des deux termes précédents celui à prédire lors des 2000 essais d’apprentissage. Une seconde étude montre que les humains sont capables d’utiliser la prédictibilité des éléments à différents niveaux de complexité, alors que les singes sont limités aux dépendances locales. Enfin notre dernière étude montre que les humains comme les singes ne bénéficient pas d’un contexte d’homogénéité de longueurs des unités par rapport à un contexte d’hétérogénéité de longueurs des unités. Ensemble, ces études montrent des continuités et discontinuités entre les primates humains et non-humains dans l’extraction, l’influence et l’organisation des unités de différents niveaux. Elles donnent des informations d’une finesse nécessaires pour contraindre les différents modèles computationnels qui ont été proposés pour décrire l’apprentissage de régularités. / In humans and animals, the repeated exposure to a sequence of stimulus creates mental representations which have the same statistical regularities as that sequence. This thesis investigates the dynamics, influence and organization of units and sub-units during sequence learning, in humans and non-human primates (Guinea baboons, Papio papio).Our results show an advantage of the final sub- unit BC over the initial sub-unit AB during learning of sequences with the form ABC in both humans and baboons. We interpret this effect as resulting from the richer contextual information AB predicting C compared to the single term A predicting B. This effect is limited to the 2 previous terms before the one term to be predicted, across 2000 learning trials. A second finding shows that in learning sequences of 3 units (i.e. ABC, DEF, HGI), humans are able to use the predictability of elements at both local (within units) and global (within sequence) levels, whereas monkeys are limited to local dependencies. A third section investigates the extraction of regularities in homogeneous (i.e., units of the same length) and heterogeneous contexts (units of different lengths). Results in humans and monkeys showed no advantage for either of these contexts.Considered together, our studies show continuities and discontinuities between human and non-human primates in the extraction, influence and organization of units of different levels. This fine-grained level of information is necessary to constrain the computational models proposed to describe the mechanisms underlying regularity extraction.

The evolutionary origins of human handedness are still unknown. The study of lateralized behaviour in our closest relatives, the nonhuman primates, is useful to clarify how this trait appeared and evolved in our species. In the present study, lateralized behaviour was assessed in a population of 32 free-ranging Mexican mantled howler monkeys (Alouatta palliata mexicana) for thirteen spontaneous motor patterns, at individual and group levels, as well as the effect that age, sex and posture have on its strength and direction. The studied population of howler monkeys displayed only few significant lateral biases at the individual level with single motor patterns (Binomial tests, p≤0.05). No biases towards the use of a particular limb or side of the body were found at a population level. Therefore, even though some individuals showed significant limb/side preference with single motor patterns, no signs of task specialization, side specialization, or true handedness were found. Similarly, no effects of sex, age or posture were found on the direction or strength of lateralized behaviour. The general absence of limb/side preferences found in this population may be due to the constraints imposed by the arboreal life and/or the type of diet. Possible causal agents of the few significant individual biases found here may be the presence of handicaps and/or experience. Further research is needed in order to assess whether the lack of human-like handedness reported in this study is only specific to the studied population, a general phenomenon of the genus Alouatta or perhaps of all the Platyrrhini.

De nombreuses études s’intéressent aux comportements décisionnels et d’apprentissage ainsiqu’aux structures qui les sous-tendent. Il a été montré que le Cortex Préfrontal (CPF) ainsiqu’un réseau de structures sous-corticales, les Ganglions de la Base (GB), étaient impliquésdans ces processus. Néanmoins, le rôle respectif de chacun n’est pas définit. Deux hypothèsessont émises. La première stipule que les deux structures fonctionnent indépendamment. LesGB seraient impliqués dans les comportements habituatifs tandis que le CPF se chargerait descomportements planifiés. La seconde hypothèse considère que les deux structures collaborent: les GB contrôleraient un processus d'apprentissage à cinétique lente dans le CPF et sedésengageraient progressivement au fur et à mesure de l’apprentissage. Ceci reviendrait d'unecertaine façon à inverser les rôles : les GB seraient nécessaires aux processus de décision tantque le CPF n'aurait pas fini son apprentissage. Celui-ci fonctionnerait ensuite sur un modeautomatique. Le principal obstacle à l’étude respectif du rôle des GB et du CPF dans cesprocessus intervient dans les paradigmes expérimentaux qui ne dissocient pas la prise dedécision per se des processus d’apprentissage. Notre premier objectif a donc été d’élaborerune tâche expérimentale qui permette de différencier les phases d’apprentissage des phases deprise de décision. Nous avons ensuite supprimé l'influence des GB sur le cortex, en inhibantleur structure de sortie, le Globus Pallidus interne (GPi) par des injections intracérébrales demuscimol chez le primate non-humain effectuant une tâche comportementale : le "two armedbandit task". Nous montrons que les animaux sont toujours capables de prendre des décisionsaprès inhibition du GPi mais qu’ils sont incapables d’apprendre la valeur de nouvelles cibles.Ces résultats confirment que, chez le primate en tous les cas, les GB et le CPF sont bienimpliqués dans un processus collaboratif : l'intégrité de l'ensemble du circuit est nécessairepour l'apprentissage alors que le cortex seul peut suffire une fois que le choix se situe dans uncontexte habituel. / Many studies are interested in decision making and learning processes and in brainareas which are engaged in. Among them, the implication Prefrontal Cortex (PFC) and a subcortical structures’ network, the Basal Ganglia (BG) has been shown. Nevertheless, theprecise role of each structure has not yet been defined. There are two main hypotheses. Thefirst one holds that GB and PFC function independently. BG would support habitualbehaviors and PFC planned behaviors. The second hypothesis proposes that both structuresare collaborating: the basal ganglia drive a low kinetic learning process in the prefrontalcortex and become less and less engaged as the task is learned. It means reversing the roles:BG would be necessary for decision making processes as soon as PFC finishes its learning.This latter would then function as an automatic mode. The main problem which avoids us todisentangle the role of each structure is the experimental paradigms used which mix uplearning and decision making. Our first aim was to design an experimental task in which therewas learning phase and decision making phase per se. Then, we blocked basal gangliainfluence on PFC by inhibiting their exit structure, the Globus Pallidus internal, withintracerebral muscimol injections in non-human primates performing a “two-armed bandittask”. Our results show that monkeys are able to do decision making after GPi inhibition butthey are unable to learn new values. These results confirm that, in non-human primates, BGand PFC are well involved as co-workers in one process: integrity of all the circuit isnecessary for learning whereas only cortex is sufficient once the choice is in habitual context.