Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Master of Science / Department of Kinesiology / Timothy I. Musch / Both cardiac and peripheral vasculature dysfunction likely contribute, in part, to elevations in TNF-[alpha] and exercise intolerance in chronic heart failure (CHF). The pharmaceutical TNF-[alpha] synthesis suppressor pentoxifylline (PTX) reduces plasma [TNF-[alpha]] and improves left ventricular (LV) function in CHF rats, but the effects of PTX on skeletal muscle blood flow (BF) and vascular conductance (VC) during exercise are unknown. We tested the hypothesis that PTX would elevate skeletal muscle BF and VC at rest and during submaximal treadmill exercise in CHF rats (coronary artery ligation). CHF rats received i.p. injections of 30 mg·kg[superscript]-[superscript]1·day[superscript]-[superscript]1 of PTX (CHF+PTX, n=13) or saline (CHF, n=8) for 21 days. Mean arterial pressure (MAP) and BF (radiolabeled microsphere infusions) were measured at rest and during treadmill exercise (20 m/min, 5% grade). Myocardial infarct (MI) size was not different between groups (CHF: 37±4, CHF+PTX: 37±3% of LV wall; p>0.05). Resting and exercising MAP was greater in CHF+PTX compared to CHF (p<0.05 for both). At rest, total hindlimb skeletal muscle BF and VC were not different between groups (p>0.05). However, during exercise PTX increased total hindlimb BF (CHF: 83±9, CHF+PTX: 114±8 ml·min[superscript]-[superscript]1·100g[superscript]-[superscript]1, p<0.05) and VC (CHF: 0.75±0.08, CHF+PTX: 0.88±0.06 ml·min[superscript]-[superscript]1·100g[superscript]-[superscript]1·mmHg[superscript]-[superscript]1, p<0.05). Furthermore, exercising BF was increased in 21, and VC in 11, of the 28 individual hindlimb muscles or muscle parts with no apparent fiber-type specificity. Thus, PTX administration augments skeletal muscle BF and VC during locomotory exercise in CHF rats, which carries important therapeutic implications for CHF patients.

Master of Science / Department of Human Nutrition / Tonatiuh Melgarejo / Late-lactation Holstein cows (n=9/treatment) were used to evaluate effects of continuous adipose tissue TNFα administration on glucose and fatty acid (FA) metabolism. Cows were blocked by feed intake and milk yield and randomly assigned within block to control or TNFα treatments. Treatments (4 mL saline or 14 μg/kg TNFα in 4 mL saline) were infused continuously over 7 d via 2 osmotic pumps in the adipose layer in the tailhead region. Plasma, milk samples, milk yield, and dry matter intake (DMI) data were collected daily. On d 7, pumps were removed and liver and contralateral tailhead adipose biopsies were collected. Results were modeled with fixed effect of treatment and random effect of block; P values > 0.10 were considered non-significant. TNFα did not alter liver TNFα mRNA abundance, plasma TNFα, IL-4, IL-6, or interferon-γ concentrations, DMI, or rectal temperature. Milk fat and lactose concentrations decreased with TNFα (P < 0.05), but milk yield was unchanged and treatments did not alter the proportion of short vs. long-chain FA in milk on d 7. Treatments did not alter plasma NEFA concentration, liver triglyceride content, or adipose mRNA abundance for hormone-sensitive lipase or perilipin. Plasma glucose turnover rate, as measured by disappearance of U-13C-glucose bolus, was not altered by treatment, nor was liver mRNA abundance for phosphoenolpyruvate carboxykinase or pyruvate carboxylase. However, TNFα tended to decrease adipose TNFα mRNA abundance (P=0.09) and increase liver IL-10 mRNA abundance (P=0.05) compared to controls. Messenger RNA expression of IL-10 in adipose and IL-37 in liver tissue increased significantly in cows treated with TNFα (Figure 1; P = .02 adipose; P < 0.05 liver). This TNFα delivery protocol may have allowed for an adaptive anti-inflammatory response to suppress systemic inflammation, which may account for the lack of metabolic responses compared with previous responses to daily subcutaneous TNFα injections.

Esquistossomose é uma das doenças parasitárias com maior incidência mundial. Schistosoma mansoni, a única espécie encontrada no Brasil, tem um ciclo de vida complexo que inclui seis estágios de desenvolvimento distintos e dois hospedeiros, sendo um deles o homem. Nosso grupo identificou um gene que codifica um receptor que possui homologia com o receptor de TNF-&#945; humano em S. mansoni (SmTNFR) e descrevemos o efeito in vitro da citocina humana TNF-&#945; sobre a expressão gênica em larga escala no parasita. A via de sinalização através da qual o TNF-&#945; atua sobre o parasita permanece desconhecida. Em humanos, o TNF-&#945;, ao se ligar à isoforma do receptor que é mais semelhante ao SmTNFR, inicia uma cascata de fosforilação que conduz à ativação de diversas proteínas quinases que levam à ativação de fatores de transcrição. A nossa hipótese é a de que esta citocina humana atuaria no parasita de forma semelhante, resultando na alteração da expressão gênica do parasita observada em nosso trabalho anterior. No presente trabalho tivemos como objetivo verificar se há aumento de proteínas fosforiladas após o tratamento in vitro de vermes com o TNF-&#945; humano, e identificá-las, elucidando a via de sinalização induzida por esta citocina em S. mansoni. A fim de identificar as proteínas que são significativamente diferencialmente fosforiladas após exposição de S. mansoni à citocina humana, vermes machos adultos foram incubados em cultura por 15 min com TNF-&#945; (20 ng / ml), ou somente com o veículo, em controles, seguido da extração de proteínas totais. Utilizamos a abordagem fosfoproteômica por meio da realização de eletroforese bidimensional, seguida de coloração com corante específico para fosfoproteínas Pro-Q Diamond (Invitrogen). Para identificar quais proteínas tiveram fosforilação alterada pelo tratamento, fizemos uma análise quantitativa dos spots (volume dos spots) nas imagens dos géis corados com Pro-Q, utilizando o software 2D Platinum (GE). Em seguida a coloração de proteína total dos géis foi realizada utilizando Coomassie Blue Coloidal, para localização de todos os spots de proteínas de cada gel. Cortamos dos géis os spots de proteínas que apresentaram mudanças estatisticamente significativas na fosforilação (n = 3 réplicas; p <0,05, teste-t), com base na comparação de seus volumes relativos nas imagens dos géis das amostras tratadas comparadas com as controles, e em seguida as proteínas das amostras foram identificadas por espectrometria de massa. Analisamos três réplicas biológicas e observamos 45 spots que tiveram fosforilação significativamente alterada, sendo que 42 deles apresentaram fosforilação aumentada e 3 apresentaram fosforilação diminuída após o tratamento com a citocina. Entre os spots identificados por espectrometria de massa, verificou-se que proteínas relacionadas a processos metabólicos, sinalização celular, citoesqueleto e contração muscular estavam entre as que sofreram aumentos de fosforilação com o tratamento com TNF-&#945; humano. Embora a abordagem experimental adotada para este estudo não tenha sido sensível o suficiente para concluir qual via canônica de transdução de sinal é ativada por TNF-&#945; humano em S. mansoni, este estudo confirmou o aumento da fosforilação de proteínas-alvo induzido por TNF-&#945; humano, abrindo novos caminhos para uma investigação mais aprofundada que caracterize o papel das proteínas identificadas como alteradas por essa via de sinalização, e permita entender melhor a importância do TNF-&#945; humano na biologia do S. mansoni e na interação parasita-hospedeiro. / Schistosomiasis remains one of the parasitic diseases with the highest incidence worldwide. Schistosoma mansoni, the only species found in Brazil, has a complex life cycle which includes six life stages and two hosts, one of them being humans. Our group has identified a gene that encodes a TNF-alpha receptor-like in S. mansoni (SmTNFR) and described the in vitro effect of human TNF-alpha cytokine on large-scale gene expression of the parasite. The signaling pathways by which TNF-alpha acts upon the parasite remain unknown. In humans, when TNF-alpha binds to the receptor isoform that is most similar to SmTNFR it initiates a phosphorylation cascade that activates a signaling pathway leading to the activation of transcription factors. Our hypothesis is that this cytokine could act in the parasite through a phosphorylation cascade that activates transcription factors resulting in the change of gene expression observed in our previous work. The aim of this work was to verify if there were proteins that showed increased phosphorylation upon the in vitro treatment of worms with human TNF-alpha, identifying them, to try and elucidate the signaling pathway by which the cytokine acts in S. mansoni. In order to identify which proteins are significantly differentially phosphorylated upon exposure of S. mansoni to the human cytokine, we incubated male adult worms for 15 min in culture with human TNF-alpha (20 ng / ml), or with vehicle only, in controls, and we extracted total proteins from the parasites. We used the phosphoproteomics approach of bidimensional gel electrophoresis followed by phospho-specific staining of gels using Pro-Q Diamond dye (Invitrogen). To identify which proteins were phosphorylated or had their phosphorylation changed due to the treatment, we quantified the spots (determined the spots volumes) from Pro-Q stained gels using Image Master 2D Platinum software (GE). A total protein staining of the gels was performed using Coomassie Brilliant Blue (CBB) in order to localize all protein spots in the gel. We excised from CBB stained gels the protein spots that showed statistically significant changes in phosphorylation (n= 3 replicates; p-value<0.05, t-test), based on the relative volumes of their spot images with respect to the total volume of spots in the gel, with samples from treated compared to control parasites, and subsequently the proteins in these samples were identified by mass spectrometry. We have analyzed three biological replicates and observed 45 spots that were differentially phosphorylated; 42 of them had their phosphorylation increased and 3 had their phosphorylation decreased upon a 15-min-treatment of male adult worms with the cytokine. Among the spots identified by mass spectrometry, we found proteins related to metabolic process, cell signaling, and cytoskeleton and muscle contraction. Although the experimental approach adopted for this study was not sensitive enough to conclude which canonical signal transduction pathway is activated by human TNF-alpha in S. mansoni, this kind of study confirmed the increase in phosphorylation of target proteins induced by human TNF-alpha, opening new paths for further investigation in order to characterize the role of the proteins identified as altered by this specific signaling, which will lead to a better understanding of the importance of this cytokine in the biology of S. mansoni and in host-parasite interaction.

Differential Display technique was used for gene profiling in trnasformed human umbilical vein endothelial cell line (ECV 304) and primary human umbilical vein endothelial cells (HUVECs) to study the cellular response to viral infection. After screening the mRNA from uninfected and infected HUVECs and ECV 304 cells with 16 different random primers we identified 8 gene targets. These genes included the human inhibitor of apoptosis-1 (h-IAP1), 2'-5' oligoadenylate synthetase (2'-5' OAS), 2'-5' oligoadenylate synthetase-like (2'-5' OAS-like), Galectin-9 (Gal-9), MxA, Mx1, Regulator of g-protein signaling (RGS2) and endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN). We found that HUVECs were a better model to study gene expression dureing dengue 2 virus infection but not the transformed cell line, ECV 304. Of the 41 primer combinations utilized in ECV 304 cells detected only one upregulated gene, h-IAP1 and 8 out of the 16 primer combinations tried for HUVECs. We hypothesize the activation of two novel signaling pathways (Tumor necrosis factor- alpha (TNF-alpha), Interleukin1-beta (IL1-beta) in endothelial cells during D2V infection. ALso, our data detected genes that are activated in the Type-I IFN (IFN alpha/beta) signaling pathway during dengue 2 virus infection in HUVEC.

Tumor necrosis factor α (TNFα) is a pleiotropic cytokine that is predominantly produced by activated macrophages. The effects of TNFα are as diverse as the cells with which it interacts, e.g., stimulating fibroblast growth, exerting cytotoxic/cytostatic; activity against various human and murine cell lines, promoting inflammation through upregulation of endothelial adhesion molecules and IL-8 production. Yet TNFα is best known, and in fact was originally described, for its role in the bacterial-induced hemorrhagic necrosis of tumors and exacerbation of septic shock in which aberrant TNFα production leads to vascular collapse, cachexia, multiple organ failure, and ultimately death in as many as 100,000 people each year in the United States alone. LPS, a component of the outer cell wall of gram-negative bacteria, is the principal inducer of macrophage TNFα production. TNFα production can be enhanced by IFNγ which also induces upregulation of TNFα receptors allowing for the establishment of a TNFα autocrine loop. It has been hypothesized that autocrine TNFα stimulation plays a critical role in the induction of macrophage effector function, e.g., nitric oxide production. This dissertation represents efforts to evaluate the respective roles of the TNFα receptors in the induction of macrophage effector function, in addition to examining the mechanism by which autocrine TNFα exerts its effects on macrophages. Exploiting the species specificity of the murine TNFα receptor type 2 (TNF-R2), splenic macrophages were stimulated with human TNFα (which binds to TNF-R1 but not TNF-R2), in the presence of IFNγ. Human TNFα was effective in the induction of nitric oxide production, albeit at concentrations 12.5-fold greater than those required by murine TNFα to achieve the same result. Addition of anti-TNF-R1 completely inhibited the murine TNFα mediated induction of macrophage effector function. However, treatment with anti-TNF-R2 resulted in partial inhibition of macrophage activation. Taken together this data suggests that the primary TNFα mediated signals involved in macrophage activation are transduced through TNF-R1, although TNF-R2 appears to contribute to the intensity of the macrophage response. To evaluate the role of autocrine TNFα signaling in the induction of macrophage effector function, immortalized macrophages from normal C57Bl/6J mice (B6/J2) and C57Bl/6J mice containing gene targeted disruptions of the TNF-R1 and TNF-R2 genes (TRN) were stimulated under CD14-dependent and CD14-independent conditions. Although the B6/J2 and TRN clones mounted similar NO responses to LPS in the presence of serum, the TRN macrophages generated a weak nitric oxide response as compared to B6/J2 when stimulated with LPS under serum-free conditions. The involvement of TNFα autocrine stimulation in the CD14-independent activation was corroborated by the ability of soluble TNF-R1 to inhibit the response of B6/J2 macrophages to LPS in serum-free medium. CD14-independent LPS stimulation of TRN and B6/J2 resulted in equivalent levels of IL-1β, TNFα, and NOS gene expression, as determined by RT-PCR, and in release of equivalent amounts of biologically active TNFα. However, western blot analysis revealed that NOS protein production by TRN was as much as 50% less than that produced by B6/J2. These results indicate that autocrine TNFα stimulation contributes to the signaling pathways initiated by ligation of CD14-independent LPS receptors and may be involved in NOS post-transcriptional regulation.

Antipsychotic medications remain the first line of treatment for individuals diagnosed with schizophrenia (SCZ). However, antipsychotic treatment is often not compliant due to dysregulation of both the central (CNS) and autonomic (ANS) nervous systems, resulting in debilitating dose-dependent side effects. Recent work suggests a new approach for treatment of SCZ that could potentially lower treatment doses and reduce side effects. Increased neuroinflammation has been shown in patients diagnosed with SCZ, particularly within the prefrontal cortex (PFC) and hippocampal (HPC) regions of the brain. Tumor necrosis factor-alpha (TNFa) is one of the key pro-inflammatory cytokines observed to be secreted during the inflammatory response. When TNFa is chronically secreted, resident CNS microglia become pro-inflammatory and toxic to the local environment. Microglial activation alongside of dopamine dysregulation thereby results in both the behavioral and neuroinflammatory aspects of SCZ. In this study, we hypothesized dietary administration of two different novel TNFamodulators (PD2024 – Experiment 1 and PD340 – Experiment 2) developed by our collaborators from P2D Bioscience, Inc. (Cincinnati, OH) would alleviate auditory sensorimotor gating deficits and reduce microglial cell activation caused by neonatal polyinosinic:polycytidylic acid (Poly I:C) treatment in rats, which is a validated rodent model of SCZ. Four groups (Experiment 1: Poly IC/PD2024, Poly IC/Control, Saline/PD2024, Saline/Control and Experiment 2: Poly IC/PD340, Poly IC/Control, Saline/PD340, Saline/Control) were intraperitoneally administered either Poly I:C (2 mg/kg) or saline (0.9% NaCl) from postnatal days 5-7. From P30-67, animals were placed on the experimental diet containing either low (10 mg/kg) or high (30 mg/kg) doses of either PD2024 or PD340, whereas the control animals remained on a normal diet. Prepulse inhibition (PPI) was used to test for auditory sensorimotor gating (behavioral abnormalities) in both adolescence (P44-46) and in adulthood (P60-66). At P67, immunohistochemistry (IHC) and confocal microscopy were used to evaluate and examine microglial cell activation using the Iba1-GFP antibody (neuroinflammatory abnormalities) in the PFC and HPC. Results revealed auditory sensorimotor gating deficits in Poly IC/Controls were alleviated in both adolescence and adulthood with either PD2024 or PD340. It was also found that both TNFa modulators significantly reduced microglial activation in the HPC, but not the PFC. The data supports our hypothesis that dietary administration of PD2024 or PD340 alleviates behavioral deficits and decreases neuroinflammation generated from the Poly I:C rodent model of SCZ. Therefore, an approach with a TNFa modulator alongside of current antipsychotic medications could treat both the behavioral and neuroinflammatory aspects of SCZ.

Le TNF-α est une cytokine pro-inflammatoire jouant un rôle délétère dans de nombreuses pathologies. Nous nous sommes intéressés à l'inhibition du TNF-α à l'aide de variants du récepteur soluble de type I du TNF-α (hTNFR-Is) administrés par thérapie génique non virale. Trois variants ont été étudiés: un monomère hTNFR-Is, correspondant au récepteur soluble physiologique, une protéine chimérique hTNFR-Is/mIgG1, dont l'efficacité par administration protéique est reconnue, et une forme dimérique obtenue par association de deux fragments hTNFR-Is à l'aide d'un espaceur polyglycine. La vectorisation des plasmides codant ces variants a été étudiée par différentes voies afin d'obtenir un effet systémique (électrotransfert intramusculaire, greffe de cellules autologues), ou un effet local (électrotransfert intra-articulaire (genou de souris), électrotransfert intra-oculaire). L'électrotransfert intramusculaire permettait d'obtenir une expression de protéine à long terme (supérieure à 6 mois) et dose-dépendante. La protéine chimérique était très stable dans la circulation, et était sécrétée à un taux élevé. Cette procédure n'induisait pas de réponse immune contre la protéine transgénique. Nous avons démontré l'obtention, par électrotransfert intra-articulaire, d'une expression dosedépendante du transgène durant deux semaines. Le passage de la protéine dans la circulation était faible, ce qui était l'objectif de cette approche locale. Aux fortes doses, une réponse immune contre la protéine chimérique était observée. Nous avons montré la faisabilité de l'électrotransfert dans un muscle lisse, le muscle ciliaire de l'œil. L'expression obtenue était uniquement localisée dans le muscle ciblé, et la procédure était sûre (pas d'inflammation ni de dommages observables). L'expression du transgène était supérieure à un mois, sans passage de la protéine dans la circulation. L'électrotransfert intramusculaire de variants du hTNFR-Is était efficace (forme chimérique principalement) dans un modèle de polyarthrite rhumatoïde (arthrite expérimentale au collagène) sur les signes cliniques et histologiques de la maladie, par un unique électrotransfert à l'apparition des signes cliniques de la maladie. La comparaison de ce traitement à l'injection répétée de protéine recombinante illustrait la potentialité d'une approche par thérapie génique, de part son efficacité à long terme. Les approches locale et cellulaire restent à tester dans ce modèle. L'électrotransfert intramusculaire de plasmide codant les hTNFR-Is ne permettait pas d'améliorer la récupération des fonctions motrices après un traumatisme crânien, dans un modèle murin, même si la protéine était détectée dans le cerveau, et capable d'inhiber le TNF-α. Ces résultats sont cependant très préliminaires. L'électrotransfert intra-oculaire du plasmide codant la forme chimérique hTNFR-Is/mIgG1 permettait d'inhiber efficacement les signes cliniques et histologiques dans un modèle expérimental d'uvéite (uvéite expérimentale aux endotoxines). Nos résultats mettent en évidence l'efficacité de l'électrotransfert pour délivrer un gène thérapeutique et obtenir une production locale ou systémique (selon la stratégie utilisée) de protéine thérapeutique. Nos résultats illustrent le potentiel de nouvelles cibles (articulation, muscle lisse de l'œil) pour cette technologie, ce qui est encourageant pour l'application future de l'électrotransfert à d'autres tissus/organes cibles et à d'autres pathologies.

La thérapie génique de myopathies telles que la dystrophie musculaire de Duchenne nécessite une approche systémique afin de traiter l'ensemble de la musculature. Le vecteur AAV est actuellement le plus efficace pour transduire le muscle. Nous montrons que la biodistribution du vecteur AAV administré par voie veineuse peut être modifiée en utilisant diverses stratégies adjuvantes chez la souris saine. La pré-injection de polymères permet ainsi d'améliorer la transduction des muscles par le vecteur AAV, ou encore de baisser la réponse immune neutralisante induite par l'injection intraveineuse du vecteur. Nous abordons également l'impact de facteurs modulateurs exogènes ou endogènes – tels que la procédure d'administration ou certains facteurs sanguins – sur la transduction systémique de l'AAV. Dans une seconde approche, nous avons évalué le transfert de gènes dans le muscle dystrophique afin de sécréter dans la circulation sanguine une protéine transgénique fusionnant le récepteur soluble I du TNF-α avec le fragment constant d'une immunoglobuline (TNFR-Is/mIgG1). La comparaison des cinétiques de sécrétion obtenu après le transfert de gène dans le muscle de souris saines ou de souris dystrophiques mdx indique que le contexte inflammatoire du muscle dystrophique favorise une réponse immune contre le transgène. Nous montrons que l'expression et la sécrétion d'un variant murin peu immunogène du TNFR-Is/mIgG1 améliore la fonction musculaire de la souris mdx sans toutefois conférer un avantage sélectif aux fibres musculaires dystrophiques qui continuent leur cycle de nécrose et de régénération.

Colon cancer and obesity are two significant and related pathological states with multiple etiological factors. In this dissertation, it was hypothesized that tumor growth is accelerated in the altered state of obesity due to their resistance towards tumor necrosis factor-alpha (TNF-alpha) mediated cytotoxicity. Physiologically elevated TNF-alpha in an obese state induces increased nuclear transcription factor-kB (NF-kB) activity, known to transcribe genes crucial to cell survival. Insulin resistance, oxidative stress, and a pro-inflammatory environment are few of the biological consequences of TNF-alpha and NF-kB pathway activation, and further contribute to disease progression. <br /><br /> Three major studies were conducted to investigate phenotypical changes in obesity associated tumors. Firstly, characteristics of the TNF-alpha resistant phenotype were preliminarily assessed by evaluating the effects of exogenous TNF-alpha treatment to HT-29 cells. Elevated levels of NF-kB in response to exogenous TNF-alpha gave an indication that this pathway is critical for cell survival. Furthermore, upregulation of TNF-alpha receptor 2 (TNFR2) suggested another strategy by which the cells were utilizing exogenous TNF-alpha for a survival advantage. Inhibition of NF-kB via St. John?s Wort treatment demonstrated that HT-29 cells may be sensitized towards TNF-alpha mediated cytotoxicity. <br /><br /> Zucker obese (Zk-Ob), Zucker lean (Zk-Ln), and Sprague Dawley (SD) animal models were used to assess tumor phenotype <em>in vivo</em>. Remarkable physiological differences between genotypes were observed. Zk-Ob rats had significantly higher body and organ weights as well as plasma TNF- alpha, insulin, leptin, and oxidative markers than Zk-Ln and SD animals. Tumor incidence and multiplicity were also notably higher in Zk-Ob rats. Protein analyses demonstrated increased levels of TNF-alpha, TNFR2, NF-kB, IkB kinase beta (IKKbeta), insulin receptor (IR), insulin like growth factor-I-receptor (IGF-IR), and mitogen activated protein kinase (MAPK) in Zk-Ob tumors than Zk-Ln counterparts. In all groups, tumors generally had higher protein expression than surrounding, normal appearing colonic mucosa. It is well known that these molecules are involved in signaling pathways that influence and co-operate with each other in rendering growth autonomy to tumor tissue. <br /><br /> A higher number of lesions in the distal than proximal colon in Zk-Ob rats was observed, supporting the emerging concept that genotype/physiological state of the host affects development and distribution of tumors. Thus, a third study was conducted to explore differences between distal and proximal tumor phenotype. Results demonstrated that expression of TNFR2, NF-kB, IR, IGF-IR, and MAPK p44 were significantly higher in distal than proximal tumors. This observation suggested that development of tumors in different regions of the colon varied under the same physiological conditions. Moreover, phenotype of distal tumors appeared to be upregulating survival pathways in comparison to proximal lesions, possibly explaining the higher tumor incidence in the distal colon. <br /><br /> Research documented in this thesis supported the hypothesis that the physiological status of the host intricately affects tumor phenotype. In particular, the TNF-alpha resistant phenotype was most prominent in Zk-Ob tumors, and appeared to be associated with upregulation of multiple signaling pathways cooperating towards tumorigenesis.