Thesis (Ph.D. in Molecular Biology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 160-175). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 163-187.

Thesis (M.S. in Biomedical Engineering)--Vanderbilt University, May 2005. / Title from title screen. Includes bibliographical references.

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

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Apr 14

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

Tumor-infiltrating myeloid-derived suppressor cells (MDSCs), are important mediators of a tumor-permissive microenvironment that contributes to tumor growth and could account for the limited success of immunotherapeutic strategies. MDSCs suppress adaptive immunity by blocking T cell activation, inducing Treg accumulation, and inhibiting natural killer cell cytotoxicity against tumor cells. We investigated the roles of MDSCs in the regeneration of the exocrine pancreas associated with acute pancreatitis and the progression of acinar to ductal metaplasia. Acute pancreatitis was induced in wild type and P48+/Cre;LSL-KRASG12D mice using caerulein and an early influx of MDSCs into the pancreas was observed flow cytometry and immunocytochemistry. Numbers of Gr1(+)CD11b(+) MDSCs increased over 20-fold in pancreata of mice with acute pancreatitis to account for nearly 15% of intrapancreatic leukocytes and have T cell suppressive properties. This marked accumulation of MDSCs returned to normal values within 24 hours of the insult in wild type mice; however, in the oncogenic KRAS mice, MDSCs levels remained elevated. When intrapancreatic MDSCs were depleted by administration of a CXCR2 antagonist (SB265610) in wild type mice the severity of acinar damage was increased. This was also accompanied by a delayed regeneration determined morphologically and with the mitotic immunomarker phospho-histone H3. Isolated intrapancreatic MDSCs from treated mice induce naïve acinar cells to undergo acinar ductal metaplasia when co-cultured in collagen 3D cultures. Purified splenic MDSCs failed to induce the phenotypic transdifferentiation. We conclude that MDSCs are required for adequate pancreatic regeneration in wild type mice with acute pancreatitis and their persistent elevation in oncogenic KRAS mice is not only associated with immune-evasion, but may also function as direct enhancer of malignant proliferation.

Dans ce travail, nous démontrons que l’expression ectopique de Pax4 dans les cellules glucagon+ adultes induit, indépendamment de l’âge, leur néogenèse et transformation en cellules «bêta-like», ce qui entraîne une hypertrophie des îlots et une néogenèse inattendue des îlots. Par l’utilisation de plusieurs approches de traçage, nous démontrons que la conversion des cellules alpha en cellules «bêta-like» médiée par l’expression de Pax4, induit également la mobilisation de précurseurs situés dans ou à proximité des canaux pancréatiques. Ces cellules ré-expriment le gène développemental Ngn3 et adoptent successivement une identité de cellules glucagon+ puis de cellules «bêta-like», suggérant le réveil des mécanismes embryonnaires. Il et à noter que ces processus sont capables de régénérer la totalité de la masse de cellules bêta après plusieurs séries d’induction chimique du diabète. Ces résultats offrent ainsi des perspectives prometteuses pour concevoir de nouvelles stratégies thérapeutiques et régénératrices dans le contexte du diabète du type I. Dans un deuxième chapitre, ce travail décrit nos résultats d'analyse par puce à ADN de pancréas transgénique d’animaux exprimant conditionnellement le gène Pax4 dans les cellules alpha adultes. Cette approche nous permis d'identifier de potentiels gènes cibles de Pax4, qui pourraient jouer un rôle important dans les processus de régénération de la masse de cellules bêta. L’analyse de la fonction de l’un de ces gènes, le facteur de croissance indépendante 1 (Gfi1) est décrite. / In this work we demonstrate that the inducible misexpression of Pax4 in glucagon+ cells age-independently provokes their conversion into beta-like cells and their glucagon shortage-mediated replacement, this process resulting in islet hypertrophy and in an unexpected islet neogenesis. Taking advantage of several lineage-tracing approaches, we show that, upon Pax4-mediated alpha-to-beta-like cell conversion, pancreatic duct-lining precursor cells are mobilized, re-express the developmental gene Ngn3, and successively adopt a glucagon+ and a beta-like cell identity through a mechanism involving the reawakening of the epithelial-to-mesenchymal transition (EMT). It is worth mentioning, that these processes can repeatedly regenerate the entire beta-cell mass, and thereby reverse several rounds of streptozotocin-mediated chemically-induced Type I diabetes. This approach thereby provides promising perspectives to design novel therapeutic regenerative strategies. Aiming to gain further insight into the molecular mechanisms underlying these regeneration and reprogramming processes, and thereby identify new putative targets of interest, a thorough micro array analysis was performed using pancreata from transgenic mice conditionally misexpressing Pax4 in adult alpha-cells. We thereby identified several promising candidate genes, whose gene expression was significantly altered in induces animals. Among these was Growth factor independent 1 (Gfi1): its expression pattern and putative function in the murine pancreas will be described in this work.

Heavily-selected livestock production traits rarely come without compromise; altered physiology arising from intensive selection often gives rise to concern of a welfare trade-off. A particularly clear example of welfare challenge caused by genetic selection in chickens is the ‘broiler-breeder paradox’, wherein breeding populations of broiler-type birds selected for fast growth are feed-restricted in order to reduce growth and maintain reproductive viability at sexual maturity. In order to better-inform management and breeding strategies for alleviating reproductive problems resulting from genetic selection for growth, it is essential to develop a better understanding of the physiological processes underpinning growth. Whereas the molecular mechanisms governing energy balance in mammals have been relatively welldescribed, analogous avian systems have not received as much research attention and remain somewhat poorly understood. The broad aim of this doctoral project was to contribute to understanding of avian energy balance, particularly in the context of selection for high growth. Using an advanced broiler-layer intercross chicken line (AIL), high- and low-growth haplotypes at the locus encoding the cholecystokinin A receptor (CCKAR), underlying the most significant QTL for growth in chickens, were characterised. Of over 300 variations detected, a select panel spaced across the CCKAR locus were tested for prediction of bodyweight in a diverse cohort of chicken populations. One intronic SNP was found to be significant (p < 0.05) and proximal to transcription factor binding sites. The effect of this locus on gross bodyweight remained significant into the 20th AIL generation (~20% at 10wk, p < 0.05). In this otherwise effectively genetically homogeneous population, several specific physiological traits were predicted by CCKAR haplotype alone, yielding some clues as to the significance of perturbed cholecystokinin (CCK) signalling in broiler strains. While birds with high-growth CCKAR haplotype (HG) did not appear to consume more, feed conversion efficiency (FCE) was improved, at least for males, compared to low-growth (LG) (p < 0.05). Visceral organ anatomies were morphologically disparate, with HG individuals exhibiting ~1/3 less gallbladder mass (p < 0.01), and ~10% shorter GI tract (p < 0.01) and metatarsal bone (p < 0.05). Further gaps in knowledge of the expression of peripheral satiety hormones in chicken are addressed in this thesis. Tissue distributions for expression of CCK, gastrin, pancreatic polypeptide (PPY) and peptide YY (PYY), were mapped and their respective dynamic responses to nutritive state examined. CCK was found to be most highly expressed in the brain, whereas PYY, PPY and gastrin were far more abundant in distinct regions of the periphery. Interestingly, peripheral CCK was not responsive to short-term (< 10h) satiety in experimental populations where PYY and gastrin were. PYY expression was found to be greatest in the pancreas and consistently upregulated within hours after feeding (p < 0.01), whereas gastrin expression was confined to the gastric antrum and paradoxically highest in fasting birds (p < 0.01). PPY expression is strictly limited to the pancreas and appears dependent on longerterm energy state. These results highlight similarities and differences to mammalian systems; notably, the avian pancreas seems to fulfil an exceptional role as a site of signal integration, perhaps unsurprising considering its disproportionate size compared to mammals. Indeed, pancreatic PYY appears to act as a primary peripheral short-term satiety hormone in birds. This body of work contributes to the understanding of avian energy balance and growth. An invaluable foundation for future research is formed by the identification of the major locations of production, and basic nutrient-responsive trends, for several peripheral avian hormones. Information on the growth role of CCKAR is consolidated and expanded upon, demonstrating a clear genetic contribution to maintenance organ morphology and overall growth. Such knowledge can be used to reliably assess and advise on selection and management of chickens to stem welfare concerns without compromising production. Comparisons between avian and other vertebrate endocrine systems make for interesting insight into the adaptive role of energy homeostatic mechanisms in divergent evolution of mammals and non-mammalian vertebrates. In some aspects, birds might better represent the ancestral phenotype from which each vertebrate clade arose.

A obesidade vem aumentando progressivamente na população canina em decorrência de distúrbios endócrinos, alimentares e de manejo, o que leva ao aparecimento de doenças a ela relacionadas, sendo a Diabetes Mellitus a mais comum. Esta enfermidade ocorre devido a alterações na secreção ou sinalização da insulina em tecidos periféricos, gerando uma resistência periférica à insulina, o que pode levar à completa exaustão beta-pancreática, processo irreversível e incompatível com a vida. Pouco se sabe sobre a patogênese da resistência à insulina causada pela obesidade em cães. A proteína C reativa está relacionada à obesidade em humanos e roedores, mas não existem dados na literatura sobre a sua relação com a obesidade e resistência à insulina em cães. O transportador de glicose GLUT4 tem importante função na entrada de glicose nas células adiposas e musculares. A alteração na expressão desta proteína já foi associada à obesidade em várias espécies, incluindo cães, porém não se sabe se esta alteração está relacionada à resistência à insulina. O objetivo deste trabalho foi elucidar alguns aspectos bioquímicos e moleculares que cercam a resistência à insulina induzida pela obesidade em cães e com isso gerar subsídios para a prevenção da diabetes mellitus na espécie canina. Para tanto, foram realizados três experimentos, onde os dois primeiros tentaram elucidar aspectos bioquímicos da obesidade (efeito sobre os níveis de proteína C-reativa) e resistência à insulina (efeito sobre os níveis de fructosamina), e o último tentou avaliar aspectos moleculares em tecido adiposo e muscular (efeito sobre a expressão de GLUT4). Os resultados mostraram que a proteína C-reativa não se altera com a obesidade canina, e que a fructosamina altera-se com o desenvolvimento da resistência à insulina em cães. Uma modificação no padrão expressão da proteína GLUT4 entre os tecidos muscular e adiposo foi observada na presença da resistência à insulina e uma alteração na translocação foi verificada nos dois tecidos, causada pela obesidade. Com base nos resultados obtidos pode-se concluir que a patogênese da resistência à insulina causada pela obesidade não é similar a outras espécies, e deve ser prevenida com medidas distintas. O presente estudo também gerou informações que podem auxiliar no diagnóstico e prevenção da resistência à insulina e diabetes mellitus na espécie canina. / endocrine, nutritional and management disturbances, what leads to the appearance of related diseases, being Diabetes Mellitus the most common. This illness occurs because of insulin secretion or signaling alterations, generating a peripheral insulin resistance, what can lead to a complete beta-pancreatic exhaustion that is considered an irreversible and life-incompatible process. Little is known about pathogenesis of insulin resistance caused by obesity in dogs. C-reactive protein is related to obesity in human and rodent species, but there is no data in the literature about its relation with obesity and insulin resistance in dogs. GLUT4 glucose transporter has an important role on adipose and muscular cells glucose uptake. Alterations on GLUT4 protein expression have been established in obesity in some species including dogs, but there is no knowledge about this mechanism in insulin resistance in canine species. The aim of this work was to elucidate some biochemical and molecular issues surrounding obesity induced insulin resistance in dogs and thus generate information leading to prevention of diabetes mellitus in canine species. To achieve that, three experiments were accomplished, where the first two tried to elucidate biochemical aspects of obesity (effect on C-reactive protein levels) and insulin resistance (effect on fructosamine levels), and the third attempted to evaluate molecular aspects in fat and muscular tissues (effect on GLUT4 expression). Results showed that C-reactive protein levels do not alter with canine obesity, and that fructosamine levels change with the onset of insulin resistance. A modification on GLUT4 protein expression pattern between adipose and muscular tissues was observed in the presence of insulin resistance and a translocational alteration was detected in both tissues, caused by obesity. Based on the obtained results it can be postulated that the pathogenesis of insulin resistance caused by obesity differs from other species, and must be prevented with distinct measures. Furthermore, the present study generated information that can help on diagnosis and prevention of insulin resistance and diabetes mellitus in canine species.