No description available.

<p>The incidence and prevalence of people suffering from end stage renal disease is increasing. Proteinuria, particularly the presence of albumin in urine is concerning because proteinuria is associated with the progression to end stage renal disease (ESRD). Understanding the mechanisms involved in damaging the glomerular filtration barrier is essential. Transforming growth factor beta (TGFB) is a key cytokine in mediating glomerulosclerosis and proteinuria. Not much is known about the downstream pathways that mediates the renal damage and proteinuria.</p> <p>I hypothesize that TGFB induces proteinuria through podocyte de-differentiation and this occurs through SMAD dependent and independent pathways.</p> <p>Methods: I used adenovirus mediated gene transfer of TGFB1 to rat renal artery to study the effects of TGFB1 on renal structure and functions. To study the importance of SMAD3 in mediating downstream effects of TGFB1 in proteinuria and podocyte effacement, I used an anti-glomerular basement membrane model in SMAD3+/+ and SMAD3-/- mice to induce glomerulonephritis and proteinuria.</p> <p>Results: Transient TGFB1 overexpression via AdTGFB1 induced significant proteinuria, podocyte foot process effacement, nephrin down-regulation, and nephrinuria. The expression of synaptopodin was also significantly down-regulated by TGFB1. TGFB1 increased the expression of the angiopoietin receptor, Tie2, in podocyte cell culture. In cultured podocytes, TGFB1 downregulated the gene and protein expression of both nephrin and synaptopodin. These findings suggest that locally produced TGFB1 can cause podocyte de-differentiation marked by a loss of synaptopodin, nephrin, and foot process effacement; this process is partly regulated by angiopoietins. This process represents a novel pathway that may explain proteinuria in a variety of common renal diseases.</p> <p>Both SMAD3+/+ and SMAD3-/- mice had proteinuria after induction of anti-GBM glomerulonephritis, though to a lesser extent in SMAD3-/- mice. SMAD3-/- and SMAD3+/+ mice developed significant glomerulonephritis with progressive interstitial fibrosis and chronic renal impairment. The SMAD3+/+ mice were found to be more prone to fibrotic changes, interstitial damage and tubular and glomerulosclerosis than the SMAD3-/- mice. This suggests that TGFB1 signals through pathways other than SMAD3 such as those triggered by hypoxia.</p> <p>Conclusion: I have shown that TGFB1 upregulation via AdTGFB1 induces proteinuria through podocyte dedifferentiation and FP effacement. Angiopoietins are essential for TGFB1 mediated podocyte injury. The effects of TGFB are partially mediated through SMAD3 as there is residual podocyte effacement and proteinuria in the SMAD3-/- mice. Hence there are SMAD3 dependent and independent pathways involved in proteinuria.</p> / Doctor of Science (PhD)

Axon degeneration can result from primary damage due to a variety of causes, and in some instances, its effects can further propagate damage to vicinal neurons. When an axon has been damaged or transected, Wallerian degeneration is the apoptotic-like mechanism that is initiated, ultimately leading to the death of the neuron. The post-injury cellular inflammatory response is recruited to clear the degraded debris and is also responsible for activating the cascade events leading to additional cell death in surrounding neurons. A unique strain of mutant mice were discovered to express what is called the Wallerian Degeneration Slow (Wld') gene, which produces a chimeric nuclear protein that has been observed to dramatically delay both the onset of axon degeneration and the initiation of the post-injury cellular inflammatory response . Recent studies seem _to indicate the neuroprotective phenotype induced by the Wld' protein is the result of it modulating levels of genetic and protein expression in the damaged neuron. This thesis will review what is known about the Wld' protein and discuss how it offers an in-depth look at the molecular mechanisms behind neurodegenerative injury and disease states. In addition, we will discuss our efforts of isolating and purifying plasmids with the Wld' gene that have been cloned in order to create a stable cell line to aid in the future study for the characterization of neuroprotective mechanisms and the molecular pathways of neurodegeneration .

<p> This work focuses on members of the Boletales. This order is comprised of a morphological and ecologically diverse set of species. While the vast majority of species are pileate-stipitate with pores and have a mutualistic nutritional strategy ectomycorrhal (ECM), there are resupinate and gilled species, and saprotrophs and mycoparasites as well. In the first chapter, we review the ecological niche occupied by <i>Boletinellus merulioides. </i> This species was originally considered to be ECM, the symbiont to <i> Fraxinus americana.</i> This hypothesis was rejected, and replaced by the possibility of a mutualism with an <i>F. americana</i> aphid pest, <i>Prociphilus fraxinifolii.</i> We present the first study that observed all three species, since the original publication, the first molecular data for each species, and isotopic fractionation results for <i> B. merulioides</i> and <i>P. fraxinifolii.</i> Additionally, we describe a new morphology for sclerotia of <i>B. merulioides.</i> In total, we are unable to reject the possibility of a facultative mutualism between <i>B. merulioides</i> and <i>P. fraxinifolii.</i></p><p> Chapters two through five review systematics in the <i>Boletineae. </i> Chapter two presents the most comprehensive phylogenetic review of the <i>Boletineae,</i> at the time publication, and remains one of the most inclusive <i>Boletineae phylogenies.</i> Three genes, nuclear large subunit, translation elongation factor 1-alpha, and DNA directed RNA polymerase II largest subunit, were used. This chapter is a summary of <i> Boletineae</i> taxonomy and morphological characteristics, with a clade by clade analysis. We present compelling evidence for the mycoparasitic nutritional mode of <i>Buchwaldoboletus lignicola.</i> Additionally, we found that <i>Chalciporus piperatus,</i> a close relative of <i> B. lignicola</i>, is likely to be a mycoparasite. We present strong evidence that the genus <i>Boletus</i> is limited to single clade that contains approximately 10% of the validly published <i>Boletus</i> species. </p><p> A subset of the taxa sampled in chapter two was used in the phylogenies presented in chapters three, four, and five. Each of these chapters reviews the phylogenetic placement of traditionally problematic species/genera; <i> Surotius eximius, Harrya chromapes</i> and allies, and the Boletaceae species with longitudinally striated spores. These groups have been in multiple. Our results show that <i>Sutorius</i> and <i>Harrya</i> species are distinct from other Boletacaea species and that the longitudinally striated species have been lumped together. By correcting taxonomic confusion and using a multigene data set we are able to resolve these problematic species, and provide a path for future systematics and evolutionary analysis.</p>

<p> Most plants harbor microbial symbionts, which often affect host performance and fitness. Endophytic <i>Epichlo&euml;</i> species are systemic fungal microbial symbionts of many cool-season pooid grasses. Benefits to the host from <i>Epichlo&euml;</i> infection include increased resistance to stressful environmental factors, such as drought and limited soil nutrients, due to morphological and physiological changes. The major benefit of <i> Epichloe</i> infection is enhanced protection against herbivory due to production of fungal alkaloids. The fungal alkaloids have varying activity against invertebrate or mammalian grazers. Although <i>Epichlo&euml; </i> endophytes are well-studied in agronomic grasses such as tall fescue and perennial ryegrass, little is known about the how the presence of different endophyte species and their frequencies and distribution are related to environmental factors in native grasses. Using two native grasses to eastern [<i>Poa alsodes</i> (Grove Bluegrass)] and western [<i>Achnatherum robustum </i> (Sleepygrass)] North America, I addressed the following questions: 1) how are endophyte species distributed among populations along a latitudinal gradient, 2) what fungal alkaloids are produced by different endophyte species, 3) how do fungal alkaloids affect insect herbivores, and 4) what are the effects of different endophytes on host plant growth? (Abstract shortened by ProQuest.) </p>

No description available.

<p> During leaf senescence nutrients are mobilized towards newly developing vegetative and reproductive structures. The IAOx pathway that produces auxin and defense molecules [indole glucosinolates (IGs) and camalexin] is up-regulated during senescence. To investigate the role of the IAOx metabolites we isolated two independent cyp79B2/cyp79B3 double mutants, which are deficient in IGs and camalexin and had reduced auxin levels. Chlorophyll, protein, and gene expression data indicate that cyp79B2/cyp79B3 mutants display early leaf senescence. Furthermore, leaves accumulated higher levels of hydrogen peroxide and seed production was significantly reduced. Auxin signaling at hydathodes and vascular tissue decreased as leaves aged, even though endogenous auxin levels increased. Since CYP79B2/CYP79B3 play only a minor role in auxin synthesis, it is most likely that IGs and/or camalexin are playing a protective role during age-induced developmental leaf senescence. Identifying molecules that slow down the rate of senescence may allow for genetic manipulation to increase nutritional value and crop yield</p>

published_or_final_version / abstract / Obstetrics and Gynaecology / Master / Master of Philosophy

SUMMARY Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by progressive destruction of insulin-producing pancreatic beta-cells. Both viral infections and the cytokines interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) have been suggested as potential mediators of beta-cell death in early T1DM. Nitric oxide (NO) is a highly diffusible, short-lived free radical gas, which plays a significant role in several physiological processes in a diversity of tissues and organisms. Prolonged exposure of rodent or human pancreatic beta-cells to combinations of cytokines induces the expression of the inducible form of nitric oxide synthase (iNOS) and Fas, NO production, and cell death. It also induces the expression of potential "defense" genes, such as manganese superoxide dismutase (MnSOD) and heat shock protein (hsp) 70. Recent studies have shown that NO, in addition to having cytotoxic actions, may also regulate gene transcription. It remains unclear whether NO mediates cytokine-induced gene expression and subsequent beta-cell death. Previous studies using NO synthase blockers yielded conflicting results, which may be due to non-specific effects of these agents. In the first part of our work, we examined the role of NO in beta-cell dysfunction and death by using an iNOS knockout mice (iNOS-/-, background C57BL/6x129SvEv). We evaluated the effects of cytokines on gene expression, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and viability, as determined by nuclear dyes, of pancreatic islet cells or fluorescence-activated cell sorter (FACS)-purified beta-cells isolated from iNOS knockout mice or their respective controls (C57BL/6x129SvEv). The combination of cytokines used was interleukin-1beta (50 U/ml) + gamma-interferon (1000 U/ml) + tumor necrosis factor-alpha (1000 U/ml). The lack of cytokine-induced iNOS activity in the iNOS-/- islet cells was confirmed by RT-PCR and nitrite determination. Cytokines induced a > 3-fold increase in Fas and MnSOD mRNA expression in wild-type (wt) and iNOS-/- islets. On the other hand, hsp 70 was induced in wt but not in iNOS-/- islets. Prolonged (6-9 days) exposure of wt islets to cytokines lead to an 80-90% decrease in islet cell viability, whereas viability decreased by only 10-30% in iNOS-/- islet cells. To determine the mode of cytokine-induced cell death, FACS-purified beta-cells were exposed to the same cytokines. After 9 days, the apoptosis index was similarly increased in wt (39 +/- 3%) and iNOS-/- (33 +/- 4 %) beta-cells. On the other hand, cytokines increased necrosis in wt (20 +/- 4 %) but not in iNOS-/- (7 +/- 3 %) beta-cells. From these data, we conclude that: 1) NO is required for cytokine-induced hsp 70 mRNA expression, but not for Fas and MnSOD expression; 2) cytokines induce both apoptosis and necrosis in mouse beta-cells; 3) cytokine-induced apoptosis is mostly NO-independent, whereas necrosis requires NO formation. In the second part of our work, we examined the role of the viral product double-stranded RNA (dsRNA) in beta-cell dysfunction and death. DsRNA is produced by many viruses during their replicative cycle. We investigated whether dsRNA (here utilized as synthetic poly IC (PIC)) modifies the effects of IL-1beta and IFN-gamma on gene expression and viability of rat pancreatic beta-cells and the role of NO in this process. FACS-purified rat beta-cells were exposed for 6-16 h (study of gene expression by RT-PCR) or 6-9 days (study of viability by nuclear dyes) to PIC and/or IL-1beta or IFN-gamma. PIC increased the expression of Fas and Mn superoxide dismutase mRNAs by 5-10-fold. IL-1beta and a combination of PIC + IFN-gamma(but not PIC or IFN-gamma alone) induced expression of iNOS and consequent NO production. Induction of iNOS expression by PIC + IFN-gamma requires NF-kappaB activation, as suggested by transfection experiments with iNOS promoter-luciferase reporter constructs into primary beta-cells. The PIC-responsive region in the Fas promoter is located between nucleotides -223 and –54. Site-directed mutations at the NF-kappaB and C/EBP binding sites prevented PIC-induced Fas promoter activity. Increased Fas promoter activity was paralleled by enhanced susceptibility of PIC + cytokine-treated beta-cells to apoptosis induced by FasL. Combinations of IL-1beta + IFN-gamma, PIC + IFN-gamma or PIC + IL-1beta induced a 2-3-fold increase in the number of apoptotic beta-cells. Blocking of iNOS activity decreased PIC + IL-1beta-, but not PIC + IFN-gamma-, induced apoptosis. beta-cell infection with an adenovirus encoding the NF-kappaB inhibitor AdIkappaB(SA)2 prevented both necrosis and apoptosis induced by PIC + IL-1beta or PIC + IFN-gamma. mRNAs for several chemokines and one cytokine were induced by PIC, alone or in combination with IFN-gamma, in pancreatic beta-cells. These included IP-10 (CXCL10), IL-15, MCP-1 (CCL2), fractalkine (CX3CL1) and MIP-3alpha (CCL20). There was not, however, induction of IL-1beta expression. PIC has an additive effect on cytokine-induced beta-cell death, by both NO-dependent (in the case of IL-1beta) and NO-independent (in the case of IFN-gamma) mechanisms. To further elucidate the molecular mechanisms involved in PIC + IFN-gamma-effects, the global profile of genes modified by these agents was analysed by high-density oligonucleotide arrays representing 8000 probes in primary rat beta-cells. Following a 6 or 24h treatment with IFN-gamma, PIC or IFN-gamma and PIC, we observed changes in the expression of 51 to 189 genes. IFN-gamma modified the expression of MHC-related genes, and also of genes involved in beta-cell metabolism, protein processing, cytokines and signal transduction. PIC affected preferentially the expression of genes related to cell adhesion, cytokines and dsRNA signal transduction, transcription factors and MHC. PIC and/or IFN-gamma up-regulated the expression of several chemokines and cytokines that could contribute to mononuclear cell homing and activation during viral infection, while IFN-gamma induced a positive feedback on its own signal transduction. PIC + IFN-gamma inhibited insulin and GLUT-2 expression without modifying pdx-1 mRNA expression. Based on these findings, we propose an integrated model for the molecular mechanisms involved in dsRNA + IFN-gamma induced beta-cell dysfunction and death. RESUME Le diabète mellitus de type 1 (DMT1) est une maladie auto-immune provoquée par la destruction progressive des cellules beta pancréatiques productrices d'insuline. Des infections virales, ainsi que deux cytokines, l’interleukine-1beta (IL-1beta) et l’interféron-gamma (IFN-gamma), ont été proposées comme médiateurs potentiels de la mort des cellules beta dans le DMT1 précoce. Le monoxyde d’azote (NO) est un gaz à radical libre, hautement diffusible, à demi-vie courte, qui joue un rôle significatif dans plusieurs processus physiologiques dans une diversité de tissus et d'organismes. L'exposition prolongée des cellules beta pancréatiques humaines ou de rongeur à des combinaisons de cytokines induit l'expression de la forme inductible de la NO synthase (iNOS) et de Fas, la production de NO, et la mort cellulaire. L'expression de gènes potentiellement impliqués dans les mécanismes de défense cellulaire, tels que la superoxyde dismutase – dépendante du manganèse (MnSOD) et la protéine ¨heat shock¨ (hsp) 70, est également induite. Des études récentes ont montré que le NO, outre le fait d'exercer des actions cytotoxiques, pourrait aussi réguler la transcription de gènes. Il n’est cependant pas encore clairement établi si le NO est un médiateur de l’induction de l'expression de gènes et de la mort de la cellule beta par les cytokines. Des études antérieures utilisant des bloqueurs de la NO synthase ont montré des résultats contradictoires, qui pourraient être dus à des effets non spécifiques de ces agents. Dans la première partie de notre travail, nous avons examiné le rôle du NO dans la dysfonction et la mort des cellules beta en utilisant une souris knockout iNOS (iNOS-/-, background C57BL/6x129SvEv). Nous avons évalué les effets des cytokines sur l'expression de gènes, par la technique de "reverse transcriptase-polymerase chain reaction" (RT-PCR), et sur la viabilité de cellules d'îlots pancréatiques ou de cellules beta purifiées par la technique de tri cellulaire basé sur la fluorescence (FACS), isolées au départ de souris knockout iNOS ou de leurs contrôles respectifs (C57BL/6x129SvEv), par l'utilisation de colorants nucléaires. La combinaison des cytokines utilisée était la suivante : interleukine-1beta (50 U/ml) + interféron-gamma (1000 U/ml) + facteur de nécrose tumorale alpha (1000 U/ml). L’absence d’induction de l’activité de l’iNOS par les cytokines dans les îlots iNOS-/- a été confirmée par RT-PCR et mesure de la formation de nitrite. Les cytokines induisaient une augmentation de trois fois de l'expression de l'ARNm de Fas et de la MnSOD dans les îlots de type sauvage (wt) et iNOS-/-. D'autre part, hsp 70 était induite dans les îlots wt mais pas dans les îlots iNOS-/-. L'exposition prolongée (6-9 jours) d'îlots wt aux cytokines a conduit à une diminution de 80-90% de la viabilité des cellules insulaires, alors que la viabilité diminuait seulement de 10-30% dans les cellules insulaires iNOS-/-. Afin de déterminer le mode d'action des cytokines dans l'induction de la mort cellulaire, des cellules beta purifiées par FACS ont été exposées à ces mêmes cytokines. Après 9 jours, l'index apoptotique augmentait de manière similaire dans les cellules beta wt (39 +/- 3%) et iNOS-/- (33 +/- 4%). D’autre part, l’index nécrotique augmentait dans les cellules beta wt (20% +/- 4%) mais pas dans les cellules beta iNOS-/- (7% +/- 3%). De ces données, nous pouvons conclure que : 1) le NO est requis pour l'expression de l'ARNm de hsp 70 induite par des cytokines; 2) les cytokines induisent à la fois l'apoptose et la nécrose dans les cellules beta de souris; 3) l'apoptose induite par les cytokines est principalement NO-indépendante, tandis que la nécrose requiert la formation de NO. Dans la seconde partie de notre travail, nous avons examiné le rôle de l'ARN viral double brin (dsRNA) dans la dysfonction et la mort des cellules beta. Le dsRNA est produit par de nombreux virus durant leur cycle de réplication. Nous avons recherché si le dsRNA (ici utilisé sous la forme de poly IC synthétique (PIC)) modifie les effets de l’IL-1beta et l’IFN-gamma sur l'expression de gènes et sur la viabilité des cellules beta pancréatiques de rat; nous avons également investigué le rôle du NO dans ce processus. Dans ce but, des cellules beta de rat purifiées par FACS ont été exposées pendant 6-16h (étude de l'expression de gènes par RT-PCR) ou 6-9 jours (étude de la viabilité par colorants nucléaires) au PIC et/ou à l’IL1-beta et l’IFN-gamma. Le PIC augmentait de 5-10 fois l'expression des ARNm de Fas et de la MnSOD. L’IL-1beta et l'association de PIC et de l' IFN-gamma (mais pas le PIC ou l’IFN-gamma seul) induisait l'expression de l’iNOS et la production de NO conséquente. L'induction de l'expression de l’iNOS par le PIC + IFN-gamma requiert l'activation de NF-kappaB, comme suggéré par des expériences de transfection avec des constructions du promoteur de l’iNOS couplé au gène rapporteur luciférase dans des cellules beta primaires. Des expériences similaires out montré que la région de réponse au PIC dans le promoteur Fas est localisée entre les nucléotides –223 et –54. La mutation des sites de liaison de NF-kappaB et de C/EBP inhibait l'activité du promoteur Fas induite par le PIC. L’induction de l'activité du promoteur Fas était parallèle à une susceptibilité plus grande à l'apoptose induite par FasL des cellules beta traitées aux cytokines + PIC. Les combinaisons IL-1beta + IFN-gamma, PIC + IFN-gamma ou PIC + IL-1beta induisaient une augmentation de 2-3 fois du nombre de cellules beta apoptotiques. Le blocage de l'activité de l’iNOS diminuait de manière significative l'apoptose induite par PIC + IL-1beta, mais pas par PIC + IFN-gamma. L'infection des cellules beta par un adénovirus exprimant IkappaB(SA)2, un inhibiteur de NFkappaB, inhibait à la fois la nécrose et l'apoptose induite par PIC + IL-1beta ou PIC + IFN-gamma. Les ARNm de plusieurs chémokines (IP-10 (CXCL10), MCP-1 (CCL2) , fractalkine (CX3CL1) et MIP-3alpha (CCL20)) et d’une cytokine (IL-15) étaient induits par le PIC, seul ou en combinaison avec l’IFN-gamma, dans les cellules beta pancréatiques. Cependant, il n'y avait pas d'induction de l'expression de l’IL-1beta. Le PIC a un effet additif sur la mort des cellules beta induite par les cytokines, par des mécanismes à la fois dépendant du NO (dans le cas de l’IL-1beta) et indépendant du NO (dans le cas de l’IFN-gamma). Nous proposons que le dsRNA, généré pendant une infection virale, pourrait contribuer au dysfonctionnement de la cellule beta à la fois en induisant l'expression de chémokines et de l’IL-15, agents potentiels du développement de l’insulite, et en agissant en synergie avec les cytokines produites localement afin d'induire l'apoptose de la cellule beta. La production de NO et l'activation du facteur de transcription NFkappaB semblent jouer un rôle central dans les effets délétères du dsRNA dans les cellules beta pancréatiques. Afin d'élucider les mécanismes moléculaires impliqués dans les effets du PIC et de l' IFNgamma, nous avons analysé le profil d'expression génique des cellules beta primaires de rat et les modifications d'expression induites par ces agents grâce à la technique des puces à ADN ("microarray"). Après une exposition de 6 ou 24h à l' IFNgamma, au PIC ou à une combinaison de ces deux agents, nous avons observé des modifications de l'expression de 51 à 189 gènes. L' IFN-gamma modifie l'expression de gènes MHC-connexes, ainsi que l'expression de gènes impliqués dans le métabolisme de la cellule beta, dans les synthèse et sécrétion des protéines ainsi que dans les voies de signalisation des cytokines. PIC affecte préférentiellement l'expression de gènes impliqués dans les processus d'adhérence cellulaire, dans les voies de signalisation des cytokines et du dsRNA, ainsi que l'expression de gènes codant pour des facteurs de transcription et les gènes MHC-connexes. L'exposition des cellules pancréatiques au PIC et/ou à l' IFN-gamma induit l'expression de plusieurs chemokines et cytokines qui pourraient contribuer à l'attraction et à l'activation des cellules mononucléaires pendant l'infection virale; l' IFN-gamma exerce un feedback positif sur sa voie de signalisation intracellulaire. Le traitement au PIC + IFN-gamma inhibe l'expression de l'ARNm de l'insuline et du GLUT-2, le transporteur du glucose spécifique aux cellules pancréatiques beta sans toutefois modifier l'expression de l' ARNm codant pour le facteur de transcription pdx-1. En se basant sur les résultats obtenus, nous proposons un modèle général pour les mécanismes moléculaires impliqués dans le dysfonctionnement et la mort de la cellule beta observés après exposition au dsRNA + IFN-gamma.

No description available.