<p>The ability to image ventilation and perfusion enables pulmonary researchers to study functional metrics of gas exchange on a regional basis. There is a huge interest in applying imaging methods to study the large number of genetic models of pulmonary diseases available in small animals. Existing techniques to image ventilation and perfusion are often associated with low spatial resolution and ionizing radiation. Magnetic Resonance Imaging (MRI) has been demonstrated successfully for ventilation and perfusion studies in humans. Translating these techniques in small animals remains challenging. This work addresses the ventilation and perfusion imaging in small animals using MRI. </p><p>Qualitative ventilation imaging in rats and mice is possible and has been demonstrated using MRI, however perfusion imaging remains a challenge. In humans and large animals perfusion can be assessed using dynamic contrast-enhanced (DCE) MRI with a single bolus injection of a gadolinium (Gd)-based contrast agent. But the method developed for the clinic cannot be translated directly to image the rat due to the combined requirements of higher spatial and temporal resolution. This work describes a novel image acquisition technique staggered over multiple, repeatable bolus injections of contrast agent using an automated microinjector, synchronized with image acquisition to achieve dynamic first-pass contrast enhancement in the rat lung. This allows dynamic first-pass imaging that can be used to quantify pulmonary perfusion. Further improvements are made in the spatial and temporal resolution by combining the multiple injection acquisition method with Interleaved Radial Imaging and 'Sliding window-keyhole' reconstruction (IRIS). The results demonstrate a simultaneous increase in spatial resolution (<200>um) and temporal resolution (<200>ms) over previous methods, with a limited loss in signal-to-noise-ratio. </p><p>While is it possible to create high resolution images of ventilation in rats using hyperpolarized ³He, extracting meaningful quantitative information indicative of changes in ventilation is difficult. In this work, we also present a signal calibration technique used to normalize the signal of ³He to volume of ³He which can then be used to extract quantitative information of changes in ventilation via normalized difference maps. Combining the techniques for quantitative ventilation and quantitative perfusion we perform studies of change in ventilation/perfusion (V/Q) before and after airway obstruction in rats. The technique is sensitive in detecting statistically significant differences in the heterogeneity of the distribution of V/Q ratio.</p> / Dissertation

New survey data indicate that injection drug users (IDU) in Victoria, BC who share syringes do so with a single person. These partnerships pose an obvious health risk to IDU, as blood borne illnesses are transmitted through the sharing of injection equipment. Here we formulate an ordinary di erential equation (ODE) model of pair formation and separation. Susceptible-infectious (SI) disease dynamics are built into this model so as to describe the syringe-mediated transmission of human immune de ciency virus (HIV) and hepatitis C virus (HCV) among IDU. We utilize a novel parameter estimation approach, and t the distribution of partnership durations observed in Victoria. The basic reproduction number is derived, and its qualitative behavior explored with both analytical and numerical techniques.

abstract: The pathophysiology of neurodegenerative diseases, such as Alzheimer’s disease (AD), remain difficult to ascertain in part because animal models fail to fully recapitulate the complex pathophysiology of these diseases. In vitro models of neurodegenerative diseases generated with patient derived human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) could provide new insight into disease mechanisms. Although protocols to differentiate hiPSCs and hESCs to neurons have been established, standard practice relies on two dimensional (2D) cell culture systems, which do not accurately mimic the complexity and architecture of the in vivo brain microenvironment. I have developed protocols to generate 3D cultures of neurons from hiPSCs and hESCs, to provide more accurate models of AD. In the first protocol, hiPSC-derived neural progenitor cells (hNPCs) are plated in a suspension of Matrigel™ prior to terminal differentiation of neurons. In the second protocol, hiPSCs are forced into aggregates called embryoid bodies (EBs) in suspension culture and subsequently directed to the neural lineage through dual SMAD inhibition. Culture conditions are then changed to expand putative hNPC populations and finally differentiated to neuronal spheroids through activation of the tyrosine kinase pathway. The gene expression profiles of the 3D hiPSC-derived neural cultures were compared to fetal brain RNA. Our analysis has revealed that 3D neuronal cultures express high levels of mature pan-neuronal markers (e.g. MAP2, β3T) and neural transmitter subtype specific markers. The 3D neuronal spheroids also showed signs of neural patterning, similar to that observed during embryonic development. These 3D culture systems should provide a platform to probe disease mechanisms of AD and enable to generation of more advanced therapeutics. / Dissertation/Thesis / Masters Thesis Bioengineering 2016

In the adult mammalian brain, neural stem cells (NSCs) in the dentate gyrus (DG) of hippocampus continuously give rise to new neurons that provide structural plasticity and playing a role in learning and memory. Alterations in this process of adult neurogenesis have been linked to the pathology of various neurodegenerative diseases. Nevertheless, our knowledge regarding adult neurogenesis in the spectrum disorder of amyotrophic lateral sclerosis (ALS)/ frontotemporal dementia (FTD) remains incomplete. Therefore, I aimed to characterize how the ALS/FTD-associated mutation within the FUS protein affects adult NSCs during aging and how this may contribute to hippocampal function. For this purpose, a heterozygous knock-in ALS/FTD mouse model was used in which one copy of FUS harbors a deletion of its nuclear localization signal and their wild-type littermates were used as a control. The obtained results suggest a strong impact of the developing FUS-associated pathology on the hippocampal NSCs in the adult brain. For the first time, an age-dependent increase in NSCs proliferation and in the production of early postmitotic neurons was observed. In addition, the hippocampal network function and the response of adult neurogenesis to external stimulations seem to be altered by FUS mutation. The expected boost of NSC activity upon external stimulation appears to be blocked at particular developmental stages, however, the effect of the used strategies on the function of the hippocampal-cortical circuitry could still be detected. For further investigation, the in vitro analyses of adult NSCs were performed. Their proliferation activity was markedly increased, in comparison to wild-type cells, confirming the in vivo results. It appears that cells harboring mutated FUS are more likely to exit the quiescence state and enter the cell cycle. Furthermore, the combined cultivation of cells of both genotypes revealed that wild-type cells accelerate their proliferation to a similar level as mutant cells. Interestingly, the proliferation behavior of mutant cells remained unchanged regardless of the presence of wild-type cells. This suggests a spreading or transfer of the pathological mechanisms between the NSCs. Additionally, a commonly known hallmark of ALS/FTD pathology, the generation and accumulation of stress granules, was examined. Understanding the pathomechanisms of ALS/FTD-FUS in relation to adult hippocampal neurogenesis clearly requires further investigation. This appears to be crucial to provide the fundamental basis for new NSC-based preventative and therapeutic strategies.:1. Introduction 8 1.1. Adult hippocampal neurogenesis 9 1.1.1. The process of generating new granule neurons within the adult dentate gyrus 9 1.1.2. Regulation and function of AHN 12 1.2. Amyotrophic lateral sclerosis/ frontotemporal dementia 17 1.2.1. Amyotrophic lateral sclerosis/ frontotemporal dementia – spectrum disorder 17 1.2.2. FUS protein characterization and its contribution to ALS/FTD pathology 20 1.2.3. Knock-in mouse model of ALS/FTD-FUS 24 2. Aims 27 3. Materials and methods 28 3.1. In vivo 28 3.1.1. Animals 28 3.1.2. Physical activity 28 3.1.3. Enrichment environment 28 3.1.4. Thymidine labeling and tissue preparation 29 3.1.5. Immunohistochemistry - DAB staining 29 3.1.6. Quantification 30 3.1.7. Electrophysiology 30 3.1.8. Statistical analysis 30 3.2. In vitro 31 3.2.1. Generation of cell line 31 3.2.2. Culturing cell lines 31 3.2.3. Western Blotting 32 3.2.3.1. Collecting protein lysates 32 3.2.3.2. Bradford method 32 3.2.3.3. Electrophoresis and transfer 32 3.2.3.4. Fluorescent immunostaining 33 3.2.4. CFSE labeling and FACS analysis 33 3.2.5. Stress treatment 34 3.2.6. Fluorescent immunostaining (without BrdU) 34 3.2.7. BrdU labeling 35 3.2.8. Fluorescent immunostaining with BrdU 35 3.2.9. Cell cycle analysis 35 3.2.10. Statistical analysis 36 4. Results 37 4.1. In vivo 37 4.1.1. General characterization of the knock-in mouse model of ALS/FTD-FUS 37 4.1.2. Proliferation of hippocampal NSCs in FusDNLS/wt mice is unchanged at young age but strongly increased at old age 38 4.1.3. Response of AHN to external stimulus, such as physical activity, is altered in aged FusDNLS/wt mice 41 4.1.4. FUS mutation causes an increased generation of DCX-positive immature neurons in the DG and an altered response to the stimulatory effects of ENR in mice at 12 months of age 45 4.2. In vitro 52 4.2.1. Partial cytoplasmic mislocalization of mutant FUS protein in FusDNLS/wt cells 52 4.2.2. General level of FUS protein remains stable regardless of genotype 54 4.2.3. FusDNLS/wt cells generated from young animals exhibit distinct proliferation pattern 56 4.2.4. Fuswt/wt cells, cultured together with FusDNLS/wt ones, exhibit FusDNLS/wt-like acceleration of proliferation activity 57 4.2.5. Stress granules dynamics in cells in vitro 59 4.2.6. FusDNLS/wt cells are more likely-activated 65 5. Discussion 67 6. Bibliography 79

<p dir="ltr">Neurodegenerative diseases are progressive, incurable conditions that affect tens of millions of people worldwide and are characterized by the aggregation of misfolded protein in the brain. Though the precise role of these amyloid aggregates in the onset and progression of these diseases is not clear at this time, there is a pressing need to understand how they form and spread in human disease. In service to these aims, I have conducted three small projects to expand knowledge in this regard. I first investigated the use of thioflavin T, a common amyloid stain, as an affinity reagent for the general purification of amyloid filaments from <i>ex vivo </i>samples, observing strong potential using a relatively simple, inexpensive magnetic bead conjugation technique. I next analyzed the formation of filaments of a truncated recombinant amyloid-beta peptide with residues 1-35, observing a new filament type formed at low pH in the wild-type sequence of this truncated peptide. Finally, I conducted structural studies on amyloid-beta(1-42) filaments prepared under different conditions consistent with traumatic brain injury to observe their effect on amyloid folding. While I found no effect of differential conditions on filament type, the low-resolution structures solved were highly consistent with aggregates found in Alzheimer’s disease patients, presenting a promising way forward for <i>in vitro</i> modeling of amyloid filaments that are true to pathology. In sum, the work here presented advances the concepts of both how amyloid aggregates from patient brains can be best prepared for structural analysis, and the factors underpinning their aggregation at the onset of neurodegenerative disease.</p>

A doença do enxerto versus hospedeiro (DEVH) é uma grave complicação do transplante de órgãos sólidos, com alta mortalidade. Seu estudo tem sido limitado pela carência de modelos experimentais apropriados. Descreve-se um modelo de DEVH baseado no aumento do quimerismo, sua evolução clínica, histopatológica, do número das células quiméricas, do perfil das citocinas e da tolerância imunológica. Ratos Lewis (LEW) foram submetidos a transplante simultâneo de intestino delgado e medula óssea provenientes de ratos ACI (grupo de estudo - E) ou LEW (grupo controle - C), tratados com FK-506 (1 mg/Kg/dia) entre o 0 e 13o PO, e uma dose semanal daí por diante. Os ratos foram divididos nos seguintes grupos: E1- 6 ratos sacrificados no 120o PO. E2- 8 ratos após apresentarem sinais clínicos graves de DEVH entre o 189o e o 271o PO. Como controle, ratos LEW foram receptores dos mesmos tipos de enxertos provenientes de ratos LEW, submetidos à mesma imunossupressão e foram assim divididos: C1- 6 ratos sacrificados no 120o PO, C2- 5 ratos sacrificados entre o 223o e o 270o PO. A citometria de fluxo foi realizada para quantificar a porcentagem das células linfóides de ACI doadores no sangue periférico nos E1, E2 em 6 períodos: 30o PO, 65o PO, 95o PO, 120o PO, 160o PO, 200o PO. Os animais foram examinados 2 vezes por semana à procura de sinais de DEVH (rash cutâneo, perda de peso, de pelo e hiperqueratose). No sacrifício dos animais do grupo E1 e C1, foram colhidas amostras de língua (LI), de linfonodos cervicais (LC), intestino delgado do receptor e do enxerto para análise das citocinas IL-2, IL-4, IL-6, IL-10, IFN-gama e TNF-alfa por meio da reação em cadeia da polimerase. Em todos os grupos foram também colhidas amostras destes órgãos para histopatologia e nos animais do grupo E2 linfonodos cervicais foram processados para análise da reatividade celular por meio da reação mista dos linfócitos (MLR). A evolução clínica e histopatológica foi graduada de 0 a 3 de acordo com a severidade dos sintomas e do infiltrado mononuclear das amostras. Os ratos dos grupos E1 e E2 iniciaram sinais da DEVH entre o 84o e 115o PO. Os ratos dos grupos C1 e C2 não apresentaram evidência de DEVH. Amostras de LI e LC dos ratos do grupo E1 apresentaram alterações histopatológicas grau 2 e do grupo E2 apresentaram alterações histopatológicas grau 3, respectivamente. Nenhuma alteração histopatológica foi encontrada nos ratos do grupo controle e em amostras do ID. Nenhuma alteração histopatológica foi encontrada no intestino delgado do receptor e do enxerto. O aumento da porcentagem de células do doador no sangue periférico do receptor foi progressivo chegando a 5,4±2.3% no 10o período, 21±4,6% no 3o período e 39,3±4% no 6o período. IL-2, IL-6, IL-10, IFN-gma e TNF-alfa estiveram aumentados em língua e IL-4, IL-6, IL-10, IFN-gama e TNF-alfa em linfonodos cervicais. Os linfócitos de ratos do grupo E2 mostraram hiporreatividade aos de ratos ACI e hiperreatividade aos de ratos PVG (terceira parte) denotando tolerância imunológica. Neste modelo experimental há uma inexorável evolução imunológica para DEVH; existe correlação direta entre o aumento do quimerismo em sangue periférico e da expressão de citocinas em língua e linfonodos cervicais e a severidade da DEVH, além da indução de tolerância imunológica do rato do grupo E2 quimérico ao rato ACI normal. / Graft-versus-host disease (GVHD) has been a major concern after small bowel transplantation (SBTX) and the lack of suitable experimental models has limited the study of GVHD after solid organ transplantation. Here we describe a re1evant experimental model of GVHD after fully allogeneic SBTX based on chimerism augmentation, its clinical and histophatological evolution, cytokine involvement, responsible donor cell and immunologic tolerance analysis. LEW rat recipients received orthotopic SBTX and simultaneous donor bone marrow cell infusion (250x106), from ACI rats (experimental group - E) or LEW (control group C). FK-506 was administered dayly at a dose of 1 mg/kg on day 0 to 13, then continued as a weekly injection of same dose until the experimental end point. The recipients were divided in the following groups: E1 - 6 rats sacrificed at 120° POD. E2 - 8 rats sacrificed with critical GVHD between DPO 189 to 271. LEW recipient of LEW grafts, under the same immunossupression were used as control and divided as: C1 - 6 rats sacrificed at POD 120; C2- 5 rats sacrificed between 223 and 270 POD the number of donor cell in the recipient circulation was determined by flowcytometry in 6 pos-operative time: 30, 65, 95, 120, 160, 200. The rats were analyzed twice a week for body weigh and searching for signs of GVHD (cutaneous rush, hiperkeratosis and loss of hair and body weigh). At the sacrificed, samples from tongue (TG), cervical lymph node (CLN), donor (SBD) and recipient (SBR) small bowel were taken from all animals for histophatology and from E1 and C1l animals for IL-2, IL-4, IL-6, IL-10, IFN-gama e TNF-alfa cytokines analysis using reverse transcription polymerase chain reaction. Samples from cervical lynph nodes of 5 animals from group E2 were used for mixed lymphocyte reaction for tolerance analysis. The clinical and histophatological evolution of the disease were evaluated from degree 0 to 3 according to the severity. GVHD in E1 and E2 animals started between 84 and 115 POD. Histophatological analysis of TG and CLN showed that E1 animals present GVHD grade 2 and E2 animals grade 3. The increase of donors cells in the recipient circulation was progressive and account for 5.4± 2.3% at POD 30, 21.4±4.6% at POD 95 and 39.3±4% at POD 200. IL-4, IL-6, IL-10, IFN-gama e TNF-alfa were upregulated in CLN and IL-2, IL-6, IL-10, IFN-gama e TNF-alfa were upregulated in TG when compared with the respective controls. The lymphocytes from E2 group showed hyporeactivety to lymphocytes of normal ACI and hypereactivety to those of PVG, meaning tolerance. No cytokines alteration was noted in SBD neither SBR. Animals from group C1 and C2 did not present any sign of disease. This result show that GVHD is a inexoravel evolution under the experimental conditions of this study and the evolution of the disease is near correlated with the augmentation of the donor cells in the recipient circulation and upregulation of cytokines gene expression in target organs. Tolerance to the same donor strain lynphocytes was also noted.

A doença do enxerto versus hospedeiro (DEVH) é uma grave complicação do transplante de órgãos sólidos, com alta mortalidade. Seu estudo tem sido limitado pela carência de modelos experimentais apropriados. Descreve-se um modelo de DEVH baseado no aumento do quimerismo, sua evolução clínica, histopatológica, do número das células quiméricas, do perfil das citocinas e da tolerância imunológica. Ratos Lewis (LEW) foram submetidos a transplante simultâneo de intestino delgado e medula óssea provenientes de ratos ACI (grupo de estudo - E) ou LEW (grupo controle - C), tratados com FK-506 (1 mg/Kg/dia) entre o 0 e 13o PO, e uma dose semanal daí por diante. Os ratos foram divididos nos seguintes grupos: E1- 6 ratos sacrificados no 120o PO. E2- 8 ratos após apresentarem sinais clínicos graves de DEVH entre o 189o e o 271o PO. Como controle, ratos LEW foram receptores dos mesmos tipos de enxertos provenientes de ratos LEW, submetidos à mesma imunossupressão e foram assim divididos: C1- 6 ratos sacrificados no 120o PO, C2- 5 ratos sacrificados entre o 223o e o 270o PO. A citometria de fluxo foi realizada para quantificar a porcentagem das células linfóides de ACI doadores no sangue periférico nos E1, E2 em 6 períodos: 30o PO, 65o PO, 95o PO, 120o PO, 160o PO, 200o PO. Os animais foram examinados 2 vezes por semana à procura de sinais de DEVH (rash cutâneo, perda de peso, de pelo e hiperqueratose). No sacrifício dos animais do grupo E1 e C1, foram colhidas amostras de língua (LI), de linfonodos cervicais (LC), intestino delgado do receptor e do enxerto para análise das citocinas IL-2, IL-4, IL-6, IL-10, IFN-gama e TNF-alfa por meio da reação em cadeia da polimerase. Em todos os grupos foram também colhidas amostras destes órgãos para histopatologia e nos animais do grupo E2 linfonodos cervicais foram processados para análise da reatividade celular por meio da reação mista dos linfócitos (MLR). A evolução clínica e histopatológica foi graduada de 0 a 3 de acordo com a severidade dos sintomas e do infiltrado mononuclear das amostras. Os ratos dos grupos E1 e E2 iniciaram sinais da DEVH entre o 84o e 115o PO. Os ratos dos grupos C1 e C2 não apresentaram evidência de DEVH. Amostras de LI e LC dos ratos do grupo E1 apresentaram alterações histopatológicas grau 2 e do grupo E2 apresentaram alterações histopatológicas grau 3, respectivamente. Nenhuma alteração histopatológica foi encontrada nos ratos do grupo controle e em amostras do ID. Nenhuma alteração histopatológica foi encontrada no intestino delgado do receptor e do enxerto. O aumento da porcentagem de células do doador no sangue periférico do receptor foi progressivo chegando a 5,4±2.3% no 10o período, 21±4,6% no 3o período e 39,3±4% no 6o período. IL-2, IL-6, IL-10, IFN-gma e TNF-alfa estiveram aumentados em língua e IL-4, IL-6, IL-10, IFN-gama e TNF-alfa em linfonodos cervicais. Os linfócitos de ratos do grupo E2 mostraram hiporreatividade aos de ratos ACI e hiperreatividade aos de ratos PVG (terceira parte) denotando tolerância imunológica. Neste modelo experimental há uma inexorável evolução imunológica para DEVH; existe correlação direta entre o aumento do quimerismo em sangue periférico e da expressão de citocinas em língua e linfonodos cervicais e a severidade da DEVH, além da indução de tolerância imunológica do rato do grupo E2 quimérico ao rato ACI normal. / Graft-versus-host disease (GVHD) has been a major concern after small bowel transplantation (SBTX) and the lack of suitable experimental models has limited the study of GVHD after solid organ transplantation. Here we describe a re1evant experimental model of GVHD after fully allogeneic SBTX based on chimerism augmentation, its clinical and histophatological evolution, cytokine involvement, responsible donor cell and immunologic tolerance analysis. LEW rat recipients received orthotopic SBTX and simultaneous donor bone marrow cell infusion (250x106), from ACI rats (experimental group - E) or LEW (control group C). FK-506 was administered dayly at a dose of 1 mg/kg on day 0 to 13, then continued as a weekly injection of same dose until the experimental end point. The recipients were divided in the following groups: E1 - 6 rats sacrificed at 120° POD. E2 - 8 rats sacrificed with critical GVHD between DPO 189 to 271. LEW recipient of LEW grafts, under the same immunossupression were used as control and divided as: C1 - 6 rats sacrificed at POD 120; C2- 5 rats sacrificed between 223 and 270 POD the number of donor cell in the recipient circulation was determined by flowcytometry in 6 pos-operative time: 30, 65, 95, 120, 160, 200. The rats were analyzed twice a week for body weigh and searching for signs of GVHD (cutaneous rush, hiperkeratosis and loss of hair and body weigh). At the sacrificed, samples from tongue (TG), cervical lymph node (CLN), donor (SBD) and recipient (SBR) small bowel were taken from all animals for histophatology and from E1 and C1l animals for IL-2, IL-4, IL-6, IL-10, IFN-gama e TNF-alfa cytokines analysis using reverse transcription polymerase chain reaction. Samples from cervical lynph nodes of 5 animals from group E2 were used for mixed lymphocyte reaction for tolerance analysis. The clinical and histophatological evolution of the disease were evaluated from degree 0 to 3 according to the severity. GVHD in E1 and E2 animals started between 84 and 115 POD. Histophatological analysis of TG and CLN showed that E1 animals present GVHD grade 2 and E2 animals grade 3. The increase of donors cells in the recipient circulation was progressive and account for 5.4± 2.3% at POD 30, 21.4±4.6% at POD 95 and 39.3±4% at POD 200. IL-4, IL-6, IL-10, IFN-gama e TNF-alfa were upregulated in CLN and IL-2, IL-6, IL-10, IFN-gama e TNF-alfa were upregulated in TG when compared with the respective controls. The lymphocytes from E2 group showed hyporeactivety to lymphocytes of normal ACI and hypereactivety to those of PVG, meaning tolerance. No cytokines alteration was noted in SBD neither SBR. Animals from group C1 and C2 did not present any sign of disease. This result show that GVHD is a inexoravel evolution under the experimental conditions of this study and the evolution of the disease is near correlated with the augmentation of the donor cells in the recipient circulation and upregulation of cytokines gene expression in target organs. Tolerance to the same donor strain lynphocytes was also noted.

<p dir="ltr">The spread and increasing prevalence of chronic wasting disease (CWD) in white-tailed deer (<i>Odocoileus virginianus</i>) has far reaching implications for natural resource management in Indiana. CWD is a transmissible spongiform encephalopathy that affects white-tailed deer and other cervids. This disease is invariably fatal in white-tailed deer, and there is concern that its continuing spread will cause populations to decline. White-tailed deer are also a culturally and economically important game species. Therefore, effective management of CWD must consider the epidemiologic, social, and economic dimensions of disease management. In Chapter One of my dissertation, I apply an agent-based model (ABM) framework to simulate how preemptive harvest increase and reactive culling affect CWD persistence and geographic spread. I found that preemptive harvest and reactive culling both had a small effect on preventing the establishment of CWD in the deer population. In Chapter Two, I test whether presenting deer hunters and non-deer hunters with results from CWD models and images of sick or healthy deer increases behavioral intention to engage in CWD mitigating behaviors. I found that using the web app did not change the behavioral intention of hunters and non-hunters. In Chapter Three, I conduct a cost-effectiveness analysis to identify the optimal combination of CWD surveillance and culling effort in terms of disease prevention. I found that testing 40% of hunter-harvested deer for CWD and culling 30% of deer within culling zones was most cost-effective in terms of disease prevention. In Chapter Four, I synthesize the results of the preceding chapters and discuss options for CWD management in Indiana.</p>

L’ascomicete Guignardia bidwellii, agente causale del marciume nero della vite, è un patogeno economicamente importante in alcuni areali viticoli. La conoscenza, disponibile sul marciume nero dell’uva, è stata recuperata dalla letteratura, analizzata e sintetizzata per sviluppare un modello meccanicistico del ciclo di vita del patogeno, guidata dalle variabili meteorologiche e dalla fenologia della vite, e basata sull'analisi dei sistemi. Il modello è stato poi valutato per la sua capacità di rappresentare il sistema reale e la sua utilità per la comprensione di epidemie di marciume nero su foglie e grappoli in un vigneto del Nord Italia, nel 2013 al 2015. Successivamente, le mancanze di conoscenza sono state analizzate, studiate e quindi colmate attraverso specifici esperimenti. In un primo passo, le dinamiche dell’inoculo primario e dei modelli di dispersione (di entrambi ascospore e conidi) da mummie svernate sono state studiate in un vigneto sperimentale per tre anni. In un secondo passo, l'effetto della temperatura e dell'umidità sulla formazione di picnidi di G. bidwellii e la successiva estrusione di cirri, nelle lesioni su foglia, la produzione e la germinazione dei conidi (inoculo secondario), e la lunghezza del periodo di latenza sono stati studiati sia in condizioni di campo che in ambiente controllato. In un terzo passo, sono stati condotti studi in ambiente controllato per studiare la produzione di conidi di G. bidwellii sulle lesioni di foglie, influenzata da lavaggi ripetuti e alternando periodi di secco ed umido. Il modello epidemiologico sviluppato in questa tesi può essere utilizzata da viticoltori come strumento predittivo per la pianificazione di trattamenti fungicidi nei vigneti. / The ascomycete Guignardia bidwellii, causal agent of black-rot on grapevines, is an economically important pathogen in some viticultural areas. The available knowledge on black-rot of grape was retrieved from literature, analyzed, and synthesized to develop a mechanistic model of the life cycle of the pathogen, driven by weather and vine phenology, and based on the systems analysis. The model was then evaluated for its ability to represent the real system and its usefulness for understanding black-rot epidemics on leaves and bunches in a vineyard of north Italy, in 2013 to 2015. Thereafter, weaknesses in our knowledge were analysed and studied through specific experiments. In a first step, dynamics of primary inoculum and dispersal patterns (both ascospores and conidia) from overwintered grape mummies were investigated in an experimental vineyard during three years. In a second step, the effect of temperature and humidity on the formation of G. bidwellii pycnidia and the extrusion of cirri in grape leaf lesions, production and germination of conidia (secondary inoculum), and the length of the latency period were studied under both environmental and controlled conditions. In a third step, environmental-controlled studies were conducted to investigate the production course of G. bidwellii conidia on grape leaf lesions as influenced by repeated washing events and alternate dry and wet periods. The model developed in this thesis can be used by vinegrowers as a predictive tool for scheduling fungicide sprays in the vineyards.

1. Unterschiedliche Definitionen der Gesundheit mit verschiedenen Betrachtungsweisen (WHO: Der Mensch eine biopsychosoziale Einheit. Schulmedizin: ohne klinischen und paraklinischen Befund mit Orientierung an kritikbedürftigen Referenzmittelwerten) führt bei Fachleuten, Behörden und Laien zu Verwirrungen, wenn es um die Beurteilung gesundheitlicher Schäden geht. 2. Es wurde die Aufgabe gestellt zu prüfen, welche der beiden Definitionen der Realität näher kommt. 3. Mittels der chronopsychobiologischen Regulationsdiagnostik, des Dreiphasenentspannungstests (Hecht und Balzer 2001), wurden unter dem Aspekt der beiden Gesundheitsdefinitionen drei Gruppen untersucht (je 40 Probanden). - klinisch Gesunde (klinisch Gesunde nach Schulmedizin ) - Gesunde nach Definition der WHO - Probanden mit nichtorganische Insomnie (ohne pathologische klinische und paraklinische Befunde) 4. Die mit den verwendeten Methoden gewonnenen Daten wiesen aus, dass zwischen den klinisch Gesunden und den Probanden mit nichtorganischer Insomnie weitgehend größere Ähnlichkeiten bestehen. Beide Gruppen zeigten aber zu der Gruppe der Gesunden nach WHO-Definition, welche die biopsychosoziale Einheit des Menschen berücksichtigt, noch hochsignifikante Unterschiede. Die Gruppe der klinisch Gesunden kann daher auf Grund unserer Ergebnisse nicht den Anspruch erheben, real gesund zu sein. 5. Mit der Bezugnahme auf die Internationale Klassifikation der Krankheiten (ICD 10F) haben die von uns untersuchten klinisch Gesunden und die nichtorganischen Insomniker eine mehr oder weniger stark ausgeprägte Symptomatik von psychischen Störungen. Dies müsste bei der Beurteilung von Schadstoff-, Lärm-, und EMF-Wirkungen auf den Menschen, wie auch bei den klinisch-pharmakoloischen Untersuchungen beachtet werden. Die in der Arbeit erzielten Ergebnisse bedürfen durch weitere Untersuchungen eine Fundierung. Sie signalisieren aber sowohl unter praktischen als auch unter theoretischen Aspekten einen dringenden Forschungsbedarf. / 1. Differing definitions of health using different criterea (WHO: The human being as a bio- psycho-social unit versus classical medicine: without clinical and paraclinical results based on suspect reference values) bring confusion to experts, authorities and laymen when assessing health damages. 2. The given task was to check which of the two definitions is closer to reality. 3. Using the chrono-psycho-biological diagnostic of regulation, the three-phase-relaxation test (Hecht and Balzer 2001), three groups were examined considering the aspects of the two health definitions (40 test subjects in the study group). - clinically healthy (clinically healthy per classical medicine definition) - healthy per definition of the WHO - test persons with non organic insomnia (i.e. no pathological or paraclinical findings) 4. The data gained from the employed methods revealed bigger similarities between clinically healthy persons and those with non organic insomnia. Both groups still showed highly significant differences to the group which fulfils the definition of the WHO regarding a human as a bio-psycho-social unit. As a result of this study, persons, though classified as "clinically healthy" might nevertheless not absolutely be healthy in reality. 5. In reference to the international classification of illnesses (ICD 10 F) the groups examined, both of clinically healthy and those with non organic insomnia, have more or less severe psychological symptoms. This should be taken into account when assessing the effects of pollution, noise, and EMF as well as clinical pharmacological studies. These present findings still need broader confirmation by further investigations. However, they clearly indicate, for practical and theoretical considerations, an urgent need for further research.