Immunotoxins (ITs), which consist of antibodies conjugated to toxins, have been proposed as a treatment for cancer and chronic infections. To develop and improve the ITs, different toxins such as ricin, have been used, aiming for higher efficacy against target cells. The toxin pulchellin, isolated from the Abrus pulchellus plant, has similar structure and function as ricin. Here we have compared two plant toxins, recombinant A chains from ricin (RAC) and pulchellin (PAC) toxins, for their ability to kill HIV Env-expressing cells. Briefly, RAC and PAC were produced in E. coli, and chromatographically purified, then chemically conjugated to two different anti-HIV monoclonal antibodies (MAbs), anti-gp120 MAb 924 or anti-gp41 MAb 7B2. These conjugates were characterized biochemically by microcapillary electrophoresis and BCA assay and immunologically by a variety of ELISA tests. We performed a side-by-side comparison of their ability to bind, enter and kill HIV infected cells (H9/NL4-3) or Env-transfected 293T cells, as well as their non-specific toxicity on uninfected or non-transfected parental cells. Cell binding and internalization were studied by flow cytometry and confocal microscopy. Results showed that PAC can function within an effective IT. The ITs demonstrated specific binding against native antigens on persistently HIV-infected cells and recombinant antigens on Env-transfected cells. An irrelevant antibody conjugated to either RAC or PAC had no effect. PAC cytotoxicity appears somewhat less than RAC, the standard for comparison. This is the first report that PAC may have utility for the design and construction of therapeutic ITs, highlighting the potential role for specific cell targeting not only for AIDS also for cancer therapy. / As toxinas imunológicas (TIs), que consistem em anticorpos conjugados com toxinas, foram propostas como tratamento para câncer e infecções crônicas. Para desenvolver e melhorar as TI, diferentes toxinas, como a ricina, foram usadas, visando uma maior eficácia contra células alvo. A toxina pulchellina, isolada da planta de Abrus Pulchellus, tem estrutura e função semelhantes à da ricina. Aqui, comparamos duas toxinas de plantas, cadeias A recombinantes de toxinas de ricina (RAC) e pulchellina (PAC), por sua capacidade de matar células que expressam HIV. Resumidamente, RAC e PAC foram produzidos em E. coli e purificados por cromatografia, depois conjugados quimicamente com dois anticorpos monoclonais anti corpo-HIV diferentes (MAcs), MAc 924 anti-gp120 ou MAc 7B2 anti-gp41. Estes conjugados foram caracterizados bioquimicamente por eletroforese microcapilar e teste BCA e imunologicamente por uma variedade de testes ELISA. Realizamos uma comparação lado-a-lado de sua capacidade de ligar, entrar e matar células infectadas pelo HIV (H9 / NL4-3) ou células 293T transfectadas com Env, bem como a sua toxicidade não específica em parentes não infectados ou não transfectados Células. A ligação celular e a internalização foram estudadas por citometria de fluxo e microscopia confocal. Os resultados mostraram que PAC pode funcionar dentro de uma TI efetiva. As TI demonstraram ligação específica contra antígenos nativos em células persistentemente infectadas pelo HIV e antígenos recombinantes em células transfectadas com Env. Um anticorpo irrelevante conjugado com RAC ou PAC não teve efeito. A citotoxicidade de PAC aparece um pouco menor que o RAC, o padrão de comparação. Este é o primeiro relatório que o PAC pode ter utilidade para o projeto e a construção de TI terapêuticas, destacando o papel potencial para o direcionamento celular específico, não apenas para a AIDS, também para a terapia do câncer.

Glaucoma is characterized by progressive optic nerve head (ONH) deformation and retinal nerve fiber layer (RNFL) thinning but the relative sequence of ONH and RNFL changes in glaucoma remains largely uncertain. It has been proposed that structural damage of the optic nerve can often be detected before detectable functional loss. Therefore, investigating structural changes of the ONH and RNFL is of importance and relevance in the monitoring and management of glaucoma progression. Spectral domain optical coherence tomography (OCT), scanning laser polarimetry (SLP) and confocal scanning laser ophthalmoscopy (CSLO) are the three prevailing digital imaging technologies for measurement of RNFL thickness, RNFL retardance and ONH parameters, respectively. Although these instruments have been extensively investigated for detection of glaucomatous damage, less is known about their relative performance for detection of change in glaucoma progression. Although previous studies on non- human primates showed that disruption of the microtubule structure of the retinal ganglion cell axons detected by SLP as reduction of RNFL retardance, as well as ONH surface deformation detected by CSLO, could be detected prior to reduction of RNFL thickness measured with OCT, clinical data corroborating this observation are lacking. The sequence of change of RNFL thickness, RNFL retardance and ONH parameters has not been investigated in human glaucoma. / This research project aimed to investigate the performance of OCT, SLP and CLSO for change detection of RNFL and ONH damages, determine the relative sequence of change of RNFL retardance and RNFL thickness and ONH deformation, and evaluate if ocular biomechanical properties, measured as corneal hysteresis by the ocular response analyzer (ORA, Reichert Inc.), influence the detection of ONH and RNFL progression in glaucoma patients. We hypothesized that ONH deformation and loss of RNFL retardance could be detected before detectable RNFL thinning and that the baseline corneal hysteresis would be a risk factor for ONH and RNFL damage in glaucoma. / In the first study, we analyzed 184 eyes of 116 patients with glaucoma and 43 normal eyes of 23 healthy individuals followed for a mean of 4.6 years. All subjects had RNFL retardance and RNFL thickness measurements obtained with GDx ECC (Carl Zeiss Meditec) and Cirrus HD-OCT (Carl Zeiss Meditec), respectively, at 4-month intervals. Progressive reduction of RNFL retardance and RNFL thickness were evaluated with Guided Progression Analysis (GPA, Carl Zeiss Meditec) with reference to the RNFL retardance change map and RNFL thickness change map, respectively. Twenty seven eyes of 26 patients showed progressive RNFL thinning whereas 8 eyes of 8 patients had RNFL retardance reduction in the latest follow-up visit. Seven eyes of 7 patients had progressive RNFL thinning and reduction of RNFL retardance detected by both instruments; all had progressive RNFL thinning evident before reduction of RNFL retardance and the mean lag time was 13.4 months (range: 4.0-37.6 months). The survival time of eyes detected with RNFL thinning was significantly shorter than the survival time of eyes detected with reduction of RNFL retardance (P=0.001). No eyes in the normal group showed progressive RNFL changes during follow-up. Collectively, we showed that at a comparable specificity (100%, 95% confidence interval: 96.3%-100%), progressive RNFL thinning was detected more often than progressive reduction of RNFL retardance and the former preceded the latter in eyes with both progressive RNFL thinning and reduction of RNFL retardance. / In the second study using a similar study design, we investigated the sequence of change of ONH surface depression detected by CSLO (HRT 3, Heidelberg Engineering) and RNFL thinning detected by OCT (Cirrus HD-OCT, Carl Zeiss Meditec) in 146 eyes of 90 glaucoma patients followed at approximately 4-month intervals for an average of 5.4 years. Significant ONH surface depression and RNFL thinning were defined with reference to Topographic Change Analysis (TCA, Heidelberg Engineering) and Guided Progression Analysis (GPA, Carl Zeiss Meditec), respectively. At a specificity of 94.3% (95% confidence interval: 86.2%-97.8%) for both RNFL thinning and ONH surface depression (determined in a normal group comprising 70 eyes from 35 normal subjects), 57 eyes (39.0%) had ONH surface depression, 46 eyes (31.5%) had RNFL thinning, and 23 eyes (15.8%) had both in the glaucoma group. Among these 23 eyes, 19 (82.6%) had ONH surface depression detected prior to RNFL thinning and the median lag time was 15.8 months (range, 4.0-40.8 months). Although only 7.0% of eyes (4/57) had RNFL thinning at the onset of ONH surface depression, 45.7% (21/46) had ONH surface depression at the onset of RNFL thinning. The survival time of eyes with ONH surface depression was significantly shorter than the survival time of eyes detected with RNFL thinning (P=0.002). With reference to the HRT TCA and OCT GPA, ONH surface depression occurred before RNFL thinning in a significant proportion of patients with glaucoma at a comparable specificity. / Of note, a significant proportion of eyes had ONH surface depression without any detectable progressive RNFL thinning in the second study, and vice versa. Investigating whether the risk factors for ONH surface depression and RNFL progression are different is therefore important. In the final study, we investigated if baseline corneal hysteresis is a risk factor for progressive ONH surface depression and RNFL thinning. Following the same cohort of 146 eyes of 90 glaucoma patients for an average of 6.8 years, we detected that 65 eyes (44.5%) had progressive ONH surface depression, 55 eyes (37.7%) had progressive RNFL thinning and 20 eyes (13.7%) had visual field progression (based on the EMGT criteria). After adjusting for ages, CCT, baseline diastolic IOP, average IOP during follow-up, baseline disc area and baseline MD in the cox proportional hazards model, baseline corneal hysteresis was significantly associated with ONH surface depression (HR=0.70, P=0.008), visual field progression (HR=0.56, P=0.019), but not with progressive RNFL thinning (HR=0.96, P=0.751). For each 1-mmHg decrease of baseline CH, the hazards for ONH surface depression and visual field progression increased by 30% and 44%, respectively. / In summary, at a comparable level of specificity, progressive ONH surface depression detected by CSLO could be observed prior to progressive RNFL thinning detected by OCT, which preceded identified reduction of RNFL retardance detected by SLP. For eyes with concomitant ONH surface depression, RNFL thinning and visual field progression, ONH surface depression always preceded visual field progression. Our finding indicates that a time window for therapeutic intervention may exist upon detection of ONH surface depression before irreversible RNFL and visual field loss and that measurement of CH would be useful to predict ONH surface depression and visual field progression. / Further studies are required to investigate the sequence of optic nerve head change and RNFL progression with the same instrument. Whether IOP lowering treatment initiated at the time of ONH deformation would be effective to prevent or slow down RNFL and visual field loss needs to be further investigated. A more reliable and accurate measure of the ocular biomechanical properties is necessary for evaluation of their contribution to glaucoma progression. / 青光眼是一種進展性視神經病變，其特徵為﹕視神經乳頭變形，神經纖維層（RNFL）的變薄以及相應的視野缺損。然而，青光眼結構性改變和功能性變化發生的相對順序仍不清楚。視神經結構性改變被認為要早於功能性改變的發生。因此，研究視乳頭的結構性改變具有重要意義，有助於早期診斷青光眼的進展及隨訪青光眼患者。目前主要用於RNFL厚度，RNFL阻滯性以及視乳頭參數的影像學掃描儀器為頻域OCT，鐳射偏振光掃描器（SLP）和共聚焦鐳射掃描眼底鏡（CSLO）。儘管這三種儀器已經廣泛用於青光眼損傷的檢測，但在青光眼患者結構性變化的應用並不常見。既往在非人靈長類動物的實驗中，通過破壞神經節細胞軸突中的微小管結構，從而發現RNFL的阻滯性以及視乳頭的變化要先於RNFL厚度變化的發生。然而在臨床研究中並未得到證實。同時，在青光眼患者中，RNFL厚度變化，RNFL阻滯減少以及視神經頭參數改變之間的先後順序並未得到證實。 / 本次實驗研究的目的在於探討OCT，SLP及CSLO在診斷青光眼病人RNFL及視乳頭進展的能力，確定RNFL厚度變化，RNFL阻滯性減少以及視神經頭參數改變之間的相對順序，以及評估眼反應分析儀（ORA）測得的角膜粘滯性（CH）是否影響視乳頭及RNFL厚度的進展。我們假設：視神經乳頭的變形，RNFL阻滯性的減少要先於RNFL厚度的變化，基線角膜粘滯性的測量會影響視乳頭及RNFL進展的檢測。116個青光眼病人的184隻眼以及23個正常對照的43隻眼被納入第一個研究中。所有受試物件均接受每4個月一次的OCT以及SLP RNFL的掃描，平均隨訪時間為4.6年。通過OCT及SLP中Guided Progression Analysis（GPA, Carl Zeiss Meditec）程式，一系列RNFL厚度及粘滯性圖被自動分析從而獲得RNFL厚度及粘滯性的變化結果。26個青光眼患者的27隻眼表現為RNFL厚度的進行性變薄，8個患者的8隻眼表現為RNFL粘滯性的減少。其中7個患者的7隻眼同時表現為RNFL厚度變薄及粘滯性的減少，所有這7隻眼的RNFL變薄的發生要早於RNFL粘滯性的減少，兩者間隔時間平均為13.4月（4.0-37.6月）。RNFL厚度變薄者的生存概率明顯小於RNFL粘滯性減少的青光眼患者（P=0.001）。隨訪中，我們未發現正常對照組中RNFL厚度變薄或者粘滯性改變者。總體說來，在同一特異性水準（100%），RNFL厚度的變化頻率高於粘滯性的改變，RNFL厚度的變薄要早於粘滯性減少的發生。 / 採用相同於第一個研究的研究方法，我們研究CSLO測得的視乳頭表面凹陷以及測得OCT的RNFL厚度變化發生的相對順序。90個青光眼患者的146隻眼以及35個正常對照物件的70隻眼被納入第二個研究中。所有受試物件均接受4個月一次的CSLO及OCT掃描從而獲得一系列的視神經頭表面的拓撲圖像以及RNFL厚度圖。CSLO TCA及OCT GPA程式自動對比基線及隨訪中所獲得的視神經頭表面的拓撲圖像以及RNFL厚度圖，從而獲得視乳頭表面凹陷及RFNL進展報告。平均隨訪5.4年後，CSLO及OCT在診斷視神經頭及RNFL進展的特異性為94.3%，57只青光眼患眼（39.0%）表現為顯著性視乳頭表面凹陷，46隻眼（31.5%）表現為RFNL厚度的進行性變薄，而23隻眼（15.8%）同時表現為視乳頭面凹陷和RFNL的進行性變薄。在這23只眼中，19隻眼（82.6%）變現為視乳頭表面凹陷先於RFNL厚度變薄的發生，間隔時間的中值為15.8個月（4.0-40.8月）。儘管在顯著性視乳頭表面凹陷發生時，僅有7.0%的患眼表現為RNFL厚度的變薄；但是，在RNFL厚度發生顯著性變薄時已有45.7%的患眼表現為視乳頭表面凹陷。視乳頭表面凹陷患眼的生存概率差於RNFL厚度變薄患眼（P=0.002）。在青光眼患者的隨訪中，CLSO TCA測得的視乳頭表面凹陷要早於OCT GPA測得的RNFL厚度的變化。 / 最後的一個研究目在於評估眼反應分析儀（ORA）測得的基線角膜粘滯性（CH）是否為視乳頭表面凹陷及RNFL厚度變薄的危險因素。平均隨訪同一人群即第二個研究中的90個青光眼患者的146眼6.8年，65隻眼（44.5%）被檢測出具有進行性視乳頭表面凹陷，55隻眼（37.7%）表現為進行性RNFL厚度的變薄，20隻眼（13.7%）表現為進行性視野的缺損（基於EMGT標準）。基線CH與視乳頭表面凹陷，視野進展間具有顯著性相關關係（HR=0.70，P=0.008及HR=0.56，P=0.019），但CH與進行性RNFL厚度變薄間並無顯著性相關關係（HR=0.96，P=0.751）。每1毫米汞柱基線CH的降低，發生視乳頭表面凹陷及視野缺損的危險性將增加30%及44%。CH的測量值與青光眼進展的危險性具有顯著相關關係。 / 總之，在具有可比性特異性水準下，CSLO檢測的進展性視乳頭表面凹陷的發生要先於OCT檢測的進行性RNFL厚度的變薄，後者的發生早於SLP測得的RNFL粘滯性的改變。對於同時有視乳頭表面凹陷，RNFL厚度變薄及RNFL粘滯性改變的青光眼患眼，視乳頭表面凹陷的發生要早於視野的進展。我們的實驗研究表明了在青光眼患者發生視乳頭表面凹陷時，治療的時間窗的存在有助於避免不可逆的RNFL缺失及視野的缺損。角膜粘滯性的測量對於預測視乳頭表面凹陷及視野進展具有重要意義。 / 展望未來的研究中，用同一種儀器進行視乳頭及神經纖維層的隨訪，從而得出相對的變化次序很有必要。研究在視乳頭或者神經纖維層發生變化時進行眼壓的干預是否能避免視功能的進一步損傷顯得尤為重要。用於測量角膜生物學特性的更為準確，可信度更高的儀器真正研發中，以及進一步探討角膜生物學特性與青光眼進展之間的關係。 / Xu, Guihua. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 116-145). / Abstracts also in Chinese. / Title from PDF title page (viewed on 18, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

La microscopie confocale classique permet d'obtenir des images à haute réso- lution de cellules en culture ou dans un tissu biologique excisé. Cette technologie peut être adaptée aux applications in vivo grâce à l'utilisation de fibres optiques et d'optiques miniaturisées. A terme, la microscopie confocale fibrée devrait permettre aux médecins et biologistes de réaliser des biopsies optiques; c'est à dire un exa- men histologique, en temps réel, des tissus biologiques à l'intérieur d'un organisme vivant et directement au contact de la zone d'intérêt. Le but premier de cette thèse est de dépasser les limites matérielles de ces in- struments d'imagerie en développant des outils de recalage d'images spécifiques et innovants. En particulier, le propos de ce manuscrit est cadré par l'objectif de pro- poser, au travers d'outils de création de mosaïques d'images, des biopsies optiques à grand champ aux médecins. Cette application est considérée, dans cette thèse, comme un système, ou un circuit, qui prendrait en entrée un flot de données brutes et délivrerait en sortie des mosaïques d'images à grand champ. Nous détaillons les éléments critiques de ce système, en particulier la reconstruction d'images en temps réel, le recalage linéaire d'images et le recalage non linéaire, avant de présenter la structure du système complet. Les données brutes produites par la microscopie confocale fibrée sont difficiles à interpréter parce qu'elle sont modulées par la structure en nid d'abeille du réseau de fibres optiques et parce qu'elle sont entachées d'artefacts géométriques. Dans ce contexte, nous montrons qu'une reconstruction en temps réel des images peut être utilisée en pré-traitement afin de produire des séquences vidéos directement interprétables. Comme la microscopie confocale fibrée est une imagerie qui se fait au contact des tissus, le mouvement relatif du tissu par rapport à la sonde optique implique qu'il est parfois difficile d'obtenir de manière robuste certaines mesures quantitatives d'intérêt. Nous avons donc attaqué le problème du recalage linéaire, efficace et robuste de paires d'images. Nous montrons que des outils ré- cents provenant du domaine du contrôle robotique par la vision peuvent surpasser les solutions standards utilisées en analyse d'images biomédicales. L'adéquation de ces outils au problème du recalage linéaire d'images nous a amenés à revisiter le problème du recalage non-linéaire. En interprétant le recalage non-linéaire comme un problème d'optimisation sur un groupe de Lie, nous développons un algorithme rapide de recalage difféomorphe non-paramétrique d'images. En plus d'être dif- féomorphe, notre algorithme produit des résultats qui sont similaires à ceux de l'algorithme des démons de Thirion mais qui sont plus lisses et plus proche de la vérité. Finalement, nous obtenons une boîte à outils de reconstruction et de recalage d'images que nous utilisons pour proposer un algorithme robuste de création de mosaïques d'images qui permette de calculer un alignement globalement cohérent à partir de résultats locaux, de compenser les distorsions liées au mouvement et de retrouver les déformations non-rigides. Par ailleurs, notre algorithme de mosaïques d'images a récemment été incorporé dans un essai clinique multicentrique. Cet essai illustre l'intérêt clinique de nos outils dans le cadre spécifique de la surveillance de l'oesophage de Barrett.

Lightly cross-linked polyelectrolyte microgels are materials with interesting properties for a range of applications. For instance, the volume of these particles can be drastically changed in response to pH, ionic strength, temperature, or the concentration of specific ions and metabolites. In addition, microgel particles can bind substantial amounts of oppositely charged substances, such as proteins and peptides, and release them upon changes in the external environment. Consequently, microgels have potential in catalysis, photonics, biomaterials, and not at least, as protective and stimuli-sensitive carriers for protein and peptide drugs. In this thesis, the interaction between anionic microgels and cationic peptides was investigated by monitoring microgel deswelling and reswelling in response to peptide binding and release using micromanipulator-assisted light microscopy. In addition, peptide distribution in microgels was analyzed with confocal laser scanning microscopy and peptide uptake determined with solution depletion measurements. The aim of the thesis was to clarify how parameters such as peptide size, charge density, pH, ionic strength and hydrophobicity influences the peptide binding to, distribution in and release from, polyelectrolyte microgels. Results obtained in this thesis show that electrostatic attraction is a prerequisite for interaction to occur although non-electrostatic contributions are responsible the finer details of the interactions. The size and charge density of the interacting peptides play a major role, as large and highly charged peptides are restricted to enter and interact with the microgel core, thus displaying a surface-confined distribution. The peptide-microgel interaction strength is highly reflected in the probability of peptides to be detached from the gel network. For instance, reducing the electrostatic interactions by adding salt induces significant peptide release of sufficiently small and moderately charged peptides, whereas longer and more highly charged peptides is retained in the microgel network due to the strong interaction, insufficient salt screening, and gel network pore size restriction. Decreasing the charge density of microgel network and/or peptides increases the probability for peptide detachment tremendously. To summarize, interactions occurring in oppositely charged microgel-peptide systems can be tuned by varying parameters such as charge density and peptide size and through this, the peptide uptake, distribution and release can be controlled to alter the performance of microgels in peptide drug delivery.

Small proline-rich protein-2 (SPRR2) functions as a determinant of flexibility and permeability in the mature cornified envelope of the skin. SPRR2 is strongly upregulated by the commensal flora and may mediate signaling to differentiated epithelia of the small intestine and colon. Yet, SPRR2 function in the GI tract is largely unexplored. Using the Caco-2 model of intestinal epithelial differentiation along the crypt-villus axis, we hypothesized that SPRR2 would be preferentially expressed in post-confluent differentiated Caco-2 cells and examined SPRR2 regulation by the protein kinase A pathway (PKA) and short chain fatty acids (SCFAs). Differentiation-dependent SPRR2 expression was examined in cytoskeletal-, membrane-, and nuclear-enriched fractions by immunoblotting and confocal immunofluorescence. We studied the effect of SCFAs, known inducers of differentiation, on SPRR2 expression in pre-confluent undifferentiated Caco-2 cells and explored potential mechanisms involved in this induction using MAP kinase inhibitors. SPRR2 expression was also compared between HIEC crypt cells and 16 to 20 week primary fetal villus cells as well as in different segments in mouse small intestine and colon. We determined if SPRR2 is increased by gram negative bacteria such as S. typhimurium. SPRR2 expression increased in a differentiation-dependent manner in Caco-2 cells and was present in human fetal epithelial villus cells but absent in HIEC crypt cells. Differentiation-induced SPRR2 was down-regulated by 8-Br-cAMP as well as by forskolin/IBMX co-treatment. SPRR2 was predominantly cytoplasmic and did not accumulate in Triton X-100-insoluble cytoskeletal fractions. SPRR2 was present in the membrane- and nuclear-enriched fractions and demonstrated co-localization with F-actin at the apical actin ring. No induction was seen with the specific HDAC inhibitor trichostatin A, while SCFAs and the HDAC inhibitor SBHA all induced SPRR2. SCFA responses were inhibited by MAP kinase inhibitors SB203580 and U0126, thus suggesting that the SCFA effect may be mediated by orphan G-protein receptors GPR41 and GPR43. S. typhimurium induced SPRR2 in undifferentiated cells. We conclude that SPRR2 protein expression is associated with differentiated epithelia and is regulated by PKA signaling and by by-products of the bowel flora. This is the first report to establish an in vitro model to study the physiology and regulation of SPRR2. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2008-04-25 12:39:06.427 / This work was funded by the CIHR GIDRU Training Grant and Aid in Research from Crohn's and Colitis Foundation of Canada

Radiata pine (Pinus radiata) is the most important plantation tree in New Zealand forestry, and factors that reduce the quality of wood cause significant economic loss. Two of the most important of these issues are compression wood and spiral grain. Compression wood is a type of reaction wood, formed when a tree moves away from the vertical, and is characterised by biochemical and structural changes within the wood that reduce its quality and value. Spiral grain, however, is the alignment of the wood grain in a helix around the tree’s axis and away from the vertical. Again, this reduces the structural qualities of the wood and thus its value. Spiral grain and compression wood are notorious for their deleterious effect on the quality of wood produced and are very important for the forest industry due to the huge economic loss they cause. The demand for reliable tools to evaluate these wood quality issues in clonal planting material at an early stage, within 3 years of germination rather than at 8 to 15 years as in current practise, is of ever increasing importance from plant breeders and other industry stake holders. Therefore this research was undertaken with an overall aim to develop quick, easy and reproducible techniques to evaluate young radiata pine clones (up to 3 years old) based on compression wood content and presence of spiral grain. This is important because a shortened breeding cycle could provide significant economic benefits to the forest industry. The incidence of these commercially important wood quality parameters has been studied in this thesis in research conducted on young trees (1 to 3 years old). The research described in this thesis used a variety of different imaging approaches to investigate wood structure, including polarised light and confocal microscopy, and X-ray tomography and circular polarised light scanning. The images achieved have been analysed using a range of different software, including Photoshop, ImageJ and Matlab bringing a quantification approach to the imaging. Compression wood was quantified in young clonal material using images collected with a commercial document scanner, and processed using image analysis tools available in Photoshop. An easy, reliable and robust, automatic image analysis protocol was successfully developed and tested for the detection and quantification of compression wood in these young trees. This new technique to detect and quantify compression wood was based on the thresholding of the blue channel of the scanned RGB image as this was demonstrated to contain the greatest image contrast. Development of this new technique may reduce the waiting time for screening clonal planting materials based on compression wood content. To understand the organisation of the grain at a cellular level within these young trees, confocal microscopy techniques were utilised. The cell wall characteristics and fluorescence properties of compression wood in comparison with normal wood were investigated using a new cellulose specific dye, pontamine fast scarlet 4B. Staining protocols for this dye for confocal microscopy were optimised, and the potential of measuring the microfibril angle of the S1 and S3 layers of the pontamine treated opposite wood was demonstrated through either direct observations of these layers, or through the property of bifluorescence where the dye is excited only when aligned parallel to the polarisation of the incident light. Despite extensive work with confocal microscopy, this technique proved to be unsuitable for investigations of spiral grain because although it provided cellular detail, imaging was limited to the surface layers of sections, and the area over which observations were required was prohibitive. Instead of confocal microscopy, the incidence of spiral grain in young stems was investigated in two completely new ways. Resin canals, which are formed from the same cambial initials as the tracheids and which align with the grain, were used as a proxy to demonstrate the grain changes. A novel technique, using circular polarised light and a professional flatbed scanner, was developed to image whole serial transverse sections of the young stems to detect the resin canals. Using ImageJ, the number and location of resin canals was measured on vertical controls, and trees that had been rocked and leaned. The number and frequency of resin canals were less in tilted trees, especially in compression wood, compared to the higher number of canals formed in the rocked trees. More importantly, a combination of serial sectioning and this approach allowed a 3-dimensional view of the orientation of resin canals inside a stem to be generated with ImageJ, and the angles of these canals could be measured using Matlab. The resin canals were oriented with a left-handed spiralling near the stem surface whereas the canals near to the pith were nearly straight, consistent with previous observations of the development of spiral grain in radiata pine. However, it was observed that while vertical trees had a symmetric pattern of grain and grain changes around the stem, this was not the case in tilted trees. In these, the opposite wood often had severe spiral grain visible through formation of twist whereas the compression wood formed on the lower side had bending. Consistent with this, grain associated with compression wood was significantly straighter than in opposite wood. This hitherto unknown link between the incidence of compression wood and spiral grain was investigated and explained on the basis of the characteristics of resin canals in these types of wood. X-ray micro-tomography was also used to investigate resin canals in the stubs from which serial sections were collected. The 3D reconstructions of the resin canals showed exactly the same patterns as observed by polarised light scanning.

Optical Confocal Holography is a combination of two well known concepts: confocal microscopy and optical (laser) holography. Confocal microscopy places an aperture at a conjugate focus to the specimen focus. This filters any rays that are not on the focus plane, allowing a 3-dimensional image of the specimen to be built up over a set of planes. Holography is the measurement of both the amplitude and phase characteristics of light. Typically most methods only measure the amplitude of the image. The phenomenon of interference allows the determination of the phase shift for a coherent source as well. The phase information is directly related to the index of refraction of a material, which in turn is a function of the temperature and composition. As a technique, confocal holography holds promise to better characterize many physical processes in materials science, such as combustion and convection. It also may contribute to the biological sciences by imaging low-contrast, weak-phase objects. Thanks to the ongoing, continued improvement in computer processing speed, it has recently become practical to interpret data from confocal holography microscopy with a computer. The objective of the microscope is to non-invasively measure the three-dimensional, internal temperatures and compositions (e.g. solute/solvent gradient) of a specimen. My contributions over the course of two years to the project were: generation and optimization of an optical design with a software package known as Zemax; sourcing and purchasing all components; formation of a CAD model of the microscope; experiments to characterize building vibrations and air currents; and the development of software in Visual Basic to simulate holograms and execute reconstruction algorithms for the specific application of confocal holography.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 8 uppsatser.

Thesis (Ph.D.)--University of Wollongong, 2005. / Typescript. Includes bibliographical references: p. 198-203.

Mucosal associated invariant T (MAIT) cells are a population of innate-like lymphocytes, with an emerging role in tuberculosis (TB). They are characterised by the expression of high levels of CD161 and IL-18Rα, possession of a Vα7.2⁺ T cell receptor (TCR), and restriction by the MHC class I-related protein (MR1). MAIT cells can be activated by MR1 presenting microbe-derived riboflavin metabolites; or, by the cytokines IL-12 and IL-18 in a TCR-independent fashion. How human MAIT cells integrate these signals for their activation in response to Mtb is unclear. Lymphatic TB (LNTB) is a common extra-pulmonary manifestation of TB; however, little is known about the status of MAIT cells in LNTB - or in other granulomatous diseases, such as sarcoidosis. In this study, an in vitro approach was used to probe MAIT cell activation by Mtb, and the roles of IL-12/-18, the TCR, cell-cell contact and the immunological synapse (IS). Following TCR ligation, TNFα expression was rapid and transient, and was enhanced following sustained IL-12/-18 exposure. IFNγ expression occurred following sustained exposure to ng/ml concentrations of IL-12/-18; however, alongside TCR stimulation, pg/ml concentrations were sufficient. Using an artificial bilayer system, CD161 was excluded from the central regions of the MAIT cell IS, whilst the distribution of IL-18Rα remained unaffected. In response to Mtb and BCG, MR1 was necessary for rapid activation and TNFα expression, IL-12/-18 were necessary for robust and sustained IFNy expression, whilst an anti-Mtb effect was indicated in an intracellular infection model. Assessment of patients with TB or sarcoid lymphadenopathy revealed a depletion of MAIT cells in the blood in sarcoidosis, but not LNTB. In both groups, MAIT cells could be detected within a proportion of sampled lymph nodes. Overall, these findings indicate the importance of inflammatory cytokine signals in the induction of high-intensity and sustained MAIT cell effector function, including in response to Mtb. The observation of a numerical deficiency of MAIT cells in sarcoidosis requires further investigation.