Primary cilia are sensory organelles that are important for initiating cell division in the brain, especially through sonic hedgehog (Shh) signaling. Several lines of evidence suggest that the mitogenic effect of Shh requires primary cilia. Proliferation initiated by Shh signaling plays key roles in brain development, in neurogenesis in the adult hippocampus, and in the generation of glial cells in response to cortical injury. In spite of the likely involvement of cilia in these events, little is known about their characteristics. Centrosomes, which are associated with primary cilia, also have multiple influences on the cell cycle, and they are important in assembling microtubules for the maintenance of the cell’s cytoskeleton and cilia. The cilia of terminally differentiated neurons have been previously examined with respect to length, incidence, and receptors present. However, almost nothing is known about primary cilia in stem cells, progenitors, or differentiated glial cells. Moreover, it is not known how the properties of cilia and centrosomes may vary with cell cycle or proliferative potential, in brain or other tissues. This dissertation focuses first on neurogenesis in the hippocampal subgranular zone (SGZ). The SGZ is one of the few brain regions in mammals that gives rise to a substantial number of new neurons throughout adulthood. The neuron lineage contains a progression of identifiable precursor cell types with different proliferation rates. This present study found that primary cilia were present in every cell type in the neuronal lineage in SGZ. Cilium length and incidence were positively correlated among these cell types. Ciliary levels of adenylyl cyclase type III (ACIII) levels relative to ADP-ribosylation factor-like protein 13b (Arl13b) was higher in neurons than in precursor cells and glia, and also changed with the cell cycle. G-protein coupled receptors, SstR3, MCHR1, and Gpr161 receptors were only found in neuronal cilia. The levels and distribution of three centrosomal proteins, γ-tubulin, pericentrin and cenexin in neurons was different from the distributions in precursors and glia. The second focus of study is glial responses to injury in the neocortex, which has been widely studied as an injury model. This study found that in the normal adult somatosensory cortex, primary cilia were present in astrocytes and polydendrocytes but not in microglia. Following injury, the incidence of primary cilia decreased in astrocytes. Also, a new cell type expressing GFAP, NG2 and Olig2 was seen 3 days following injury, but was not present in normal mice. The characteristics of primary cilia and centrosome described here suggest that in stem cells and progenitors their characteristics may be well suited for proliferation, whereas in neurons, the cilia and centrosomes are important for other sensory functions.

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L’analyse du mouvement, ou l’analyse d’une séquence d’images, est l’extension naturelle de l’analyse d’images à l’analyse de séries temporelles d’images. De nombreuses méthodes d’analyse de mouvement ont été développées dans le contexte de la vision par ordinateur, incluant le suivi de caractéristiques, le flot optique, l’analyse de points-clef, le recalage d’image, etc. Dans ce manuscrit, nous proposons une boite a outils de techniques d’analyse de mouvement adaptées à l’analyse de séquences biomédicales. Nous avons en particulier travaillé sur les cellules ciliées qui sont couvertes de cils qui battent. Elles sont présentes chez l’homme dans les zones nécessitant des mouvements de fluide. Dans les poumons et les voies respiratoires supérieures, les cils sont responsables de l’épuration muco-ciliaire, qui permet d’évacuer des poumons la poussière et autres impuretés inhalées. Les altérations de l’épuration mucociliaire peuvent être liées à des maladies touchant les cils, pouvant être génétiques ou acquises et peuvent être handicapantes. Ces maladies peuvent être caractérisées par l’analyse du mouvement des cils sous un microscope avec une résolution temporelle importante. Nous avons développé plusieurs outils et techniques pour réaliser ces analyses de manière automatiques et avec une haute précision, à la fois sur des biopsies et in-vivo. Nous avons aussi illustré nos techniques dans le contexte d’éco-toxicité en analysant le rythme cardiaque d’embryons de poissons / Motion analysis, or the analysis of image sequences, is a natural extension of image analysis to time series of images. Many methods for motion analysis have been developed in the context of computer vision, including feature tracking, optical flow, keypoint analysis, image registration, and so on. In this work, we propose a toolbox of motion analysis techniques suitable for biomedical image sequence analysis. We particularly study ciliated cells. These cells are covered with beating cilia. They are present in humans in areas where fluid motion is necessary. In the lungs and the upper respiratory tract, Cilia perform the clearance task, which means cleaning the lungs of dust and other airborne contaminants. Ciliated cells are subject to genetic or acquired diseases that can compromise clearance, and in turn cause problems in their hosts. These diseases can be characterized by studying the motion of cilia under a microscope and at high temporal resolution. We propose a number of novel tools and techniques to perform such analyses automatically and with high precision, both ex-vivo on biopsies, and in-vivo. We also illustrate our techniques in the context of eco-toxicity by analysing the beating pattern of the heart of fish embryo

Foi estudada a ação do fator neurotrófico ciliar (CNTF) e da interleucina 6 (IL-6) exógenos na regeneração nervosa periférica. Aplicação de CNTF recombinante humano: o nervo ciático de 12 camundongos adultos C57BL/6J (3 grupos, n=4) foi seccionado e os cotos proximal e distal ancorados com ponto único de sutura no interior de tubo de polietileno (TP) com 6 mm d e comprimento e 0,76 mm de diâmetro interno, com intervalo de 4 mm entre os cotos. Os TP foram implantados vazios ou preenchidos com uma das seguintes soluções: (1) colágeno (col.) purificado (Vitrogen, 2,4 mg/ml) em tampão fosfato (0,2M), na proporção d e 1:1; (2) col.+CNTF (1:1, com 100 ng/ml de CNTF/tubo). Após 6 semanas os tubos contendo ao cabos nervosos regenerado s (CR) foram processados e incluídos em Epon. Fibras nervosas mielínicas foram contadas na porção média dos CR com um sistema controlado por computador (Biographics). Camundongos que receberam CNTF apresentaram número significativamente maior de axônios regenerados (3027±62, média±erro padrão) em relação aos animais implantados com tubos vazios (1384±128) ou preenchidos apenas com colágeno (1639±104). Outros 12 animais adicionais tiveram o nervo ciático seccionado e reparado da mesma maneira. Após 6 semanas, um tubo contendo solução do traçador neuronal HRP foi implantado no coto distal do nervo. Decorrido um período suplementar de 3 dias, neurônios marcados foram contados na medula espinhal e nos gânglios das raízes dorsais L4,5,6- Não houve diferença significante no número de motoneurônios entre os diferentes grupos experimentais (vazio=740±21; col.=749±44; CNTF=790±14) e o s animais não-operados (794±30). Não houve também diferença significante no número de neurônios sensitivos entre os diferentes grupos experimentais (vazio=1920±59; co!.=2262±152; CNTF=2124+96) e todos apresentaram número menor de neurônios sensitivos quando comparados com os animais não operados (4211+96) Aplicação de IL-6 recombinante murina: 12 camundongos C57BL/6J adultos foram divididos em dois grupos (n=6) e implantados com TP preenchidos com: (1) Vitrogen (2,4 mg/ml) e tampão fosfato (0,2 M), na proporção d e 1:1; (2) Vitrogen + IL-6 (1:1, com 100 |ig/ml de IL-6/tubo). Após tempo d e sobrevida de 6 semanas , os tubos com os CR foram processados da mesma forma que no experimento anterior. Fibras nervosas mielínicas foram contadas na porção média d o s CR. Os resultados mostraram que os animais implantados com col.+IL-6 tinham número significativamente maior de axônios mielínicos (2025+143) que os animais injetados apenas com col. (1542±122). O gânglio L5 foi também removido dos mesmos animais e cortado seriadamente (5|am), para posterior contagem do número de neurônios sensitivos. Não houve diferença significante no número de neurônios sensitivos entre os grupos experimentais (col.=528+42, col.+IL-6=554±37); todos, no entanto, apresentaram número significantemente menor de neurônios sensitivos quando comparados com os animais não operados (1112+63). Esses resultados indicam que a aplicação local de CNTF ou IL-6 estimula a regeneração de nervos seccionados e que este efeito é devido, provavelmente, a um aumento da taxa de brotamento dos axônios em regeneração / We studied the actions of exogenous ciliary neurotrophic factor (CNTF) and interleukin-6 (IL-6) on peripheral nerve regeneration. The sciatic nerve of 12 adult C57BL/6J mice (3 groups; n=4) w a s transected and both proximal and distal nerve stumps were secured by a single 10-0 suture into a 6-mm of a polyethylene tube (PT) (0,76 mm ID) to give a final gap length of 4mm. The PT were implanted empty or filled with one of the following solutions: (1) a purified preparation of collagen (Vitrogen, 2,4 mg/ml) plus phosphate buffer solution (0,2M), in 1:1 ratio; (2) Vitrogen + human recombinant CNTF (1:1, with 100 ng/ml of CNTF/tube). After 6 weeks the tubes containing the regenerated nerve cables (NC) were processed for Epon embedding. Myelinated nerve fibers were counted from the mid-portion of the cab les with a computer-controlled system (Biographics). CNTF injected mice regenerated significantly more myelinated axons (3027±62, mean±SEM) compared to the animals implanted with tubes left empty (1384±128) or filled with collagen alone (1639±104). Next, 12 additional animals had the sciatic nerve transected and repaired a s above. After 6 weeks, HRP was applied to the distal stump of the regenerated nerves and labeled neurons were counted in the spinal cord an d L4.5.6 dorsal root ganglia (DRG). No significant difference in the number of motoneurons was detected between the experimental (empty=740±21; collagen=749±44; CNTF=790±14) and non-operated (794±30) animals. No difference was also found in the n umber of labeled DRG neurons among the experimental groups (empty=1920±59; collagen=2262±152; CNTF=2124±96) and all had fewer labeled sensory neurons compared to the non-operated group (4211+96). For the IL-6 experiments, 12 C57BL/6J mice were divided into 2 groups (n=6) and implanted with PT filled with one of the following solutions: (1) Vitrogem (2,4 mg/ml) plus phosphate buffer solution (0,2 M), in 1:1 ratio; (2) Vitrogen + murine recombinant IL-6 (1:1, with 100 |ig/ml of IL-6/tube). Following a survival time of 6 weeks, the tubes with the regenerating nerve cables were processed for Epon embedding and myelinated nerve fiber counting. The results showed a significant difference in the number of myelinated axons between the collagen+IL-6 group (2025±143) and the collagen alone group (1542±122). The L5 DRG was also removed from the same mice, and serially sectioned (5^m) for sensory neuron counts. No significant difference was found in the number of DRG neurons between the experimental groups (collagen=528±42; collagen+IL-6=554±37). However, all had significantly fewer sensory neurons compared to the non-operated group (1112+63). These results indicate that locally applied CNTF and IL-6 stimulate peripheral nerve regeneration in adult animals, and that the effects are due to a neurite-promoting activity on axotomized neurons

Accommodation is the change in the dioptric power of the eye altering the focus from distance to near. Presbyopia is the loss of accommodative function that occurs with age. There are many techniques used to measure accommodation, however, there is little consensus as to how clinical data should be collected and analysed. The overarching theme of this thesis is the in vivo examination of accommodation and how lifestyle can affect the onset of presbyopia. An open-field autorefractor with badal adaption was used to examine accommodative dynamic profiles under varying demands of vergence. From this data a new metric for assessing the time for accommodative change was derived. Furthermore this thesis describes a bespoke automated accommodative facility instrument that was developed to provide further assessment of accommodative speeds. Defocus curves are used for assessing accommodation and depth-of-focus; the work presented explores the use of non-linear regression models to define the most appropriate method of assessing defocus curves in phakic subjects, and pseudophakic subjects implanted with an extended depth-of-focus intraocular lens. Using an absolute cut-off criteria of +0.30logMAR improved the repeatability and reliability of the depth-of-focus metrics over a cut-off criteria relative to the best corrected visual acuity. A swept-source anterior segment optical coherence tomographer (AS-OCT) was used to image the morphology of the ciliary muscle during accommodation. The accuracy of ciliary muscle measurements was improved when using reference points on the sclera to align the AS-OCT scan. The use of a ciliary muscle area metric demonstrated poor repeatability and reliability when compared to the traditional assessment of muscle morphology via thickness measurements. Physiological ageing in the crystalline lens occurs in line with ageing in other structures in the body. The methods for assessing accommodative function examined in previous chapters, were used to examine whether lifestyle factors which affect the rate of systemic ageing, such as smoking, also affect accommodative function. Although being a current smoker and having greater central adiposity was associated with a slower time for accommodative change, further research is required before these findings can be applied to the target population.

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