Suspensory desmitis is a common problem and affects a broad cross section of equine athletes in various disciplines. For this study, the proximal portion of the suspensory ligament was collected from 6 horses without suspensory ligament injury (16 ligaments) and 4 horses with degeneration of the suspensory ligament (11 ligaments). Specimens were collected immediately after euthanasia and placed in neutral-buffered 10% formalin. The tissue was fixed, sectioned, and stained with hematoxylin and eosin (H&E), Masson's trichrome, and for apoptosis by the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technique. Histological changes in the abnormal ligaments included mineralization, fibroplasias, neovascularization, collagen degeneration, and significant architecture disruption in 2 ligaments. There was a trend for increased apoptosis in the injured ligaments compared to the normal ligaments. / Master of Science

Intramammary infections (IMI) are common in non-lactating heifer and dry cow mammary glands and occur during periods of appreciable mammary growth and development. The presence of these infections is expected to negatively impact mammary growth and development but has yet to be investigated. The works reported here investigated how IMI affects mammary tissue structure, cellularity, and the expression of integral mammogenic hormone receptors implicated in mammary growth. Non-pregnant non-lactating cows (n = 19) were administered estradiol and progesterone to stimulate mammary growth and 2 quarters of each cow were subsequently infused with either saline (n = 19) or Staphylococcus aureus (n = 19). Intramammary infusion of Staphylococcus aureus increased the number of immune cells present in gland secretions and also increased the proportion of neutrophils comprising these secretion somatic cells. Mammary tissues from quarters infused with Staphylococcus aureus contained more immune cells, less mammary epithelial tissue area, and greater tissue areas of intralobular stromal tissue than saline quarters. Staphylococcus aureus quarters also contained more apoptotic mammary epithelial cells and a lower proportion of apoptotic cells in the intralobular stroma compartment than saline infused quarters; this signified that Staphylococcus aureus quarters had less epithelial growth and experienced an expansion and/or lack of regression of stromal tissues. The number of cells expressing estrogen receptor α (ESR1) and progesterone receptor (PGR), as well as staining characteristics of ESR1 and PGR positive nuclei was also examined in these tissues. No appreciable differences were observed in any of the examined ESR1 and PGR measures between Staphylococcus aureus and saline mammary glands, but myoepithelial cells from Staphylococcus aureus glands had a greater nuclear staining area than saline quarters, indicating that these cells were affected by IMI. The results of these investigations indicate that IMI, in mammary glands that are concurrently stimulated to grow and develop, limits the growth of mammary epithelium and impairs regression of the stromal tissue, both of which are necessary for successful lactational performance. / PHD / Successful growth and development of the dairy cow udder (mammary gland) is important and has long-term impacts on milk production. Most mammary growth occurs during the first pregnancy but, at this same time, a bacterial infection can be present within the mammary gland and is expected to hinder normal growth and development. The studies conducted here sought to examine how a bacterial infection, within a cow’s udder, affects mammary gland growth and development. Overall, it was observed that a bacterial infection in the mammary gland reduced the amount of functional tissue that would eventually produce milk while simultaneously increasing the amount of connective tissue. Infected mammary glands also had a greater number of dying mammary cells, reducing the number of cells that would eventually produce milk. Estrogen and progesterone are known to be integral in supporting mammary growth, so an examination of the number of cells being able to receive signals from estrogen and progesterone was also undertaken; presence of an infection did not alter the number of cells able to receive estrogen and progesterone’s signal. This work furthered our understanding of how bacterial infections affect mammary tissue and alter normal developmental processes.

Apoptosis is a form of "planned cell death" and is an essential component of normal tissue differentiation and functional regulation. Neutrophil apoptosis facilitates down regulation of the inflammatory response while minimizing "by stander" injury to normal tissue, and disruption of this process by various diseases may have a significant negative impact on patient recovery. Consequently, neutrophil apoptosis has been the focus of research in many species. However, methods for measuring apoptosis have not been evaluated in the horse. The goal of this study was to adapt previously reported methods for inducing and measuring both neutrophil apoptosis and necrosis in non-equine species for use in equine peripheral blood neutrophils. To achieve this goal the experiment was divided into three parts: 1. Induce apoptosis and necrosis in equine peripheral blood neutrophils using previously used known inducers and examine the relationship between exposure time and percentage of affected cells; 2. Measure percentage of apoptosis and necrosis using three methods of detection: a) Annexin-V Fitc PI assay, b) Homogenous caspase 3/7 assay and c) Light microscopy and; 3. Compare the results between the three methods of apoptosis detection to determine if results are comparable The hypothesis was that previously reported methods for inducing and measuring both neutrophil apoptosis and necrosis in non-equine species can be adapted for use in equine peripheral blood neutrophils. Venous blood samples were collected aseptically from the jugular vein of eight horses. Isolation of neutrophils was performed using density gradient centrifugation on percoll. In part 1 of the experiment aliquots of the neutrophil suspension were cultured in the presence of four known inducers of apoptosis; actinomycin D, staurosporin, cycloheximide and sodium hypochlorite, at four different concentrations (table 2). A fifth population was to induce necrosis using a freeze-thaw cycle and bleach. A control sample was examined (no inducer) to determine spontaneous rate of apoptosis. The aliquots were cultured and the percentage of apoptosis determined at two sequential time points for each horse. Apoptosis was measured at either 30 minutes and 3 hours or 6 and 12 hours by three simultaneous methods: (1) annexin-V FITC PI assay (AVF), (2) homogenous caspase assay (HC) and (3) light microscopy (MS). The AVF and HC methods detect events associated with early apoptosis whilst MS detects nuclear changes which are late events of apoptosis. Using AVF and MS apoptotic cells are able to be differentiated from necrotic cells. In part two of the experiment the agreement and reproducibility between AVF and MS was further examined. In this part of the experiment neutrophils were isolated from the peripheral blood of 10 normal healthy adult horses. Each isolated sample was cultured with 80µM Actinomycin D for 12 hours and a control sample (no inducer) also prepared. Three triplicate samples were next set up from both the induced and control sample and apoptosis was determined using both AVF and MS. In part 3 of the experiment, data was analyzed using the mixed model ANOVA following log transformation of the data. Main effects of treatment, concentration and time were analyzed. Statistical significance was considered if P was < 0.05. The relationship between the three techniques; light microscopy, flow cytometry and the fluorescent plate reader, was investigated using Spearman rank correlation coefficients (Fisher's Z transformation). The Bland-Altman approach for method analysis was used to further characterize the correlation between results obtained via light microscopy and flow cytometry. Statistical significance was considered if P < 0.05. All inducers increased the percentage of apoptotic cells at either one or more time point and results were most comparable between AVF and MS. Increasing exposure time increased percentage of apoptotic neutrophils for all inducers using AVF and MS (p<0.0001). For both AVF and MS, cycloheximide and staurosporin induced apoptosis significantly above control levels at 3, 6 and 12 hours; actinomycin D at 6 and 12 hours and bleach at 3 and 6 hours as well was 12 hours for AVF only. With HC induction of apoptosis was detected earlier with bleach at 30 minutes and 3 hours and staurosporin at 30 minutes, 3 and 6 hours. Apoptosis was detected only at 6 hours for cycloheximide. Increasing concentration of inducer significantly increased the percentage apoptotic cells for staurosporin and cycloheximide between the lowest and highest concentration using AVF (p<0.001). For both AVF and MS, increasing concentration of bleach decreased the percentage of apoptotic cells (p<0.05). Increasing the concentration of staurosporin resulted in an increase in apoptosis at 30 minutes and 3 hours. Both bleach and the freeze-thaw cycle induced necrosis at all time periods excluding 30 minutes for the freeze-thaw cycle (p<0.0001). Spearman rank correlation coefficients revealed a very high correlation for percentage apoptosis and necrosis between AVF and MS (r2 = 0.91, 95% CI 0.89 – 0.93). A high correlation was also present for AVF and HC (r2 = 0.75, 95% CI 0.69 – 0.79) and MS and HC (r2 = 0.76, 95% CI 0.71 – 0.81). The lower limit of the confidence intervals suggests there is some concern about the similarity between AVF, HC and MS, HC. The Bland and Altman statistical approach indicates that both AVF and MS are highly reproducible methods with minimal variation between the triplicate samples (AVF: 8.9%, 95% CI 6.25 – 11.6%, MS 7.9%, 95% CI 6 – 9.8%). The mean difference between the two methods is 6.7% (95% CI 3.89 – 9.42%). The 95% limits of agreement indicate that results from MS can be 8.7% below to 22% above results from AVF (95% CI -13.41 – 26.7%). These findings indicate that caspase activation may occur prior to phosphatidylserine externalization and visible nuclear changes, which is in accordance with previously published data. We discovered that actinomycin D induces significant and reproducible equine peripheral blood neutrophil apoptosis in a time dependant fashion. Similarly, necrosis results from a freeze-thaw cycle or high concentration of bleach and is suitable as a positive control for necrosis. Apoptosis was effectively detected using AVF assay and results indicate good correlation between AVF and MS with an acceptably low mean difference. MS could serve as an inexpensive, simple and quick on site method to rapidly verify results attained from AVF. Induction of apoptosis using the HC was not consistent and can not be recommended based on the results of this study. Future investigation aimed at evaluating assays multiplexed to the AVF which detect other aspects of the apoptotic pathway would lead to increased confidence of results and further evidence of the mode of cell death prior to undertaking clinical studies. / Master of Science

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Programmed cell death (apoptosis) is the most common mechanism of cell death in eukaryotes. The ability of cancer cells to evade and inhibit apoptosis has become a hallmark feature of cancer. This is accomplished through a family of proteins known as the inhibitor of apoptosis proteins (IAPs). X-Linked inhibitor of apoptosis protein (XIAP) is one of the best characterized IAPs. XIAP suppresses apoptosis by forming complexes with cysteine-aspartic proteases (caspase), through one of its baculovirus IAP repeat (BIR) domains. Its activity is endogenously antagonized by a second mitochondria derived activator of caspase (Smac). The anti-apoptotic behaviour of XIAP and the critical role it plays in the apoptotic program makes the Smac-XIAP interaction an important drug target. To this end, our laboratory is interested in synthesizing biologically related Smac mimetics which can induce apoptosis in a MDA-MB-231 cell line. Efforts have focused on (1) understanding BIR domain binding sites which allow for this interaction, and (2) the design and synthesis of molecules which are much more effective at inducing apoptosis compared to other well known analogues. Through the synthesis and evaluation of various divalent Smac mimetics we have been able to support the hypothesis that the likely binding site on XIAP is the BIR3 domain. As well, through the synthesis of a library of novel compounds, as described in the thesis, we have been able to assess the nature of the linker which joins the two tetrapeptide units. In our effort to understand which domains Smac binds with, various divalent analogues were synthesized containing MeAVPI-linker-IPVMeA (forward-reverse) and MeAVPI-linker-MeAVPI (forward-forward) sequence, which incorporated linkers with varying degrees of flexibility. We hypothesized that the forward-forward divalent mimetics would have decreased activity compared to the peptides synthesized in a forward-reverse fashion. Lastly, information gathered from structure activity relationship (SAR) studies have shown that substituting the lysine (P2) and isoleucine residues (P4) in the AVPI protein can create more potent inducers of apoptosis than its native AVPI sequence. As one of the most potent Smac mimetic that has been previously made known contains an alkyne bridge at P2 and a large hydrophobic moiety at P4, we hypothesized that similar Smac mimetics containing a propargyl glycine residue at P2 and a bulky hydrophobic moiety at P4 will be much more potent in inducing apoptosis.

Diss. (sammanfattning) Stockholm : Karol. inst., 2007. / Härtill 3 uppsatser.

Résumé : Les maladies cardiovasculaires représentent, par une large mesure, la première cause de morbidité et de mortalité chez les diabétiques. L’activation de RAGE par les produits de glycation avancée (AGE) générés en conditions hyperglycémiques est associée à une multitude de complications diabétiques vasculaires, notamment par une signalisation favorisant l’inflammation chronique ainsi que la mort des cellules formant les tissus et les organes exposés aux AGE. La surexpression de RAGE dans les cellules musculaires lisses des plaques athérosclérotiques vulnérables suggère que le récepteur pourrait contribuer à la survenue des accidents vasculaires. Nous avons donc émis l’hypothèse que l’activation de RAGE dans les cellules musculaires lisses était impliquée dans leur apoptose. Pour le vérifier, nous avons, dans un premier temps, mis au point une nouvelle méthode de détection sans marqueur basée sur le principe de la résonance des plasmons de surface (SPR) pour mesurer l’apoptose d’une monocouche cellulaire en temps réel et caractériser avec précision les paramètres cinétiques des phases d’initiation et d’exécution. Cet essai a permis de montrer que l’activation de RAGE induit l’apoptose dans plus de 75,6% des cellules musculaires lisses stimulées avec le CML-HSA pendant 20 heures. De surcroît, nous avons remarqué que l’activation de RAGE générait un fort stress du réticulum endoplasmique, indiqué par la formation d’un grand nombre de granules de stress ainsi que par l’augmentation de l’expression du marqueur de stress réticulaire HuR et de la caspase-9, deux importants régulateurs de l’apoptose induite par le stress réticulaire endoplasmique. Afin de vérifier le potentiel d’un antagoniste à bloquer l’activation du récepteur, nous avons ensuite synthétisé le peptide iRAGE dont la séquence est dérivée d’un site de liaison du CML-HSA ayant la particularité de posséder de nombreuses charges négatives à pH physiologique. Le prétraitement avec iRAGE s’est montré efficace pour prévenir l’activation de NF-κB, l’induction de l’apoptose et l’augmentation du stress réticulaire endoplasmique. Nous suggérons un modèle de fonctionnement par lequel iRAGE inhibe la signalisation de RAGE en empêchant la liaison des ligands multimériques et en stabilisant les récepteurs sous forme de monomères. À terme, la synthèse d’un antagoniste de RAGE utilisable en clinique pourrait constituer une avancée majeure dans la prévention des complications vasculaires et l’amélioration de la qualité de vie chez les diabétiques. / Abstract : Cardiovascular diseases represent, to a large extent, the first cause of morbidity and mortality among people with diabetes. RAGE activation by advanced glycation end products (AGE) generated in hyperglycemic conditions is associated to a multitude of vascular diabetic complications, in particular by a signaling promoting chronic inflammation as well as death of cells forming tissues and organs exposed to AGE. Overexpression of RAGE in smooth muscle cells of vulnerable atheromatous plaques suggests the receptor could contribute to heart attacks and strokes. Therefore, we hypothesize that RAGE activation in smooth muscle cells is involved in apoptosis. To verify this hypothesis, we first designed a new label-free assay based of surface plasmon resonance (SPR) to measure apoptosis of a cell monolayer in real-time and to characterize precisely the kinetic parameters of the initiation and execution phases. This assay showed that RAGE activation induces apoptosis of more than 75.6% of smooth muscle cells stimulated with CML-HSA for 20 hours. Moreover, we noticed that RAGE activation generated strong endoplasmic reticular stress, indicated by the formation of a great number of stress granules as well as the increased expression of stress marker HuR and caspase-9, two important regulators of reticular stress-induced apoptosis. In order, to assess the potential of an antagonist to block RAGE activation, we then synthesized the iRAGE peptide whose sequence is derived from a binding site of CML-HSA that has the particularity of owning numerous negative charges at physiological pH. Pretreatment with iRAGE was successful to prevent activation of NF-κB, induction of apoptosis and generation of endoplasmic reticular stress. We suggest a model by which iRAGE inhibits RAGE signaling by hindering the binding of multimeric ligands and by stabilizing the receptors in a monomer state. Ultimately, the synthesis of a RAGE antagonist usable in clinic could constitute a major progress in the prevention of vascular complications and in the quality of life of people with diabetes.

Retinal degenerative disease, which includes age-related macular degeneration and retinitis pigmentosa, is the main cause of blindness in developed countries. Degeneration of the retinal pigment epithelium (RPE) and photoreceptor cells through apoptosis is believed to be the main mechanism of cell death. X-linked inhibitor of apoptosis (XIAP) is an endogenous anti-apoptotic protein that mediates its effects through the inhibition of caspases - the proteins regulating the final stages of apoptosis. The neuroprotection of XIAP has been demonstrated in various neurodegenerative models. Retinal gene therapy based on adeno-associated virus (AAV) has recently been proven safe and effective in clinical trials of Leber's congenital amaurosis. However, studies are very limited so far about AAV-mediated XIAP effect on degeneration of the RPE and photoreceptor cells. In this thesis, a comprehensive study of AAV-mediated XIAP was performed in the RPE and photoreceptor degenerative models. First, an oxidative stress model was investigated using H2O2 in a human RPE cell line. Second, AAV2-mediated XIAP conferred marked protection on the RPE cells against H2O2 induced apoptosis. Third, an in vivo analysis using confocal scanning laser ophthalmoscope was applied to the NaIO3 induced retinopathy in two transgenic mice (NRL-GFP and B6TGOPN1LW-EGFP). However, subretinal injection of AAV2-XIAP did not rescue photoreceptor cells in the NaIO3-treated animals. Finally, AAV8-XIAP was tested in a rhodopsin mutant mouse line with retinal degeneration (the Rho-/- B6TGOPN1LW-EGFP mouse) but did not reveal any protection on cone photoreceptors. Overall the work in this thesis indicates a limited protection of AAV-mediated XIAP on the RPE and photoreceptor cells in the degenerative models used. XIAP based gene therapy may be helpful for RPE preservation in atrophic AMD, but it needs further research.