No description available.

Beatmungsinduzierte Lungenschädigung stellt eine häufige Komplikation bei der Behandlung von ALI / ARDS dar. Selbst kleinere, bereits als protektiv bezeichnete Tidalvolumina können so auf dem Boden einer bereits stark beeinträchtigten Lunge zur einem starken lokalen Entzündungsgeschehen im alveolären Gewebe führen, in dessen weiteren Verlauf es zu einer mechanisch induzierten Apoptose und Nekrose kommen kann. Die Apoptose, auch als programmierter Zelltod bezeichnet, kann auf ihrem extrinsischen Weg über eine Ligand / Rezeptor Interaktion ausgelöst oder gehemmt werden. Dies macht das Zellsterben auf molekularer Ebene in gewisser Weise steuerbar, was sich präventiv und therapeutisch-medikamentös zu Nutze machen ließe. Daher soll in dieser Arbeit untersucht werden, ob bei der mechanisch induzierten Apoptose von Typ II-Pneumozyten durch unphysiologische Beatmung ebenfalls klassische Marker einer extrinsischen Apoptose zu finden sind und welche Rolle sie im Prozess der dehnungsinduzierten Apoptose spielen. Diese Untersuchungen wurden an frisch isolierte Typ II-Pneumozyten aus der Sprague-Dawley-Ratte durchgeführt. Die Zellen wurden auf elastischen 6-Well Platten jeweils 24h bei unphysiologischen (Frequenz 40 / Amplitude 30) auf der Flexercell FX-3000 Dehnmaschine gedehnt und im Anschluss mittels ELISA und Western-Blot auf Marker der extrinsischen Apoptose hin untersucht. Dabei konnte eine mäßige Erhöhung der für den extrinsischen Apoptoseweg typischen, jedoch mit ihm nicht zwangsläufig assoziierten Caspase-8 und TNF-α ermittelt werden. Für die Marker FAS, FAS-L und FADD, die eindeutig für den extrinsischen Apoptoseweg stehen, konnte keine Konzentrationserhöhung nach mechanischer Dehnung nachgewiesen werden. Diese Ergebnisse, frühere Forschungsergebnisse aus unserer Forschungsgruppe und die weltweite Studienlagen lassen somit den Schluss zu, dass die extrinsische Apoptose bei mechanischer Dehnung von Typ II-Pneumozyten keine entscheidende Rolle spielt.

The overall objective of this thesis was to evaluate the effects of apoptosis-like membrane and DNA changes in bull sperm, and to relate these changes to a bull’s fertility potential. This thesis hypothesis is that apoptosis-like changes occurring in fresh or cryopreserved bull sperm have a negative effect on a bull’s fertility potential. Two studies were conducted, the objectives of study 1 were to confirm the relationship of apoptosis-related membrane and nuclear changes in bull sperm with fertility, to predict the fertility of beef bulls used for natural mating; and to evaluate the effect of sperm with nicked-DNA on cleavage and blastocyst formation in vitro. In Experiment 1, phosphatidylserine (PS) translocation, from the inner to the outer plasma membrane, and DNA nicks in the sperm from 50 dairy bulls were determined using Annexin-V/PI and TUNEL assays, respectively. Relationships between the parameters of the assays and the known fertility levels of the bulls were calculated. In Experiment 2, fertility levels of 15 beef bulls used for natural mating were estimated using a regression model of DNA nicks developed in Experiment 1. In Experiment 3, the effect of DNA nicked sperm on cleavage and blastocyst rates were evaluated in in vitro produced embryos, using high and low sperm concentrations (30,000 and 300,000 sperm per IVF droplet) to fertilize the mature oocytes. In Experiment 1, there were significant relationships of fertility with live sperm (P<0.05) and necrotic sperm (P<0.01) (Annexin-V/PI assay), and with DNA-nicked sperm (P<0.001) (TUNEL assay). In Experiment 2, the fertility level of bulls used for natural breeding was estimated and ranged from -7.3 to 2.4. In Experiment 3, the cleavage rate was significantly affected by the number of sperm with nicked DNA, regardless of sperm concentration. At the low sperm concentration, blastocyst rate was significantly lower when higher DNA nicked sperm were used (51% vs 32%; high vs low DNA nicks) (P<0.05). Blastocyst rate was non significant at the higher sperm concentration regardless of DNA nicks. The second objective of this study was to evaluate the effect of apoptosis inhibitors added to post-thaw sperm samples on their longevity, to increase the availability of viable sperm to oocytes for fertilization. Frozen semen from seven bulls was used; six straws from each bull were pooled. Samples included, untreated control (sperm remaining in extender), treated control (washed sperm), and four treatments (inhibitors) each at two concentrations. Apoptosis inhibitors assessed included; Bax channel blocker, z-VAD-FMK, Coenzyme Q10, and XIAP. Motility related characteristics were evaluated using computer assisted sperm analysis (CASA). Membrane intactness and normal acrosomes were evaluated using fluorescein isothiocyanate-peanut agglutination (FITC-PNA)/propidium iodide (PI) assay. Mitochondrial membrane potential was evaluated using Mitotracker Deep Red (MtDR). Sperm parameters were evaluated at 0, 3, 6, and 12 hours of incubation. Our results showed, no significant effect of apoptosis inhibitors on post-thaw sperm motility and structural characteristics. The decline in sperm motility and structural characteristics at 6 h of incubation was lower (P<0.05) in treated control and treatment groups than untreated control group. In conclusion, the presence of nicked DNA in sperm may be used as an estimate of the fertility level of a breeding bull. The levels of sperm with DNA nicks have a negative effect on cleavage rates and subsequent blastocyst development. The second conclusion indicates that the addition of an apoptosis inhibitor post-thaw to semen samples does not improve longevity or fitness, in any of the parameters evaluated. The simple removal of extender showed to be beneficial to sperm longevity and fitness. Further studies are needed to evaluate the cleavage and blastocyst rate of embryos fertilized with a single sperm known to carry DNA nicks. As well, the effect of the addition of apoptosis inhibitors before cryopreservation of bull semen needs to be evaluated.

Programmed cell death (PCD or apoptosis) is an evolutionarily conserved, genetically controlled suicide mechanism for cells, which when deregulated, can lead to developmental defects, cancers and degenerative diseases. In C. elegans, DNA damage induces germ cell death by signaling through cep-1/p53 ultimately leading to the activation of the CED-3/caspase. It has been hypothesized that the major regulatory events controlling cell death occur by cell autonomous mechanisms, that is within the dying cell. In support of this, genetic studies in C. elegans have shown that the core apoptosis pathway genes ced-4/APAF1 and ced-3/caspase are required in cells fated to die. However, it is not known whether the upstream signals that activate apoptosis function in a cell autonomous manner. Here I show that two genes, kri-1, an ortholog of KRIT1/CCM1 that is mutated in the human neurovascular disease cerebral cavernous malformations (CCMs) and daf-2, an insulin-like receptor, are required to activate DNA damage-dependent cell death independently of cep-1/p53. Interestingly, I found that both genes can regulate cell death in a non-autonomous manner, revealing a novel role for non-dying cells in eliciting death in response to DNA damage.

The roundworm Caenorhabditis elegans possesses a single, conserved phosphatidylinositol 3-kinase (PI3K) signaling pathway that regulates somatic developmental decisions and lifespan through the Insulin-like receptor tyrosine kinase (RTK) DAF-2, the class I PI3K AGE-1 and the 3-phosphoinositide-dependent protein kinase 1 (PDK1) homologue PDK-1. This pathway ultimately controls the action of Akt homologues on the forkhead transcription factor DAF-16. The C. elegans Akt orthologue akt-1 also negatively regulates the DNA damage-dependent apoptosis of worm germ cells by indirectly interfering with activation of the key transcription factor CEP-1, the sole homologue of p53 in the worm. Because upstream regulation by RTK/PI3K signaling is known to couple with downstream Akt kinase activity, I hypothesized that the worm daf-2/age-1/pdk-1 pathway would function upstream of akt-1/Akt in response to DNA damage. Surprisingly, this was not the case: daf-2/InsR and pdk-1/PDK1 do not function upstream of akt-1/Akt and instead promote DNA damage-induced germ cell apoptosis independently of CEP-1/p53 by regulating the B cell lymphoma (Bcl2) homologue CED-9 and the Apoptotic protease-activating factor 1 (Apaf1)-like adapter protein CED-4, respectively. Furthermore, PDK-1/PDK1 promotes germ cell apoptosis by a mechanism that does not include changes in the subcellular localization or absolute levels of CED-4/Apaf1, but does require the presence of CEP-1/p53. Therefore, daf-2/RTK, pdk-1/PDK1, and cep-1/p53 co-operate from independent pathways to drive germ cell death. The separation of worm Akt function from canonical RTK/PI3K regulation is consistent with the ability of AKT-1 to function without major changes in phosphorylation at threonine 350, a site homologous to Thr308 in mammals. Since this modification is an essential step in the activation of Akt proteins by PDK1, it is likely that damage-dependent germline activity of AKT-1 is controlled by a novel mechanism that does not involve phosphorylation by PDK-1 on key regulatory sites. These data argue that C. elegans re-arranges single homologous components of a signalling pathway to respond to different stimuli in vivo. Finally, I present data identifying the C. elegans ataxia and telangectasia and Rad3-related (ATR) kinase homologue ATL-1 as a potential target of AKT-1. Collectively, my work has uncovered a novel DNA damage-dependent pathway that allows AKT-1 to control CEP-1/p53-dependent apoptosis.

Myc is an oncogene that is commonly deregulated in human cancers. Mechanistic studies reveal that Myc is a transcription factor that interacts with a protein partner called Max. Heterodimerization and the formation of the Myc:Max complex enables Myc:Max to bind to the E-box and subsequently regulate the activation and repression of Myc target genes. Since regulation of its target genes are essential for Myc to drive transformation, the Myc and Max interaction has been targeted in mouse model studies to determine whether the oncogenic activity of Myc can be inhibited. Surprisingly, these studies reveal that targeting Myc is not only possible but a powerful way to suppress tumour growth. Since a better understanding of how Myc carries out its biological functions makes the possibility of targeting Myc a reality, it is essential to investigate and study the mechanisms of how Myc promotes tumourigenesis. In the first part of this thesis, we investigate the idea that the N-terminal end of Myc may be post-translationally modified and this modification may dictate Myc activities. Indeed we report here that the N-terminal end of Myc can be ubiquitylated as well as acetylated, and that the loss of these modifications results in a decrease in Myc activities. Furthermore, we characterize Mdm2 as a potential E3 ubiquitin ligase that may ubiquitylate the N-terminal end of Myc. In the second part of this thesis, we investigate CUL7 as a novel oncogene that may inhibit Myc-potentiated apoptosis and cooperate with Myc in transformation. Indeed, CUL7 is a novel p53 interacting protein that inhibits Myc potentiated apoptosis through the inhibition of p53. We have also characterized CUL7 to be overexpressed in primary human lung cancers, and a higher level of CUL7 expression associates with short-term survival of lung cancer patients. Through the better understanding of the enzymes that post-translationally modify the N-terminal end of Myc and proteins, such as CUL7, that can cooperate with Myc to drive tumourigenesis, we may begin to devise ways to target and control deregulated Myc in cancer cells.

Programmed cell death (PCD or apoptosis) is an evolutionarily conserved, genetically controlled suicide mechanism for cells, which when deregulated, can lead to developmental defects, cancers and degenerative diseases. In C. elegans, DNA damage induces germ cell death by signaling through cep-1/p53 ultimately leading to the activation of the CED-3/caspase. It has been hypothesized that the major regulatory events controlling cell death occur by cell autonomous mechanisms, that is within the dying cell. In support of this, genetic studies in C. elegans have shown that the core apoptosis pathway genes ced-4/APAF1 and ced-3/caspase are required in cells fated to die. However, it is not known whether the upstream signals that activate apoptosis function in a cell autonomous manner. Here I show that two genes, kri-1, an ortholog of KRIT1/CCM1 that is mutated in the human neurovascular disease cerebral cavernous malformations (CCMs) and daf-2, an insulin-like receptor, are required to activate DNA damage-dependent cell death independently of cep-1/p53. Interestingly, I found that both genes can regulate cell death in a non-autonomous manner, revealing a novel role for non-dying cells in eliciting death in response to DNA damage.

The roundworm Caenorhabditis elegans possesses a single, conserved phosphatidylinositol 3-kinase (PI3K) signaling pathway that regulates somatic developmental decisions and lifespan through the Insulin-like receptor tyrosine kinase (RTK) DAF-2, the class I PI3K AGE-1 and the 3-phosphoinositide-dependent protein kinase 1 (PDK1) homologue PDK-1. This pathway ultimately controls the action of Akt homologues on the forkhead transcription factor DAF-16. The C. elegans Akt orthologue akt-1 also negatively regulates the DNA damage-dependent apoptosis of worm germ cells by indirectly interfering with activation of the key transcription factor CEP-1, the sole homologue of p53 in the worm. Because upstream regulation by RTK/PI3K signaling is known to couple with downstream Akt kinase activity, I hypothesized that the worm daf-2/age-1/pdk-1 pathway would function upstream of akt-1/Akt in response to DNA damage. Surprisingly, this was not the case: daf-2/InsR and pdk-1/PDK1 do not function upstream of akt-1/Akt and instead promote DNA damage-induced germ cell apoptosis independently of CEP-1/p53 by regulating the B cell lymphoma (Bcl2) homologue CED-9 and the Apoptotic protease-activating factor 1 (Apaf1)-like adapter protein CED-4, respectively. Furthermore, PDK-1/PDK1 promotes germ cell apoptosis by a mechanism that does not include changes in the subcellular localization or absolute levels of CED-4/Apaf1, but does require the presence of CEP-1/p53. Therefore, daf-2/RTK, pdk-1/PDK1, and cep-1/p53 co-operate from independent pathways to drive germ cell death. The separation of worm Akt function from canonical RTK/PI3K regulation is consistent with the ability of AKT-1 to function without major changes in phosphorylation at threonine 350, a site homologous to Thr308 in mammals. Since this modification is an essential step in the activation of Akt proteins by PDK1, it is likely that damage-dependent germline activity of AKT-1 is controlled by a novel mechanism that does not involve phosphorylation by PDK-1 on key regulatory sites. These data argue that C. elegans re-arranges single homologous components of a signalling pathway to respond to different stimuli in vivo. Finally, I present data identifying the C. elegans ataxia and telangectasia and Rad3-related (ATR) kinase homologue ATL-1 as a potential target of AKT-1. Collectively, my work has uncovered a novel DNA damage-dependent pathway that allows AKT-1 to control CEP-1/p53-dependent apoptosis.

Myc is an oncogene that is commonly deregulated in human cancers. Mechanistic studies reveal that Myc is a transcription factor that interacts with a protein partner called Max. Heterodimerization and the formation of the Myc:Max complex enables Myc:Max to bind to the E-box and subsequently regulate the activation and repression of Myc target genes. Since regulation of its target genes are essential for Myc to drive transformation, the Myc and Max interaction has been targeted in mouse model studies to determine whether the oncogenic activity of Myc can be inhibited. Surprisingly, these studies reveal that targeting Myc is not only possible but a powerful way to suppress tumour growth. Since a better understanding of how Myc carries out its biological functions makes the possibility of targeting Myc a reality, it is essential to investigate and study the mechanisms of how Myc promotes tumourigenesis. In the first part of this thesis, we investigate the idea that the N-terminal end of Myc may be post-translationally modified and this modification may dictate Myc activities. Indeed we report here that the N-terminal end of Myc can be ubiquitylated as well as acetylated, and that the loss of these modifications results in a decrease in Myc activities. Furthermore, we characterize Mdm2 as a potential E3 ubiquitin ligase that may ubiquitylate the N-terminal end of Myc. In the second part of this thesis, we investigate CUL7 as a novel oncogene that may inhibit Myc-potentiated apoptosis and cooperate with Myc in transformation. Indeed, CUL7 is a novel p53 interacting protein that inhibits Myc potentiated apoptosis through the inhibition of p53. We have also characterized CUL7 to be overexpressed in primary human lung cancers, and a higher level of CUL7 expression associates with short-term survival of lung cancer patients. Through the better understanding of the enzymes that post-translationally modify the N-terminal end of Myc and proteins, such as CUL7, that can cooperate with Myc to drive tumourigenesis, we may begin to devise ways to target and control deregulated Myc in cancer cells.

Cellular Inhibitor of Apoptosis 2 (cIAP2) belongs to the IAP family, a group of endogenous proteins that inhibit apoptosis. However, the physiological role of cIAP2 remains poorly defined. Knock-out (KO) and wild type (WT) mice were used to examine the effect of cIAP2 protein on the progression of atherosclerosis in apoE -/- mice. Following the high-fat diet period of 4 and 12 wks, tissues were harvested and analysis focused on the aortic root, the aortic arch, the descending aorta, and the blood. Ex vivo results show a significant decrease in aortic arch lesion area in KO vs. WT in both study groups. Results also show a decrease in aortic root lesion size in KO vs. WT in both study groups. These results support that cIAP2 is an important survival factor for lesion-associated macrophages, since loss of cIAP2 expression in this mouse model reduced atherosclerotic lesion development.