No description available.

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.

Islet transplantation is an attractive method to achieve blood glucose homeostasis. However, β-cell function declines over time. Therefore, it is necessary to explore strategies to enhance the β-cell mass and function. Also, because there is a severe shortage of human cadaver tissue, alternative sources of insulin secreting tissue need to be examined. Neonatal porcine islet (NPI) tissue has emerged as an attractive alternative source of β-cells. The aim of this thesis was to optimize the culturing conditions of NPIs pre-transplantation so that the available tissue can be used as efficiently and economically as possible. The results from this study indicate that the treatment of NPI cultures with z-VAD-FMK, a pan caspase inhibitor and general protease inhibitor significantly enhances β-cell survival. Additionally, the optimum length of culturing NPIs pre-transplantation appears to be 3-5 days. Since widespread cell death stimulates immunogenic response, this treatment also has the potential benefit of reducing immunosuppression needs in the recipient. / Experimental Surgery

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / This systematic review and meta-analysis tries to determine whether there is an association between the use of protease inhibitors (PIs) and the incidence of diabetes mellitus (DM) and/or metabolic syndrome (MS) in HIV-infected patients. A systematic literature search was performed using MEDLINE/PubMed, CENTRAL, LILACS, and EMBASE. Included articles were observational studies published on or prior to November 2015 that met specific inclusion criteria. Pooled relative risks (RRs) and hazard ratios (HRs) were calculated. Nine articles met the inclusion criteria, describing 13,742 HIV patients. Use of PIs was associated with the development of MS (RR: 2.11; 95% CI 1.28–3.48; p-value 0.003). No association between the use of PIs and development of DM was found: the HR for the incidence of DM among patients using PIs was 1.23 (95% CI 0.66–2.30; p-value: 0.51) and the RR was 1.25 (95% CI 0.99–1.58; p-value 0.06). Use of PIs in HIV-infected patients is associated with an increased risk of MS. No evidence of an increased risk of DM was found. However, because MS is a precursor to DM, it is possible that studies with a longer follow-up duration are needed in order to detect an association between PI use and onset of DM. / First, we would like to thank our families for all their support. Second, we would like to thank the Universidad Peruana de Ciencias Aplicadas, the Health Sciences Department, and the School of Medicine for their support and for all the tools they have provided throughout this process. Finally, we want to thanks to Dr Gwenyth O. Lee and Dr Daniela E. Kirwan for their comments. / Revisión por pares

Ewing Sarcoma (ES) is an aggressive pediatric solid tumor. Even though overall-survival for localized patients is approximately 70%, the overall-survival for high risk ES patients has not improved in the last 20 years. Therefore, there is a need for exploration of new therapeutic agents in ES. Recent evidence has demonstrated that ES cells behave like BRCA-deficient tumor types which renders them sensitive to PARP inhibitors in vitro and in vivo. However, a phase II study of the efficacy of single-agent PARP inhibition in patients with relapsed ES did not significantly improve outcome. As single-agent therapy is rarely expected to result in significant clinical responses, in this study, we plan to validate potential targeted combination therapies with PARP inhibitors in ES. Since ES appears to demonstrated BRCA-deficient biology with impaired homologous recombination, cells are expected to be sensitive to both PARP inhibitors and ATR inhibitors, drugs which have a role in regulating DNA damage and impairing homologous recombination. In breast cancer and ovarian cell lines with genetic BRCA-deficiency, PARP and ATR inhibitors have synergistic activity. We hypothesize that these inhibitors will also have synergistic anti-Ewing activity. Furthermore, we recognize that ES cells demonstrate remarkably quiet genomes suggesting that there is minimal ongoing DNA-damage when cells are growing unperturbed. Therefore, we also plan to test the effect of adding low-dose genotoxic chemotherapy to induce additional sensitivity to the combination of PARP and ATR inhibitors in ES. The specific aims of this study were to explore the possible anti-tumor effect of PARP inhibitors combined with ATR inhibitors in ES cell lines, and to explore whether low dose genotoxic chemotherapy with SN38 can potentiate the anti-tumor effect of combined PARP and ATR inhibition in ES cell lines. We studied the anti-Ewing Sarcoma effect of the combination of a PARP inhibitor, AZD2281, and an ATR inhibitor, AZD6738, across a range of doses with and without low doses of a DNA damaging agent, SN38 (irinotecan metabolite), in two ES cell lines. We analyzed synergy by determining the Combination Index (CI) and Fractional Inhibition (FA) of each combination. We found that the ATR inhibitor, AZD6738, was synergistic across large range of concentrations when combined with the PARP inhibitor, AZD2281, in ES cell lines. We also found that treatment of cells with low doses of SN38 increases ES cell sensitivity to treatment with the PARP inhibitor and ATR inhibitor combination. This study provides preclinical support for additional studies exploring these combinations in ES. Given the low number of pediatric patients with ES compared to adult cancer patients, there will be limited attempts in combining these agents in clinical trials. Therefore, the development of an in vivo trial testing the safety and efficacy of this combination in ES mouse models is proposed. / 2020-07-03T00:00:00Z

Hormonally induced artificial propagation of individually marked broodstock was performed identically at two sequential reproductive seasons. In both years, four separate groups of females were intraperitonealy injected by carp pituitary extrakt, Supergestran (containing GnRH analogue Lecirelin), Ovopel and Dagin (containing GnRH analogue and dopamine inhibitor) at 21 °C. In control group, no injection was carried out in broodstock. It observed the effect of treatment on the ovulation ratio, survival and growth of broodfisch, the ability of stripping in the next season and the effect on eggs.

As neoplasias mieloproliferativas (NMPs) negativas para o rearranjo t(9;22)/BCRABL1, incluindo Policitemia Vera (PV), Trombocitemia Essencial (TE) e Mielofibrose Primária (MFP), são doenças hematopoéticas clonais e estão frequentemente associadas à mutação JAK2V617F. Apesar dos avanços no conhecimento da fisiopatologia após a descoberta da mutação JAK2V617F e do desenvolvimento de inibidores da JAK2, o tratamento permanece não curativo. Sabe-se que as célulastronco mais primitivas nas NMPs são responsáveis pela iniciação da doença e que a expansão dos precursores mieloeritróides contribui para o fenótipo clínico. Dados recentes obtidos com ensaios in vitro mostram que as proteínas da família BCL2, reguladoras da apoptose mitocondrial, desempenham um papel relevante na patogênese das NMPs. Acreditamos que a expressão anômala de BCL2 nas células progenitoras hematopoéticas (CPH) das NMPs pode contribuir para a patogênese desse grupo de doenças. Avaliamos a expressão gênica, por meio de PCR em Tempo Real, da família BCL2 (genes antiapoptóticos BCL-xL e BCL2 e o pró-apoptótico BIM) nas diferentes subpopulações de progenitores hematopoéticos murinos (de um modelo condicional knockin de expressão heterozigótica condicional da Jak2V617F) e de pacientes portadores de NMPs bem como sua contribuição para o fenótipo da doença e resposta ao inibidores da JAK2 (com a droga ruxolitinibe) e/ou inibição da família BCL2 (com o inibidor de BCL2 obatoclax). Não encontramos diferença de expressão basal dos genes BCL2, BCL-xL e BIM nas células CD34+ bem como nas subpopulações de células CD34+38-/+ de pacientes com NMPs, independente da presença da mutação JAK2V617F, em relação às células CD34+ e subpopulações CD34+38-/+ dos controles (p>0.05). Nas células CD34+ de pacientes com TE encontramos aumento de expressão de BCL2 em relação às células CD34+ pacientes com MFP (p=0.03). No modelo transgênico de camundongos Jak2 wt/VF (que apresentam uma NMP semelhante à PV) e Jak2 wt/wt (controles), comparamos a expressão diferencial dos genes da família Bcl2 em precursores hematopoéticos imaturos (LSKs) e progenitores mieloides mais maduros (MPs). A expressão do BclxL em MPs de camundongos wt/VF foi maior em relação à subpopulação de células LSKs e em relação as duas subpopulações de células dos controles (p=0.0011). Não houve diferença significativa de expressão do Bcl2 nas subpopulações de células LSKs e MPs de animais wt/VF e wt/wt (p=0.12). Observou-se menor expressão de Bim em LSKs em relação às células MPs dos animais mutados (p=0.026), diferença essa não observada entre os controles Jak2 wt/wt. O tratamento isolado com inibidor de JAK2 ou de BCL2 resultou em aumento de expressão do Bim nas CPH (LSKs e MPs) de camungongos Jak2 wt/VF em relação aos animais Jak2 wt/wt. Este aumento da expressão de Bim foi ainda mais evidente após o tratamento das células com a combinação das duas drogas quando comparadas às células não tratadas ou tratadas com um dos dois inibidores, sendo maior em animais doentes do que em animais controles (p<0.0001). A análise do efeito do tratamento com os inibidores de JAK2 e BCL2 na indução de apoptose por meio de citometria de fluxo (marcação com anexina/7-AAD) revelou que as células LSKs foram mais resistentes à apoptose tardia do que as células MPs independentemente da mutação da JAK2 (p<0.05). O tratamento com obatoclax resultou em indução de apoptose diferentemente do que foi observado com o tratamento com ruxolitinibe (p=0.594) nas células MPs de animais Jak2 wt/VF. Ademais, o tratamento combinado com ruxolitinibe e obatoclax resultou no aumento da apoptose nas células MPs dos animais com fenótipo de PV (Jak2 wt/VF) em relação aos animais Jak2 wt/wt (p=0.05). Em conclusão, demonstramos que a resistência à apoptose nas NMPs ocorre desde as CPH iniciadoras da doença. Nossos resultados sugerem que a modulação da apoptose mitocondrial pode ser uma nova estratégia terapêutica para pacientes com NMP em combinação aos inibidores de JAK2, na medida em que atua tanto nas CPH que iniciam a doença como nos MPs, responsáveis pelos sinais e sintomas de mieloproliferação. / Myeloproliferative Neoplasms (MPNs) negative for t(9;22)/BCR-ABL1 rearrangement, including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF), are clonal hematopoietic diseases and are often associated with the JAK2V617F mutation. Despite advances in the pathophysiology knowledge after the discovery of the JAK2V617F mutation and the development of JAK2 inhibitors, treatment remains non-curative. It is known that MPN primitive stem cells are essential for the initiation of the disease and that the expansion of the myeloeritroid precursors contributes to the clinical phenotype. Recent data, obtained with in vitro assays, showed that BCL2 family proteins, regulators of mitochondrial apoptosis, play a relevant role in the pathogenesis of MPNs. We believe that the anomalous expression of BCL2 in hematopoietic progenitor cells (HPCs) of MPNs may contribute to their pathogenesis. We evaluated BCL2 family (antiapoptotic genes BCL-xL and BCL2 and the pro-apoptotic BIM) gene expression by real-time PCR in different subpopulations of hematopoietic progenitors from a conditional Jak2V617F knockin murine model and from patients with MPNs as well as their contribution to the disease phenotype and response to JAK2 inhibitors (with ruxolitinib) and/or to the inhibition of the BCL2 family (with the BH3-mimetic obatoclax). We found no difference in the basal expression of the BCL2, BCL-xL and BIM in CD34+ cells as well as in subpopulations of CD34+ 38-/+ cells from patients with MPNs, regardless of the presence of the JAK2V617F mutation. In CD34+ cells obtained from patients with ET, we found an increase of BCL2 expression when compared to CD34+ cells with PMF (p=0.03). In the Jak2 wt/VF transgenic mice (that develop a MPN similar to PV) and Jak2 wt/wt controls, we compared the differential expression of Bcl2 family genes in immature hematopoietic precursors (LSKs) and more mature myeloid progenitors (MPs). Expression of Bcl-xL in MPs of wt/VF mice was greater when compared to LSKs and to the two progenitor subpopulations of control cells (p=0.0011). There was no significant difference in Bcl2 expression between the subpopulations of LSKs and MPs from wt/VF and wt/wt animals (p=0.12). Lower Bim expression in LSKs than in MPs was observed in samples from JAK2-mutated animals (p=0.026). Such difference was not observed between the Jak2 wt/wt subpopulations. Treatment with JAK2 or BCL2 inhibitors alone resulted in increased Bim expression in LSKs and MPs of the Jak2 wt/VF mice when compared to Jak2 wt/wt animals. This increase in Bim expression was even more evident when these cells were treated with the combination of the two drugs as compared to single treatment with one of the two inhibitors, being higher in mutaded than control animals (p<0.0001). The analysis of apoptosis by flow cytometry (annexin / 7-AAD labeling) revealed that LSK cells were more resistant to late apoptosis than MP cells regardless of the JAK2 mutation (p<0.05). Treatment with obatoclax resulted in greater apoptosis induction than it was observed with ruxolitinib treatment (p=0.594) on MP cells of Jak2 wt/VF animals. In addition, the combined treatment with ruxolitinib and obatoclax resulted in increased apoptosis in MP cells of animals with the PV phenotype (Jak2 wt/VF) as compared to the Jak2 wt/wt animals (p=0.05). In conclusion, we demonstrated that resistance to apoptosis in MPNs occurs at the level of the hematopoietic progenitors that initiate the disease. Our results suggest that modulation of mitochondrial apoptosis may be a new therapeutic strategy for MPN patients in combination with JAK2 inhibitors, as it acts on both the disease initiating and more mature progenitors, responsible for the clinical findings of myeloproliferation.

No description available.

Novel conserved amino acid variations of papaya cystatin (PC) were investigated by amino acid substitutions using oryzacystatin-I (OCI) as a model plant cystatin for comparison. These amino acid residues in the conserved motifs are involved in binding with cysteine proteases, these include the GG (Gly-Gly) in the N-terminal region for both OCI and PC, the (Q)QVVAG (Gln-Val-Val-Ala-Gly) motif for OCI and (Q)AVVEG (Ala-Val-Val-Glu-Gly) motif for PC in the first inhibitory loop, and the PW (Pro-Trp) motif for OCI and LW (Leu-Trp) motif for PC in the second inhibitory loop. Recombinant OCI and PC mutant proteins were expressed in Escherichia coli and were tested for altered inhibitory activity against commercial cysteine proteases (papain and cathepsin L) and extracts from Colorado potato beetle (Leptinotarsa decemlineata) larvae, from banana weevil larvae (Cosmopolites sordidus) and tobacco leaf extracts (Nicotiana benthamiana). In all tests higher amounts of PC had to be used to obtain similar inhibition levels as OCI. Changing the amino acid Q at position 52 to E in OCI in the first inhibitory loop, had lowered the Ki value of the mutant against the commercial proteases. Concurrently the same amino acid string (EQ) in PC had resulted in a significantly decreased Ki value compared to PC wild-type and other mutants. All other OCI mutants were less efficient than the wild-type OCI, whereas all PC first inhibitory loop mutants had improved inhibitory activity against protease activity with the highest improvement against the protease extracts was found for the substitution of E with A at position 55. This study has shown the importance of the three conserved motifs and that it is possible to improve the binding capacity of a plant cystatins to cysteine protease activity by amino acid substitution using site-directed mutagenesis. By mutating individual amino acid residues in the first binding loop of the relatively “weak” papaya cystatin to amino acid residues found in OCI caused a significant improvement in inhibitory potency of PC. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Plant Science / unrestricted

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.