Force production by a muscle is critical to maintaining proper function and overall health of a human or animal. Muscle adapts to increased loading with hypertrophy by activating a number of intracellular signaling cascades that regulate protein synthesis. The overall hypothesis is that force-dependent processes acutely activate growth-related signaling during active force generation. This project took two approaches. The first employed a general survey of muscles in which age-dependent changes in muscle activity differed. No conclusive activity-dependent signaling emerged however coordinated signaling among kinases broke down with age. The second approach utilized an in situ muscle preparation in which force production or metabolic costs were specifically controlled. Similar sub-maximal force levels generated by different methods found that force, per se, is not a primary modulator of growth-related signaling but that ERK phosphorylation is dependent on fiber-activation. Prolonging the duration of electrical stimulation applied to the nerve or increasing the frequency at which stimulations are applied was expected to increase the metabolic stress associated with contraction. Several growth-related kinases correlated with markers of metabolic stress, i.e. increased AMPK activity and decreased glycogen content, which were decoupled from force decline. This suggests energy depletion, specific to stimulation pattern, strongly influences the immediate response to high force contraction signaling. The overall conclusion is that signaling molecules previously implicated in force-dependent signaling lie much too downstream to relay strict force-dependent signaling.

published_or_final_version / Surgery / Master / Master of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy

The 33 kilodalton (kD) manganese stabilizing protein (MSP) is intimately associated with the photolysis of water to molecular oxygen. The two main purposes of this study were: 1) to analyze previously constructed MSP mutants and 2) to subclone, express, and purify the wild-type MSP in Escherichia coli in order to investigate the relationship between structure and function of this protein.Growth rates were compared between bacterial cells harboring only the vector, the vector plus the wild-type MSP gene, and the vector plus a mutant MSP gene. No significant differences were seen. This result implies that the expression of the wild-type MSP or mutant MSP is not toxic to the cells. Plasmid DNA isolation and restriction analyses of several of the mutant clones also confirmed the presence of the correct size inserts in the vector. However, upon sequencing several mutant clones, it appeared that losses and/or rearrangements of sequences was occurring. Thus, it was concluded that MSP was not being stably maintained in E. coli.Expression of the wild-type gene was achieved in E. coli by IPTG induction of the gene in pUC120 cloned under the control of the lac promoter. The expressed protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and confirmed by western blotting. Purification of the wild-type protein was obtained by membrane fractionation over a DEAE ion exchange column and the expression product was detected by western blotting. However, the expression product was lost in the concentration procedure and therefore is not available for reconstitution experiments.The wild-type MSP gene was also subcloned in a hybrid shuttle vector pTNTV, previously constructed in our laboratory (1). This construct was used to permit constitutive highlevel expression of the cloned gene and may prove to be an alternative vector to better express the MSP and mutant MSP in future investigations.These results demonstrate that it is possible to express the wild-type MSP gene from cyanobacteria in E. coli, but the problems of instability and recombination of the mutant genes in the vector have to be addressed before proper expression of these genes can be obtained. / Department of Biology

[Truncated abstract] It is becoming increasingly obvious that cell signalling pathways are more complicated than we originally perceived. Research is revealing that, not only is there a multitude of new proteins involved in signalling cascades, but also that previously identified proteins may have additional, alternate roles in intracellular trafficking. Gonadotropin-releasing hormone (GnRH) in conjunction with its receptor (GnRHR), the primary regulator of reproduction in all species, is no exception. In the past few years it has become readily accepted that the classic linear GnRHR-Gαq/11 signalling pathway is not universal and that this receptor is involved in a far greater range of cellular activities than was previously considered. In particular, it is widely accepted that continuous administration of GnRH analogs results in an inhibition of growth of a number of reproductive-derived tumours and that this may, in part, be mediated by direct activation of GnRHs expressed on these cells. However, it is not fully understood how the GnRHR mediates these growth effects or whether such effects are unique to reproductive-derived cancer cells. Research within this thesis aimed to determine how the presence or absence of this receptor in different cell types might affect the ability of GnRH to directly mediate growth effects. We demonstrate that continuous treatment with a GnRH agonist (GnRHA) induces an anti-proliferative effect in a gonadotropederived cell line (LβT2) and also in HEK293 cells stably expressing either the rat or human GnRHR. The anti-proliferative effect was time- and dose-dependent and was specifically mediated via the GnRHR, as co-treatment of the GnRHRexpressing cell lines with a GnRH antagonist blocked the growth suppressive effect induced by GnRHA treatment. Cell cycle analysis revealed that the GnRHA treated HEK/GnRHR cell lines induced an accumulation of cells in the G2/M phase while a G0/G1 arrest was observed in LβT2 cells. Previous identification by our group of a potential interaction between the GnRHR and the transcription factor E2F4, an integral cell cycle regulatory protein, prompted further investigation as to the nature of this interaction. Bioluminescence energy transfer (BRET) was utilised to demonstrate that the GnRHR also interacts with E2F5, another member of the E2F family of cell cycle proteins that shares a high level of homology to E2F4. In addition, it was determined that the interaction between human GnRHR and E2F4, detected using BRET, was influenced by cell density.

Made available in DSpace on 2014-11-10T11:09:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-03-06Bitstream added on 2014-11-10T11:57:44Z : No. of bitstreams: 1 000784225.pdf: 760734 bytes, checksum: c70c8782d6352b14ba371a32c3549c66 (MD5) / O sistema de cultivo in vitro de embriões apresenta menor eficiência em relação ao in vivo, sendo caracterizado por menores taxas de desenvolvimento e de adesão, além de uma menor tolerância à certas alterações que provocam estresse, como agentes físicos, químicos, térmicos, osmóticos, oxidativos, radioativos, entre outros. De acordo com a intensidade do estresse, pode-se estimular uma resposta celular favorecendo o desenvolvimento embrionário, adquirindo maior tolerância através de aceleração no metabolismo, proliferação e crescimento celular. O objetivo do presente trabalho foi analisar o efeito do estresse óptico em diferentes momentos ao longo do cultivo e conhecer as possíveis aplicações, a partir do estresse subletal e da bioestimulação promovida pela irradiação com diodo emissor de luz (LED). Com isto, os resultados demonstraram que em alguns estágios do desenvolvimento embrionário, tal como em blastocistos, o uso do estresse óptico pode ser benéfico ao embrião, protegendo-o de eventos como a apoptose. Os principais resultados demonstram que na produção de blastocistos houve aumento na produção dos mesmos nos grupos irradiados, apesar de não haver diferença estatística (Controle 23,2; IRD3inf 31,3; IRD6Infra 33,3). O estímulo por LED prévio à vitrificação mostrou que a irradiação por infravermelho em D3 pode estimular o aumento, em média, do numero de células (Controle: 91,9; IRD3inf: 112,8). Em relação às células apoptóticas, grupos irradiados com luz infravermelha mostraram menos número de células envolvidas neste processo (Controle: 11,6; IRD6inf: 6,6; IRFIVinf: 7,8), e a irradiação por luz vermelha pareceu aumentar a apoptose (IRD3verm: 15,6; IRD6verm: 16,1). Em relação à taxa de reexpansão, o a irradiação por luz vermelha teve efeito sobre o grupo irradiado em D3, mostrando uma diminuição significativa para o mesmo (Controle: 70,5 IRD3 verm; 39,3). De ... / The in vitro embryo culture system has lower efficiency compared to in vivo, because the in vitro system is characterized by lower rates of development, deployment, and a lower tolerance to certain changes that cause stress, such as physical, chemical, thermal, osmotic, oxidative and radioactive among others. According to the stress intensity it is possible to stimulate a cellular response promoting embryonic development and acquiring greater tolerance by the acceleration of metabolism, cell proliferation and growth. The objective of this study was to analyze the effect of the optical stress at different periods throughout the embryo development and understand the possible applications for this stimulation, from sublethal stress and biostimulation promoted by irradiation with lasers. Therefore, the results showed that optical stress can be beneficial to the embryos at certain stages of their development, such as blastocysts stage, protecting them from apoptosis. The main results show that an increase in blastocysts production for infrared irradiated groups, although there was no statistical difference (control 23.2; IRD3inf 31.3; IRD6Inf 33.3). The infrared LED stimulus prior to vitrification showed a higher number of cells for embryos in D3 (control: 91.9; IRD3inf: 112.8). Also, infrared irradiation appeared to decrease the number of apoptotics cells, when compared to controls (control: 11.6; IRD6inf: 6.6; IRFIVinf: 7.8), and red light irradiation appeared to increase apoptosis (IRD3verm: 15.6; IRD6verm: 16.1). Regarding re-expansion rate, the red light irradiation in D3 is related a significant decrease in the rate (control: 70.5; IRD3verm: 39.3). According to the results, irradiation with infrared light effect is shown in blastocyst production, which is economically important to embryos IVP. Moreover, the apoptosis analysis showed that the association between the techniques applied in this study are important for the in ...

A description of two techniques used extensively in this study namely cell counting with a "cell counting plate" and argentation TLC for the separation of ω -6 -fatty acids is given. Zn supplementation into GM of two malignant (BL-6 and Hep- 350) and a non-malignant (LLC-MK) cell line/s resulted in an increased uptake of Zn by the cells and progressively suppressed proliferation of particularly the malignant cells. Zn chelation by EDTA suppressed in vitro proliferation of all 3 cell line, this effect being more pronounced in the malignant cells. A dietary Zn deficiency resulted in alopecia in mice and both a dietary Zn deficiency and Zn excess reduced growth of BL-6 tumours implanted subcutaneously in mice. Zn supplementation into GM progressively increased the uptake of [1-¹⁴C]-LA by BL-6 and LLC-MK cells but had a very slight though irregular effect on this parameter in the Hep- 350 cells. Zn supplementation also stimulated desaturase activity in the BL-6 cells. These results suggested that there are select cell lines whose Δ⁶-desaturase activity responds positively to Zn supplementation (e.g. the BL-6 cells). Delta-6-desaturase activity was also assayed in microsome preparations from different tissues. No enzyme activity was detected in the microsomes prepared from the BL-6 tumours. There was no significant effect with the addition of Zn or EDTA, on Δ⁶-desaturase activity of the regenerating liver microsomes. In the resting liver microsomes this enzyme activity was reduced only when EDTA and Zn were added together and when EDTA was added to the reaction medium as well as to the microsome preparations 2 hr before the enzyme activity assay was initiated. The results of these experiments suggested that the Δ⁶-desaturase enzyme in the microsome preparations may have had an adequate amount of Zn with further additions having no stimulatory effect on the enzyme. Two independent mechanisms of control of cell proliferation by low and high Zn are suggested to operate.

Anti-oestrogens act via the oestrogen receptor whether they compete with the hormone for binding to the receptor and therefore interfere with DNA binding or inhibit transcriptional activity. These receptors exist as a large 85 complex and/or a small 45 form on sucrose density gradients. High performance ion-exchange chromatography has confirmed that the oestrogen and progestin complex is present in various isoforms. Progestin receptor heterogeneity could be influenced by the presence of oestrogens and anti-oestrogens in the culture media of hormone-dependent neoplastic cells. Cell culture methods offer the opportunity to test effects of specified components in repeated experiments on a homogeneous population of cells. MCF-7 and T47-D human breast cancer cell lines were conditioned to grow in a serum-free environment. There was no difference in cell proliferation rates, nor in their oestrogen or progestin receptor levels when compared to the same cells grown in conventional media. Receptors were present mainly in the large molecular 85 form. Both the MCF-7 and T47-D breast cancer cells showed an increase in proliferation rate with the addition of oestrogen or diethylstilbestrol. There was a corresponding loss of progestin receptor levels and an alteration in the high performance ion-exchange isoforms. Flow cytometry confirmed differences in the S-phase components of the cells following exposure to oestrogens. The proliferation rates of the cell lines as well as their progestin receptor levels decreased when treated with tamoxifen or the hydroxylated tamoxifen. There were marked changes on high performance ion-exchange chromatography profiles. DNA ploidy and S-phase showed signs of toxicity and there was an increase in cellular debris. The MCF-7 and T47-D human breast cancer cell line retained response to antioestrogen saturation.

The glycoprotein stanniocalcin-1 (STC1) is a paracrine factor in mammals which plays roles in various (patho)physiological functions, such as inflammation and carcinogenesis. Considerable numbers of studies showed dysregulation of STC1 expression in different types of human cancers. A previous study from our group, using clinicopathological data of 216 hepatocellular carcinoma (HCC) patients revealed greater STC1 gene expression in tumors than the paired normal samples. However, patient samples with greater STC1 level exhibited smaller tumor size. In fact, multiple cell types, growth factors and matrix components in tumor microenvironment (TME) control cancer progression. Emerging evidence support the important role of infiltrating immune cells on tumor progression. Among those, tumor associated macrophages (TAM) in TME is known to be an essential driver of tumor inflammation and progression, exerting a yin-yang influence to determine if the tumor is suppressed or paving the way to metastasize. Hepatocellular carcinoma (HCC) is mainly caused by chronic inflammation. With hindsight, the roles of STC1 in inflammation and carcinogenesis were documented. However, the observation on the negative correlation of STC1 expression with tumor size in HCC patients and the roles of STC1 on the interactions between tumor cells and macrophages are not clear. In Chapter 2, the inverse correlation of STC1 expression with tumor size was addressed. Human metastatic HCC cell line, MHCC97L which was stably transfected with empty vector (P) and STC1 (S1) were used. Nude mice xenograft model showed that tumor size and volume formed from S1 cells were significantly smaller than that from P cells. The observation agreed with the clinical data aforementioned. In vitro studies demonstrated S1 cells had lower plating efficiency, migratory and proliferative potential, illustrating a lower tumorigenicity. Biochemical analyses on the rate of glycolysis, extracellular O2 consumption, ATP production and Western blot studies on mTOR/p70S6K/rpS6 pathway showed the S1 cells adopted a lower energy metabolism. The data may explain the negative correlation between STC expression level and tumor size. In cancer microenvironment, infiltration of host immune cells, especially macrophages, contributes to inflammation and tumor progression. In Chapter 3, it was hypothesized that cancer cell-derived STC1 alter macrophage functions. Therefore, the effects of STC1-overexpressing MHCC97L on macrophages were studied. To mimic their interactions, Boyden chamber insert model was adopted to co-culture MHCC97L (97L/P and 97L/S1) and THP-1. Our data illustrated 97L/S1 suppressed migratory response of THP-1, with or without the addition of monocyte chemoattractant protein 1 (MCP-1) as the chemoattractant. Quantitative PCR showed downregulation of cytokine/chemokine receptors (CCR2, CCR4, CSF-1R) in THP-1 when co-cultured with 97L/S1. This prompted us to study the alterations of pathways related to cell motility in THP-1 by 97L/S1. Transcriptomic analysis detected 1784 differentially expressed genes (DEGs) between THP-1 cells co-cultured with 97L/P and 97L/S1. Ingenuity Pathway Analysis (IPA) prioritized an inhibition of RhoA signaling, which is known to stimulate cell motility. Western blotting analysis supported the IPA prediction and the cell migration data to show a significant reduction of MLC2 phosphorylation, leading to impaired formation of stress fibers, cell contraction and cell motility. The preceding chapters focused on cancer cell-derived STC1 on HCC cells or THP-1 derived macrophages. In Chapter 4, it was hypothesized that macrophage-derived STC1 may also play a role in macrophage differentiation and inflammation, which modulate tumorigenicity of HCC during macrophage-cancer cell interactions. Thus, the roles of endogenous STC1 in macrophage differentiation and functions were investigated. Using human leukemia monocytic cell line THP-1, a pilot study showed a treatment with phorbol 12-myristate 13-acetate (PMA) significantly upregulated STC1 expression and pro-inflammatory cytokines. In follow-up studies, THP-1 was pharmacologically stimulated to differentiate into (i) classically activated macrophages (CAM)/ M1 state, and (ii) alternatively activated macrophages (AAM)/ M2 state. Greater STC1 expression was found to be associated with CAM. To examine the role of STC1 in CAM, siRNASTC1 was used for gene knockdown. Conditioned medium collected from siRNASTC1-treated CAM inhibited migration of HCC cell line Hep3B. Transcriptomic analysis of siRNASTC1-treated CAM revealed an upregulation on TBC1D3G gene, which is involved in the release of extracellular vesicles (EVs) in macrophage to mediate inflammation. This study demonstrated the association between STC1 and macrophage-mediated inflammation. Collectively, the above studies elucidated the influence of STC1 on cancer cell metabolism, macrophage differentiation and function. It warrants further investigations to unravel the therapeutic potential of STC1 in inflammation and carcinogenesis.

Indiana University-Purdue University Indianapolis (IUPUI) / Phosphatase and tensin homolog, PTEN, is a key tumor suppressor. Mutation of PTEN is associated with both sporadic cancers and a cluster of familial cancer predisposition syndromes called PTEN hamaratoma syndromes. These germline mutations span the length of the PTEN gene with a mutational hot spot localized in exon 5. This exon encodes the catalytic domain of PTEN, which is critical for its tumor suppressor activity. PTEN function is most commonly attributed to lipid phosphatase activity on Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) that leads to inhibition of a cascade with downstream pro-survival effectors including Akt, but PTEN also has phosphatase activity on a small number of proteins. Recently, a mutation, G129E, has been described as a gain of function (GOF) mutation in PTEN knockin mice. This mutant only retains protein phosphatase activity while it completely lacks lipid phosphatase activity. Collectively (in the mouse and in vitro studies), there is no clear mechanism to explain the GOF nature of this mutant. Understanding how mutants of PTEN function in the cells to provide a growth advantage will provide insight into what pathway to therapeutically target. Our central hypothesis is that mutations of PTEN promote tumorigenesis through gain of function activities that result in cell cycle progression. We will determine the signaling pathways that are affected by the gain of function mutant PTEN G129E to better understand the mechanism by which mutants of PTEN confer a growth advantage.