Fas-FasL expression and interactions in mouse tumor cell lines: Implications for tumor immune escape

The Fas system, comprising the Fas receptor (Fas/Apo-1/CD95) and its ligand, Fas ligand (FasL), is a central mediator of programmed cell death in various physiological and pathological processes. FasL exists as transmembrane and soluble forms and induces apoptosis on crosslinking with Fas receptor. Recent evidence indicated that tumor cells exploit this system for their immunologic escape that includes the loss of Fas and the gain of FasL expression. In the present study, nine mouse tumor cell lines of diverse origin were examined immunocytochemically for the expression of Fas and FasL. Nine of nine cell lines expressed FasL, and five of nine cell lines expressed Fas. FasL expression in these tumor cell lines was demonstrated to be functional by its induction of apoptosis in Fas-sensitive target cells in coculture experiments. These results suggest that FasL may be a prevalent mediator of immune privilege in mouse malignancies, and support the recently proposed "counterattack model" for local elimination of tumor-reactive immune cells by tumor cell-derived FasL.^ Culture supernatant of four cell lines expressing FasL showed cytotoxic effect on Fas-sensitive target cells, indicating the possibility of secreted FasL in the medium. The Fas-expressing cell lines were sensitized to anti-Fas antibody cytotoxicity following treatment with IL-2 and IFN-$\gamma$, suggesting cytokine stimulation as an effective target for future immunotherapeutic strategies. ^

Cytokine analysis in Atlantic bottlenose dolphins: Molecular characterization of IL-4, IL-6, and IL-10

The health status of wild and captive Atlantic Bottlenose dolphins ( Tersiops truncatis) is difficult to ascertain. Mass strandings of these animals have been attributed to pollutants, as well as bacterial infections. Using human Enzyme Linked Immuno-Assays (ELISA) for immunological cytokines, I measured soluble cytokine levels with respect to their health status. In a retrospective analysis of dolphin sera, there was a trend of higher cytokine levels in “sick” animals. I cultured dolphin lymphocytes in the presence of a mitogen (PHA), a super antigen (Staph-A), Lipopolysaccharide (LPS), and a calcium flux inducer (PMA). Levels of messenger RNA, from these cultured cells, were assayed with Polymerase Chain Reaction (PCR) using primers for the human cytokines IL-2, IL-4, IL-6, IL-10, Tumor Necrosis Factor, and Interferon gamma. Only IL-4, IL-6, and IL-10 messages were obtained, inferring similar nucleotide homology to the human primer sequences. The PCR products were sequenced. Sixteen IL-4 sequences, twelve IL-6 sequences and seven IL-10 sequences were obtained and analyzed. Each cytokine exhibited the same nucleotide sequence in all dolphins examined. There was no difference in the cytokine profile in response to the various stimuli. The derived amino acid composition for each of the dolphin cytokines was used for molecular modeling, which showed that dolphin IL-4, IL-6, and IL-10 were structurally similar to the corresponding proteins of Perissodactyla. ^

Immunomodulation by shark cartilage extracts

The immune system is composed of innate and adaptive mechanisms. Innate immune responses are significantly modulated by immunomodulatory factors that act through the induction of specific patterns of cytokine production in responding cells. Human leukocytes have been shown to respond to substance(s) present in acid extracts of commercial shark cartilage (SC). Shark cartilage is a food supplement taken by consumers as a prophylaxis and for the treatment of conditions ranging from arthritis to cancer. No reliable scientific evidence in the literature supports the alleged usefulness of shark cartilage supplements, but their use remains popular. Cartilage extracts exhibit immunomodulatory properties by inducing various inflammatory, Th1-type cytokines and potent chemokines in human peripheral blood leukocytes (HPBL) in vitro. The objectives of the study were to (1) to determine the nature of the active component(s), (2) to define the scope of cellular response to SC extract, and (3) to elucidate the molecular mechanisms underlying bioactivity. Results showed that there are at least two cytokine-inducing components which are acid stable. One anionic component has been identified as a small (14-21 kDa) glycoprotein with at least 40% carbohydrate content. Shark cartilage stimulated HPBL to produce cytokines resembling an inflammatory, Th1 polarized response. Leukocyte-specific responses consist of both initial cytokine responses to SC directly (i.e., TNF-α) and secondary responses such as the IFN-γ response by lymphocytes following initial SC stimulation. Response of RAW cells, a murine macrophage cell line, indicated that TNF-á could be induced in macrophages of another mammalian species in the absence of other cell types. The results suggest that the human monocyte/macrophage is most likely to be the initial responding cell to SC stimulation. Stimulation of cells appears to engage at least one ligand-receptor interaction with TLR 4, although the role of TLR 2 cannot be ruled out. Initial activation is likely followed by the activation of the JNK and p38 MAPK signal transduction pathways resulting in activation, release, and translocation of transcription factor nuclear factor κB (Nf-κB). This dissertation research study represents the first in-depth study into characterizing the bioactive component(s) of commercial shark cartilage responsible for its immunomodulating properties and defining cellular responses at the molecular level.

Anti-diarrheal plants of central Anatolia: Do they inhibit diarrhea-causing bacteria?

Infectious diarrhea results in 2 to 5 million deaths worldwide per year, and treatments that are safe, effective, and readily available are under investigation. The field of medicinal ethnobotany focuses on plants that are used by different cultural groups for treating various diseases and evaluates these plants for efficacy and cytotoxicity. In the present study, ethnobotanical research was conducted with Central Anatolian villagers in Turkey. Folk concepts and etiologies surrounding diarrhea were analyzed, as were salient plant-based remedies for diarrhea. Reviewing the literature, 91 plant species were described as anti-diarrheal in all of Turkey. In Central Anatolia, villagers described 35 species. For continued research via bactericidal and bacteriostatic bioassays, 15 plants were selected. Methanolic and aqueous extracts of medicinally used plant parts were evaluated for inhibitory properties against 10 diarrhea-causing bacteria in the first bioassay, and later 21 bacteria in a second assay utilizing spectrophotometry. The cytotoxic properties were also evaluated in an Alamar Blue Assay using HepG-2, PC-3, and SkMEL-5 human cell lines. While several extracts showed bactericidal and bacteriostatic properties, the methanolic extract of R. canina galls inhibited the most bacteria at the lowest concentrations. They were not cytotoxic. Thus, R. canina methanolic gall extracts were selected for bio-assay guided fractionation. Antibacterial activity was maintained in the third fraction which was composed of almost pure ellagic acid. The bioassay was repeated with standard ellagic acid, and the polyphenol retained potency in inhibiting multiple bacterial strains. Several other extracts showed promise for safe, effective anti-bacterial remedies for diarrhea.

4-hydroxy estradiol-induced oxidant-mediated signaling is involved in the development of breast cancer

Breast cancer is a disease associated with excess exposures to estrogens. While the mode of cancer causation is unknown, others have shown that oxidative stress induced by prolonged exposure to estrogens mediates renal, liver, endometrial and mammary tumorigenesis though the mechanism(s) underling this process is unknown. In this study, we show that 4-hydroxyl 17β-estradiol (4-OHE2), a catechol metabolite of estrogen, induces mammary tumorigenesis in a redox dependent manner. We found that the mechanism of tumorigenesis involves redox activations of nuclear respiratory factor-1 (NRF1); a transcriptions factor associated with regulation of mitochondria biogenesis and oxidative phosphorylation (OXPHOS), as well as mediation of cell survival and growth of cells during periods of oxidative stress. Key findings from our study are as follows: (i) Prolonged treatments of normal mammary epithelial cells with 4-OHE2, increased the formation of intracellular reactive oxygen species (ROS). (ii) Estrogen-induced ROS activates redox sensitive transcription factors NRF1. (iii) 4-OHE2 through activation of serine-threonine kinase and histone acetyl transferase, phosphorylates and acetylate NRF1 respectively. (iv) Redox mediated epigenetic modifications of NRF1 facilitates mammary tumorigenesis and invasive phenotypes of breast cancer cells via modulations of genes involved in proliferation, growth and metastasis of exposed cells. (v) Animal engraftment of transformed clones formed invasive tumors. (vi) Treatment of cells or tumors with biological or chemical antioxidants, as well as silencing of NRF1 expressions, prevented 4-OHE2 induced mammary tumorigenesis and invasive phenotypes of MCF-10A cells. Based on these observations, we hypothesize that 4-OHE2 induced ROS epigenetically activate NRF1 through its phosphorylation and acylation. This, in turn, through NRF1-mediated transcriptional activation of the cell cycle genes, controls 4-OHE2 induced cell transformation and tumorigenesis.^

The role of redox signaling in the molecular mechanism of tamoxifen resistance in breast cancer.

The emergence of tamoxifen or aromatase inhibitor resistance is a major problem in the treatment of breast cancer. The molecular signaling mechanism of antiestrogen resistance is not clear. Understanding the mechanisms by which resistance to these agents arise could have major clinical implications for preventing or circumventing it. Therefore, in this dissertation we have investigated the molecular mechanisms underlying antiestrogen resistance by studying the contributions of reactive oxygen species (ROS)-induced redox signaling pathways in antiestrogen resistant breast cancer cells. Our hypothesis is that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with a progressive shift towards a pro-oxidant environment of cells as a result of oxidative stress. The hypothesis of this dissertation was tested in an in vitro 2-D cell culture model employing state of the art biochemical and molecular techniques, including gene overexpression, immunoprecipitation, Western blotting, confocal imaging, ChIP, Real-Time RT-PCR, and anchorage-independent cell growth assays. We observed that tamoxifen (TAM) acts like both an oxidant and an antioxidant. Exposure of tamoxifen resistant LCC2 cell to TAM or 17 beta-estradiol (E2) induced the formation of reactive oxidant species (ROS). The formation of E2-induced ROS was inhibited by co-treatment with TAM, similar to cells pretreated with antioxidants. In LCC2 cells, treatments with either E2 or TAM were capable of inducing cell proliferation which was then inhibited by biological and chemical antioxidants. Exposure of LCC2 cells to tamoxifen resulted in a decrease in p27 expression. The LCC2 cells exposed to TAM showed an increase in p27 phosphorylation on T157 and T187. Conversely, antioxidant treatment showed an increase in p27 expression and a decrease in p27 phosphorylation on T157 and T187 in TAM exposed cells which were similar to the effects of Fulvestrant. In line with previous studies, we showed an increase in the binding of cyclin E-Cdk2 and in the level of p27 in TAM exposed cells that overexpressed biological antioxidants. Together these findings highly suggest that lowering the oxidant state of antiestrogen resistant LCC2 cells, increases LCC2 susceptibility to tamoxifen via the cyclin dependent kinase inhibitor p27.

A novel 3-D segmentation algorithm for anatomic liver and tumor volume calculations for liver cancer treatment planning

Three-Dimensional (3-D) imaging is vital in computer-assisted surgical planning including minimal invasive surgery, targeted drug delivery, and tumor resection. Selective Internal Radiation Therapy (SIRT) is a liver directed radiation therapy for the treatment of liver cancer. Accurate calculation of anatomical liver and tumor volumes are essential for the determination of the tumor to normal liver ratio and for the calculation of the dose of Y-90 microspheres that will result in high concentration of the radiation in the tumor region as compared to nearby healthy tissue. Present manual techniques for segmentation of the liver from Computed Tomography (CT) tend to be tedious and greatly dependent on the skill of the technician/doctor performing the task. ^ This dissertation presents the development and implementation of a fully integrated algorithm for 3-D liver and tumor segmentation from tri-phase CT that yield highly accurate estimations of the respective volumes of the liver and tumor(s). The algorithm as designed requires minimal human intervention without compromising the accuracy of the segmentation results. Embedded within this algorithm is an effective method for extracting blood vessels that feed the tumor(s) in order to plan effectively the appropriate treatment. ^ Segmentation of the liver led to an accuracy in excess of 95% in estimating liver volumes in 20 datasets in comparison to the manual gold standard volumes. In a similar comparison, tumor segmentation exhibited an accuracy of 86% in estimating tumor(s) volume(s). Qualitative results of the blood vessel segmentation algorithm demonstrated the effectiveness of the algorithm in extracting and rendering the vasculature structure of the liver. Results of the parallel computing process, using a single workstation, showed a 78% gain. Also, statistical analysis carried out to determine if the manual initialization has any impact on the accuracy showed user initialization independence in the results. ^ The dissertation thus provides a complete 3-D solution towards liver cancer treatment planning with the opportunity to extract, visualize and quantify the needed statistics for liver cancer treatment. Since SIRT requires highly accurate calculation of the liver and tumor volumes, this new method provides an effective and computationally efficient process required of such challenging clinical requirements.^

Black band disease: Elucidating origins and disease mechanisms

Coral diseases were unknown in the scientific community fifty years ago. Since the discovery of a coral disease in 1965, there has been an exponential increase in the number of known coral diseases, as the abundance, prevalence, distribution, and number of host species affected has also significantly increased. Coral diseases are recognized as contributing significantly to the dramatic losses of coral cover on a global basis, particularly in the Caribbean. The apparent sudden emergence of coral diseases suggests that they may be a symptom of an overall trend associated with changing environmental conditions. However, not much evidence has been gathered to address this question. The following studies were designed to build a comprehensive argument to support this hypothesis for one important coral disease—black band disease (BBD). A meta-analysis of clone libraries identifying the microbial communities associated with BBD reveal important information including that a single cyanobacterial operational taxonomic unit (OTU) was by far the most prevalent OTU in diseased samples, and that the alphaproteobacteria, which include some of the most common bacteria in marine waters, were the most diversely represented. The analysis also showed that samples exhibited regional similarities. An fine and ultrastructural characterization of the disease revealed that the cyanobacteria are prolific borers through the coral skeleton, and that the cyanobacteria penetrate coral tissue, leading to their presence ahead of the main migrating disease band. It was further found that apparently healthy corals exposed to toxins found in BBD, exhibited similar tissue degradation to those infected with BBD. Comparing the disease progression to biofilm formation, it was determined that scouting cyanobacteria may contribute to the migration of the disease through progressive biofilm development over intact coral tissue. Together, these studies provide significant evidence for the hypothesis that BBD is an opportunistic disease, caused by common environmental bacteria, facilitated by the changing environmental conditions associated with climate change.

Positron emission tomography (PET) tumor segmentation and quantification: Development of new algorithms

Tumor functional volume (FV) and its mean activity concentration (mAC) are the quantities derived from positron emission tomography (PET). These quantities are used for estimating radiation dose for a therapy, evaluating the progression of a disease and also use it as a prognostic indicator for predicting outcome. PET images have low resolution, high noise and affected by partial volume effect (PVE). Manually segmenting each tumor is very cumbersome and very hard to reproduce. To solve the above problem I developed an algorithm, called iterative deconvolution thresholding segmentation (IDTS) algorithm; the algorithm segment the tumor, measures the FV, correct for the PVE and calculates mAC. The algorithm corrects for the PVE without the need to estimate camera's point spread function (PSF); also does not require optimizing for a specific camera. My algorithm was tested in physical phantom studies, where hollow spheres (0.5-16 ml) were used to represent tumors with a homogeneous activity distribution. It was also tested on irregular shaped tumors with a heterogeneous activity profile which were acquired using physical and simulated phantom. The physical phantom studies were performed with different signal to background ratios (SBR) and with different acquisition times (1-5 min). The algorithm was applied on ten clinical data where the results were compared with manual segmentation and fixed percentage thresholding method called T50 and T60 in which 50% and 60% of the maximum intensity respectively is used as threshold. The average error in FV and mAC calculation was 30% and -35% for 0.5 ml tumor. The average error FV and mAC calculation were ~5% for 16 ml tumor. The overall FV error was ∼10% for heterogeneous tumors in physical and simulated phantom data. The FV and mAC error for clinical image compared to manual segmentation was around -17% and 15% respectively. In summary my algorithm has potential to be applied on data acquired from different cameras as its not dependent on knowing the camera's PSF. The algorithm can also improve dose estimation and treatment planning.^

Synthesis and Biological Evaluation of Novel Folic Acid Receptor-Targeted, β-Cyclodextrin-Based Drug Complexes for Cancer Treatment

Drug targeting is an active area of research and nano-scaled drug delivery systems hold tremendous potential for the treatment of neoplasms. In this study, a novel cyclodextrin (CD)-based nanoparticle drug delivery system has been assembled and characterized for the therapy of folate receptor-positive [FR(+)] cancer. Water-soluble folic acid (FA)-conjugated CD carriers (FACDs) were successfully synthesized and their structures were confirmed by 1D/2D nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS), high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and circular dichroism. Drug complexes of adamatane (Ada) and cytotoxic doxorubicin (Dox) with FACD were readily obtained by mixed solvent precipitation. The average size of FACD-Ada-Dox was 1.5–2.5 nm. The host-guest association constant Ka was 1,639 M−1 as determined by induced circular dichroism and the hydrophilicity of the FACDs was greatly enhanced compared to unmodified CD. Cellular uptake and FR binding competitive experiments demonstrated an efficient and preferentially targeted delivery of Dox into FR-positive tumor cells and a sustained drug release profile was seen in vitro. The delivery of Dox into FR(+) cancer cells via endocytosis was observed by confocal microscopy and drug uptake of the targeted nanoparticles was 8-fold greater than that of non-targeted drug complexes. Our docking results suggest that FA, FACD and FACD-Ada-Dox could bind human hedgehog interacting protein that contains a FR domain. Mouse cardiomyocytes as well as fibroblast treated with FACD-Ada-Dox had significantly lower levels of reactive oxygen species, with increased content of glutathione and glutathione peroxidase activity, indicating a reduced potential for Dox-induced cardiotoxicity. These results indicate that the targeted drug complex possesses high drug association and sustained drug release properties with good biocompatibility and physiological stability. The novel FA-conjugated β-CD based drug complex might be promising as an anti-tumor treatment for FR(+) cancer.

Aedes aegypti pharate first instar aseasonal quiescence cues anticipatory plasticity with implications for urban vector ecology and control

The eggs of the dengue fever vector Aedes aegypti possess the ability to undergo an extended quiescence period hosting a fully developed first instar larvae within its chorion. As a result of this life history stage, pharate larvae can withstand months of dormancy inside the egg where they depend on stored reserves of maternal origin. This adaptation known as pharate first instar quiescence, allows A. aegypti to cope with fluctuations in water availability. An examination of this fundamental adaptation has shown that there are trade-offs associated with it. ^ Aedes aegypti mosquitoes are frequently associated with urban habitats that may contain metal pollution. My research has demonstrated that the duration of this quiescence and the extent of nutritional depletion associated with it affects the physiology and survival of larvae that hatch in a suboptimal habitat; nutrient reserves decrease during pharate first instar quiescence and alter subsequent larval and adult fitness. The duration of quiescence compromises metal tolerance physiology and is coupled to a decrease in metallothionein mRNA levels. My findings also indicate that even low levels of environmentally relevant larval metal stress alter the parameters that determine vector capacity. ^ My research has also demonstrated that extended pharate first instar quiescence can elicit a plastic response resulting in an adult phenotype distinct from adults reared from short quiescence eggs. Extended pharate first instar quiescence affects the performance and reproductive fitness of the adult female mosquito as well as the nutritional status of its progeny via maternal effects in an adaptive manner, i.e., anticipatory phenotypic plasticity results as a consequence of the duration of pharate first instar quiescence and alternative phenotypes may exist for this mosquito with quiescence serving as a cue possibly signaling the environmental conditions that follow a dry period. M findings may explain, in part, A. aegypti's success as a vector and its geographic distribution and have implications for its vector capacity and control.^

The role of the transcription factor Ets1 in melanocyte development

Melanocytes, pigment-producing cells, derive from the neural crest (NC), a population of pluripotent cells that arise from the dorsal aspect of the neural tube during embryogenesis. Many genes required for melanocyte development were identified using mouse pigmentation mutants. The deletion of the transcription factor Ets1 in mice results in hypopigmentation; nevertheless, the function of Ets1 in melanocyte development is unknown. The goal of the present study was to establish the temporal requirement and role of Ets1 in murine melanocyte development. In the mouse, Ets1 is widely expressed in developing organs and tissues, including the NC. In the chick cranial NC, Ets1 is required for the expression of Sox10, a transcription factor critical for the development of melanocytes, enteric ganglia, and other NC derivatives. ^ Using a combination of immunofluorescence and cell survival assays Ets1 was found to be required between embryonic days 10 and 11, when it regulates NC cell and melanocyte precursor (melanoblast) survival. Given the requirement of Ets1 for Sox10 expression in the chick cranial NC, a potential interaction between these genes was investigated. Using genetic crosses, a synergistic genetic interaction between Ets1 and Sox10 in melanocyte development was found. Since Sox10 is essential for enteric ganglia formation, the importance of Ets1 on gut innervation was also examined. In mice, Ets1 deletion led to decreased gut innervation, which was exacerbated by Sox10 heterozygosity. ^ At the molecular level, Ets1 was found to activate a Sox10 enhancer critical for Sox10 expression in melanoblasts. Furthermore, mutating Ets1 at a site I characterized in the spontaneous variable spotting mouse pigmentation mutant, led to a 2-fold decrease in enhancer activation. Overexpression and knockdown of Ets1 did not affect Sox10 expression; nonetheless, Ets1 knockdown led to a 6-fold upregulation of the transcription factor Sox9, a gene required for melanocyte and chondrocyte development, but which impairs melanocyte development when its expression is prolonged. Together, these results suggest that Ets1 is required early during melanocyte development for NC cell and melanoblast survival, possibly acting upstream of Sox10. The transcription factor Ets1 may also act indirectly in melanocyte fate specification by repressing Sox9 expression, and consequently cartilage fate.^

Étude exploratoire du recours à des interventions médicales de type "lourd' pour soulager la souffrance existentielle en fin de vie

Au cours du siècle dernier, des améliorations au niveau des conditions de vie ainsi que des avancées importantes dans les sciences biomédicales ont permis de repousser les frontières de la vie. Jusqu’au début du XXe Siècle, la mort était un processus relativement bref, survenant à la suite de maladies infectieuses et avait lieu à la maison. À présent, elle survient plutôt après une longue bataille contre des maladies incurables et des afflictions diverses liées à la vieillesse et a le plus souvent lieu à l’hôpital. Pour comprendre la souffrance du malade d’aujourd’hui et l’aborder, il faut comprendre ce qu’engendre comme ressenti ce nouveau contexte de fin de vie autant pour le patient que pour le clinicien qui en prend soin. Cette thèse se veut ainsi une étude exploratoire et critique des enjeux psychologiques relatifs à cette mort contemporaine avec un intérêt premier pour l’optimisation du soulagement de la souffrance existentielle du patient dans ce contexte. D’abord, je m’intéresserai à la souffrance du patient. À travers un examen critique des écrits, une définition précise et opérationnelle, comportant des critères distinctifs, de ce qu’est la souffrance existentielle en fin de vie sera proposée. Je poserai ainsi l’hypothèse que la souffrance peut être définie comme une forme de construction de l’esprit s’articulant autour de trois concepts : intégrité, altérité et temporalité. D’abord, intégrité au sens où initialement l’individu malade se sent menacé dans sa personne (relation à soi). Ensuite, altérité au sens où la perception de ses conditions extérieures a un impact sur la détresse ressentie (relation à l’Autre). Et finalement, temporalité au sens où l’individu souffrant de façon existentielle semble bien souvent piégé dans un espace-temps particulier (relation au temps). Ensuite, je m’intéresserai à la souffrance du soignant. Dans le contexte d’une condition terminale, il arrive que des interventions lourdes (p. ex. : sédation palliative profonde, interventions invasives) soient discutées et même proposées par un soignant. Je ferai ressortir diverses sources de souffrance propres au soignant et générées par son contact avec le patient (exemples de sources de souffrance : idéal malmené, valeurs personnelles, sentiment d’impuissance, réactions de transfert et de contre-transfert, identification au patient, angoisse de mort). Ensuite, je mettrai en lumière comment ces dites sources de souffrance peuvent constituer des barrières à l’approche de la souffrance du patient, notamment par l’influence possible sur l’approche thérapeutique choisie. On constatera ainsi que la souffrance d’un soignant contribue par moment à mettre en place des mesures visant davantage à l’apaiser lui-même au détriment de son patient. En dernier lieu, j'élaborerai sur la façon dont la rencontre entre un soignant et un patient peut devenir un espace privilégié afin d'aborder la souffrance. J'émettrai certaines suggestions afin d'améliorer les soins de fin de vie par un accompagnement parvenant à mettre la technologie médicale au service de la compassion tout en maintenant la singularité de l'expérience du patient. Pour le soignant, ceci nécessitera une amélioration de sa formation, une prise de conscience de ses propres souffrances et une compréhension de ses limites à soulager l'Autre.

Étude exploratoire du recours à des interventions médicales de type "lourd' pour soulager la souffrance existentielle en fin de vie

Au cours du siècle dernier, des améliorations au niveau des conditions de vie ainsi que des avancées importantes dans les sciences biomédicales ont permis de repousser les frontières de la vie. Jusqu’au début du XXe Siècle, la mort était un processus relativement bref, survenant à la suite de maladies infectieuses et avait lieu à la maison. À présent, elle survient plutôt après une longue bataille contre des maladies incurables et des afflictions diverses liées à la vieillesse et a le plus souvent lieu à l’hôpital. Pour comprendre la souffrance du malade d’aujourd’hui et l’aborder, il faut comprendre ce qu’engendre comme ressenti ce nouveau contexte de fin de vie autant pour le patient que pour le clinicien qui en prend soin. Cette thèse se veut ainsi une étude exploratoire et critique des enjeux psychologiques relatifs à cette mort contemporaine avec un intérêt premier pour l’optimisation du soulagement de la souffrance existentielle du patient dans ce contexte. D’abord, je m’intéresserai à la souffrance du patient. À travers un examen critique des écrits, une définition précise et opérationnelle, comportant des critères distinctifs, de ce qu’est la souffrance existentielle en fin de vie sera proposée. Je poserai ainsi l’hypothèse que la souffrance peut être définie comme une forme de construction de l’esprit s’articulant autour de trois concepts : intégrité, altérité et temporalité. D’abord, intégrité au sens où initialement l’individu malade se sent menacé dans sa personne (relation à soi). Ensuite, altérité au sens où la perception de ses conditions extérieures a un impact sur la détresse ressentie (relation à l’Autre). Et finalement, temporalité au sens où l’individu souffrant de façon existentielle semble bien souvent piégé dans un espace-temps particulier (relation au temps). Ensuite, je m’intéresserai à la souffrance du soignant. Dans le contexte d’une condition terminale, il arrive que des interventions lourdes (p. ex. : sédation palliative profonde, interventions invasives) soient discutées et même proposées par un soignant. Je ferai ressortir diverses sources de souffrance propres au soignant et générées par son contact avec le patient (exemples de sources de souffrance : idéal malmené, valeurs personnelles, sentiment d’impuissance, réactions de transfert et de contre-transfert, identification au patient, angoisse de mort). Ensuite, je mettrai en lumière comment ces dites sources de souffrance peuvent constituer des barrières à l’approche de la souffrance du patient, notamment par l’influence possible sur l’approche thérapeutique choisie. On constatera ainsi que la souffrance d’un soignant contribue par moment à mettre en place des mesures visant davantage à l’apaiser lui-même au détriment de son patient. En dernier lieu, j'élaborerai sur la façon dont la rencontre entre un soignant et un patient peut devenir un espace privilégié afin d'aborder la souffrance. J'émettrai certaines suggestions afin d'améliorer les soins de fin de vie par un accompagnement parvenant à mettre la technologie médicale au service de la compassion tout en maintenant la singularité de l'expérience du patient. Pour le soignant, ceci nécessitera une amélioration de sa formation, une prise de conscience de ses propres souffrances et une compréhension de ses limites à soulager l'Autre.

The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin

Les anthracyclines tels que la doxorubicin et la daunorubicin sont une famille de médicaments anticancéreux hydrophiles qui doivent être transportés dans les cellules afin d’exercer leur action par intercalation à l’ADN dans le noyau cellulaire. Ceci mène à la perturbation du métabolisme de l’ADN et entraine la mort cellulaire. Les anthracyclines sont utilisés pour le traitement d’une variété de cancers incluant la leucémie, les lymphomes, le cancer du sein, le cancer des poumons et le cancer des ovaires. Étant donné que le transport actif des anthracyclines dans les cellules a partiellement été démontré, le transporteur spécifique impliqué dans ce processus n’est pas encore connu. En utilisant un modèle de cancer des ovaires, la lignée cellulaire TOV2223G, nous avons démontré que des substrats spécifiques au transporteur de cations organiques 1 (OCT1), notamment la ergothionéine, la thiamine et la phenformin, ont partiellement inhibé l’absorption de la daunorubicin en différence de la carnitine qui est un substrat de haute affinité des transporteurs CT2 et OCTN2. Ces résultats suggèrent que les transporteurs organiques spécifiques au transport de la carnitine ne sont pas impliqués dans le transport des anthracyclines. Ainsi, nos résultats ont démontré que l’absorption de la daunorubicin est orchestrée par le transporteur OCT1 dans les cellules TOV2223G (Km ~ 5 μM) et des concentrations micromolaires de choline ont complètement abolies l’absorption de la drogue. De plus, un ARN sh dirigé contre OCT1 a réprimé son expression protéique, ce qui a été confirmé par la technique d’immuno-buvardage en utilisant un anti-OCT1 anticorps. Les cellules déficientes en OCT1 n’ont pas été capables d’absorber la daunorubicin et ont été plus résistantes à l’action de la drogue par rapport aux cellules contrôle. La transfection des cellules HEK293T avec un plasmide construit de façon à faire exprimer OCT1 comme protéine de fusion avec la protéine fluorescente EYFP a montré que celle-ci est localisée dans la membrane plasmique. Les cellules transfectées ont été capables d’absorber cinq fois plus de daunorubicin comparé aux cellules contrôles. Cette étude est, selon nous, la première à démontrer que OCT1 est un transporteur de haute affinité des anthracyclines. Ainsi, nous avons émis l’hypothèse que des défauts de OCT1 peuvent contribuer à l’efficacité de la réponse des cellules cancéreuses à la chimiothérapie avec les anthracyclines.