Breast cancer metastasis to the brain is one of the most lethal forms of metastases. Metastasis is regarded as a non-random process governed by several biomechanical factors including tissue stiffness. As brain tissue is ultrasoft and extremely heterogeneous compared to breast cancer primary sites; how are breast cancer cells able to colonize the vastly different microenvironment of the brain? As a key protein of the Hippo pathway, YAP is regarded as a mechanotransducer that is sensitive to changes in substrate stiffness. Its biochemical activity is intertwined with Piezo1, a mechanosensitive ion channel activated through plasma membrane deformation. To impact cellular function, YAP enters the nucleus and binds to the TEAD transcription domain triggering downstream expression of proteins involved in cell motility, wound healing, and metastasis. In this work, triple-negative breast cancers (TNBC) were shown to experience greater migration rates on stiff surfaces compared to soft PDMS substrates. Concurrently, cells showed YAP nuclear localization in a stiffness dependent manner. Then, mechanical characterization of human brain tissue was performed to characterize the stiffness heterogeneity in the brain associated with region specific metastasis. Five to six regions of the brain from two different patients showed similar patterns of stiffness heterogeneity with the anterior regions being generally stiffer than posterior regions. As Piezo1 is directly linked with detecting changes in biomechanical stimuli, it was used as a readout of surface stiffness to examine if cells in the brain could detect different regional stiffnesses. Comparisons of grey and white matter showed no significant difference in Piezo1 expression. As a drug screening framework, molecular dynamic simulations were performed to evaluate drug efficacy on well-characterized inflammatory mediators that are implicated in metastasis. These findings contribute to understanding the gap in knowledge surrounding the interplay between tissue stiffness and YAP mechanotransduction in the context of breast-to-brain metastasis. / Thesis / Master of Applied Science (MASc) / Breast cancer is the most common cause of cancer related deaths in women particularly when it spreads to the brain. The brain is composed of many different sub-locations comprised of different proteins that can change the tissue’s stiffness. Breast cancer can detect these changes and become more aggressive in its growth using a combination of proteins such as yes associated protein (YAP) and Piezo1. How these proteins interact in the context of breast to brain cancer metastasis however is poorly understood. This project examined the effects of surface stiffness, on YAP, and Piezo1 activity to understand how breast cancer and brain cells react to changes in surface stiffness. Results showed that on stiff surfaces YAP activity affects cancer cell migration. Also, human brain tissue was found to vary in stiffness depending on the region examined. Future investigations may shed light on therapies that could take advantage of learnings in this area to better target the spread of breast cancer.

Yes-associated protein (Yap) transcriptional co-activator, a major downstream effector of Hippo signaling pathway, controls organ size by modulating cell proliferation and apoptosis. The Hippo signaling cascade phosphorylates Yap, and this phosphorylation inhibits the nuclear retention of Yap, which is essential for cell proliferation. Thus, the loss of Hippo pathway components leads to enlarged organs through increased Yap activity in the nucleus. Our initial study showed that Yap was expressed in the developing retina and retinal pigment epithelium (RPE), suggesting Yap's tissue-specific roles during the eye development. Intriguingly, Yap proteins were localized at the apical junctions in addition to the nucleus and cytosol of the retinal progenitor cells, adding another level of regulation. To uncover the tissue- and localization-specific functions of Yap, we generated a Yap conditional knockout mouse with Rx-Cre for the ablation of the Yap gene in the developing retina and RPE. Upon deletion of Yap, the retina showed severe lamination defects with numerous folding, which is reminiscent of the polarity and adhesion loss. The RPE, a single pigmented cell layer overlying the retina, lost pigmentation and changed into a multi-layered epithelium. The marker analysis revealed that 1) in the retina, the localization of the polarity complex proteins such as Pals1, Crb1 and atypical PKC were disrupted, suggesting Yap's indispensable role in junctional stability, and 2) the level of Otx2 in RPE decreased while those of Chx10 and beta-tubulin increased, suggesting transdifferentiation of RPE into the retina. In addition, the deletion of Yap induced a decrease in proliferation and an increase in apoptosis, ultimately resulting in microphthalmia. In conclusion, our results are consistent with the model that Yap functions in the stabilization of apical proteins for maintenance of the laminar organization, determination of RPE territory, and regulation of proliferation and apoptosis during the eye development. / Cell Biology

Heart failure after myocardial infarction (MI) remains the leading cause of mortality among all cardiovascular diseases globally. Angiogenesis plays a critical role in cardiac functional recovery after MI. Heat shock protein A12B (HSPA12B) is predominately expressed in endothelial cells and required for angiogenesis. Yes-associated protein (YAP) has been reported to promote tumor angiogenesis. In the present study, we investigated the cooperative role of HSPA12B and YAP in angiogenesis following myocardial ischemic injury. Endothelial specific deficiency of HSPA12B (eHspa12b-/-) or YAP (eYap-/-) impairs angiogenesis and exacerbates cardiac dysfunction after MI, when compared with wild type (WT) mice. In addition, MI induced angiogenesis and the expression of angiogenic factors (angiopoietin-1, VEGF and VEGFR2) were impaired in both eHspa12b-/- and eYap-/- hearts. MI increased YAP expression and nuclear translocation in WT hearts, but not in eHspa12b-/- myocardium. Similarly, MI also markedly increased HSPA12B expression and nuclear translocation in WT mice but not in eYap-/- hearts. In vitro data shows that overexpression of HSPA12B upregulated hypoxia induced endothelial cell proliferation, migration and angiogenesis. On the contrary, deactivation of YAP by verteporfin attenuates endothelial cell proliferation, migration and angiogenesis after hypoxic challenge. In accordance, silencing of either HSPA12B or YAP suppressed endothelial cell proliferation and angiogenesis promoted by hypoxia. Importantly, YAP inhibition abrogates HSPA12B induced endothelial cell proliferation and angiogenesis. Deficiency of HSPA12B suppresses YAP expression and nuclear translocation following hypoxia while knockdown of YAP attenuates hypoxia stimulated HSPA12B expression and nuclear translocation. Mechanistically, hypoxia induced an interaction between endothelial HSPA12B and YAP. Of note, ChIP assay shows that HSPA12B is a target gene of YAP/transcriptional enhanced associated domain 4 (TEAD4). Further investigation indicates that HSPA12B also acts as a co-activator in YAP associated proliferation and angiogenesis. HSPA12B can stabilize YAP and prevent YAP from degradation. Therefore, our results delineated a previously unrecognized role of endothelial HSPA12B as a novel target and co-activator for YAP/TEAD4 and cooperates with YAP to promote endothelial cell proliferation, migration and angiogenesis following myocardial ischemia.

Recent evidence from cancer research indicates that lactate exerts a suppressive effect on innate immune responses in cancer. This study investigated the mechanisms by which lactate suppresses macrophage pro-inflammatory responses. Macrophages [Raw 264.7 and bone marrow derived macrophages (BMDMs)] were treated with LPS in the presence or absence of lactate. Pro-inflammatory cytokines, NF-κB and YAP activation and nuclear translocation were examined. Our results show that lactate significantly attenuates LPS stimulated macrophage TNF-α and IL-6 production. Lactate also suppresses LPS stimulated macrophage NF-κB and YAP activation and nuclear translocation in macrophages. Interestingly, YAP activation and nuclear translocation are required for LPS stimulated macrophage NF-κB activation and TNFα production. Importantly, lactate suppressed YAP activation and nuclear translocation is mediated by GPR81 dependent AMKP and LATS activation which phosphorylates YAP, resulting in YAP inactivation. Finally, we demonstrated that LPS stimulation induces an interaction between YAP and NF-κB subunit p65, while lactate decreases the interaction of YAP and NF-κB, thus suppressing LPS induced pro-inflammatory cytokine production. Our study demonstrates that lactate exerts a previously unknown role in the suppression of macrophage pro-inflammatory cytokine production via GPR81 mediated YAP inactivation, resulting in disruption of YAP and NF-κB interaction and nuclear translocation in macrophages.

No description available.

Increasing the milking frequency of early lactation dairy cows increases their milk yield, both during increased milking and after the cows are returned to a normal twice-daily milking schedule. When milked four times a day (4X) for only the first twenty-one days of lactation, the right udder half produces 3 kg/d more milk than the left half, which is milked only two times a day (2X) over the course of lactation (Hale et al., 2003). Alterations to this increased milking frequency (IMF) approach have been investigated in order to maximize production of the animals and determine the most efficient practice for producers. The aim of this study was to determine the appropriate duration of early lactation IMF treatment by increasing milking frequency of early lactation cows for various lengths of time, and subsequently increasing the use of this management practice on Virginia dairy farms. The right udder half of twenty-three primiparous and multiparous Holstein cows were milked 4X for 10, 20, or 40 days at the beginning of lactation, and the left udder half 2X for the entire lactation. Udder-half milk yields were measured at various time points throughout lactation and used to calculate the difference between right (4X) and left (2X) udder halves. Overall, treatment did not have a significant effect on milk yield difference throughout the entire lactation; the udder half differences for each group were -0.45 kg, 1.92 kg and 4.62 kg for the 10 d, 20 d and 40 d treatments (P > 0.05). In addition to the IMF portion of the experiment, two different methodologies were used to investigate the possible mechanism of local regulation of milk yield in response to IMF treatment. Milk fatty acid analysis was performed on milk samples obtained from the above experiment. Three different groups of fatty acids were analyzed to detect potential changes in the right udder half (4X) when compared to the left (2X); the three groups were denovo, C16, and preformed fatty acids. There was no significant effect of treatment on fatty acid composition of right and left udder halves for any of the three groups (P > 0.05). The second methodology used to explore a possible mechanism behind increased milk yield following IMF treatment was immunohistochemistry of mammary gland tissue samples obtained after IMF treatment in a previous experiment. The key target investigated was a component of the Hippo signaling pathway, Yes-associated protein (YAP). Intensity of YAP staining in the cytoplasmic area of mammary epithelial cells (MEC) and number of YAP-positive stained nuclei located in the MEC were quantified for each of the images obtained. There was no effect of treatment or day on intensity of staining (P > 0.05) with no difference in the intensity of staining between 4X and 2X samples or d 21 and d 60 samples. However, the interaction for treatment x day tended to be significant (P < 0.06), with the d 60 samples tending to have higher intensity of staining than d 21 samples. For YAP-positive nuclei, there was a significant effect of day (P < 0.05), with d 60 samples having significantly more YAP positive nuclei. There was not a significant effect of treatment or treatment x day interaction (P > 0.05) with 2X and 4X samples having the same number of YAP positive nuclei. Results from the first experiment reveal that 40 d of IMF during early lactation is sufficient to produce an increase in milk and component yields throughout lactation. This practice could be implemented on Virginia dairy farms as a way to increase efficiency and milk yield per cow. Results from the second half of this research indicates that further research is needed to investigate the fatty acid content of milk from cows subject to IMF treatment during early lactation. In addition, YAP potentially plays a role in the changes occurring in the mammary gland, with increased intensity of YAP staining and increased number of YAP positive nuclei observed at 60 DIM. Understanding of this protein and its involvement in the mammary gland could lead to identifying a mechanism for which this increase in milk yield and components following IMF is occurring. Further research needs to be done to provide results supporting the current experiment. / MSLFS / The focus of this study is increasing milk yield by increasing milking frequency of early lactation cows. We will use a technique where each half of the cow's udder is milked with different milking frequencies. Previous studies have shown an increase in milk yield due to increased milking frequency. When milked four times a day for 21 days, the right udder half produced 2,032 lb. more milk than the left over the course of a lactation. This project will investigate modifications to the 21-day increase in milking frequency that might increase the use of this management practice on Virginia dairy farms. This experiment will determine the increase in milk yield when cows are milked four times a day for 10, 20, or 40 days. Farmers are concerned about return on investment - including time invested engaging extra milkings. This experiment will determine the effective duration of increased milking frequency needed to gain an increase in milk production with the minimum investment of time necessary to apply increased milking frequency - can the increase achieved with 20 days be achieved in only 10 or does increasing to 40 days provide an even greater benefit than 20-day application?

Le cancer colorectal est la première pathologie cancéreuse de la sphère digestive, tant en terme de fréquence que de mortalité par an. Chaque année, 41 000 nouveaux cas sont diagnostiqués et 17 000 décès sont dus à ce cancer en France. Deux paramètres cliniques expliquent la mortalité de ce cancer; d'une part le fait qu'un patient sur deux est diagnostiqué au stade métastatique ou va présenter des lésions métastatiques durant l'histoire de sa pathologie, d'autre part le fait que les patients après traitement vont fréquemment présenter une récidive de leur pathologie. L'utilisation de régimes de chimiothérapies avant et après résection métastatique améliore la survie sans récidive à court terme, mais à 2 ans post chirurgie l'avantage apporté est perdu. Ainsi, la compréhension des mécanismes d'échappement à la chimiothérapie et régissant la croissance tumorale est d'intérêt pour tenter de limiter la récidive tumorale. L'objectif de ce travail de thèse a consisté en l'analyse de sous-populations obtenues sous pression de chimiothérapie au 5-Fluorouracile (5FU) dérivées de la lignée cancéreuse colique HT29, ainsi que les mécanismes moléculaires associés. Notre clone le plus chimiorésistant isolé, le modèle cellulaire 5F31, quitte le compartiment prolifératif sous traitement à fortes doses de 5FU, ceci étant associé à une perturbation de la voie de signalisation de la Src kinase c-Yes et de son partenaire, le co-activateur transcriptionnel YAP. Sous traitement, les cellules chimiorésistantes entrent en quiescence, le complexe protéique entre c-Yes et YAP est perdu et la quantité totale et nucléaire de YAP diminue de manière significative (Igoudjil, Touil, Corvaisier et al. 2014 Clinical Cancer Research). Dès lors, la suite des travaux a consisté en l'étude du rôle potentiel de YAP sur la balance quiescence/prolifération sous 5FU. L'inhibition pharmacologique ou l'inhibition transitoire de l'expression de YAP et de son paralogue, la protéine TAZ, dans plusieurs lignées cancéreuses coliques induit l'augmentation de la fraction de cellules quiescentes, associée au ralentissement significatif de la croissance tumorale. A l'inverse, la surexpression d'une forme constitutivement active de YAP demeurant nucléaire sous 5FU maintient les cellules 5F31 en prolifération et sensibilise les cellules à la chimiothérapie. Au niveau des effecteurs protéiques, l'induction de quiescence (par traitement à la chimiothérapie ou inhibition de YAP/TAZ) est associée à la perte d'expression de la Cycline E1 et du facteur de transcription c-Myc. A l'inverse, la surexpression du dominant constitutivement actif de YAP dans les cellules 5F31 conduit à l'expression soutenue de la Cycline E1 sous 5FU, expression nécessitant l'activation du facteur de transcription CREB. L'inhibition de la Cycline E1 permet d'induire la quiescence cellulaire, proposant cette protéine comme l'un des effecteurs des protéines YAP/TAZ dans la régulation entre la quiescence et la prolifération cellulaire (Corvaisier et al, Oncotarget, 2016). En conclusion, nos données montrent l'importance du rôle des protéines YAP/TAZ dans le maintien des cellules en prolifération via l'expression notamment de la Cycline E1. Nos résultats sur cohorte de patients atteints de métastases hépatiques de cancers colorectaux montrent que l'expression des co-activateurs YAP/TAZ est liée à un index prolifératif plus important, confortant nos données sur le rôle de ces protéines dans la croissance tumorale. De plus, l'expression élevée de YAP et TAZ est associée en analyses multivariées à une récidive plus précoce et à une survie globale plus faible. Ainsi, l'étude de l'expression et du niveau d'activation de ces acteurs serait un marqueur pronostic intéressant dans l'anticipation de la récidive métastatique ; ainsi que des cibles thérapeutiques intéressantes pour tenter de limiter la rechute tumorale. / Colorectal cancer is the most frequent and lethal cancerous pathology from the digestive system. Each year in France, 41 000 new cases are diagnosed and 17 000 patients die due to this pathology. This high mortality is mainly due to the rate of patients with liver metastatic lesions and the early relapse of those metastases after treatment. The use of chemotherapy prior to surgery induces a decrease of early relapse, however 2 years after resection this advantage is lost. Thus, understanding the mechanisms underlying escape to treatment is required to try to delay or prevent tumor recurrence.The aim of this doctoral work was to analyze clonal chemoresistant subpopulations derived from the colorectal cancer cell line HT29 after chronic exposure to 5-Fluorouracil (5FU) and molecular mechanisms associated with chemoresistance. The most chemoresistant clonal subpopulation, 5F31, stops its proliferation after treatment with high dose of 5FU, this behavior being associated with the modulation of the c-Yes/YAP axis. After treatment, 5F31 cells enter quiescence, interaction between c-Yes and YAP is lost and total and nuclear YAP protein expression reduces significantly (Igoudjil, Touil, Corvaisier et al. 2014, Clinical Cancer Research). The next step was to study functions of YAP protein in this chemotherapy- induced quiescence.Pharmacological or transient inhibition of YAP and its homolog TAZ, induces quiescence and reduces cellular growth in several colorectal cancer cell lines. On the other hand, overexpression of a constitutively active form of YAP in 5F31 cells forces cells to remain proliferative under 5FU treatment, enhancing 5F31 cell chemosensitivity to 5FU.Regarding proteic effectors, quiescence (either induced by 5FU or YAP/TAZ inhibition) is associated with loss of expression of the transcription factor c-Myc and Cyclin E1. In 5F31 cells expressing the active mutant form of YAP, Cyclin E1 expression is sustained after 5FU treatment through the activation of the transcription factor CREB. Cyclin E1 inhibition is sufficient to induce quiescence, therefore introducing this protein as one of the final effectors of YAP/TAZ co-activators in the regulation of the proliferation/quiescence switch in colorectal cancer cells (Corvaisier et al. 2016, Oncotarget).To conclude, our work reveals the importance of YAP/TAZ proteins for the maintenance of colorectal cancer cells proliferation through Cyclin E1 expression. Our work on liver metastases from patients with colorectal cancer shows that high expression of YAP/TAZ is connected to a higher proliferative index in metastatic lesions. Moreover, high YAP/TAZ expression is associated with shorter patient progression-free survival and shorter overall survival. Studying the expression and level of YAP/TAZ activation could be an interesting prognosis marker to anticipate metastatic relapse and potent druggable target to delay tumoral recurrence.

Molecular dynamics (MD) is a powerful tool that can be applied to protein folding and protein structure. MD allows for the calculation of movement, and final position, of atoms in a biomolecule. These movements can be used to investigate the pathways that allow proteins to fold into energetically favorable structures. While MD is very useful, it still has its limitations. Most notable, computing power and time are of constant concern. Protein structure is inherently important due to the direct link between the structure of a protein and its function. One of the four levels of protein structure, the secondary structure, is the first level to accommodate for the three-dimensional shape of a protein. The main driving force behind secondary structure is hydrogen bonding, which occurs between the carboxyl oxygen and the amine hydrogen of the backbone of a peptide. Determining a greater link between hydrogen bond patterns and types of secondary structure can provide more insight on how proteins fold. Because molecular dynamics allows for an atomic level view of the dynamics behind protein folding/unfolding, it becomes very useful in observing the effects of particular hydrogen bond patterns on the folding pathway and final structure formed of a protein. Using molecular dynamic simulations, a series of experiments in an attempt to alter structure, hydrogen bonding, and folding patterns, can be performed. This information can be used to better understand the driving force of secondary structure, and use the knowledge gained to manipulate these simulations to force folding events, and with that, desired secondary structure features.

Electrospun nanofibers have been researched extensively in the culturing of stem cells to understand their behavior since electrospun fibers mimic the native extracellular matrix (ECM) in many types of mammalian tissues. Here, electrospun nanofibers with defined structure (orientation/alignment) and pore size could significantly modulate human mesenchymal stem cell (hMSC) behavior. Controlling the fiber membrane pore size was predominantly influenced by the duration of electrospinning, while the alignment of the fiber membrane was determined by parallel electrode collector design. Electric field simulation data provided information on the electrostatic interactions in this electrospinning apparatus.hMSCs on small-sized pores (~3-10 µm²) tended to promote the cytoplasmic retention of Yes-associated protein (YAP), while larger pores (~30-45 µm²) promoted the nuclear activation of YAP. hMSCs also displayed architecture-mediated behavior, as the cells aligned along with the fiber membranes orientation. Additionally, fiber membranes affected nuclear size and shape, indicating changes in cytoskeletal tension, which coincided with YAP activity. The mechanistic understanding of hMSC behavior on defined nanofiber structures seeks to advance their translation into more clinical settings and increase biomanufacturing efficiencies.

<p>The rotator cuff is a group of four muscles and tendons in the shoulder that function to lift and rotate the arm. Rotator cuff tendon tears are increasingly common: more than 545,000 rotator cuff surgeries occur annually in the US. However, treatment is often complicated by disorganized collagen matrix formed via fibrosis and results in high re-tear rates. Tendon tissue engineering seeks to solve the problem using biomaterials to promote neo-tendon formation to augment repair or regenerate tendon. However, while current biomaterials provide the opportunity to improve tendon healing, they frequently still exhibit fibrosis in preclinical studies. Therefore, a critical need exists to understand the mechanisms of aligned collagen formation when designing biomaterials for tendon tissue engineering. Matrix architecture and transient receptor potential cation channel subfamily V member 4 (TRPV4) regulate aligned collagen formation during tenogenesis in vitro, but the mechanism remains to be determined. Recently, TRPV4 stimulation was found to induce nuclear localization and activation of transcriptional co-activators Yes-associated protein (YAP). YAP expression is upregulated during tendon development, a process characterized by aligned collagen formation, and in response to physiological mechanical stimulation, suggesting it could play an important role in tendon. The objective of this work is to improve tissue engineering strategies and progress toward making a device that regenerate tendon after injury. Aim 1 incorporates tendon-derived matrix into synthetic polymer scaffolds to add biological signaling cues to induce tenogenesis. Aim 2 uses a 2D photolithography system (microphotopatterning) to optimize architectural and structural cues to promote stem cell differentiation toward tenogenic, chondrogenic, and osteogenic lineages. Aim 3 investigates dynamic tensile loading protocols to promote collagen matrix synthesis and improve engineered tendon mechanical function. Aim 4 investigates the role of TRPV4 and YAP in collagen alignment during engineered tendon development.</p>