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Tesis por compendio / Los objetivos generales de esta tesis fueron desarrollar nuevos diluyentes de inseminación artificial (IA) suplementados con un análogo de GnRH y caracterizar el proteoma del semen de conejo. En el capítulo I, se evaluó la inclusión de un cóctel de inhibidores de proteasas en el diluyente de inseminación (DI) para evitar parte de la actividad proteasa del plasma seminal de conejo. La calidad seminal y la fertilidad no se vieron afectadas por el cóctel. Sin embargo, la prolificidad fue significativamente menor en el grupo experimental en comparación con los grupos de control positivo y negativo (8,2±0,22 vs. 9,3±0,23 y 9,2±0,26 gazapos por parto, respectivamente). De este capítulo, se puede concluir que la adición de una amplia variedad de inhibidores de proteasas en el diluyente de semen de conejo afecta negativamente la tasa de prolificidad y que en el futuro sería aconsejable probar inhibidores específicos de aminopeptidasas (AMIs). En el capítulo II, suplementamos el DI con AMIs (bestatina y EDTA), y estudiamos su efecto sobre la calidad seminal y el rendimiento reproductivo. La inclusión de AMIs no afectó la calidad seminal, ni la fertilidad (85,3 vs. 88,6%), ni la prolificidad (10,12 vs. 10,51 gazapos por parto) en comparación con el grupo control. Por lo tanto, concluimos que los AMIs se pueden utilizar en los DIs de conejo para inhibir parte de la actividad aminopeptidasa del plasma seminal (PS). En el capítulo III, probamos nuevos DIs de conejo que contenían AMIs con o sin nanopartículas de quitosano (CS)-sulfato de dextrano (DS) que atrapan el análogo de GnRH. Se estudiaron los siguientes diluyentes: C4 (4 µg de buserelina/coneja en medio control (MC): tris-ácido cítrico-glucosa suplementado con AMIs), C5 (5 µg de buserelina/coneja en MC), Q4 (4 µg de buserelina/coneja en nanopartículas CS-DS en MC) y grupo Q5 (5 µg de buserelina/coneja en nanopartículas CS-DS en MC). La fertilidad fue significativamente menor en el grupo C4 en comparación con los grupos C5, Q5 y Q4 (0,7 frente a 0,85, 0,85 y 0,82, respectivamente). Por el contrario, la prolificidad fue similar en los cuatro grupos experimentales. Por lo tanto, las nanopartículas de CS-DS como transportador de acetato de buserelina permiten reducir la concentración de la hormona en diluyentes con AMIs sin afectar la fertilidad ni la prolificidad. Por ello, la nanoencapsulación parece ser un sistema prometedor para proteger el análogo de GnRH en los diluyentes de IA de conejos. Por otro lado, el objetivo de los últimos tres capítulos fue caracterizar las proteínas del semen de conejo. En los capítulos IV y V, se estudiaron las proteínas del PS de conejo. Las muestras de semen se recuperaron utilizando 6 machos de cada línea genética (A y R) y seleccionando una muestra heteroespérmica del comienzo, del medio y del final de cada estación y de cada línea (24 muestras en total). En el capítulo IV, utilizamos la técnica de electroforesis en gel de poliacrilamida 1D. Siete bandas proteicas fueron significativamente diferentes entre las líneas genéticas y tres bandas entre las estaciones. En el capítulo V, se sometió el PS a nano LC-MS/MS y se identificaron y cuantificaron 402 proteínas. 23 proteínas se expresaron diferencialmente entre genotipos. Con respecto al efecto de la estación en el proteoma del PS de conejo, los resultados mostraron que no hubo un patrón claro de variación de proteína a lo largo del año. En el capítulo VI, se caracterizaron las proteínas del espermatozoide de conejo. Se recuperaron 6 muestras espermáticas utilizando 5 machos de cada genotipo y se sometieron a nano LC-MS/MS, identificándose y cuantificándose 487 proteínas. 40 proteínas se expresaron diferencialmente entre genotipos. En conclusión, los resultados de los tres últimos capítulos evidencian que el genotipo está relacionado con una abundancia específica de proteínas del PS y del espermatozoide. Finalmente, se creó la / The general objectives of this thesis were to develop new artificial insemination (AI) extenders supplemented with a GnRH analogue and to characterise the proteomic profile of rabbit semen. In chapter I, the inclusion of a protease inhibitors cocktail in the insemination extender (IE) to avoid part of the rabbit seminal plasma protease activity was evaluated. Seminal quality and fertility rate were not affected by the cocktail, having similar values between experimental and control groups. However, prolificacy rate was significantly lower in experimental group compared to positive and negative control groups (8.2 ±0.22 vs. 9.3 ±0.23 and 9.2 ±0.26 total born per litter, respectively). From this chapter, it may be concluded that the addition of a wide variety of protease inhibitors in the rabbit semen extender negatively affects prolificacy rate and it in the future it would be advisable to test specific aminopeptidase inhibitors (AMIs). Therefore, in chapter II, we supplemented the IE with AMIs (bestatin and EDTA), and we studied their effect on rabbit seminal quality and reproductive performance. Seminal quality was not affected by AMIs. Regarding reproductive performance, the inclusion of AMIs, did not affect fertility (85.3 vs. 88.6%), nor the prolificacy rate (10.12 vs. 10.51 kits per delivery) in comparison with control group. Thus, we concluded that AMIs can be used in rabbit IEs to inhibit part of the seminal plasma aminopeptidase activity. In chapter III, we test new rabbit IEs containing AMIs with or without chitosan (CS)-dextran sulfate (DS) nanoparticles entrapping the GnRH analogue. The following experimental extenders were studied: C4 (4 µg buserelin/doe in control medium (CM): Tris-citric acid-glucose supplemented with AMIs), C5 (5 µg of buserelin/doe in CM), Q4 (4 µg of buserelin/doe into CS-DS nanoparticles in CM) and Q5 group (5 µg of busereline/doe into CS-DS nanoparticles in CM). Results showed that fertility was significantly lower in C4 group compared to C5, Q5 and Q4 groups (0.7 vs. 0.85, 0.85 and 0.82, respectively). On the contrary, prolificacy was similar in the four experimental groups studied. Thus, CS-DS nanoparticles as carrier for buserelin acetate allow reducing the hormone's concentration in extenders supplemented with AMIs without affecting the fertility and prolificacy of rabbit females. Therefore, nanoencapsulation seems to be a promising system to protect the GnRH analogue in rabbit AI extenders. On the other hand, the aim of the last three chapters was to characterize rabbit semen proteins. In chapters IV and V, rabbit seminal plasma (SP) proteins were studied. Semen samples were recovered using 6 males from each genetic line (A and R). For each genotype, one pooled sample at the beginning, middle and end of each season was selected to develop the experiment (24 pools in total). In chapter IV, we used a 1D polyacrylamide gel electrophoresis approach. Seven protein bands were significantly different between genetic lines and three protein bands were significantly different between seasons. In chapter V, SP was subjected nano LC-MS/MS and 402 proteins were identified and quantified. Twenty-three proteins were differentially expressed between genotypes. Regarding the effect of season on rabbit SP proteome, results showed that there was no clear pattern of protein variation throughout the year. The results obtained in both chapters evidence that genotype is related to a specific abundance of SP proteins. In chapter VI, rabbit sperm proteins were characterised. Six samples were recovered during two months using 5 males from each genotype. Sperm proteins were subjected to nano LC-MS/MS and 487 proteins were identified and quantified. Forty proteins were differentially expressed between genotypes. In conclusion, rabbit sperm proteins showed that genotype has also a huge impact on their abundance. Finally, with these data, the first publicly accessible database of rabbit semen proteome was c / Els objectius generals d'aquesta tesi van ser desenvolupar nous diluents d'inseminació artificial (IA) suplementats amb un anàleg de GnRH i caracteritzar el proteoma del semen de conill. En el capítol I, es va avaluar la inclusió d'un còctel d'inhibidors de proteases en el diluent d'inseminació (DI) per evitar part de l'activitat proteasa del plasma seminal de conill. La qualitat seminal i la fertilitat no es van veure afectades pel còctel. No obstant això, la prolificitat va ser significativament menor en el grup experimental en comparació amb els grups de control positiu i negatiu (8,2 ± 0,22 vs. 9,3 ± 0,23 i 9,2 ± 0,26 catxaps per part, respectivament). D'aquest capítol, es pot concloure que l'addició d'una àmplia varietat d'inhibidors de proteases en el diluent de semen de conill afecta negativament la taxa de prolificitat i que en el futur seria aconsellable provar inhibidors específics de aminopeptidasas (AMIS). En el capítol II, suplementàrem el DI amb AMIs (bestatina i EDTA), i vam estudiar el seu efecte sobre la qualitat seminal i el rendiment reproductiu. La inclusió de AMIs no va afectar la qualitat seminal, ni la fertilitat (85,3 vs. 88,6%), ni la prolificitat (10,12 vs. 10,51 catxaps per part) en comparació amb el grup control. Per tant, concloem que els AMIs es poden utilitzar en els DIs de conill per inhibir part de l'activitat aminopeptidasa del plasma seminal (PS). En el capítol III, vam provar nous DIs de conill que contenien AMIs amb o sense nanopartícules de quitosà (CS)-sulfat de dextrà (DS) que atrapen l'anàleg de GnRH. Es van estudiar els següents diluents: C4 (4 µg de buserelina/conilla en medi control (MC): tris-àcid cítric-glucosa suplementat amb AMIs), C5 (5 µg de buserelina/conilla en MC), Q4 (4 µg de buserelina/conilla en nanopartícules CS-DS en MC) i grup Q5 (5 µg de buserelina/conilla en nanopartícules CS-DS en MC). La fertilitat va ser significativament menor en el grup C4 en comparació amb els grups C5, Q5 i Q4 (0,7 enfront de 0,85, 0,85 i 0,82, respectivament). Per contra, la prolificitat va ser similar en els quatre grups experimentals. Per tant, les nanopartícules de CS-DS com a transportador d'acetat de buserelina permeten reduir la concentració de l'hormona en diluents amb AMIs sense afectar la fertilitat ni la prolificitat. Per això, la nanoencapsulació sembla ser un sistema prometedor per protegir l'anàleg de GnRH en els diluents d'IA de conills. D'altra banda, l'objectiu dels últims tres capítols va ser caracteritzar les proteïnes del semen de conill. En els capítols IV i V, es van estudiar les proteïnes del PS de conill. Les mostres de semen es van recuperar utilitzant 6 mascles de cada línia genètica (A i R) i seleccionant una mostra heteroespérmica del començament, del mitjan i del final de cada estació i de cada línia (24 mostres en total). En el capítol IV, utilitzàrem la tècnica d'electroforesi en gel de poliacrilamida 1D. Set bandes proteiques van ser significativament diferents entre les línies genètiques i tres bandes entre les estacions. En el capítol V, es va sotmetre el PS a nano LC-MS / MS i es van identificar i quantificar 402 proteïnes. 23 proteïnes es van expressar diferencialment entre genotips. Pel que fa a l'efecte de l'estació en el proteoma del PS de conill, els resultats van mostrar que no hi havia un patró clar de variació proteica al llarg de l'any. En el capítol VI, es van caracteritzar les proteïnes de l'espermatozoide de conill. Es van recuperar 6 mostres espermàtiques utilitzant 5 mascles de cada genotip i es van sotmetre a nano LC-MS / MS, identificant i quantificant 487 proteïnes. 40 proteïnes es van expressar diferencialment entre genotips. En conclusió, els resultats dels tres últims capítols evidencien que el genotip està relacionat amb una abundància específica de proteïnes del PS i de l'espermatozoide. Finalment, es va crear la primera base de dades d'accés públic del proteoma / Casares Crespo, L. (2018). Development of new artificial insemination extenders supplemented with GnRH analogues to induce ovulation and proteomic characterization of rabbit semen [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/100854 / Compendio

Thesis advisor: Eranthie Weerapana / Reactive oxygen species (ROS) modulate protein function through cysteine oxidation. Identifying protein targets of ROS can provide insight into uncharacterized ROS-regulated pathways especially within ROS generating organelles such as the mitochondria. There are several known examples of mitochondrial cysteine targets that alter protein and pathway activity resulting in pathological effects. Several chemoproteomic workflows, including ABPP and OxICAT, can be used to identify sites of cysteine oxidation. However, determining ROS targets localized within subcellular regions and ROS hotspots remains challenging with existing workflows. Here, we present combined cysteine- monitoring chemoproteomic platforms (isoTOP-ABPP and OxICAT) with mitochondrial enrichment (organelle isolation and proximity labeling) to monitor cysteine oxidation events within the mitochondria. First, we profile redox- sensitive cysteines under exogenous and endogenous peroxide in isolated mitochondria using isoTOP-ABPP and OxICAT. Next, we introduce PL-OxICAT which combines enzymatic proximity labeling (PL) (TurboID/APEX) and OxICAT to monitor localized cysteine oxidation events within subcellular compartments such as the mitochondrial matrix and intermembrane space as well as ROS hotspots. Together, these platforms further hone our ability to monitor cysteine oxidation events within specific subcellular locations and ROS hotspots and provide a deeper understanding of the protein targets of endogenous and exogenous ROS. / Thesis (PhD) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Cancer is characterized by a deregulation of the cell cycle resulting in abnormal proliferation of cells that can bypass tightly regulated molecular checkpoints. Breast cancer is the most common cancer diagnosed in women, ~70% of cases displaying an estrogen receptor positive (ER+) phenotype. The aim of the present work was to generate a comprehensive overview of the biological mechanisms, molecular pathways and specific proteins involved in cell cycle progression in ER+ breast cancer cells. We focused on the G1-to-S phase transition of the cell cycle because major differences in cell proliferation mechanisms between normal and cancerous cells are observed at this point. We developed a large-scale proteomics strategy to enable the comparison of MCF-7 ER+ (cancer) and MCF-10A (non-tumorigenic) epithelial breast cells. Samples were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) followed by a label-free quantitation approach, i.e., spectral counting, for differential protein expression analysis. The study was divided into three distinct parts: 1) qualitative profiling of MCF-7 cells arrested in the G1-phase and released into the S-phase of the cell cycle, 2) differential expression profiling of MCF-7 cells in G1 and S, and 3) differential expression profiling of the G1-phases of MCF-7 and MCF-10A cells. The qualitative evaluation of MCF-7 proteomic data resulted in the identification of >2700 proteins (p-score<0.001). A large number of these proteins were involved in cell cycle relevant processes, being representative of all hallmarks of cancer. Differential expression analysis of the MCF-7 G1 and S-phases resulted in the identification of >250 proteins with roles in DNA repair, transcription, translation, chromatin maintenance and signaling. The MCF-7/MCF-10 comparison revealed that major cellular processes that require DNA access, such as the ones identified in the MCF-7 analysis, are up-regulated in the nucleus of MCF-7 cells during starvation, possibly allowing these cancerous cells to bypass the restriction point. Several proliferative and anti-proliferative markers were identified in both MCF-7 and MCF-10A cells. / Ph. D.

The discovery of cancer biomarkers is crucial in the clinical setting to facilitate early diagnosis and treatment, thereby increasing survival rates. Proteomic technologies with mass spectrometry detection (MS) have the potential to affect the entire spectrum of cancer research by identifying these biomarkers. Simultaneously, microfabricated devices have evolved into ideal analysis platforms for minute amounts of sample, with promising applications for proteomic investigations and future biomarker screening. This thesis reports on the analysis of the proteomic constituents of the MCF7 breast cancer cell line using a shotgun 2-D strong cationic exchange/reversed phase liquid chromatography electrospray ionization tandem mass spectrometry (SCX/RP-LC-ESI-MS/MS) protocol. A series of optimization strategies were performed to improve the LC-MS experimental set-up, sample preparation, data acquisition and database searching parameters, and to enable the detection and confident identification of a large number of proteins. Over ~4,500 proteins were identified using conventional filtering parameters, and >2000 proteins using a combination of filters and p-value sorting. Of these, ~1,950 proteins had p<0.001 (~90%) and more than half were identified by &#8805; 2 unique peptides. About 220 proteins were functionally involved in cancer related cellular processes, and over 100 proteins were previously described in the literature as potential cancer markers. Biomarkers such as PCNA, cathepsin D, E-cadherin, 14-3-3-sigma, antigen Ki-67, TP53RK, and calreticulin were identified. These data were generated by subjecting to mass spectrometric analysis ~42 µg of protein digest, analyzing 16 SCX peptide fractions, and interpreting ~55,000 MS2 spectra. Total MS time required for analysis was 40 h. Selective SCX fractions were also analyzed by using a microfluidic LC platform. The performance of the microchip LC was comparable to that obtained with bench-top instrumentation when similar experimental conditions were used. The identification of 5 cancer biomarkers was enabled by using the microchip LC platform. Furthermore, this device was also capable to analyze phosphopeptides. / Master of Science

Proteomics is defined as a tool to explore how proteins control and regulate important molecular and physiological processes. Further, peptide-centric approaches, or bottom-up methods, provide more comprehensive coverage of a proteome compared to whole-protein approaches. This body of work assesses the technical feasibility of several bottom-up proteomics technologies applied to Arabidopsis thaliana, including gel-based methods, those that require peptide derivitization, and those that do not. Selected-reaction monitoring (SRM) for targeted proteomics, and data-independent acquisition (MSE) was also evaluated. In addition to assessing the capabilities of these technologies, we then applied them to the context of uncovering new insights into the flavonoid biosynthetic pathway and the auxin and ethylene signaling pathways. Chapter one provides background information related to secondary metabolism, phytohormone signaling, and the status of proteomics in plants. In Chapter 2 and Appendix A, we establish the methodology to apply traditional and DiGE-based 2D-GE strategies to global proteomics in Arabidopsis. Our results suggest that while 2D-GE is applicable to Arabidopsis, there are practical and conceptual limitations that must be understood. Further, our results suggest that pertubations in the flavonoid pathway do not affect the abundance of proteins in Arabidopsis seedlings, roots, or flowers that can be studied using 2D-GE and DiGE. Additionally, we demonstrated the first parallel comparison of the effects of auxin and ethylene on the Arabidopsis root proteome and observed no overlap among the proteins regulated by the two phytohormones, at least for the most abundant proteins observed by 2D-GE. Chapter 3 explores the efficacy of selected reaction monitoring for relative peptide quantification in Arabidopsis roots. Our results suggest that while the technology parallels application in yeast and humans, there are substantial analytical challenges that much be addressed. In Chapter 4 we explore the MSE data acquisition scheme for global proteomics in Arabidopsis. We observe that treatment with exogenous auxin affects the abundance of many proteins representing diverse biological processes. Interestingly, we observe minimal overlap among genes and proteins regulated by exogenous auxin. Appendix B explores the efficacy of iTRAQ labeling for relative peptide quantification in Arabidopsis roots. / Ph. D.

Energy balance includes energy intake and energy expenditure. Either excessive food intake or insufficient physical activity will increase the body mass and cause obesity, a worldwide health problem. In the US, more than two-thirds of people are obesity or overweight. Conversely, it is well accepted that reducing energy intake can increase the life span and the resistance to age-related diseases. MicroRNAs are highly conserved non-coding RNA molecules with a length of 21-23 nucleotides. Recent studies show that numerous microRNAs are associated with the regulation of oxidative stress, inflammation, insulin signaling, apoptosis, and angiogenesis that relate to obesity. However, the role of microRNAs in the regulation of energy balance in central nervous system remains unknown, especially within the hypothalamus, a primary site of energy balance control. In this project, microRNA, and mRNA were profiled using microarray technology. Furthermore, quantitative proteomics were used to identify differential protein levels during fasting, and in a genetically obese mouse model, Mice were given either a 24-hour fast, or ad libitum access to food. Hypothalamic RNA and microRNA samples were analyzed by microarray, using both the Affymetrix and Toray 3D mRNA and microRNA platforms. No microRNAs were found to be differentially expressed between two treatments, whereas numerous mRNAs were significantly regulated by fasting, including 7 cell cycle related genes. Hypothalamic protein samples from WT and N2KO mice treated either to ad lib feeding or 24-hour fasting were analyzed by MSE quantitative proteomics. Over 650 proteins were identified with some proteins showing significantly different abundances between or among the four groups. Between ad lib fed WT and N2KO mice, 53 proteins were differentially expressed, with some of these linked to neurodegeneration, NAD synthesis, and the citrate acid cycle (TCA). Overall, the results of this study suggest that while microRNA-mediated mechanisms are not significant modulators of hypothalamic gene expression upon a 24 hour fast, cell cycle gene expression changes represent a major contributor to the fasting response. Moreover, Nlhl2 might play an important role in the neurodegeneration and mitochondrial metabolism. / Ph. D.