INTRODUÇÃO. A proteína Fator Inibitório da Migração de Macrófagos (MIF) é frequentemente observada com expressão elevada em tecidos tumorais quando comparados aos tecidos equivalentes normais e estudos têm sugerido seu papel como marcador prognóstico de neoplasias malignas, incluindo carcinomas hepatocelular, de ovário, de esôfago e também de cabeça e pescoço. Adicionalmente, alguns de seus mecanismos de ação já demonstrados, como a indução da proliferação e migração celular permitem implicar essa expressão diferencial no desenvolvimento tumoral e, consequentemente, no prognóstico das neoplasias malignas. OBJETIVO. Esse estudo objetivou avaliar o papel diagnóstico e prognóstico da proteína MIF em carcinoma epidermóide da cavidade bucal. METODOLOGIA. O estudo foi composto por 50 pacientes com carcinoma epidermoide da cavidade bucal coletados prospectivamente e 57 casos retrospectivos admitidos nos Serviços de Cirurgia de Cabeça e Pescoço do Hospital Heliópolis e da Faculdade de Medicina da Fundação ABC. As análises foram feitas por meio de imunoistoquímica dos tecidos tumorais e margens epiteliais normais e de ELISA das amostras de soro e saliva, coletadas pré e pós-tratamento cirúrgico, dos pacientes participantes. Os resultados foram correlacionados aos achados clínicos e histopatológicos. RESULTADOS. A expressão da proteína MIF e seu receptor CD74 mostrou-se elevada em tecido tumoral quando comparado ao tecido epitelial livre de neoplasia (p < 0,0001). Associação entre a alta expressão da MIF no tumor e infiltração vascular linfática foi observada (p=0,005) e alta expressão da MIF no epitélio livre de tumor apresentou correlação marginalmente significativa com ocorrência de segundo tumor primário (p=0,072). A expressão positiva do receptor CD74 não apresentou associação com variáveis clínicas ou histopatológicas. A concentração sorológica da proteína MIF apresentou associação inversa com metástase linfonodal (p=0,018) e estádios patológicos avançados (p=0,040) e foi significativamente reduzida após a ressecção do tumor (p=0,001). A concentração da MIF na saliva não apresentou redução significativa após o tratamento cirúrgico, mas foi associada aos estágios pT3 e pT4 (p=0,001) e a estádios patológicos avançados (p=0,032). CONCLUSÕES. A redução significante da concentração da MIF observada no soro dos pacientes após a ressecção cirúrgica do tumor permite sugerir papel potencial dessa proteína como biomarcador para a detecção precoce de recorrência do carcinoma epidermoide da cavidade bucal. A expressão tecidual da proteína MIF e seu receptor CD74 apresentou papel controverso, mas a concentração salivar da proteína MIF parece relacionar-se a um possível papel pró-tumoral em carcinoma epidermoide da cavidade bucal / INTRODUCTION. The Macrophage Migration Inhibitory Factor (MIF) overexpression is frequently observed in tumor tissues compared to normal tissues and some previous studies have suggested its role as a prognostic marker of malignancies, including hepatocellular, ovarian, esophageal and also head and neck carcinoma. Additionally, some of its mechanisms of action, as migration and cell proliferation induction, have been demonstrated, which allow imply a differential expression in tumor progression and therefore in the prognosis of malignant neoplasms. OBJECTIVES. This study aimed to evaluate the role of MIF protein and its receptor CD74 in prognosis and diagnostic of oral squamous cell carcinoma. METHODS. The study consisted of 50 patients with oral squamous cell carcinoma prospectively collected and 57 patients retrospectively collected admitted at the Head and Neck Surgery Service from Heliópolis Hospital and ABC Medical School. The analysis were performed by Imunohistochemistry of tumor and normal tissues and by ELISA of serum and saliva samples collected pre and post-surgical treatment. Results were correlated to clinical and histopathological data. RESULTS. The expression of MIF protein and of its receptor CD74 was higher in OSCC than in normal epithelium (p < 0,0001). Association between overexpression of MIF in tumor tissue and lymphatic vessel invasion was observed (p=0,005) and higher concentration of MIF in normal epithelium showed correlation of marginal significance with second primary tumor occurrence (p=0,072). The positive expression of the receptor CD74 did not presented association with clinical or histopathological variables. Serum MIF concentration presented inverse association with lymph node metastasis (p=0,018) and advanced pathological stage (p=0,040) and it was significantly reduced after the surgery (p=0,001). The salivary MIF concentration was not significantly reduced after the surgery, but it was associated with pT3 and pT4 stages (p=0,001) and advanced pathological findings (p=0,032). CONCLUSIONS. The results showing significant reduction of MIF concentration in post-surgical serum of patients suggest its potential role as a biomarker to early detection of oral squamous cell carcinoma recurrence. The MIF and CD74 expression presented controversial role, but the salivary concentration of MIF seems to develop a possible pro-tumoral role

This study investigated characteristics of algal organic matter (AOM) derived from four species (cyanobacteria Microcystis aeruginosa and Merismopedia tenuissima, diatom Fragilaria crotonensis and green alga Chlamydomonas geitleri) dominating phytoplanktonic populations in the reservoirs supplying drinking water treatment plants - the water reservoirs Švihov (the Želivka water treatment plant) and Josefův Důl (the Jizerské Mountains), Czech Republic. The four mentioned microorganisms were cultivated under laboratory conditions and the algal growth was quantified by a number of cells, chrorophyll-a and optical density. Production of AOM was evaluated by dissolved organic carbon concentration measurements, by specific UV absorbance, the amount of peptide/protein and non-peptide (carbohydrate) contents, water affinity and molecular weight (MW). AOM of all considered microorganisms are formed predominantly by hydrophilic substances with low values of specific UV absorbance (< 2 L/(m.mg)). Considerable portions of both types of AOM, i.e. extracellular and cellular organic matters are represented by substances with MW < 1 kDa or with MW > 100 kDa. However, the total amount and composition of AOM significantly depend on algal species and their growth. The obtained results imply that the knowledge of AOM...

<div>Delayed fracture healing is a major health issue involved with aging. Therefore, strategies to improve the pace of repair and prevent non-union are needed in order to improve patient outcomes and lower healthcare costs. In order to accelerate bone fracture healing noninvasively, we sought to develop a drug delivery system that could safely and effectively be used to deliver therapeutics to the site of a bone fracture. We elected to pursue the promising strategy of using small-molecule drug conjugates that deliver therapeutics to bone in an attempt to increase the efficacy and safety of drugs for treating bone-related diseases.</div><div>This strategy also opened the door for new methods of administering drugs. Traditionally, administering bone anabolic agents to treat bone fractures has relied entirely on local surgical application. However, because it is so invasive, this method’s use and development has been limited. By conjugating bone anabolic agents to bone-homing molecules, bone fracture treatment can be performed through minimally invasive subcutaneous administration. The exposure of raw hydroxyapatite that occurs with a bone fracture allows these high-affinity molecules to chelate the calcium component of hydroxyapatite and localize primarily to the fracture site.</div><div>Many bone-homing molecules (such as bisphosphonates and tetracycline targeting) have been developed to treat osteoporosis. However, many of these molecules have toxicity associated with them. We have found that short oligopeptides of acidic amino acids can localize to bone fractures with high selectivity and with very low toxicity compared to bisphosphonates and tetracyclines.</div><div>We have also demonstrated that these molecules can be used to target peptides of all chemical classes: hydrophobic, neutral, cationic, anionic, short, and long. This ability is particularly useful because many bone anabolics are peptidic in nature. We have found that acidic oligopeptides have better persistence at the site of the fracture than bisphosphonate-targeted therapeutics. This method allows for a systemic administration of bone anabolics to treat bone fractures, which it achieves by accumulating the bone anabolic at the fracture site. It also opens the door for a new way of treating the prevalent afflictions of broken bones and the deaths associated with them.</div><div>We further developed this technology by using it to deliver anabolic peptides derived from growth factors, angiogenic agents, neuropeptides, and extracellular matrix fragments. We found several promising therapeutics that accelerated the healing of bone fractures by improving the mineralization of the callus and improving the overall strength. We optimized the performance of these molecules by improving their stability, targeting ligands, linkers, dose, and dosing frequency.</div><div>We also found that these therapeutics could be used to accelerate bone fracture repair even in the presence of severe comorbidities (such as diabetes and osteoporosis) that typically slow the repair process. We found that, unlike the currently approved therapeutic for fracture healing (BMP2), our therapeutics improved functionality and reduced pain in addition to strengthening the bone. These optimized targeted bone anabolics were not only effective at healing bone fractures but they also demonstrated that they could be used to speed up spinal fusion. Additionally, we demonstrated that acidic oligopeptides have potential to be used to treat other bone diseases with damaged bone.</div><div>With these targeted therapeutics, we no longer have to limit bone fracture healing to casts or invasive surgeries. Rather, we can apply these promising therapeutics that can be administered non-invasively to augment existing orthopedic practices. As these therapeutics move into clinical development, we anticipate that they will be able to reduce the immobilization time that is the source of so many of the deadly complications associated with bone fracture healing, particularly in the elderly.</div>

<p>Urine-based biomarkers offer numerous advantages for clinical analysis, including non-invasive collection, a suitable sample source for longitudinal disease monitoring, a better screenshot of disease heterogeneity, higher sample volumes, faster processing times, and lower rejection rates and costs. They will be extremely useful in a clinical trial context, which can be applied alone or in combination with other methods as long as they demonstrate clear reproducibility across cohorts. While biofluids such as urine present enormous challenges with a wide dynamic range and extreme complex typically dominated by a few highly abundant proteins, we have demonstrated that the analytical issue can be efficiently addressed by focusing on extracellular vesicles (EVs), tiny packages released by all kinds of cells. These tiny packages contain different kinds of molecules from inside the cells. Here, we established a robust EV isolation and characterization platform to screen and validate Parkinson’s Disease (PD) and Renal Cell Carcinoma (RCC) biomarkers from urine. PD is a progressive neurological disorder affecting body movement because some brain cells stop producing dopamine. PD is often not diagnosed until it has advanced, making early detection crucial. We investigated urinary EVs from 138 individuals to enable early detection and found several proteins involved in PD development that could be biological indicators for early disease detection. Several biochemical techniques were applied to verify our findings. In the second project, we attempted to develop a novel diagnostic technique for early intervention of RCC. Here, we made our efforts to develop a quantitative method based on data-independent acquisition (DIA) mass spectrometry to analyze urinary EV phosphoproteomics for non-invasive RCC biomarker screening. Combined with our in-house EVtrap method for EV isolation and PolyMAC enrichment of phosphopeptides, we quantified 2,584 unique phosphosites. We observed unique upregulated phosphosites and pathways differentiating healthy control (HC), chronic kidney disease (CKD), low-grade, and high-grade clear cell RCC. These applications have a significant promise for early PD and RCC diagnosis and monitoring based on actual functional proteins with urine as the source. These studies might provide a viable path to developing urinary EV-based disease diagnosis.</p>

<p> </p> <p>Learning about ‘behavior’ has always been at the heart of my research endeavors. While my undergraduate work in evolution and ecology exposed me to the science behind why a behavior exists, in my graduate work, I intended to explore how to use something’s behavior to widen its applicability. In this thesis, <em>I will present three works that utilize some of the fundamental</em></p> <p><em>behaviors (i.e., properties) of elastin-like polypeptides (ELP) to improve existing protein purification methods or explore their applicability in bladder cancer imaging and immunotherapy. </em></p> <p>Bladder cancer has high recurrence rates (60-70 % annually) that necessitate multiple follow-up therapies making it one of the costliest cancers per patient. In this work, we have attempted to address two leading causes of the recurrence. First is a low sensitivity (62-84 %) and variable specificity (43-95 %) of white light cystoscopy used to diagnose and remove tumors. We aimed to address the heart of this problem, i.e., the non-specific mode of detection using white light. Only the trained eyes can discern abnormal from normal-appearing tissues even then, leaving up to 45% of tumors unresected to colonize and spread. <em>We developed and characterized near infrared dye-peptide-ligand conjugates (NIR-ELP-ligand) that undergo receptor-mediated binding and internalization to human bladder cancer cells in vitro and tissues ex vivo.</em> By using a molecular target-based probe in combination with NIR imaging, we can aid in improving the detection limit via selective binding to the tumor and reduction in background autofluorescence.</p> <p>Bacillus-Calmette Guérin (BCG) instillation in the bladder is the gold-standard</p> <p>immunotherapy used after surgical removal of bladder tumors. This was approved as a response to the inefficiency of surgery alone in improving cancer status. It has succeeded by reducing the recurrence rate to 30-50 %. But it comes with the complications of putting a live mycobacterium</p> <p>in the human body and giving a patient a urinary tract infection right after surgical tumor resection. <em>Thus, we aimed to deliver nucleic acid as immunotherapeutic cargo in a selective manner to elicit robust anti-tumor immune responses while minimizing the side effects due to its carrier.</em> Towards</p> <p>this goal, we have developed a highly modular and adaptable ELP-ligand fusion protein-based nucleic acid delivery carrier targeted toward bladder cancer. Before developing targeted peptide-based cancer imaging and nucleic acid delivery modalities, we addressed the Achilles heel of peptide-based approaches. The peptide and protein industry suffers</p> <p>through complex, time-consuming, inconsistent, and low-yielding purification methods. <em>We have developed a scalable, facile, and reproducible protein purification method that delivers ELP and ELP fusion proteins free of host cell proteins and nucleic acids and has low lipopolysaccharide</em></p> <p><em>content in just 3 h starting from a bacterial pellet. </em>Thus, for a coherent narrative, the thesis is structured as follows:</p> <p>1. Introduction</p> <p>2. ELP as a protein purification tag: Development of a rapid purification method for ELPs and ELP fusion proteins.</p> <p>3. ELP as a cancer imaging agent: Development of NIR-ELP-Ligand imaging probe targeting bladder cancer.</p> <p>4. ELP as a drug delivery agent: Utilizing ELP-ligand fusion protein in the formulation of targeted nucleic acid delivery carrier to bladder cancer.</p>

<p>This dissertation describes the effects of a <i>de novo</i> mutation of CaMKII found in children with learning disabilities and describes its effect on catalytic activity. We develop a malachite green assay for the measurement of CaMKII activation and use it for high-throughput chemical screening to identify CaMKII inhibitors and enhancers. We also propose a new mechanism of regulation of CaMKII activity by ADP.</p><p><br></p>