The Development of Peptide Analogues as Potential Fluorescent and PET Imaging Probes.

Molecular imaging is a non-invasive way to diagnose disease. Molecular imaging probes consist of a targeting entity that has a high affinity for a biological target and a signaling source for external detection. Multimodality imaging combines the advantages of different imaging modalities, at the same time eliminating their shortcomings, thereby providing more accurate results. This thesis focuses on the development of novel dual PET/fluorescence imaging agents based on peptides as targeting entities. Porphyrins are suitable for multimodality imaging applications due to their innate characteristics such as an ability to fluoresce and to coordinate metals. A gallium-protoporphyrin IX(PPIX)-ghrelin based analogue was designed as a potential dual PET/fluorescence imaging probe for detection of growth hormone secretagogue receptor 1a (GHS-R1a) and it was found that fluorescence was not quenched upon coordination to Ga. The PPIX-ghrelin analogue was also successfully radiolabeled with 68Ga. The specific uptake of this analogue by the receptor target, GHS-R1a, was demonstrated through confocal fluorescence microscopy using OVCAR-8 cells transfected with GHS-R1a. Thus, these dual modality probes can aid in bridging the gap and extend research from in vitro fluorescence imaging to in vivo PET imaging. 69/71Ga-hematoporphyrin-bombesin analogues were synthesized as potential PET/fluorescence imaging probes for the gastrin releasing peptide receptor (GRP-R), but the reaction yields were low and all the components of the hematoporphyrin-bombesin mixture could not be coordinated with gallium and this mixture of coordinated and the uncoordinated analogues could not be analyzed by optical methods. Nonetheless, the ability of hematoporphyrin to act as a chelator for Ga and to couple to peptides was demonstrated. BODIPY dyes are widely used for fluorescence imaging but suffer from drawbacks such as complicated synthesis and small Stokes shifts. However, novel BF2-formazanate dyes have favourable properties such as convenient synthesis and optical properties that can be tuned by modifying the structure. BF2-formazanate- peptide based analogues were developed as fluorescence imaging agents for GHS-R1a and GRP-R. Use of a modified ghrelin sequence led to an improvement in the IC50 value. Specific uptake of the probes could be visualized through fluorescence microscopy. Keywords Optical imaging, positron emission tomography, protoporphyrin, hematoporphyrin, GHS-R1a, GRP-R, BF2-formazanat

Identification of resistance mechanism to targeting of the ADAM 17/ EGFR axis in triple negative breast cancer in vivo

Triple-negative breast cancers (TNBCs) (ER/PR/HER2 negative) represent 15% of invasive\nbreast cancers and occur at a higher rate in young and African-American women. Exploration of\nnovel therapeutic approaches is critical, since only 30% of woman with metastatic breast cancer\nwill survive and virtually none with metastatic TNBC. The status quo as it pertains to the\ntreatment of TNBCs can be summarized as: no effective therapies available. In part, the lack of\ntherapeutic success is due to high genetic heterogeneity of TNBCs, challenging single drug\napproaches.\nMany targeted strategies to treat TNBC are being explored, including the inhibition of kinase\npathways (e.g. PI3K/Akt, MEK, VEGFR and PDGFR), the inhibition of DNA repair, of survival\npathways and androgen receptor blockade. In most cases, such single-drug targeted therapy is\ncombined with systemic genotoxic chemotherapy. For example, although about 60% of basallike\nTNBCs over express EGFR, EGFR targeted therapy, including kinase inhibition, has been\ndisappointing due to the development of resistance.\nVarious resistance mechanisms allow cancer cells to evade single-drug targeted therapies:\nmutations in the targeted molecules, extensive crosstalk/pathway redundancy and the upregulation\nof alternate growth or survival pathways. Design of combinatorial approaches of\ntherapeutics for TNBC that overcome resistance is therefore critical. The contribution of the\nproposed research is expected to be the identification of signaling network perturbations that\noccur in response to single targeted therapies, in particular in ADAM17/EGFR axis inhibition,\nand confer resistance. Based on published results, inhibition of the ADAM17/EGFR ligand axis\nin TNBC should provide therapeutic benefit with reduced tumor growth and decreased\nmetastasis, if not possibly cure.\nIn our in vitro studies on PKC? and PPP1R14D gene knockout in MDA-MB-231 cells indeed led\nto decreases in cellular growth and migration. However, to our surprise, when the same cells\nwere injected into mice through orthotropic fat pad transplantation, they produced aggressive,\nmetastatic tumors that showed activation of alternate growth signaling pathways, namely of the\nmitogen-activated protein kinase ERK and of the PI3kinase target Akt, also a mitogen activated\nkinase. This suggested that TNBC cells were developing resistance to EGF ligand regulator\nknockdown by rewiring their growth factor signaling pathways. To determine where these\nadditional growth signals come from, we first considered the tumor cells themselves.\nIn this context we discovered that when kept in culture, MDA-MB-231 cells expressing sh-\nRNAs targeting either PKCa or PPP1R14D maintained knockdown of the target for up to 35days\ntested. At the same time EGFR and ERK showed low activity as expected due to a decrease in\nEGF ligand cleavage; Akt activity was undetectable. Since we observed strong reactivation of\nERK and new activation of Akt in tumors in vivo, we considered possible up-regulation of other\ngrowth factor receptors on the cell surface that would be engaged by factors released from the\ntumor stroma once cells are inserted in vivo. Indeed, we found that FGFR2 and Erbb4 were\nupregulated. It is therefore likely that reactivation of ERK and new activation of Akt was due to\nFGFR2 and Erbb4. This would suggest that combination therapy of EGF ligand release regulator\ninhibition and FGFR inhibition would decrease growth of these tumors in vivo.Fil: Sharma, Neha. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaEl cáncer de mama triple negativo (TNCB) es aquel que no expresa el receptor de estrógenos\n(ER), ni el de progesterona (PR) o el HER2. Esta patología representa el 15% de los tumores de\nmama invasivos y tiene una alta incidencia en mujeres jóvenes Afro-Americanas. Es responsable\nde una alta tasa de mortalidad por cáncer de mama ya que generalmente el TNCB causa\nmetástasis; además, responde pobremente a las terapias con quimioterápicos a largo plazo y\ngeneralmente desarrolla resistencia a las terapias dirigidas, incluyendo las que implican al EGFR.\nPor todo ello, es fundamental el desarrollo de terapias alternativas, dado que solo el 30% de las\nmujeres con cáncer de mama metastásico sobrevive pero ninguna de las que presentan TNBC\nmetastásico.\nActualmente, no existe una terapia adecuada y efectiva para el TNBC. En parte, esto se debe a la\nalta heterogeneidad genética que presentan estos tumores, lo cual redunda en la inefectividad de\nterapias basadas en una única droga. Terapias basadas en blancos terapéuticos específicos están\nen investigación y desarrollo, como aquellas basadas en la inhibición de quinasas implicadas en\nseñalización (ejemplo: /Akt, MEK, VEGFR, PDGFR), reparación del DNA, supervivencia\ncelular o acciones androgénicas. Mayormente, estas terapias específicas son combinadas con\nquimioterapia sistémica. Sin embargo, hasta el momento, los beneficios de tales propuestas\nterapéuticas no son claros. Aproximadamente el 60% de los TNBC de tipo basal sobreexpresan\nEGFR; sin embargo, las terapias que implican la inhibición del receptor son mayormente\ninefectivas debido al desarrollo de resistencia. Distintos mecanismos están involucrados en el\ndesarrollo de resistencia a las terapias dirigidas, como ser mutaciones en la proteína blanco o la\nredundancia y sobreactivación de vías de señalización compartidas con otros factores de\ncrecimiento.\nPor lo tanto, es fundamental diseñar terapias combinadas para TNBC que contemplen el posible\ndesarrollo de resistencia. El trabajo de investigación propuesto intenta identificar alteraciones de\nvías de señalización intracelular ocasionadas por las terapias dirigidas, particularmente en lo que\nrespecta al eje ADAM17/EGFR, con el fin de establecer su posible implicancia en el desarrollo\nde resistencia.\nDado que se desconoce como es regulada la actividad y selectividad de ADAM17, se realizó un\namplio estudio mediante shRNA para dilucidar como se regula el clivaje de and PPP1R14D\nregulan el clivaje de TGFa, AREG y HB-EGF sin afectar la actividad proteasa de ADAM17. La\ninhibición del eje ADAM17/EGFR sería beneficioso para el tratamiento del TNBC. Nuestros\nestudios in vitro revelaron que células MDA MB 231 knockout para PKC? and PPP1R14D no\npresentan sobreactivación de RTKs, sugiriendo que en estos modelos podría verse potenciada la\neficacia terapéutica de la inhibición del eje ADAM17/EGFR.\nSin embargo, cuando las mismas células fueron inyectadas a ratones, produjeron un fenotipo de\ntumor agresivo y metastásico, asociado a la reactivación de vías de señalización intracelular\ncomo las mediadas por ERK y PI3K/Akt. Ello se asoció a un aumento de la expresión y\nactivación de distintas RTKs, incluido el EGFR como así también de Akt.\nEstos resultados sugieren la activación alternativa de vías de señalización que permiten que las\ncélulas tumorales proliferen y produzcan metástasis

Future of Nanoparticles in the Field of Medicine

The chapter deals with the application of iron oxide nanoparticles in the field of medicine. It focuses on the treatment of cancerous cells in the body as a case study. Cancer as we all know is a disease which is spreading at the speed of light across the nations, primarily due to the lifestyles and heredity. The human war against the disease is on, and many cures are in practice or under research, so as to limit the deaths due to it. Most of the research is focused on finding alternative and effective techniques in conquering cancer, so that the stigma attached with it can be diminished; the researchers are also focusing on lowering the side effects of the currently practiced cures. We all hope that a day will come when it will come under the category of conquerable diseases. It has been shown that cancer deaths in the world have declined considerably, but it is still unconquerable. It is still one of the leading causes of death around the globe. Usual therapy like radiation, surgery, and immunotherapy in addition to chemotherapy has shown challenges like ease of access to the tumor cells, danger of operating on a vital organ to name some. Off late, research laboratories are using nanoparticles for the detection in addition to drug delivery in treatment of various diseases. It gives boost to minimizing the side effects encountered in conventional therapies at the cellular and tissue level. Nanoparticles’ widespread use is accounted by their size

Assessment and Analysis of QuikSCAT and COAMPS Model Vector Wind Products for the Gulf of Mexico: A Long-Term and Hurricane Perspective

Global weather changes have become a matter of grave concern in hurricane prone areas as intensities of hurricanes are observed to be increasing every year, necessitating improved monitoring capabilities. NASA’s QuikSCAT satellite sensor has provided significant support in analyzing and forecasting winds for the past 8 years. In this study, the performance of QuikSCAT products, including JPL’s latest L2B 12.5km swath winds, was evaluated against buoy-measured winds in the Gulf of Mexico. The long-term study period was 1/2005 – 2/2007. The Coupled Ocean/Atmospheric Mesoscale Prediction System (COAMPS) was also assessed. The regression analyses showed very good results for QuikSCAT products, with the best results obtained from L2B winds. R2 values for moderate wind speeds were 0.75 and 0.89, 0.88 and 0.93, 0.66 and 0.77 for speed and direction and for L3, L2B and COAMPS respectively. The National Weather Product (NWP) model winds provided in the L2B dataset were also studied. Hurricanes that took place from 2002 to 2006 were studied individually to obtain regressions of QuikSCAT and COAMPS versus buoys for those events. The correlations were very high indicating that QuikSCAT is at par with buoys during hurricanes. These measurements were compared with the NHC best track analyses to determine the accuracy and found to be almost half those obtained by NHC, possibly due to rain contamination. Sea Surface Height Anomaly (SSHA) measurements by Jason-1 and sea surface temperature (SST) measurements by the Moderate Resolution Imaging Spectroradiometer (MODIS) Aqua and GOES-12 (Geostationary) were compared with wind fields during hurricanes to study the effects of the Loop Current and Warm Core Rings on the intensification of the hurricanes. A preliminary study was conducted in which the regions of enhanced wind speeds were observed by studying the longitudinal and latitudinal transects across the hurricane for two hurricanes, namely Hurricanes Ivan and Katrina. This study would act as a precursor to further analysis of the radius of maximum wind and critical wind radii

Survey: Trust-based Approaches to Solve Routing Issues in MANET

A mobile ad hoc network is a wireless network. The ability to work without any central controlling authority without any requirement of established infrastructure makes it need of the present scenario. This dynamicity comes with a downside of security. Since the nodes may act maliciously and pose threat to the working condition of the MANET. Trust approaches are well suited in these situations. Here we discuss TRUST; the meaning, characteristics and different schemes

Web usage Mining: Web user Session Construction using Map-Reduce

Web Usage Mining deals with the understanding of user behavior while interacting with the website by using various log files The whole process of Web Usage Mining gets completed in three phases namely Data Preprocessing Pattern Discovery and Pattern Analysis Data Preprocessing is important because it takes 80 of the time of the whole process of Web Usage Mining Data Preprocessing involves Data Cleaning User Identification and Session Identification In Session Identification we find out the set of pages visited by a user within the duration of one particular visit to a website also called as Sessionization In paper 1 we proposed a new method for session construction As the size of log files are very large so there is a requirement of an approach for Session Identification by which processing time of our proposed method will be reduced to a great extent In this paper we used Map-reduce method to calculate sessions in which we combine both time and user navigation method This approach is faster than the existing approach because we have performed the whole process in distributed environmen

Anaerobic Reductive Bioleaching of Manganese Ores

The increasing demand of manganese in the industries and various hindrances in its production from low grade ores by conventional method has made it imperative for researchers around the world to develop a method of manganese extraction from low grade ores that is both environment friendly and economical. Bioleaching has shown significant potential in manganese extraction and efficiencies of extraction have been found to be 70-98% with the help of various bacteria and fungi. This study focuses on extraction of manganese with the help of mixed bacterial strains that have been collected from their natural anaerobic environment. The extraction of manganese from reagent grade manganese dioxide and high grade manganese ore has been studied over 130 days at room temperature and pH around 5. Highest concentrations of dissolved manganese have been found to be 866.7 mg Mn/L for reagent grade manganese dioxide and 545.7 mg Mn/L for ore grade manganese

Financial Literacy and Financial Inclusion: A Case Study of Punjab

The successful inclusion of the financial services to the people of the country requires good level of financial literacy among them.By looking the strong relationship between financial literacy and financial inclusion, the present study aims to identify the main factors affecting the level of financial literacy in India with special reference to Punjab State.The study is based on the primary data collected from three main districts (Amritsar, Jalandhar, & Ludhiana),one each from all threeregions of Punjab namely, Majha, Doaba, and Malwa. Data have been collected from both rural and urban areas of each districts and comparative analysis has been conducted to show the differences in level of financial literacy within the district and between the districts. Chi-square test has been utilized to check the association between respondents’ characteristics and their existing level of financial knowledge. The whole analysis reveals that there exist differences in the level of financial literacy between male and female respondents and it is less in female respondents. These differences are also lies between urban and rural respondents. The level of financial literacy is higher in urban areas than in rural areas. Finally, the respondent’s qualification and its parental qualification also affect the level of financial knowledge in Punjab. On the basis of results, the study recommends the adoption of policies related to the women empowerment in both urban and rural areas which may help in reducing the gap between financial literacy among two areas and improve the overall level of financial literacy. The improvement in the level of financial literacy will further enhance financial inclusion as per the linkage between two

Unveiling the prospects of point-of-care 3D printing of Polyetheretherketone (PEEK) patient-specific implants

Additive manufacturing (AM) or three-dimensional (3D) printing is rapidly gaining acceptance in the healthcare sector. With the availability of low-cost desktop 3D printers and inexpensive materials, in-hospital or point-of-care (POC) manufacturing has gained considerable attention in personalized medicine. Material extrusion-based [Fused Filament Fabrication (FFF)] 3D printing of low-temperature thermoplastic polymer is the most commonly used 3D printing technology in hospitals due to its ease of operability and availability of low-cost machines. However, this technology has been limited to the production of anatomical biomodels, surgical guides, and prosthetic aids and has not yet been adopted into the mainstream production of patient-specific or customized implants. Polyetheretherketone (PEEK), a high-performance thermoplastic polymer, has been used mainly in reconstructive surgeries as a reliable alternative to other alloplastic materials to fabricate customized implants. With advancements in AM systems, prospects for customized 3D printed surgical implants have emerged, increasing attention for POC manufacturing. A customized implant may be manufactured within few hours using 3D printing, allowing hospitals to become manufacturers. However, manufacturing customized implants in a hospital environment is challenging due to the number of actions necessary to design and fabricate the implants. The focus of this thesis relies on material extrusion-based 3D printing of PEEK patient-specific implants (PSIs). The ambitious challenge was to bridge the performance gap between 3D printing of PEEK PSIs for reconstructive surgery and the clinical applicability at the POC by taking advantage of recent developments in AM systems. The main reached milestones of this project include: (i) assessment of the fabrication feasibility of PEEK surgical implants using material extrusion-based 3D printing technology, (ii) incorporation of a digital clinical workflow for POC manufacturing, (iii) assessment of the clinical applicability of the POC manufactured patient-specific PEEK scaphoid prosthesis, (iv) visualization and quantification of the clinical reliability of the POC manufactured patient-specific PEEK cranial implants, and (v) assessment of the clinical performance of the POC manufactured porous patient-specific PEEK orbital implants. During this research work, under the first study, we could demonstrate the prospects of FFF 3D printing technology for POC PEEK implant manufacturing. It was established that FFF 3D printing of PEEK allows the construction of complex anatomical geometries which cannot be manufactured using other technologies. With a clinical digital workflow implementation at the POC, we could further illustrate a smoother integration and faster implant production (within two hours) potential for a complex-shaped, patented PEEK patient-specific scaphoid prosthesis. Our results revealed some key challenges during the FFF printing process, exploring the applicability of POC manufactured FFF 3D printed PEEK customized implants in craniofacial reconstructions. It was demonstrated that optimal heat distribution around the cranial implants and heat management during the printing process are essential parameters that affect crystallinity, and thus the quality of the FFF 3D printed PEEK cranial implants. At this stage of the investigation, it was observed that the root mean square (RMS) values for dimensional accuracy revealed higher deviations in large-sized cranial prostheses with “horizontal lines” characteristics. Further optimization of the 3D printer, a layer-by-layer increment in the airflow temperature was done, which improved the performance of the FFF PEEK printing process for large-sized cranial implants. We then evaluated the potential clinical reliability of the POC manufactured 3D printed PEEK PSIs for cranial reconstruction by quantitative assessment of geometric, morphological, and biomechanical characteristics. It was noticed that the 3D printed customized cranial implants had high dimensional accuracy and repeatability, displaying clinically acceptable morphologic similarity concerning fit and contours continuity. However, the tested cranial implants had variable peak load values with discrete fracture patterns from a biomechanical standpoint. The implants with the highest peak load had a strong bonding with uniform PEEK fusion and interlayer connectivity, while air gaps and infill fusion lines were observed in implants with the lowest strength. The results of this preclinical study were in line with the clinical applicability of cranial implants; however, the biomechanical attribute can be further improved. It was noticed that each patient-specific reconstructive implant required a different set of manufacturing parameters. This was ascertained by manufacturing a porous PEEK patient-specific orbital implant. We evaluated the FFF 3D printed PEEK orbital mesh customized implants with a metric considering the design variants, biomechanical, and morphological parameters. We then studied the performance of the implants as a function of varying thicknesses and porous design constructs through a finite element (FE) based computational model and a decision matrix based statistical approach. The maximum stress values achieved in our results predicted the high durability of the implants. In all the implant profile configurations, the maximum deformation values were under one-tenth of a millimeter (mm) domain. The circular patterned design variant implant revealed the best performance score. The study further demonstrated that compounding multi-design computational analysis with 3D printing can be beneficial for the optimal restoration of the orbital floor. In the framework of the current thesis, the potential clinical application of material extrusion-based 3D printing for PEEK customized implants at the POC was demonstrated. We implemented clinical experience and engineering principles to generate a technical roadmap from preoperative medical imaging datasets to virtual surgical planning, computer-aided design models of various reconstructive implant variants, to the fabrication of PEEK PSIs using FFF 3D printing technology. The integration of 3D printing PEEK implants at the POC entails numerous benefits, including a collaborative team approach, quicker turnaround time of customized implants, support in pre-surgical and intraoperative planning, improved patient outcomes, and decreased overall healthcare cost. We believe that FFF 3D printing of customized PEEK implants could become an integral part of the hospitals and holds potential for various reconstructive surgery applications