Interrogation and Manipulation of the IGG FC Glycan

Antibody signaling is a cardinal feature of successful adaptive immune responses. The magnitude and direction of this signaling is determined by the structure of a given antibody’s fragment crystallizable (Fc) domain, which interfaces with cells of the immune system. Often considered a constant region, the immunoglobulin G (IgG) Fc protein backbone and conserved N-linked glycan combine to introduce structural diversity in IgG molecules that in turn, triggers divergent humoral responses. These N-glycans are variably constructed, ultimately leading to families of highly related, but non-equivalent glycoproteins known as glycoforms. Despite burgeoning interest in understanding the complexities of IgG Fc glycoforms and their functions, there is an evident scarcity of tools available to distinguish and target them. In addition, the highly conserved nature of the glycan and its presence on the B cell receptor (BCR) provokes questions of its possible role in the generation of antibody responses. In the first part of this thesis, I identify a novel class of synthetic nanobodies that can distinguish glycoforms without reactivity to off-target glycoproteins or glycans. Applying this technology to Fc glycoforms defines nanobodies that specifically recognize either IgG lacking its core-fucose or IgG bearing terminal sialic acid residues. Solving the structure of a nanobody-Fc complex via x-ray crystallography reveals a unique mode of recognition of IgG glycoforms. By adapting nanobodies to standard biochemical methods, I clinically stratify dengue virus and SARS-CoV-2 infected individuals based on their Fc glycan profile, selectively disrupt IgG-Fcγ receptor (FcγR) binding both in vitro and in vivo, and interrogate BCR glycan structure on living cells. In the second part of this thesis, I develop mouse models and biochemical tools to help define the role of the IgG Fc glycan in developing antibody responses. Preliminary studies show that following immunization, mice lacking the N-glycan acceptor residue, Asn 297, exhibit deficiencies in germinal center (GC) responses. In this model, B cells with an aglycosylated BCR participate less frequently in the GC reaction, bind antigen less avidly, and show signaling deficits downstream of BCR engagement. Development of an aglycosylated mouse IgG1-specific nanobody shows that in allelic competition experiments, B cells with glycosylated BCRs are favored. To aid future studies, an additional mouse model null for endogenous Fc-FcγR binding is generated. Finally, a mouse expressing human IgG1 in the heavy chain locus proves useful for chronic administration of human antibodies

Response of leuprolide on gonadal functions of women with overt hypothyroidism: a tertiary care centre study from Uttar Pradesh, India

Background: Hypothyroidism can cause menstrual disturbances mainly oligoanovualtory cycles and sometimes menorrhagia. It has also been seen to cause subfertility and pregnancy related complications. Various studies have been done to evaluate gonadal dysfunctions in overt hypothyroidism but very few studies are there which have done using a gonadotrophin response in that subset of patients. Present study evaluates the response of leuprolide on gonadal functions of women with overt hypothyroidism in a tertiary care centre at Meerut.Methods: In this study 50 females of age 20 to 40 years with newly diagnosed overt hypothyroidism were taken as cases and age and Body Mass Index (BMI) matched healthy females were taken as controls. Both in cases and controls, basal FSH, LH, estradiol was measured on 2nd day to 5th day of menstrual cycle. Thereafter Leuprolide 20 mcg/kg was given subcutaneously on the same day. Post leuprolide test, stimulated LH, FSH and estradiol were measured. Basal and stimulated values were compared between both groups.Results: Basal LH was significantly higher in controls (8.2±3.2 mIU/L) when compared to cases (6.45±2.75 mIU/L) with a p value 0.03(<0.05). Basal estradiol and FSH levels were found to be nearly similar and non-significant in cases and controls. No significant differences were found between stimulated mean LH and estradiol in both the groups. Leuprolide response after stimulation test was found to be sluggish in patients with overt hypothyroidism compared to normal euthyroid controls. This study is the rare one done on human subject in tertiary care centre of India, however large sample and multicentric trials are necessary before establishing the biochemical results.Conclusions: Pituitary and gonadal (ovarian) response to leuprolide was found to have impaired (decreased) in overt hypothyroidism cases. This is the first study to be done in overt hypothyroid subjects to asses both basal and stimulated gonadotropin levels

Body and Tail Coordination in the Bluespot Salamander (\u3cem\u3eAmbystoma laterale\u3c/em\u3e) During Limb Regeneration

Animals are incredibly good at adapting to changes in their environment, a trait envied by most roboticists. Many animals use different gaits to seamlessly transition between land and water and move through non-uniform terrains. In addition to adjusting to changes in their environment, animals can adjust their locomotion to deal with missing or regenerating limbs. Salamanders are an amphibious group of animals that can regenerate limbs, tails, and even parts of the spinal cord in some species. After the loss of a limb, the salamander successfully adjusts to constantly changing morphology as it regenerates the missing part. This quality is of particular interest to roboticists looking to design devices that can adapt to missing or malfunctioning components. While walking, an intact salamander uses its limbs, body, and tail to propel itself along the ground. Its body and tail are coordinated in a distinctive wave-like pattern. Understanding how their bending kinematics change as they regrow lost limbs would provide important information to roboticists designing amphibious machines meant to navigate through unpredictable and diverse terrain. We amputated both hindlimbs of blue-spotted salamanders (Ambystoma laterale) and measured their body and tail kinematics as the limbs regenerated. We quantified the change in the body wave over time and compared them to an amphibious fish species, Polypterus senegalus. We found that salamanders in the early stages of regeneration shift their kinematics, mostly around their pectoral girdle, where there is a local increase in undulation frequency. Amputated salamanders also show a reduced range of preferred walking speeds and an increase in the number of bending waves along the body. This work could assist roboticists working on terrestrial locomotion and water to land transitions

Efficient Computation of Map-scale Continuous Mutual Information on Chip in Real Time

Exploration tasks are essential to many emerging robotics applications, ranging from search and rescue to space exploration. The planning problem for exploration requires determining the best locations for future measurements that will enhance the fidelity of the map, for example, by reducing its total entropy. A widely-studied technique involves computing the Mutual Information (MI) between the current map and future measurements, and utilizing this MI metric to decide the locations for future measurements. However, computing MI for reasonably-sized maps is slow and power hungry, which has been a bottleneck towards fast and efficient robotic exploration. In this paper, we introduce a new hardware accelerator architecture for MI computation that features a low-latency, energy-efficient MI compute core and an optimized memory subsystem that provides sufficient bandwidth to keep the cores fully utilized. The core employs interleaving to counter the recursive algorithm, and workload balancing and numerical approximations to reduce latency and energy consumption. We demonstrate this optimized architecture with a Field-Programmable Gate Array (FPGA) implementation, which can compute MI for all cells in an entire 201-by-201 occupancy grid ({\em e.g.}, representing a 20.1m-by-20.1m map at 0.1m resolution) in 1.55 ms while consuming 1.7 mJ of energy, thus finally rendering MI computation for the whole map real time and at a fraction of the energy cost of traditional compute platforms. For comparison, this particular FPGA implementation running on the Xilinx Zynq-7000 platform is two orders of magnitude faster and consumes three orders of magnitude less energy per MI map compute, when compared to a baseline GPU implementation running on an NVIDIA GeForce GTX 980 platform. The improvements are more pronounced when compared to CPU implementations of equivalent algorithms