Sleep has roles in memory consolidation. Memory consolidation is thought to include systems consolidation which reflects communication among different brain regions, as well as synaptic consolidation at the local circuitry level. I found systems consolidation during sleep among separated regions in cerebral cortex (Miyamoto et al., Science, 2016), however, synaptic consolidation pattern is still unknown. Net cortical synaptic strength changes homeostatically through wake/sleep cycle: average synaptic strength increases during wakefulness and decreases during sleep (Tononi & Cirelli, Neuron, 2014). While skill learning is known to potentiate subsets of synapses in motor cortex for encoding skill memory (Hayashi-Takagi et al., Nature, 2015), plasticity of those synapses through sleep is unknown. If sleep depresses memory-encoding potentiated synapses, it may result in impairing memory consolidation. If sleep depresses other non-potentiated synapses relatively, it may enhance signal/noise ratio and memory consolidation. Here I will use complex-wheel task for studying synaptic mechanisms of sleep-dependent skill memory consolidation in mice. Through skill learning and sleep, I will perform in vivo two-photon imaging in motor cortex to visualize fluorescence tagged AMPA receptors, since the expression of these excitatory glutamate receptors is an established index of synaptic strength. To visualize plasticity in synaptic population, I will analyze time-course of fluorescence intensity change at single-synapse resolution. This study will help clarifying how sleep favors both memory consolidation and synaptic homeostasis.

The formation of higher order chromatin structure is crucial for DNA replication, transcription, recombination, repair and chromosome segregation. The cohesin-complex is thought to contribute to formation of chromatin loops and of topologically associating domains (TADs) by entrapping two different regions of the same chromatids. Its binding along genomic DNA is thought to be controlled by two of cohesin’s cofactors, CTCF and Wapl. Cohesin has been proposed to regulate the long-range chromosomal cis-interaction by a “loop extrusion” mechanism, in which cohesin can regulate chromatin loop formation by translocating along genomic DNA. However, how cohesin controls chromatin structure remains unclear. To address this question, I will first establish assays based on FISH, Cas9-FISH in living cells, oligopaint strategy and super resolution microscopy (SRM) to visualize chromatin loops and TADs. Second, I will employ these assays and Hi-C to analyze chromatin structure in cells in which the genomic distribution of cohesin is experimentally altered. For this, I will use knowledge from my host lab which recently discovered that the distribution of cohesin in mammalian genomes depends on the DNA binding protein CTCF, the cohesin release factor Wapl and transcription. Third, I will monitor the transition of cohesin re-location and of conformational change of chromatin induced by rapid degradation of Wapl and CTCF in real time. I will then test prediction of the loop extrusion model by comparing my experimental data with the simulations recently described in the literature.

How genetic programs regulate mammalian morphogenesis is one of the central problems in developmental biology. Digit patterning, a representative model to study mechanisms of morphogenesis, is regulated by multiple factors including cytokines and Homeobox-containing genes, or Hox genes, particularly Hoxa13 and Hoxd13. Recently, it has been illustrated that a reaction-diffusion model can explain this patterning in which the interactions of Sox9, a transcription factor, and WNT and BMP, cytokines, generate the periodic digit pattern. Interestingly, this periodic wave is modulated by FGF signaling and Hox genes. However, little is known about the molecular basis how Hox genes contribute to the proper digit patterning by regulating these interactions. In this project, to elucidate the role of Hox genes in mammalian digit patterning and autopod tissue identity, I will conduct integrative analyses including genetic, biochemical, and computational approaches. Firstly, single-cell RNA sequencing will be performed on both of wild type and Hoxa13 and/or Hoxd13 knockout mice to discover a gene network consisting the periodic wave pattern and analyze dosage effects of Hox genes on it. Secondly, HOXD11/D12/D13 will be epitope-tagged with genome editing tools. Thirdly, they will be immunoprecipitated to comprehensively analyze their binding specificity to the genome and other proteins. Lastly, these data are integrated with the reaction-diffusion model and investigate mechanisms of the modulation of BMP and WNT signaling by Hox genes to generate the periodic digit pattern, and consequently, establish the identity in autopod regions.

Controlling the direction of cell expansion is one of the key aspects of environmental and developmental signaling responses in plants. The direction of cell expansion largely relies on the microtubule network just beneath the plasma membrane. These cortical microtubules are dynamic and their organization is rearranged by a wide range of signals such as gravity, mechanical stress and light, but the molecular mechanisms that carry out these signal transmissions and microtubule array rearrangement are poorly understood. Our recent studies have demonstrated that Phototropin signaling regulates microtubule nucleation and severing activities. However, it is not clear how Phototropin regulates these activities. Phototropin is thought to have kinase activity and control wide range physiological responses. While our preliminary data with minus-end regulator suggest microtubule reorientation in response to blue light also requires the regulation of minus end dynamics, we have almost no information on how the remarkable minus end dynamics are orchestrated in plant cell. To tackle this linking question between plant physiology and cell biology, I propose to study about how the microtubule organization are regulated by transmitted blue light signaling through Phototropin. Specifically, I will propose following 3 projects: 1) identify factors being downstream of Phototropin kinase and upstream of microtubule organization, 2) investigate the molecular mechanism of the microtubule minus ends, and 3) introduce a new technique enabling us to conditionally manipulate target proteins to understand their spatiotemporal function during live-cell imaging.

A myocardial infarction in mammals typically displays limited regeneration and instead produces significant cardiomyocyte death. This loss of contractile cells is instead replaced with a permanent non-contractile fibrotic scar. In contrast, the pro-regenerative zebrafish displays increased cardiomyocyte proliferation and fibrotic scar resolution following injury. In order to find candidate genes, other labs have RNA sequenced whole mammalian hearts prior to and after injury. The spatial resolution however is lost, and other unrelated genes may mask candidate genes responsible for regeneration. This project will use a cardiac injury zebrafish model and at critical stages during regeneration, apply a novel genomic technique “RNA Tomography (Tomo-seq)” to spatially resolve gene expression at a genome-wide level. In addition, Tomo-seq concepts will be applied to develop a new genomic technique to examine the regulatory regions surrounding genes. Bioinformatic analysis will identify candidate genes and their regulatory regions associated with different phases of regeneration, and spatially correlate them with different areas in the injury site. Finally potential targets are functionally validated in zebrafish. By identifying pro-regenerative candidate genes at a spatial-temporal resolution in zebrafish, we can further understand and possibly modulate signals to increase the limited regenerative capacities in mammals.

The gut is under constant surveillance by immune cells, which ensures the homeostasis of commensal gut microbiota whilst conferring protection from invading enteric pathogens. B lymphocytes are central to maintaining balanced responses through production of huge quantities of the Immunoglobulins IgA and IgG. Ig production is the outcome of specialized germinal center (GC) responses, which form spontaneously in gut-associated lymphoid tissues in response to chronic exposure to microbiota. Despite being the precursor to Ig secretion, gut GC responses are entirely uncharacterized. Recently, when mesenteric lymph nodes (mLNs) were visualized using multicolor fate mapping (MCFM) large proportions of spontaneous GCs became monoclonal, an unexpectedly specific outcome in the face of exposure to such a diverse range of antigens. We intend to combine MCFM with in vivo models of commensal colonization to track the kinetics and clonal diversity of GC and Ig responses in mLNs through multiphoton microscopy. After GC isolation, we will use sequencing approaches to determine which bacterial species predominantly induce clonal GCs and map subsequent Ig specificity. Following this, we will investigate GC responses during enteric infection. We are especially interested in whether newly activated pathogen-specific B cells utilize pre-existing spontaneous GCs for their own affinity maturation, and how this is altered by composition of the current or past bacterial community. These data could have wide-reaching implications for both the fundamental biology of gut immunology as well as universal principles of GC dynamics in the presence of multiple antigens.

Previous studies have suggested that embryogenesis is robust to environmental, genetic, or stochastic perturbations. However, little is understood about the mechanisms that enable multicellular organisms to develop robustly, and about the extent to which – and the way in which – they changes their developmental patterns in response to harmful perturbations. Especially, how organisms adapt to environmental stresses during embryogenesis is not well defined, partly because in most cases experimental organisms have been cultured under favorable conditions. Considering that organisms usually live in harsh conditions in their wild habitat, it is highly likely that they have evolved the undiscovered ability to deal with environmental stresses.
In this project, I will employ a high-performance cell tracing system and sophisticated computational techniques to accurately track the positions of ALL cell nuclei in developing embryos of the nematode Caenorhabditis elegans. By this approach, I will comprehensively examine the effects of nutrition deficiency on cell division and differentiation during embryogenesis. Additionally, I will elucidate the developmental basis of transgenerational starvation response. After the detection of the developmental differences caused by nutritional stress, I will clarify how the differences are induced (or suppressed) at the molecular level using powerful molecular genetics and mathematical analyses.
The success of this project will provide fundamental insights into embryogenesis and might lead to understanding of the mechanisms underlying teratogenesis, spontaneous abortion, and neonatal death in nutritionally challenged humans.

The spectacular manoeuvres of flocking birds and schooling fish are among the most dramatic and mysterious sights in the natural world. How can hundreds or thousands of individuals coordinate their movements so perfectly, behaving almost as a single super-organism? The answer to this puzzle began to be uncovered through mathematical models showing that collective order can emerge as a by-product if all individuals within a group follow simple rules to align with and stay close to their neighbours. However, unlike the simulated agents in these models, real animals are not identical, and can differ both in their individual characteristics and in their relationships with one another. A group’s composition is therefore likely to affect its overall structure and cohesion as well as its ability to reach consensus decisions when responding to the environment. Understanding these effects has important implications, from determining how animal groups respond to threats, to mitigating the impacts of crop pests, managing crowd safety and developing intelligent systems in robotics. We will use mixed-species flocks of rooks and jackdaws (birds of the crow family, or corvids) to understand the effects of group composition on collective behaviour in nature. Combining field experiments with cutting-edge imaging and computational techniques, we will produce 3D reconstructions of the movements of every bird within flocks of varying composition and examine how a flock’s composition affects its structure and movements, and its responsiveness when avoiding or mobbing predators. Are more homogeneous groups better able to respond as a coherent unit, or does diversity enhance group responses as in human social institutions? Our 3D reconstructions will also allow us to determine the fine-scale internal structure of flocks. Do corvid flocks, like human crowds, contain sub-groups, reflecting flock members' social preferences? Finally, we will use our data to understand how individuals' flight decisions are influenced by who their neighbours are. By building mathematical models based on these measurements and testing the models using flocks of robot-controlled drones, we can find out how local interactions and social preferences among neighbours generate both internal sub-structure and collective order in complex societies.

The control of phenotypic variance, known as phenotypic robustness, is a fundamental characteristic of living organisms. This assures a relative stability of phenotypes even when organisms are exposed to stressful internal or external environments. However, it has been extensively documented that stressful conditions decrease organismal robustness resulting in an increase of phenotypic variance at the population level. Although ubiquitous in biology, the underlying genetic factors driving such shift in variance are poorly understood. This project specifically addresses this question by studying the genetic basis of gene expression variability in Drosophila melanogaster exposed to dietary stress. For this, genomic loci associated with transcriptional variability will be identified (variance-eQTL or v-eQTL); these are loci whose allelic state predicts the amount of variability around the expected mean. Recently developed outbred Drosophila populations will be used; this will allow the exploration of more genetic diversity than ever before. Thousands of flies will be exposed to two dietary conditions, standard food and high-sugar diet. v-eQTL maps will be generated for both conditions and will be compared with two objectives: 1) to reveal cryptic genetic variants only relevant under stress; and 2) to explore how stress disturbs the co-expression networks present in normal conditions. The analysis of this data will reveal the genomic loci associated with variation in transcriptional robustness between individuals, and will ultimately offer a deeper evolutionary and medical understanding of phenotypic robustness.

Synaptic vesicle (SV) is a vital cell organelle in neuronal communication via the exocytosis process and it corresponds to the basis of cognition, memory and neural diseases. The overall objective of this project is to develop new micro- and nano-analytical approaches to discover how cognitive drugs (CDs) modulate chemical communication processes of neurons by SVs. At first, I aim to use the electrochemical cytometry (EC) to realize the changes in the neurotransmitter content of SVs after taking individual CDs. But the orientation of vesicle opening in EC is unclear. Realizing this orientation is important as it lets us accurately measure the vesicle content, and it is also a model for the pore opening at the membrane in exocytosis. I propose two new methods, a nanogap electrode and nanohole system, to trap and measure vesicle contents with EC. Then I will carry out intracellular EC inside living cells and in Drosophila Melanogaster after administration of CD, by ultrasmall nanometer electrode tips. I hypothesize that a vital part of drug action in the brain, beyond to the change in the synaptic vesicle content, can be alteration of lipid distribution and change in the neuron membrane composition following the fusion of SVs to the cell membrane via the exocytosis. I propose imaging experiments by the state of the art instruments like nano secondary ion mass spectrometry, to prob the entire contents of vesicles and cell membrane; and to compare the changes observed with the dynamic electrochemical detection of exocytotic release in intracellular EC. All these experiments lead to fascinating new ideas about how the release of transmitters from vesicles is regulated by CDs.

Tissue morphogenesis is a process by which the embryo is reshaped into the final form of a developed animal. Tissues are constituted by cells that are interconnected one another: local changes of cell mechanical properties and shape drive consequent tissue shape change. Nevertheless the knowledge per se of the mechanisms and mechanics at the cell level which drive cell shape changes is insufficient to explain how tissues change their shape. Emerging properties arise at higher scales resulting from the interaction of cells within tissues and of tissues coordinating and interacting with one another. Studying this is a great challenge both technologically and conceptually. From the technological perspective, new tools are needed to be able to visualize cells and to provide quantifiable data at high temporal and spatial resolution over large regions and across the entire embryo. New techniques are required to manipulate tissues with temporal and spatial specificity and to measure mechanical properties in different regions of an embryo. Conceptually, it is challenging to understand how different sheets of cells with different mechanical properties interact. I will use the Drosophila embryo as a model system and focus on the process of tissue folding, process that is vital for the animal since folding defects can impair neurulation in vertebrates and gastrulation in all animals which are organized into the three germ layers. Thanks to my interdisciplinary background and expertise in biology, engineering and physics, my work will provide new knowledge on how biophysical forces sculpt functional living organisms.

Despite its significance, the transgenerational inheritance adaptive behaviors through germline modifications are poorly established and understood in terms of the underlying biological mechanisms. Here, by employing a novel social learning paradigm in Drosophila I aim to understand how predator-threat information is successfully inherited by subsequent generations through an epigenetic reprogramming of parental germline cells. In fruit flies, exposure to an endoparasitoid wasp elicits preferences for alcohol containing food and oviposition depression. Visual input mediated predator presence induces germline apoptosis resulting in both acute and learned oviposition depression in wasp-exposed females. Strikingly, daughters of these exposed females are preprogrammed to prefer egg-laying in alcohol enriched media even in the absence of predator. In addition, the altered food preference behavior in response to an anticipated predator-threat persists for up to five generations. These interesting observations set us to understand the mechanisms for predator-threat induced social learning in terms of (i) cellular and molecular signals, which trigger apoptosis in developing oocytes of wasp-exposed females, and (ii) epigenetic reprogramming in exposed flies that alters germline information conferring transmission of behaviors to subsequent generations. While the current proposal questions the classical conception of learning, the outcomes of this study in terms of the neurophysiological basis for inheritance of social behaviors will radically transform the way we think about learning, memory and behavior.

Learning to associate fear with stimuli predicting aversive outcomes is key for adaptation and survival. However, experiencing fear in maladaptive contexts can be crippling and disabling. Around 4.5% of the global population suffers from anxiety disorders such as post-traumatic stress disorder, generalized anxiety disorder and specific phobia. One of the most influential ways to overcome feared associations is extinction, which consists of exposure to previously feared stimuli in neutral circumstances, thus reducing the fear response. Extinction, frequently manipulated in the laboratory context, putatively stands at the base of effective psychological exposure therapy. In this proposal, I plan to adapt a new experimental paradigm, targeted memory reactivation (TMR), to strengthen fear extinction in humans. TMR, which has effectively facilitated several different types of memory, involves the subliminal presentation of previously associated cues during slow-wave sleep. Employing the lab's expertise in TMR procedures, I will try to strengthen extinction in a controlled setting and reveal the underlying neural mechanism by which TMR affects learnt memories using neuroimaging techniques (electroencephalogram, functional MRI) and electrophysiological recordings (electrocorticography). With this project, I aspire to bridge between the cognitive literature on the features and limitations of human memory and the more mechanistic animal-based literature on consolidation and memory formation. This can only be achieved by combining a well-controlled and manipulated behavioral paradigm together with state-of-the-art high-resolution brain-imaging techniques.

Recent genome-wide methods of mapping functional DNA elements have uncovered a vast landscape of genomic loci that have the characteristics of enhancers. However, the identification and mapping of this complex putative enhancer landscape does not link specific genes to their enhancers, nor does it prove any causal relation between the putative enhancers and gene activation or repression. In this project I will combine a powerful screening platform that harnesses CRISPR-cas9-based screens to test the function of hundreds of enhancers, in isolation and combination, which putatively regulate gene expression of genes associated with ricin sensitivity. In the first part of the project I will identify all potentially "active" enhancers during the cell response to ricin. In the second stage I will generate a CRISPR-cas9 library to disrupt the activity of all enhancers in isolation or in designed combinations, and measure the change in the cell response to ricin. In the third phase I will focus my attention on dozens of individual enhancers that affect the cell response to ricin, and using ATAC-seq, RNA-seq and 4C-seq methods I will determine the regulatory mechanism for these individual enhancers. In the final part of the project I will build a model trying to explain enhancer behavior in gene regulation. This work will provide the first genome-wide, high-throughput investigation of enhancer function on gene regulation, and will begin to unravel the combinatorial effects of enhancers by modulating them in their native genomic location. In short, this work will put regulatory genomics on firm functional or mechanistic footing.

Precise and coordinated regulation of tyrosine phosphatases and kinases is crucial to mount an effective immune response and to prevent autoimmune diseases. Lyp, a lymphoid-specific phosphatase, is a negative regulator of T-cell receptor (TCR) signaling. Some studies point to a gain-of-function when Lyp interacts with the tyrosine kinase Csk, while others suggest that Lyp/Csk complex limits its activity. This project aims to elucidate how the spatiotemporal coordinated arrangement of Lyp, Csk, and the transmembrane adaptor phosphoprotein PAG to which both associate, down-regulates TCR signaling by exploiting the potential of super-resolution fluorescence microscopy. Super-resolution imaging has already proven to be a powerful tool to study the immunological synapse. Still, multiplexed interrogation of distinct target proteins remains challenging. I will implement a recently reported multiplexed super-resolution imaging technique named DNA-PAINT (Points Accumulation for Imaging in Nanoscale Topography) to image the target proteins with high spatial and temporal precision during T-cell activation using a single-laser. I will further develop a 3-color co-localization clustering analysis for unveiling the complex pathways involved in signaling termination. Given the strong association of a single-nucleotide polymorphism in the gene encoding Lyp with various autoimmune disorders, I will also address the regulatory mechanism involved in the disease-associated Lyp variant.

Large-scale monitoring of the brain demonstrates that neuronal computation arises from interactions of numerous neuronal populations. Yet, methods that allow for large-scale recording of activity at high spatial resolution and for extended time periods are lacking. To meet this challenge, we propose to design, develop and test novel large-scale neural interface devices that will allow long-term, high spatiotemporal resolution, stable recording and stimulation of neural activity by injecting polymers into the scalp. These devices, and the data generated by them, will be used to address key questions in systems neuroscience. We aim to investigate the coupling mechanisms of neural oscillations between functionally distinct brain regions, and identify how different oscillations generate unique spatial and temporal patterns of activity. We will establish electrical stimulation protocols to modulate the occurrence rate, power, and frequency of specific brain oscillations to be able to assess the function role of such oscillations. Lastly, we will combine these recording and stimulation interfaces in form of closed-loop electrophysiological experiments to be able to manipulate and observe the effect of these oscillations on physiological and behavioral levels. The ability to acquire, stimulate, and analyze neural network simultaneously from multiple brain regions will enhance comprehension of neural network processes and has implications for brain disorders characterized by disordered network function.

I seek to understand how the patterns of animal movement seen in nature emerge from the combined actions of many individuals. For group-living species, this process occurs over two scales of organization: individual decisions emerge into coordinated group movement, and the movements of groups drive population-level patterns of space use. To understand how these processes connect, we need to collect and analyze data at both of these scales, yet doing so within a single system has remained a challenge. I will address this challenge using the meerkats of the Kalahari Desert as a model system. Meerkats (Suricata suricatta) live in stable groups that remain together as they forage over large distances. Leveraging recent technology, I will collect data on the fine-scale movements and vocalizations of all individuals within meerkat groups. Through probabilistic modeling, I aim to determine what rules govern where individuals move, and how these rules give rise to coordinated group movement. Key questions include how meerkats’ calls allow groups to maintain cohesion and decide where to go, and whether certain individuals have disproportionate influence over group movements. At the group level, I will use movement and demographic data collected over 20 years at the Kalahari field site to determine what factors drive group movement, including habitat features, memory, and interactions with other groups, to understand how aggregate space-use patterns emerge. Bringing together a wealth of data from a long-term field study with newly-available high-resolution tracking methods will enable us to link the multiple spatial and temporal scales driving animal movement patterns in the wild.

The ability to precisely edit and manipulate the genome is critical for biological research and the treatment of human disease. The power of such tools is highlighted by advances in CRISPR-Cas9 mediated gene editing, but there is continual need to develop new tools. The discovery of diverse genome manipulating enzymes throughout nature highlights the importance of basic biological findings and the wealth of natural systems that may be amenable for repurpose in human cells.

One compelling biological system is the programmed genome rearrangement of the ciliate Oxytricha which involves the fragmentation and joining of over 225,000 DNA pieces to form functional genes. Remarkably, gene coding sequences are often scrambled and must be reordered during reassembly, a process thought to be mediated by RNA templates. This phenomenon warrants further investigation and hints at the existence of remarkable genome manipulating tools. Despite our knowledge of the outcome of rearrangement, how this process happens and the proteins involved is not well understood.

The objective of my research is to identify the key proteins involved in the programmed genome rearrangements of Oxytricha and to elucidate the molecular mechanism of this process.
I aim to:
1) characterize changes in the transcriptome during programmed genome rearrangement,
2) develop functional genetic tools to screen for required factors, and
3) to recapitulate genome rearrangement in vitro using cell extracts.

This work aims to elucidate the identity and basic biological properties of the enzymes involved in this process with the long-term objective to adapt and harness identified components for use in human cells.

Neurons are characterized by their unique morphology, which requires efficient sorting and transport of cargoes over long distances. One class of molecules that affect neuronal development, function and survival are neurotrophins. Neurotrophic growth factors are target tissue-derived cues that bind to their cognate receptors on the pre-synaptic membrane and are transported retrogradely to the soma, where they induce signalling events which control transcriptional programs governing neuronal differentiation and survival. Despite their importance in neuronal homeostasis, the regulation of uptake and transport of neurotrophins and related growth factors is presently unknown. Recently, the host laboratory reported that the tetanus neurotoxin requires the synaptic basement membrane protein nidogen for its entry and axonal transport in mammalian motor neurons (Bercsenyi K, et al., 2014). Importantly, the tetanus toxin is known to enter the same intracellular trafficking pathway entered by neurotrophins (Salinas S, et al., 2010). In this context, this proposal is aimed at investigating the question: Does the synaptic basement membrane play a role in regulating the uptake, transport and biological activity of neurotrophins and related growth factors? As part of the research plan, I intend to identify growth factors that bind to nidogens and other components of the basement membrane at the mammalian neuromuscular junction, genetically modulate this binding and study its effects on neurotrophic factor endocytosis and trafficking, with a view to understanding the physiological relevance of this biological process.

Immunogen specific elicitation of HIV-1 broadly neutralizing antibodies has been proposed as the key step of an AIDS vaccine. Unfortunately, such vaccination strategy remains elusive. Priming B cell receptor germline precursors with specific engineered immunogens has been already proposed to be a potential strategy to generate bNAbs. Hence, characterizing and identifying potential germline precursors of bNAbs in the B cell repertoire of patients that has been exposed to native HIV-1 or a HIV-1 mimicking immunogen for a short period of time is a necessary step for the iterative design of successful bNAbs generating HIV-1 immunogens. In this project, using the PGT121-class germline-targeting Env SOSIP trimer, I will isolate low-affinity binding germline precursors of the bNAb PGT121 via particulate presentation from samples from (1) HIV-1 freshly infected patients and from (2) HIV-1 vaccinated patients. Using single cell RNA-seq, I will compare the antibody sequence of the isolated PGT121-class germline precursors with the sequence of their corresponding mature bNAb PGT121. I aim to obtain unprecedented information about the potential generation of germline precursors of bNAbs in patients after short exposure to HIV-1. The information obtained in this project will be crucial to design vaccine immunogens that can generate immune protection against HIV-1.